Apis Mellifera) Produce Honey from Flowers of Tea Plants (Camellia Sinensis) Kieko Saito, Rieko Nagahashi, Masahiko Ikeda and Yoriyuki Nakamura

Advances and Trends in Agricultural Sciences Vol. 1

India . United Kingdom

Editor(s)

Dr. Ahmed Medhat Mohamed Al-Naggar,

Professor of Plant Breeding, Department of Agronomy, Faculty of Agriculture, Cairo University, Egypt Email: [email protected], [email protected], [email protected];

FIRST EDITION 2019

ISBN 978-81-934224-3-4 (Print) ISBN 978-93-89246-17-9 (eBook) DOI: 10.9734/bpi/atias/v1

______© Copyright 2019 The Author(s), Licensee Book Publisher International, This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Contents

Preface i

Chapter 1 Reproducible Agrobacterium-mediated Transformation of Nigerian 1-11 Cultivars of Tomato (Solanum lycopersicum L.) S. O. A. Ajenifujah-Solebo, I. Ingelbrecht, N. R. Isu and O. Olorode

Chapter 2 12-16 Honeybees (Apis mellifera) Produce Honey from Flowers of Tea Plants (Camellia sinensis) Kieko Saito, Rieko Nagahashi, Masahiko Ikeda and Yoriyuki Nakamura

Chapter 3 17-26 Bio-pesticidal Properties of Neem (Azadirachta indica) B. E. Agbo, A. I. Nta and M. O. Ajaba

Chapter 4 27-37 Postharvest Heat Treatments to Extend the Shelf Life of Banana (Musa spp.) Fruits P. K. Dissanayake

Chapter 5 38-44 Development and Properties of Green Tea with Reduced Caffeine Kieko Saito and Yoriyuki Nakamura

Chapter 6 45-59 Productivity of Some Hausa Potato Accessions (Solenostemon rotundifolius (Poir) J. K. Morton in Jos-Plateau Environment O. A. T. Namo and S. A. Opaleye

Chapter 7 60-64 Roots of Hydroponically Grown Tea (Camellia sinensis) Plants as a Source of a Unique Amino Acid, Theanine Kieko Saito and Yoriyuki Nakamura

Chapter 8 65-80 Genetic Variability of Sugarcane Clones as Affected by Major Endemic Diseases in Ferké, Northern Ivory Coast Yavo M. Béhou and Crépin B. Péné

Chapter 9 81-89 Riparian Buffer Strip Width Design in Semiarid Watershed Brazilian Victor Casimiro Piscoya, Vijay P. Singh, Jose Ramon Barros Cantalice, Sergio Monthezuma Santoianni Guerra, Moacyr Cunha Filho, Cristina dos Santos Ribeiro, Renisson Neponuceno de Araújo Filho and Edja Lillian Pacheco da Luz

Chapter 10 90-100 Phenotypic Plasticity: The Best Approach for Stress Selection Ciro Maia, Paulo Mafra de Almeida Costa, Cleverson de Freitas Almeida, Luiz lexandre Peternelli and Márcio Henrique Pereira Barbosa

Chapter 11 101-109 Abundance and Incidence of Zucchini (Cucurbita pepo L) Flies in the Korhogo Department of Northern Côte d’Ivoire and Pest Control Methods Used by Farmers Yalamoussa Tuo, Klana Kone, Michel Laurince Yapo and Herve Kouakou Koua

Chapter 12 110-122 Soluble Bases and CEC Variation across Undisturbed and Disturbed Coastal Forests in Tanzania Elly Josephat Ligate and Can Chen

Chapter 13 123-132 Surface Water Nitrogen Load Due to Food Production-Supply System in South Asian Megacities: A Model-based Estimation Syeda Jesmin Haque, Shin-ichi Onodera and Yuta Shimizu

Chapter 14 133-143 Nutrient Solution: Agronomic Characteristics and Quality of Strawberry Fruits Cultivated in Substrate Dalva Paulus and Anderson Santin

Preface

This book covers all areas of agricultural sciences and other related fields. The contributions by the authors include tomatoes, genetic transformation, GUS gene, tea, Camellia sinensis, flower, honey, bio- pesticides, efficacy, food production, neem, pesticides, banana, postharvest life, green tea, Hausa potato, roots, hydroponics, leaf scald, smut, pokkah boeng, agro-ecology, erosion, soil conservation, abiotic stress, root system, Saccharum spp., Zucchini, attacked fruits, coastal forests, forest ecosystem, nitrogen load, nutrient etc. This book contains various materials suitable for students, researchers and academicians in the field of agricultural sciences.

Chapter 1 Print ISBN: 978-81-934224-3-4, eBook ISBN: 978-93-89246-17-9

Reproducible Agrobacterium-mediated Transformation of Nigerian Cultivars of Tomato (Solanum lycopersicum L.)

S. O. A. Ajenifujah-Solebo1*, I. Ingelbrecht2,3, N. R. Isu4 and O. Olorode4

DOI:10.9734/bpi/atias/v1

ABSTRACT

This study was carried out to develop transformation protocol for the possible improvement of local cultivars of tomatoes in Nigeria using complete randomized design (CRD). The research was conducted at the Plant Biotechnology Centre, International Institute of Tropical Agriculture (IITA), Ibadan, Oyo State, Nigeria between May 2009 and December 2009. Seeds of three promising farmer preferred varieties of cultivars of tomatoes namely Ibadan local, Ife and JM94/46 were selected and cultivated in-vitro. Sterile cotyledon and leaf explants were transformed using Agrobacterium tumefaciens strain LBA4404 with plasmid (pOYE153). Transformed plants were analyzed using GUS assay and PCR methods. Results showed that leaf explants had higher transformation efficiency than cotyledon explants in the three cultivars. Ife cultivar had the best transformation efficiency in both explant types - leaf 42.5% and cotyledon 8.89%. Histochemical GUS assay of transgenic plants showed blue coloration in leaves, stems and roots. PCR analysis showed amplification of 600 bp fragments of GUS and nptII genes in the transgenic plants on 1.0% agarose gel. The GUS and nptII genes were successfully integrated into the three cultivars of tomatoes thereby providing a reliable transformation protocol for the genetic improvement of local cultivars of tomatoes for desirable traits such as longer shelf-life, pest and disease resistance, enhanced nutrients, higher soluble solids, etc. The GUS and nptII genes were successfully integrated into the three cultivars of tomatoes thereby providing a reliable transformation protocol for the genetic improvement of local cultivars of tomatoes for desirable traits such as longer shelf-life, pest and disease resistance, enhanced nutrients, higher soluble solids, etc.

Keywords: Nigeria; tomatoes; Agrobacterium tumefaciens; genetic transformation; GUS gene.

1. INTRODUCTION

Tomato is one of the most important vegetable crops grown all over Nigeria. It is the world’s largest vegetable crop after potato and sweet potato but it tops the list of canned vegetables. In Nigeria, tomato is regarded as the most important vegetable after onions and pepper [1]. Nigeria is the largest producer of tomatoes in tropical Africa, with an annual production of 1,504,670 tons out of the estimated annual production of 16.55 million tons in Africa [2]. A total area of one million hectares is reportedly used for tomato cultivation in Nigeria [3,4]. The use of tomato is about 18 percent of the average daily consumption of vegetables in Nigeria [5] and is the most popular vegetable crop in Nigeria dominating the largest area under production among vegetable crops [6].

A substantial volume of the tomatoes in Nigeria are usually transported over long distances from the Northern part of the country to other parts and from the hinter lands to towns and cities. In Nigeria, as most other developing countries, efficient storage, packaging, transport and handling techniques are ______

1National Biotechnology Development Agency, Abuja, Nigeria. 2International Institute of Tropical Agriculture, Ibadan, Oyo State, Nigeria. 3Department of Plant Biotechnology and Genetics, University Gent, Belgium. 4Department of Biological Sciences, University of Abuja, Abuja, Nigeria. *Corresponding author: E-mail: [email protected];

Advances and Trends in Agricultural Sciences Vol. 1 Reproducible Agrobacterium-mediated Transformation of Nigerian Cultivars of Tomato (Solanum lycopersicum L.)

practically non-existent for perishable crops [7] resulting in considerable loss of produce. Postharvest loss is a major challenge hampering tomatoes production in most developing countries [8]. Tomato being a perishable crop as a result of its high moisture content has short shelf life of about 48 hours [9] under tropical conditions. Specialised postharvest handling practices and treatment methods are needed in order to extend the shelf life of the crop after harvest [10]. Also cultivated tomatoes suffer from a myriad of problems ranging from diseases caused by bacteria, fungi, viruses and nematodes to post harvest losses due to biochemical processes. Therefore improvements such as longer shelf-life, resistance to biotic and abiotic stresses, nutrient enhancement, higher soluble solids, etc are desirable in the local cultivars of tomato. Losses of up to 50% can be recorded in tomatoes between the harvesting and consumption stages of the distribution chain in tropical countries [11] which is in line with estimates by Gustavo et al. [12] that between 49 and 80% of all agricultural commodities end up with the consumer whilst the remainder is lost. However, the introduction of genes that confer these qualities to commercial cultivars by conventional breeding techniques often encounters serious difficulties due to high incompatibility barriers to hybridization [13]. To overcome these problems certain more recent approaches of gene manipulation might be required.

The cultivated tomato (Solanum lycopersicum) has been a subject of research because of the commercial value of the crop and its potential of amenability to further improvement through genetic engineering strategy [14]. The tomato is an excellent model for both basic and applied research programs. This is due to it possessing a number of useful features, such as the possibility of growing under different cultivation conditions, its relatively short life cycle, seed production ability, relatively small genome (950 Mb), lack of gene duplication, high self-fertility and homozygosity, easy way of controlling pollination and hybridization, ability of asexual propagation by grafting and possibility to regenerate whole plants from different explants [15]. Advances in agricultural biotechnology have provided the opportunity to expand the genetic resources available for tomato improvement. The goals of tomato genetic engineering have been to protect the tomato crop from environmental and biological assaults and to improve the quality of tomato fruit in order to deliver greater value in processed tomato products or more healthful and attractive fresh fruit.

Well characterized ripening mutants, high density genetic maps, small genome size, short life cycle, efficient and stable transformation made tomato an excellent model for studying viruses, development and fruit ripening process through genetic modification [16,17]. Cold, heat and soil salinity are the major environmental factors that significantly affect the productivity and quality of tomato and other crops [18,19]. There is however paucity of documented work on the genetic improvement of Nigerian cultivars of tomato; such work that would provide the background work for the application of genetic engineering in solving these problems. This study gives the report of the genetic transformation of the leaf and cotyledon explants from three Nigerian cultivars with Agrobacterium strain LBA4404 (pOYE513) containing uidA (GUS) and nptII genes using established in-vitro regeneration protocols for the three Nigerian cultivars of tomatoes [20,21]. This protocol can provide insight for the production of improved and more stress tolerant local cultivars and therefore reduce the importation and introduction of new varieties that would have to be adapted to the local environment. There is therefore an urgent need to domesticate these technologies for the improvement of Nigerian indigenous cultivars of tomatoes that are already adapted to the local environment.

The choice of cultivars is based on agronomic studies carried out at the National Institute for Horticultural Research and Training (NIHORT). Ibadan local and Ife cultivars are farmer preferred varieties in the south-western part of Nigeria which are reported to be resistant to certain diseases and relatively high yielding [22,23]. ICS-Nigeria [24] also reported Ife cultivar to be high yielding with fruits and is a determinate bushy plant; and that other local cultivars are fairly resistant to virus, have round and irregularly shaped fruits but are soft and prone to cracking.

2. MATERIALS AND METHODS

2.1 In-vitro Cultivation of Tomato Seedling

Seeds of three promising farmer preferred varieties of cultivars of tomatoes namely Ibadan local, Ife and JM94/46 were obtained from the National Institute for Horticultural Research and Training

Advances and Trends in Agricultural Sciences Vol. 1 Reproducible Agrobacterium-mediated Transformation of Nigerian Cultivars of Tomato (Solanum lycopersicum L.)

(NIHORT), Ibadan, Nigeria. The seeds were sterilized in 3.5% (v/v) sodium hypochlorite (NaOCl) with a drop of Tween-20 for 20 min and were rinsed with sterile distilled water three times. Seeds were germinated on MS medium [25] with 30 gL-1 sucrose, 8 gL-1 agar gel at pH 5.8. Germination medium, 50-100 ml was dispensed into the culture jars and was autoclaved at 120°C at 15psi for 15 min and then allowed to cool and solidify. Each culture jar containing the germination medium was inoculated with ten sterilized seeds and were placed in the dark at 25±2°C for 3-5 d to germinate and then transferred to culture conditions of 16 h photoperiod with light intensity of 1500 lux for 10-13 d at the same temperature to produce the tomato seedlings.

2.2 Preparation of Agrobacterium tumefaciens Culture

Agrobacterium tumefaciens strain LBA4404 containing plasmid (pOYE153) consisting of kanamycin resistant gene (nptII -neomycin phosphotransferase II) as a selectable marker being controlled by the 35S Cauliflower mosaic virus (CaMV) promoter and β-glucuronidase (GUS) gene (UidA) as the reporter gene being controlled by the Cassava vein mosaic virus (CsVMV) promoter, with the nopaline synthase (nos) gene at the 3’ terminator end (IITA, Ibadan) was used for the transformation of the three local tomato cultivars. Agrobacterium tumefaciens [LBA4404 (pOYE153)] (unpublished) culture was initiated from pure glycerol cultures of the Agrobacterium stored at -80°C and grown in 5 ml cultures in Luria broth (LB) medium with no selection for two (2) nights in 250 ml Erlenmeyer flasks at 29°C, shaking on an orbit shaker at 250 rpm [26]. Single colonies from the 48 h cultures of Agrobacterium tumefaciens strain LBA4404 (pOYE153) were streaked on solid yeast extract broth (YEB) medium containing selective concentrations of rifampicin (100 mg/L) and kanamycin (100 mg/L) and grown for 24 h at 28°C in petri dishes. Colonies from the plate were scraped and re- suspended in 1 ml of sterile inoculation medium (IM) containing MS medium with 30 g L–1 sucrose, 100 μM acetosyringone (AS) and 10 μM 2-mercaptoethanol. The optical density at 600 nm (OD600) of the bacterial suspension was determined by spectrophotometry (DU 530 Beckman Coulter). The bacterial suspension had an OD600 = 3.8 and 1 ml was diluted with 3.8 ml of IM to obtain a concentration of OD600 = 1. This suspension was used for explants inoculation [27]. For genetic transformation experiments, the plasmid pOYE153 was utilized (unpublished).

2.3 Agrobacterium-mediated Transformation

Cotyledon and leaf pieces were excised from 10-13 d in-vitro seedling under aseptic conditions and the ends of each was cut off, sectioned into two halves at the mid-vein region and cut into 5x5 mm2 pieces explants to allow it adsorb the bacterial suspension. The explants were inoculated with Agrobacterium tumefaciens strain LBA4404 (pOYE153) by dipping into the bacterial suspension and continuously shaken on shaker (Labnet-Orbit LS) for 45 min. The bacterial suspension was pipetted out and the explants were blotted dry on a sterilized paper towel. The explants were placed with the abaxial surface of the leaf in contact with the co-cultivation medium in petri-plates. The co-cultivation medium (CCM) contained MS salts, MS vitamins, MS iron, 30 g L-1 sucrose, 8 g L–1 agar gel, 1.5 mg L–1 zeatin and 100 µM acetosyringone, at pH 5.8. The plates were wrapped with aluminum foil and incubated for 3–4 d at 25±2°C. After 4 days of co-cultivation, GUS assay was carried out on the explants to determine transient expression of the GUS gene.

2.4 Regeneration and Selection of Transformed Explants

After 3-4 d on the co-cultivation medium, the explants were washed in IM without acetosyringone and mercaptoethanol and then subcultured in callus induction medium (CIM) containing MS salts, MS vitamins, MS iron, 1.5 mg L–1 zeatin, 100 mg L–1 kanamycin, and 150 mg L–1 timetin (GlaxoSmithKline, Uxbridge, UK) at 25±2°C for 16 h photoperiod with light intensity of 1500 lux in the growth room [28]. Explants commenced callus development at the edges about 4 weeks after Agrobacterium inoculation. The explants were subcultured onto fresh CIM every two weeks. After about 8 wks in culture, well-developed calli with mass of shoot buds were transferred to callus shooting medium (CSM) containing MS salts, MS vitamins, MS iron, 30 g L–1 sucrose, 8 g L–1 agar gel, 1.0 mg L–1 zeatin, 100 mg L–1 kanamycin, and 150 mg L–1 timetin at pH 5.8. Elongated multiple shoots were excised from the calli and transferred to non-selective rooting medium containing MS

Advances and Trends in Agricultural Sciences Vol. 1 Reproducible Agrobacterium-mediated Transformation of Nigerian Cultivars of Tomato (Solanum lycopersicum L.)

salts, MS vitamins, iron, 15 g L–1 sucrose, 8 g L–1 agar gel and 0.1 mg L–1 NAA [29] at pH 5.8. After 10 –14 d, rooted shoots were selected as putative transgenic plants and were transferred to peet pellet and vermiculite medium in humidity chamber for 2 weeks before planting in top soil, also under humidity chamber and gradually acclimatized.

A B

Sterilization of Sterilization of seeds seeds

Germination of Germination of sterilized seeds sterilized seeds

Preparation of Preparation of explants explants

10-13 d

Culture in CM Agrobacterium MS medium with 100 uM infection 30-45 min mercapto, 100 uM AS, Ag OD600 = 1 3-4 wks

Culture in SRM Co-cultivation

2-3wks GUS Assay

Culture in RM Callus induction

10-14 d 4-6 wks GUS Assay

Hardening in peet Culture in pellet and vermiculite selective SRM

3-4 wks GUS Assay 10-14 d

Culture in RM Transfer to soil

10-14 d GUS Assay

Hardening in peet pellet and vermiculite

10-14 d

Transfer to soil

Fig. 1. (A) Schematic representation of tomato regeneration (B) Schematic representation of Agrobacterium-mediated transformation of Nigerian cultivars of tomato

Advances and Trends in Agricultural Sciences Vol. 1 Reproducible Agrobacterium-mediated Transformation of Nigerian Cultivars of Tomato (Solanum lycopersicum L.)

2.5 GUS Histochemical Assay

Histochemical assay to determine GUS activity was carried out at the callus, shooting and rooting stages to determine the expression of the GUS gene in the plants. Histochemical staining to detect β- glucuronidase (GUS) gene expression in the explants was carried according to [30] using 0.33 g ferricyanide and 0.04 g ferrocyanide mixed in 10 ml sterile distilled water and the addition of 40 µl of triton in a solution. In separate vacuum tubes, 1.39 g NaH2PO4 and 2.42 g of Na2HPO4 respectively were dissolved in sterile distilled water to prepare 50 ml stock solutions each. 10 ml GUS assay buffer was prepared consisting of 5 ml sodium phosphate solution (2 ml of NaH2PO4 solution and 3 ml of Na2HPO4 solution) and 5 ml of ferrous solution. To carry out the assay, 0.0125 X-Gluc (5-bromo-4- chloro-3-indolyl β-D-glucuronide) was dissolved in 100 µl of Dimethyl sulfoxide (DMSO – used under fume hood) and added to 2.5 ml of GUS assay buffer solution.

Placing the explants in eppendorf tubes, the GUS assay solution was added and placed in the oven at 37°C for 1-2 h. The solution was pipetted out and 70% ethanol was added to the explants and incubated in the ethanol for 4 d. The ethanol was replaced every day. The explants were observed under the microscope (Zeiss model Stemi 2000-C) for GUS activity. GUS assay was carried out on explants after co-cultivation with Agrobacterium for transient expression and on calli, shoots and roots of regenerated plants for putative expression.

2.6 PCR Analysis of Genomic DNA of Transgenic Plants

DNA was extracted from callus and different tomato tissues using established procedures [31]. Forward and reverse primers OLIV9 (5ʹ-GGTGATCGGACGCGTCG-3ʹ) and OLIV13 (5ʹ- CCGCTTCGCGTCGGCATC-3’) and ARAJI1k (5ʹ-ATGACTGGGCACAACAGACAATCGG-3ʹ) and ARAJI2k (5ʹ-CGGGTAGCCAACGCTATGTCCTGATA-3ʹ) were used for the amplification of UidA and nptII genes respectively in PCR reactions. PCR was carried out using Peltier thermal cycler-PTC200. According to a modified procedure of [32], 10 µl of PCR mixes contained 1.0 µl 10X reaction buffer (100 mM Tris pH 9, 15 mM MgCl2, 500 M KCl and 0.1% Gelatin), 0.8 µl dNTPs 200 mM, 1 µl of each forward and reverse primers 5 µM / µl primer, 1 µl of 20 ng/µl genomic DNA and 0.4 U/ ul red Taq polymerase (Sigma) and remaining water. Only one DNA sample and both forward and reverse primer were added to any single reaction. The thermocycling programme used for UidA was: one cycle (initial denaturing) at 96°C for 3 min; 30 cycles at 95°C for 1 min (denaturing); 60°C for 1 min (annealing); 72°C for 2 min and one cycle (final extension) at 72°C for 5 min, kept at 4°C. The nptII gene amplification program consists of 1 cycle at 94°C for 4 minutes (initial denaturing), then 35 cycles at 94°C for 30 seconds (denaturing), 60°C for 30 seconds (annealing), then 72°C for 30 seconds (extension) and kept at 4°C. Bands were resolved on 1% agarose gel at 100 V for 3 h and the size of the band was determined by comparison with λPst ladder (Bioline) loaded on adjacent gel tracks.

2.7 Agarose Gel Electrophoresis of DNA of Transgenic Plants

Using modified procedures of [33], 1.0% agarose gel was prepared by weighing 1.5 g of agarose powder and melting in 150 ml 1% TBE buffer (10x -10.8 g Tris-base; 5.5 g boric acid; 20 mM EDTA in 1 L) in the micro-wave (100°C) until completely dissolved. The gel was allowed to cool slightly to about 40°C by continuous stirring on the magnetic stirrer (Thermolyne Cimarec 2) and then poured into the gel tank to set with the combs fixed. Then, 2 µl of gel loading dye (bromophenol blue) was added to 3 µl of PCR product and spun down in the centrifuge to mix thoroughly. The amplified DNA samples of the transgenic plants were loaded on the gel; with λPst (Bioline) loaded on adjacent gel tracks to determine the size of the bands by comparison and allowed to run for 2-3 hr at 100 volts (Voltmeter EC 105). The gels were dipped into ethidium bromide solution (10 mg/ml) for 1 min; de- stained for 5-10 min in tap water and then visualized at 302 nm on UV transilluminator and photographed with UVP bioimaging system (GDS-800).

2.8 Statistical Analysis

This study on the establishment of transformation protocol for three cultivars of Nigerian tomatoes was carried out using complete randomized design (CRD) via an intermediate callus phase from leaf

Advances and Trends in Agricultural Sciences Vol. 1 Reproducible Agrobacterium-mediated Transformation of Nigerian Cultivars of Tomato (Solanum lycopersicum L.)

explants. The average of pooled data from triplicate experiments was used to obtain the transformation frequency and Chi square test was used to determine the dependence of the transformation frequencies on the explant type (age of explant).

3. RESULTS AND DISCUSSION

3.1 Agrobacterium-mediated Transformation of Nigerian Tomato Cultivars

Results of the transformation experiment using cotyledon explants showed that JM had the highest number of explants forming calli (29); followed by Ife (20) and IbL (19). Ife however had the highest shooting frequency of 40.0%; IbL 15.79% and JM 8.16%. The highest value of 2.33 for average number of plants per callus was recorded for IbL; and 0.43 and 0.25 for Ife and JM respectively. Ife had the highest transformation frequency at 8.99% (Table 1). Using the true leaves, IbL had the highest number of explants forming calli, followed by Ife and JM with values of 59, 55 and 37 respectively. The value of 2.41 was recorded as the highest for average number of shoots per callus in Ife; IbL 1.52 and JM 1.10. Ife cultivar had the highest shooting frequency with the true leaves at 69.01%, with JM and Ife having 56.76% and 47.46% respectively (Table 2). Ife cultivar had the highest shooting frequency in both true leaves (69.01%) and in the cotyledon explants (8.99%). Highest transformation frequency was also recorded with Ife at 42.5%. Transformation frequencies of 3.33-8.89% were recorded from cotyledon explants while frequencies of 23.8-42.5 was recorded from secondary or leaf explants.

Table 1. Transformation frequency of tomato cultivars with *GUS gene using cotyledon explants based on pooled data of three experiments

Cultivar No. of No. of No. of Shooting Av. Transformation explants explants shooting frequency plants/callus frequency (%) forming calli (%) calli (rooted plants) IbL 90 19 3(7) 15.79 2.33 3.33 Ife 90 20 8(3) 40.00 0.43 8.89 JM 90 29 4(1) 8.16 0.25 4.44

Table 2. Transformation frequency of tomato cultivars with *GUS gene in true leaves or secondary leaves explants based on pooled data of three experiments

Cultivar No. of No. of No. of shooting Shooting Av. Transformation explants explants calli frequency plants/callus frequency (%) forming (rooted plants) (%) calli IbL 90 59 28(43) 47.46 1.52 31.3 Ife 90 55 38(92) 69.01 2.41 42.5 JM 90 37 21(23) 56.76 1.10 23.8 *GUS, β-glucuronidase gene

Higher transformation efficiencies were recorded in true leaves than in cotyledon explants in these experiments, which agrees with the report of [34] that leaf explants showed higher organogenesis capacity (> 90%) than cotyledon explants. Higher transformation rates with tomato cotyledons than leaf was however reported by [26,35]. Rooted plants appeared morphologically true to type of tomato plants in the screen house and flowered.

3.2 Regeneration of Transformed Plants

The responses recorded with the various cultivars and the two explant types (cotyledon and leaves) used in the experiments indicate that plant regeneration is often genotype dependent. Other factors such as explants type, plant growth hormones and other environmental factors also play a critical role. This is the first report of the regeneration of transformed plants from these local cultivars and it would pave way for further research on these and other local tomato cultivars that requires improvement.

Advances and Trends in Agricultural Sciences Vol. 1 Reproducible Agrobacterium-mediated Transformation of Nigerian Cultivars of Tomato (Solanum lycopersicum L.)

Well developed calli were observed after about 6-8 wks on selective callus culture and Ife cultivar gave the highest shooting frequency in the two explants types used. Whole transformed plants were regenerated from the calli in about 4-5 months. [26] had opined that if there are no visible calluses or green bumps during the first three weeks of incubation, it was not worth continuing the experiment. In our experiments, visible callus growth was observed after about four to five weeks of incubation. Higher regeneration rate was obtained on the selective shoot regeneration medium containing 1 mg L-1 zeatin. The effectiveness of zeatin in the regeneration of kanamycin-resistant shoots was also reported by [36]. Although hypocotyls can be used for transformation, they are not as efficient in generating transgenic shoots and the shoots take longer to develop [26]. A regeneration frequency of 37-38% was reported [37] from six independent transformation experiments.

A B

C D

E F

Plate A. +ve GUS assay in leaf explant after 4 d co-cultivation Plate B. Shoots from transgenic callus (3 wks in XSRM4) – Ife Plate C. Seedlings from transgenic plants (6 wks in XSRM4) Plate D. GUS expression in leaves of independent transgenic plants (jbf) Plate E. GUS expression in stems - IbL Plate F. GUS expression in roots – Ife Plate G. Flowering transgenic tomato plant Key: b- Ibadan local; j– JM/94/46; f-ife

Advances and Trends in Agricultural Sciences Vol. 1 Reproducible Agrobacterium-mediated Transformation of Nigerian Cultivars of Tomato (Solanum lycopersicum L.)

3.3 GUS Histochemical Assay

Both cotyledon and leaf explants were susceptible to transformation by A. tumefaciens strain LBA4404 (pOYE), however higher numbers of GUS positive explants were recorded from leaf explants than cotyledons. Blue coloration was observed in all tissues of assayed regenerated transformed plants i.e. leaves, stems and roots; which was absent in the control. This indicates that the transgene was constitutively expressed in all parts of the transformed plants.

3.4 PCR Analysis of Transgenic Plants

The presence of transgenes in the transformed plants was further confirmed by the amplification of genomic DNA from thirteen transformed plants; originating from the calli of seven independent transformation events, using specific GUS and nptII primers. The agarose gel electrophoresis of the amplified DNA products is shown in Plate 1 and Plate 2. For expression of the amplified GUS gene (Plate 1), distinct bands of about 600 bp fragments are seen in all thirteen transformants (lanes 1 – 13) and the positive control on lane 15. No band was seen in the negative control on lane 14 in Plate 1. A 600 bp fragment of the amplified nptII gene was also present in all the transformed plants (Plate 2). The band was however absent in the positive control despite several PCR runs, probably due to low GUS gene expression or silencing. The two genes were expressed about the same loci and their amplification and resolution on gel is an indication of the presence of the genes in the transgenic plants and is. These results are a further confirmation of the constitutive expression observed in the histochemical GUS assay that showed blue coloration in the leaves, stems and leaves of the transformed plants.

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 M

805 bp

514 bp

Plate 1. Amplified GUS gene in transformed plants Lane M-Pst I Lambda marker; Lane 1-13-DNA of transgenic plants; Lane 14-ve control; Lane 15+ve control

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 M

805 bp

514 bp

Plate 2. Amplified nptII gene in transformed plants Lane M-Pst I Lambda marker; Lane 1-13-DNA of transgenic plants; Lane 14-ve control; Lane 15+ve control

Advances and Trends in Agricultural Sciences Vol. 1 Reproducible Agrobacterium-mediated Transformation of Nigerian Cultivars of Tomato (Solanum lycopersicum L.)

4. CONCLUSION

The GUS and nptII genes were successfully integrated into the three cultivars of tomatoes thereby providing a reliable transformation protocol for the genetic improvement of local cultivars of tomatoes for desirable traits such as longer shelf-life, pest and disease resistance, enhanced nutrients, higher soluble solids, etc.

ACKNOWLEDGEMENTS

This work was supported by the STEP-B Grant to the National Biotechnology Development Agency (NABDA). We wish to acknowledge the support of Prof. Bamidele O. Solomon, Director-General/CEO, NABDA; Dr. Olagorite Adetula (NIHORT) for providing seeds of the tomato cultivars; Mr. Femi Oyelakin for his technical assistance; Mrs. Oluwasoga for her assistance with statistical analysis of the data and Dr. (Mrs.) Nike Adeyemo for her invaluable support.

COMPETING INTERESTS

Authors have declared that no competing interests exist.

REFERENCES

1. Olaniyi JO, Akanbi WB, Adejumo TA, Ak OG. Growth, fruit yield and nutritional quality of tomato varieties. African Journal of Food Science. 2010;4(6):398-402. 2. FAOSTAT; 2011. Available:http://faostat.fao.org/site/567/DesktopDefault.aspx?PageID=567#ancor 3. Anonymous. Fertilizer use and management practices for crops in Nigeria. Federal Ministry of Agriculture, Water Resources and Rural Development, Lagos. Series No 2. 1989;163. 4. Bodunde JG, Erinle ID, Eruotor PG, Amans EB. Recommendation for the release of four heat tolerant tomato varieties. Paper Approved by the Professional and Academic board, IFAR, ABU, Zaria, Nigeria. 1993;165. 5. Olayide SO, Olatunbosun D, Idusogie EO, Abiagom JD. A quantitative analysis of food requirement, supplies and demand in Nigeria, 1968-1985. 1972;113. 6. Ramalan AA. Irrigation and environment: The state of research and development at the Institute for Agricultural Research, IAR, Samaru. NOMA Magazine. 1994;11:16-19. 7. Babalola DA, Makinde YO, Omonona BT, Oyekanmi MO. Determinants of post harvest losses in tomato production: A case study of Imeko-Afon local government area of Ogun state. Acta Satech. 2010;3(2):14-18. 8. Arah IK, Kumah EK, Anku EK, Amaglo H. An overview of post-harvest losses in tomato production in Africa: Causes and possible prevention strategies. Journal of Biology, Agriculture and Healthcare. 2015;5(16):78–88. 9. Muhammad RH, Bamisheyi E, Olayemi FF. The effect of stage of ripening on the shelf life of tomatoes (Lycopersicon esculentum) stored in the evaporative cooling system (E.C.S). Journal of Dairying, Foods & Home Sciences. 2011;30(4):299–301. 10. Arah IK, Ahorbo GK, Anku EK, Kumah EK, Amaglo H. Postharvest handling practices and treatment methods for tomato handlers in developing countries: A mini review. Advances in Agriculture; 2016. 11. Pila N, Gol NB, Rao TVR. Effect of post harvest treatments on physicochemical characteristics and shelf life of tomato (Lycopersicon esculentum Mill.) fruits during storage. American- Eurasian Journal of Agricultural & Environmental Science. 2010;9(5):470–479. 12. Gustavo BCV, Juan FMJ, Stella M, Maria ST, Aurelio LM, Jorge WC. Handling and preservation of fruits and vegetables by combined methods for rural areas. Technical Manual FAO Agricultural Services Bulletin 149, FAO, Rome, Italy; 2003. 13. Kaul M. Reproductive biology of tomato. In: Kalloo G. (Eds). Mono. in Theor Appl Genet, 14, Genetic Improvement of Tomato. Springer-Verlag, Berlin, Heidelberg, New York. 1991;1-9. 14. Evans DA. Somaclonal varaiation-genetic basis and breeding applications. Trends Genet. 1989;5:46-50.

Advances and Trends in Agricultural Sciences Vol. 1 Reproducible Agrobacterium-mediated Transformation of Nigerian Cultivars of Tomato (Solanum lycopersicum L.)

15. Bai Y, Lindhout P. Domestication and breeding of tomatoes: What have we gained and what can we gain in the future? Ann. Bot. 2007;100:1085–1094. 16. Pech JC, Bouzayen M, Latché A. Climacteric fruit ripening: ethylene-dependent and independent regulation of ripening pathways in melon fruit. Plant Science. 2008;175(1):114- 120. 17. Moore S, Vrebalov J, Payton P, Giovannoni J. Use of genomics tools to isolate key ripening genes and analyse fruit maturation in tomato. Journal of Experimental Botany. 2002;53(377): 2023-2030. 18. Movahedi S, Tabatabaei BS, Alizade H, Ghobadi C, Yamchi A, et al. Constitutive expression of Arabidopsis DREB1B in transgenic potato enhances drought and freezing tolerance. Biologia Plantarum. 2012;56(1):37-42. 19. Ali A, Muzaffar A, Awan MF, Din S, Nasir IA, Husnain T. Genetically modified foods: Engineered tomato with extra advantages. Adv. Life Sci. 2014;1(3):139-152. 20. Ajenifujah-Solebo SOA, Isu NA, Olorode O, Ingelbrecht I. Effect of cultivar and explants type on tissue culture regeneration of three Nigerian cultivars of tomatoes. Sustain Agri Res. 2013;2(3):58-64. 21. Ajenifujah-Solebo SOA, Isu NA, Olorode O, Ingelbrecht I, Abiade OO. Tissue culture regeneration of three Nigerian cultivars of tomatoes. Afr J. Plant Sci. 2012;6(14):370-375. 22. Badra T, Denton O, Anyim O. Tomato germplasm evaluation. National Institute for Horticultural Research and Training (NIHORT) Annual Report. 1984;22-23. 23. Anno-Nyako F, Ladunni A. Evaluation of tomato germplasm under field conditions for reaction to tomato virus disease. National Institute for Horticultural Research and Training (NIHORT) Annual Report. 1984;23-24. 24. Anonymous. Growing tomatoes in Nigeria. Commercial crop production guide series. A Publication of International Institute of Tropical Agriculture (IITA) Supported by United States Agency for International Development (USAID) Information and Communication Support for Agricultural Growth in Nigeria (ICS-Nigeria). 2000;1-4. Available:http://dx.doi.org/10.1016/0168-9525(89)90021-8 25. Moorashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant. 1962;15:473-497. 26. McCormick S. Transformation of tomato with Agrobacterium tumefaciens. Plant Tiss Culture Manual. B6: 1-9, Kluwer Academic Publishers, Netherlands; 1991. 27. Soma P, Sikdar SR. Expression of nptII marker and gus reporter genes and their inheritance in subsequent generations of transgenic Brassica developed through Agrobacterium-mediated gene transfer. Plant Mol. Cell Genet, Bose Institute, P-1/12 CIT Scheme VII M, Calcutta 700 054, India; 1999. 28. Sun H, Uchii S, Watanabe S, Ezura H. A highly efficient transformation protocol for Micro-Tom, a model cultivar for tomato functional genomics. Plant Cell Physiol. 2006;47(3):426–431. 29. Davis DG, Breiland KA, Frear DS, Secor GA. Callus initiation and regeneration of tomato (Solanum lycopersicon) cultivars with different sensitivities to metribuzin. Plant Growth Reg Soc of America Quarterly. 1994;22:65-73. 30. Jefferson RA, Kavanagh TA, Bevan MW. GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 1987;6:3901-3907. 31. Dellaporta SJ, Wood J, Hicks JB. A plant DNA mini preparation: Version II. Plant Mol Biol Rep. 1983;1:19–21. 32. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning. A laboratory manual. (2nd Edition). Cold Spring Harbor; Cold Spring Harbor Laboratory Press. 1989;23-44. 33. Rajput SG, Wable KJ, Sharma KM, Kubde PD, Mulay SA. Reproducibility testing of RAPD and SSR markers in Tomato. Afr J Biotech. 2006;5(2):108-112. 34. Majoul H, Gharsallah-Chouchane S, Gorsane F, Fakhfakh H, Lengliz R, Marrakchi M. In-vitro regeneration plants of two cultivated tomato Solanum lycopersicon Mill. Acta Hort (ISHS). 2007;758:67-71. Available:http://www.actahort.org/books/758/758_6.htm 35. Rashid R, Bal SS. Agrobacterium-mediated genetic transformation of tomato (Solanum lycopersicum L.) with Cry1Ac gene for resistance against fruit borer. J Trop Agric. 2011;49(1-2): 110-113.

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36. Song G, Walworth A. Agrobacterium tumefaciens-mediated transformation of Atropa belladonna. Plant Cell Tiss Org Cult. 2013;15:107-113. DOI: 10.1007/s11240-013-0342-y 37. Pino LE, Lombardi-Crestana S, Azevedo MS, Scotton DC, Borgo L, Quecini V, Figueira A, Peres LEP. The Rg1 allele as a valuable tool for genetic transformation of the tomato “Micro- Tom” model system. Plant Methods. 2010;6:23.

Biography of author(s)

Dr. S. O. A. Ajenifujah-Solebo National Biotechnology Development Agency, Abuja, Nigeria.

She is presently a Deputy Director at the National Biotechnology Development Agency (NABDA), Abuja, Nigeria where she established a functional tissue culture laboratory with temporary immersion bioreactor system (TIBS) for mass production of elite planting materials. The laboratory has established in-vitro regeneration protocols for fruit and tree crops, and other indigenous species. She obtained her Ph.D. in Microbiology (Plant Biotechnology) from the prestigious University of Abuja with research in the in-vitro regeneration and Agrobacterium-mediated transformation of some Nigerian cultivars of tomatoes. She is a member of the Nigerian Institute of Food Science & Technology, Biotechnology Society of Nigeria and the American Society of Microbiology. She has pioneered different research ranging from tissue culture regeneration of Solanum lycopersicum, development of agrobacterium-mediated transformation protocol for local cultivars of tomato, to the development & evaluation of Maize-“Oncom” mixes as a “Tuwo” meal. She has a very vast knowledge in GMO testing and analysis, and molecular biology. She has hosted meetings/workshops with grants from International Centre for Genetic Engineering and Biotechnology (ICGEB). She has attended scientific trainings in USA, India, Italy, South Africa, Belgium, Nigeria. In 1998 she worked with the team of professionals that implemented the National Science and technology Policy on agriculture and natural sciences including biotechnology. She is currently developing the capacity of the Agricultural Biotechnology Department in NABDA for molecular biology research in agriculture. Her current research is on the improvement of local cultivars of tomato using genetic engineering. ______© Copyright 2019 The Author(s), Licensee Book Publisher International, This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

DISCLAIMER This chapter is an extended version of the article published by the same authors in the following journal with CC BY license. American Journal of Experimental Agriculture, 4(7): 797-808, 2014.

Reviewers’ Information (1) Anonymous, Egypt. (2) Anonymous, India.

Chapter 2 Print ISBN: 978-81-934224-3-4, eBook ISBN: 978-93-89246-17-9

Honeybees (Apis mellifera) Produce Honey from Flowers of Tea Plants (Camellia sinensis)

Kieko Saito1,2*, Rieko Nagahashi3, Masahiko Ikeda3 and Yoriyuki Nakamura2

DOI:10.9734/bpi/atias/v1

ABSTRACT

We obtained honey from the blooming flowers of tea plants (Camellia sinensis L.) pollinated by honeybees (Apis mellifera L.). Functional amino acids, theanine, which is a unique ingredient to tea, was determined using reversed-phase chromatography. We also determined the main ingredients: caffeine and catechins. The obtained honey contained theanine, which shows that it was derived from tea flowers. The theanine concentration of the nectar of the tea flowers exceeded that of the honey. Caffeine was detected (but no catechins) in both the honey and the nectar of the tea flowers. Our results refute the previously held view that tea nectar is toxic to honeybees. Our new finding reveals that it is possible to obtain honey from the nectar of tea flowers. The obtained honey and the nectar of tea flowers contained a very rare amino acid, theanine, indicating that the honey was derived from tea flowers. Furthermore, the nectar of tea flower contained the best caffeine concentration that activated the brain function of honeybees to produce the honey.

Keywords: Tea; Camellia sinensis; theanine; flower; honey.

1. INTRODUCTION

Green tea (Camellia sinensis L.) leaves provide beneficial effects for human health, and the functions of the main components of their leaves have been widely studied [1]. Recently several physiological functions (e.g. antioxidant, antimicrobial, immunomodulatory and antitumor activities) of tea flowers have been reported [2-5], and the flowers have received attention as a natural healthy material for food and cosmetics. The health-promoting effects of green tea are mainly attributed to its polyphenol content [6], particularly flavanols and flavonols, which represent 30% of fresh leaf dry weight [7]. It is not well known that the fragrant tea flowers have sweet nectar. The tea nectar may be attractive to honeybees. One study of bee pollen collected from the flowers of tea plants suggests that honeybees like the pollen of tea (Camellia sinensis L.) [8]. However, the honey from tea flowers has not been studied, even though in autumn, many tea fields are filled with blooming flowers in almost all the tea production areas around the world. The most utilized part of the tea plant is the leaves. Thus, less attention has been paid to tea flowers. Since the application of asexual propagation to tea plants, tea flowers have become a “waste resource”, competing with tea leaves for water and nutrients. To promote the yield and quality of tea leaves, some chemicals, such as ethephon and α-naphthalene acetic acid, have been used to suppress tea plant blossoming [9], Sharma et al. reported that tea nectar exhibited toxicity to honeybees (Apis mellifera L.) [10]. Healthy broods and larvae were fed the nectar of tea flowers in the laboratory and were killed. Sharma’s report discouraged beekeepers from harvesting the honey of tea flowers whose nectar might have been toxic to physiologically immature broods and larvae, even though they could eat the nectar by themselves. Some other workers also reported toxic nature of the Camellia sinensis nectar [11,12,13].

It remains unclear whether tea nectar is toxic to honeybees. In this study, we took actual tea honey from the flowers to investigate whether the honeybees collected tea nectar to produce honey. To ______

1Institute for Environmental Sciences, University of Shizuoka, Yada, Shizuoka 422-8526, Japan. 2Tea Science Center, University of Shizuoka, Yada, Shizuoka 422-8526, Japan. 3Faculty of Social Environment, Tokoha University, Yayoi, Shizuoka 422-8581, Japan. *Corresponding author: E-mail: [email protected];

Advances and Trends in Agricultural Sciences Vol. 1 Honeybees (Apis mellifera) Produce Honey from Flowers of Tea Plants (Camellia sinensis)

determine whether the honey was derived from tea flowers, theanine (γ-ethylamide-L-glutamic acid), which is a specific amino acid of tea plants [14-17]. Furthermore, we investigated the concentration of catechin and caffeine, which are the main ingredients in tea plants. We also analyzed the theanine, the catechin, and the caffeine of the tea nectar to compare them with the obtained honey.

2. MATERIALS AND METHODS

2.1 Beekeeping

We used honeybees (Apis mellifera L.) to obtain honey from tea flowers according to Japan’s beekeeping association’s manual [18]. The honey was collected from September to November 2013 around tea fields. Samples were obtained from individual beehive cells with pipettes.

2.2 Plant Materials

Tea plants (Camellia sinensis L.) were cultured in hydroponics to obtain the nectar of tea flowers in quality and quantity [19]. The plants were cultured in a nutrient solution under controlled condition for several months until the tea flowers bloomed [20]. The nectar of the tea flowers was carefully collected with pipettes at the bottom of pistil just after blooming and kept at 4°C until it was used.

2.3 Analytical Reversed-phase High-performance Liquid Chromatography (HPLC)

We determined the theanine, catechin, and caffeine content of the honey or nectar using an Agilent 1100 HPLC system (Agilent Technologies, Palo Alto, Calif.) that was equipped with a C18 column (4.6 i.d. x 150 mm, 5 μm, Tokyo Chemical Industry Co. Ltd., Tokyo, Japan) [20]. The HPLC column was maintained at 30°C in an oven. The mobile phase for the detection was 0.1 M NaH2PO4 buffer/acetonitrile (87:13) at a flow rate of 1.0 ml/min.

Each peak was identified by comparing the UV-Vis spectral characteristics and retention times with those from commercial standards supplied by Wako Pure Chemicals Industry, Ltd., Japan.

2.4 Statistical Analysis

Data are expressed as mean ± standard deviation. Analyses were performed using Student’s t-test (Microsoft Excel Version 14.5.2) for comparison between honey and nectar.

3. RESULTS AND DISCUSSION

We collected actual honey from tea flowers that contained theanine, which is a very rare amino acid and ingredient of green tea that has only been found in several camellia species and one mushroom, Xerocomus badius [21,22]. Bees normally continue flying in a 3 km area to collect flower nectar, although during this experiment, there were no plants with theanine in the vast area around the beehives. Theanine was detected from the honey collected in our experiment, and the nectar of the flowers also included theanine, indicating that it was actually derived from the tea flowers. Honeybees, especially, Apis mellifera L., tend to collect the nectar of a single species of flower, such as acacia and lotus. We placed beehives in the middle of a vast expanse of a tea field, so the honeybees could collect the nectar of tea flowers. Recently, Wright et al. [23] reported that caffeine appears to have a secondary advantage that attracts honeybees and enhances their long-term memory [24], which suggests that honeybees learn to seek the nectar of flowers that possess caffeine. They also argued that 0.1 mM (0.019 mg/mL) of caffeine activated the brains of honeybees, supporting the data of Table 1 where the tea nectar included about 0.02 mg/mL of caffeine. Such definite evidence suggests that honeybees collect nectar from tea plants. Caffeine tastes bitter to mammals and is toxic and repellent to pollinators at high doses; however, tea nectar, which includes a low dose of caffeine, attracts honeybees to it. Even though Sharma et al. demonstrated the toxicity of tea nectar, they failed to experimentally show that it affected adult honeybees; it only affected the broods and larvae. In addition, their nectar was derived from pollen collected by adult honeybees [10]. The tea nectar obtained in this study did not include catechins (Table 1), but the pollen included catechins (0.5 mg/g) and caffeine (0.345 mg/g) [25], where the LD50 values for a rat (oral) are 2 g/kg and 192 mg/kg, respectively [26]. Catechins and caffeine in tea pollen are probably nontoxic for mammals. However, their LD50 values in honeybees are unclear because no data exists for them. Catechins and/or the

Advances and Trends in Agricultural Sciences Vol. 1 Honeybees (Apis mellifera) Produce Honey from Flowers of Tea Plants (Camellia sinensis)

caffeine of the pollen may affect honeybees, especially broods, larvae, and immature bees, even though the tea nectar did not include catechins. Recent reports suggest that such agricultural chemicals as pesticides, herbicides, and fungicide pollute pollen and nectar and kill honeybees [27- 32]. In this study, after obtaining honey from tea flowers, we conclude that the nectar of tea flowers is attractive to honeybee, but not toxic. Our new finding, which presents significant information on the relationship of honeybees (Apis mellifera L.) and tea flowers, might activate tea and beekeeping industry, leading to develop the production of honey from tea nectar. Moreover, the honey from tea flower might be a novel honey with additional function.

Table 1. Concentration of main ingredients of the tea nectar and the obtained honey

4. CONCLUSION

In this study, we showed honeybees produced honey from flowers of tea plants. The obtained honey and the nectar of tea flowers contained a very rare amino acid, theanine, indicating that the honey was derived from tea flowers. Furthermore, the nectar of tea flower contained the best caffeine concentration that activated the brain function of honeybees to produce the honey.

COMPETING INTERESTS

Authors have declared that no competing interests exist.

REFERENCES

1. Eto H, Tomita I, Shinmura J, Isemura M, Hara M, Yokogoshi H, Yamamoto M, Editors. Health science of tea (Cha no Kinou); 2013. No-Bun-Kyo, Tokyo (in Japanese). 2. Yoshikawa M, Morikawa T, Yamamoto K, Kato Y, Nagatomo A, Matsuda H. Floratheasaponins A–C, acylated oleanane-type triterpene oligoglycosides with anti -hyperlipidemic activities from flowers of the tea plant (Camellia sinensis). J. Nat. Prod. 2005;68:1360–1365. 3. Xu R, Ye H, Suna Y, Tu Y, Zeng X. Preparation, preliminary characterization, antioxidant, hepatoprotective and antitumor activities of polysaccharides from the flower of tea plant (Camellia sinensis). Food Chem Toxicol. 2012;50:2473-2480. 4. Matsuda H, Nakamura S, Morikawa T, Muraoka O, Yoshikawa M. New biofunctional effects of the flower buds of Camellia sinensis and its bioactive acylated oleanane-type triterpene oligoglycosides. J. Natural Med. 2016;70:689–701. 5. Chen Y, Zhou Y, Zeng L, Dong F, Tu Y, Yang Z. Occurrence of functional molecules in the flowers of tea (Camellia sinensis) plants: Evidence for a second resource. Molecules. 2018;23:790. 6. Naghma K, Hasan M. Tea polyphenols for health promotion. Life Sciences. 2007;81:519-533. 7. McKay DL, Blumberg JB. The role of tea in human health: An update. J Am Coll Nutr. 2002;21: 1-13. 8. Lin H, Chang SY, Chen SH, Lin S. The study of bee-collected pollen load in Nantou, Taiwan. Taiwania. 1993;38:117-133. 9. Lin YS, Wu SS, Lin JK. Determination of tea polyphenols and caffeine in tea flowers (Camellia sinensis) and their hydroxyl radical scavenging and nitric oxide suppressing effects. J. Agric. Food Chem. 2003;51:975–978. 10. Sharma OP, Raj D, Garg R. Toxicity of nectar of tea (Camellia Thea L.) to honeybee. J. Apicultural Res. 1986;25:106-108. 11. Atkins EL. Injury to honey bees by poisoning from the hive and the honey bee. Eds Dadant & Sons, Inc. Hamilton, IL, USA: Dadant & Sons, Inc. 1975;665-696. 12. Maurizio A. How bees make honey from honey: A comprehensive survey. Ed. E. Crane. London: Heinemann in cooperation with International Bee Research Association. 1975;96-97. 13. Majak W, Neufeld R, Corner J. Toxicity of Astragalus miser v. serotinus to the honeybee. J. Apic. Res. 1980;19:196-199.

Advances and Trends in Agricultural Sciences Vol. 1 Honeybees (Apis mellifera) Produce Honey from Flowers of Tea Plants (Camellia sinensis)

14. Juneja LR, Chu DC, Okubo T, Nagato Y, Yokogoshi H. L-theanine—a unique amino acid of green tea and its relaxation effect in humans. Trends in Food Sci. & Tech. 1999;10:199-204. 15. Kimura K, Ozeki M, Juneja LR, Ohira H. L-Theanine reduces psychological and physiological stress responses. Biol. Psychol. 2007;74:39–45. 16. Unno K, Fujitani K, Takamori N, Takabayashi F, Maeda K, Miyazaki H, Tanida N, Iguchi K, Shimoi K, Hoshino M. Theanine intake improves the shortened lifespan, cognitive dysfunction and behavioural depression that are induced by chronic psychosocial stress in mice. Free Radic. Res. 2011;45:966–974. 17. Vuong QV, Bowyer MC, Roach PD. L-Theanine: Properties, synthesis and isolation from tea. J. Sci. Food Agric. 2011;91:1931–1939. 18. Japanese Society for Honeybees. A mannual for apiculture. Japanese Council for Beekeeping, Tokyo, Japan; 2011. 19. Saito K, Ikeda M. The function of roots of tea plant (Camellia sinensis) cultured by a novel form of hydroponics and soil acidification. Am. J. Plant Sci. 2012;3:646-648. 20. Saito K, Furue K, Kametani K, Ikeda M. Roots of hydroponically grown tea (Camellia sinensis) plants as a surce of a unique amino acid, theanine. Am. J. Exp.Agr. 2014;4:125-129. 21. Casimir J, Jadot J, Renard M. Separation and characterization of N- ethyl-g-glutamine in Xerocomus badius (Boletus ladius). Biochim. Biophys. Acta. 1960;39:462–468. 22. Wei-Wei D, Shinjiro O, Hiroshi A. Distribution and biosynthesis of theanine in Theaceae plants. Plant Phys. Biochem. 2010;47:70-72. 23. Wright GA, Baker DD, Palmer M, Stabler JD, Mustard JA, Power EF, Borland AM, Stevenson PC. Caffeine in floral nectar enhances a Pollinator's Memory of Reward. Science. 2013;339: 1202-1204. 24. Chittka L, Peng F. Cafffeine boosts bees’memories. Science. 2013;339:1157-1159. 25. Ueno J, Konishi S, Ishikawa F. Caffeine and catechins in tea pollens. Japanese J. Palynol. 1985;31:39-43. 26. SAFTY DATA SHEET [Internet]. Cayman Chemical Company. [Cited 2019 May 25] Available:https://www.caymanchem.com/msdss/70935m.pdf 27. Balayiannis G, Balayiannis P. Bee honey as an environmental bioindicator of pesticides’ occurrence in six agricultural areas of Greece. Arch Environ Contam Toxicol. 2008;55:462–470. 28. Spivak M, Mader E, Vaughan M, et al. The plight of the bees. Environ. Sci. Technol. 2011;45:34-38. 29. Blacquière T, Smagghe G, van Gestel CA, Mommaerts V. Neonicotinoids in bees: A review on concentrations, side-effects and risk assessment. Ecotoxicol. 2012;21:973–992. 30. Zhelyazkova I. Honeybees – bioindicators for environmental quality. Bulgarian J. Agricul. Sci. 2012;18:435-442. 31. Rundlof M, Andersson GKS, Bommarco R, Fries I, Hederstrom V, Herbertsson L, Jonsson O, Klatt BK, Pedersen TR, Yourstone J, Smith HG. Seed coating with a neonicotinoid insecticide negatively affects wild bees. Nature. 2015;521:77-80. 32. Hesselbach H, Scheiner R. The novel pesticide flupyradifurone (Sivanto) affects honeybee motor abilities. Ecotoxicol. 2019;28:354–366.

Biography of author(s)

Dr. Kieko Saito Institute for Environmental Sciences, University of Shizuoka, Yada, Shizuoka 422-8526, Japan and Faculty of Social Environment, Tokoha University, Yayoi, Shizuoka 422-8581, Japan

Advances and Trends in Agricultural Sciences Vol. 1 Honeybees (Apis mellifera) Produce Honey from Flowers of Tea Plants (Camellia sinensis)

She is the Assistant Professor of School of Food and Nutritional Sciences, University of Shizuoka, Shizuoka, Japan. She received her master degree from Graduate School of Agriculture, Nihon University in 1990. After working at RIKEN (Saitama, Japan) and Gerontology Research Center, NIH (USA) as a research associate, she started her career at the University of Shizuoka in 1996. She has Been at present position since 2008. In 2008, she received her PhD degree based on the thesis of Oxidative stress and Aging in 1991 from Nihon University. Her specialization is in Functional Food and Environmental Science. She joined Tea Science Center of University of Shizuoka in 2014 to assist research related with the tea industry. Her current research interests center on the physiological function of fermented tea and honey from tea flower (Camellia sinensis).

Dr. Yoriyuki Nakamura Tea Science Center, University of Shizuoka, Yada, Shizuoka 422-8526, Japan

He is the project professor and director of Tea Science Center, University of Shizuoka, Shizuoka, Japan since 2013. He graduated from Graduate School of Agriculture, Iwate University in March 1979 and joined the Shizuoka prefectural government in April. Worked at Shizuoka Tea Research Center and Shizuoka Research Institute of Agriculture & Forestry for 36 years. During this period, he obtained his PhD from Gifu University in 2006 and became the director of Shizuoka Tea Research Center in 2008. His specialization is in tea propagation and breeding. Given the Japanese Society of Tea Science and Technology Award in 1991 and The Society of Tea Science of Japan Award in 2013. He is also an international expert commissioner to evaluate tea quality. ______© Copyright 2019 The Author(s), Licensee Book Publisher International, This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Reviewers’ Information (1) Ronaldo De Carvalho Augusto, Oswaldo Cruz Institute, Fiocruz, Brazil. (2) Bozena Denisow, University of Life Sciences in Lublin, Poland. (3) Adalberto Alves Pereira Filho, Universidade Federal de Minas Gerais, Brazil.

Chapter 3 Print ISBN: 978-81-934224-3-4, eBook ISBN: 978-93-89246-17-9

Bio-pesticidal Properties of Neem (Azadirachta indica)

B. E. Agbo1*, A. I. Nta2 and M. O. Ajaba3

DOI:10.9734/bpi/atias/v1

ABSTRACT

Consequence upon the geometrically rising world population and the increasing pressure on food items, it has become increasingly necessary to increase food production from the present level. The possibility of achieving this is not only to increase production but also to protect the crops cultivated. Crop protection can be achieved through several means. One of such is the use of pesticides. This paper therefore reviews the use of neem extracts as bio-pesticides among other plant species with inherent pesticidal activities. It is no doubt that the chemical pesticides or insecticides possess inherent toxic substances that endangers the ecological environment, operators of application equipment, soil microbiota and consumers of the agricultural products. It is therefore important that we encourage the use of biological pesticides as they affect only target pest, are easily biodegradable, increase farm land fertility, environmentally friendly, cost effective and ease of availability. It is also important that because of the low cost of production of bio-pesticides it should be encouraged as an option in African countries especially Nigeria in agricultural practices. The practice of farmers making their own neem-based products for pest control would reduce their dependence on external inputs for agriculture. It would also reduce their cost of pest control to almost zero, leaving only labour as a potential expenditure item. Pests can also be controlled without the use of toxic chemical pesticides, which will reduce the harm posed to humans and the environment alike. There is wide scope for innovation in developing neem as an efficient bio-pesticide. There is enough information to encourage the use of different neem extracts. With the increasing trend of using bio fertilizers, insecticides and pesticides, neem should be increasingly cultivated and grown all over the world to get active ingredient-azadirachtin, responsible for stopping the growth cycle of pests. Neem is also assuming a lot of importance in crop management. Considering the fact that neem is not only a cheaper, naturally occurring product and an effective method to control pests and insects, but also has no side effects on plants or other living beings especially soil micro biota.

Keywords: Bio-pesticides; efficacy; food production; neem; pesticides.

1. INTRODUCTION

Pesticides are substances or mixture of substances used to prevent, destroy, repel, attack, sterilize, or mitigate pests. The heavy use of these chemicals has already caused grave damage to health, ecosystems, soil micro biota and ground water. It is therefore increasingly urgent that environment friendly methods of improving soil fertility and pests and disease control are used [1,2]. Bio-pesticides are a type of pesticide derived from natural materials as animals, plants, bacteria, and certain minerals [2]. Although chemical pest control agents are extensively used in all countries of the world but they are regarded as ecologically unacceptable. Bio-pesticides or biological pesticides based on pathogenic microorganisms are specific to a target pest, offer an ecologically sound and effective solution to pest problems [4].Therefore, there is an increased social pressure to replace them gradually with bio-pesticides which are safe to humans and non-target organisms [5]. The neem tree (Azadirachta indica) is indigenous to India, it belongs to the family maliceae. All the parts of the neem

______

1Department of Microbiology, University of Calabar, P. M. B. 1115, Calabar, Nigeria. 2Department of Zoology and Environmental Biology, University of Calabar, P. M. B. 1115, Calabar, Nigeria. 3Department of Science laboratory Technology, University of Calabar, P. M. B. 1115, Calabar, Nigeria. *Corresponding author: E-mail: [email protected], [email protected];

Advances and Trends in Agricultural Sciences Vol. 1 Bio-pesticidal Properties of Neem (Azadirachta indica)

tree is medicinal [6,7,8,9,10]. The neem tree is an incredible plant that has been declared the “Tree of the 21st century” by the United Nations (UNEP, 2012). The US National Academy of Science published a report in 1992 entitled “Neem: A tree for solving global problems” (National Academy of Science, 1993). It can easily grow to an average height of 15-20m but rarely to 35-40m. Neem grows on altitudes up to 1500 m [11]. It can grow well in wide temperature range of 0 to 49°C [12]. It is evergreen but under severe drought it may shed most or nearly all of its leaves. For thousands of years the beneficial properties of neem have been recognized in the Indian tradition [13]. It is known to co-exist with other vegetation but deleterious to insects. Both the bark and leaves also contain biologically active molecules but not high levels of azadirachtin which is found mainly in the seed kernels [14]. Both leaves and fruit of neem plant are known to have bitter taste having fungicidal, insecticidal and nematicidal properties [15]. Indians have revered the neem tree for a very long time. For centuries, millions have cleaned their teeth with neem twigs, smeared skin disorders with neem leaf juice, taken neem tea as a tonic and placed neem leaves in their beds, books, grain bins, cupboards, closets to keep away troublesome bugs [16]. To millions of Indians, neem has miraculous powers. Indian farmers have kept away insects with different neem extracts. The tree is considered so invaluable that it is found in every part of the country, every roadside, every field and almost every house. Indian farmers used neem leaves and seed for the control of stored grain pests [17]. India has shared its “free tree” and knowledge of its utilisation with the world community. It is because of its tremendous therapeutic, domestic, agricultural and ethno-medical significance, and its proximity with human culture and civilization, that it has been called ‘’the wonder tree’’ and ‘’nature’s drug store’’ [6,18]. Numerous plant species in the family Meliaceae exhibit promising bioactivity against a variety of insects, only neem extract is approved for use and sold as a botanical insecticide in the USA [19]. The oil and purified product of every part of the tree, particularly the leaves, bark, seed are widely used for treatment of cancer, bacterial and fungi infections [18]. Over 60 different types of biochemical products including, Nimbolide, Margolone, Mahoodin, Margolonone have been purified from neem [20, 21]. Several active chemical compounds are present in the plant, including glycosides, dihydrochalcone, coumarin, tannins, zadirachtin, nimbin, nimbidine, diterpenoids, triterpenoids, proteins, carbohydrates, sulphurous compounds, polyphenolics, among others [22]. This review outlines the current state of knowledge on the potential use of bio-pesticides in global control of pests.

2. BIO-PESTICIDES USE IN PEST CONTROL

The harmful environmental implications of the synthetic chemicals have compelled researchers to search for some alternative naturally occurring pest control agents: bio-pesticides. Bio-pesticides include a broad array of microbial pesticides, biochemically derived from micro-organisms, plant extracts and processes involving the genetic modification of plants to express genes encoding insecticidal toxins [23].

(1) Entomopathogenic fungi

The entomopathogenic fungi have potential as myco-insecticide against diverse insect pests attacking agricultural crops as they moderate the insect populations. These fungi infect their hosts by penetrating through the cuticle, gaining access to the hemolymph and producing toxins. They grow by utilizing nutrients present in the haemocoel to circumvent insect immune responses [24]. Example of fungal bio pesticides are Muscodor albus used in fields, greenhouses, and warehouses [25] and Aspergillus flavus targeted for Aedes fluviatilis and Culex quinquefasciatus [26,27]. Entomopathogenic fungi may be applied in the form of conidia or mycelium which sporulates after application. The use of fungal entomopathogens as alternative to synthetic insecticides or applied in combination could be very useful for insecticide resistant management [28].

The commercial myco-insecticide ‘Boverin’ based on Beauveria bassiana with reduced doses of trichlorophon have been used to suppress the second-generation outbreaks of Cydia pomonella (Ferron). Mordue and Nisbet [29] detected higher insect mortality when B. bassiana and sublethal concentrations of insecticides were applied to control Colorado potato beetle (Leptinotar sadecemlineata), attributing higher mortality rates to between the two agents synergism. The combined application of the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin and neem was experimented against sweet potato whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), on eggplant [30]. The combination of B. bassiana and neem yielded the highest

Advances and Trends in Agricultural Sciences Vol. 1 Bio-pesticidal Properties of Neem (Azadirachta indica)

mortality level of B. tabaci egg and nymph mortalities at alowest LT50 value. Therefore, neem was used along with B. bassiana suspension as an integrated pest management method against B. tabaci. Other insects that have been successfully control by the use of fungicides singly or in synergy with sub lethal doses of synthetic agents include cassava green mite (Mononychellus tanajoa), potato red spider mite, Ceratitis capitata and sweet potato whitefly.

(2) Viral bio pesticides

Before, World War 2, the first suppression of pest by viral bio-pesticides, baculovirus, occurred accidentally. Thereafter, viruses were used and studied widely as bio-pesticides in 1940s [31]. According to Mazid et al. [23], baculoviruses has been used along with a parasitoid imported from Canada to control Spruce sawfly, Diprion hercyniae using the Negative Predictive Value (NPV) for spruce sawfly.

At present, the number of registered viral bio-pesticides based on baculovirus, though slowly, increases steadily [32]. Among the known viruses use are Cydia pomonella granulovirus that protects against pest resistant to Spinosad for organic agriculture [31] and bacteriophage omnilitics to kill Xanthomonas, a bacterium [32].

(3) Bacterial bio-pesticides

Most bio-pesticides formulations are bacterial based, this is because it is cheaper. Many bacterial species are insecticidal but members in the genus Bacillus are most widely used in bio-pesticide formulations. One of the Bacillus species, Bacillus thuringiensis, has developed many molecular mechanisms to produce pesticidal toxins; most of the toxins are coded for by several cry genes [33]. Since its discovery in 1901 to date, over one hundred Bt based bio insecticides have been developed from it which are mostly used against lepidopteran, dipteran and coleopteran larvae. In addition, the genes that code for the insecticidal crystal proteins have been successfully transferred into different crops plants which have led to significant economic benefits. Because of their high specificity and their safety in the environment, Bt and Cry proteins are efficient, safe and sustainable alternatives to chemical pesticides for the control of most insect pests [34,35]. The mode of action of the cry proteins have traditionally been explained. The protein create trans-membrane pores or ion channels that results in osmotic cell lysis [34]. For Bacillus subtilis and Pseudomonas flourescens to be effective against insect pests, they must come into contact with the target pest [36]. The lethality of Bacillus thuringiensis (Bt) endotoxins is highly dependent upon the alkaline environment of the insect gut, a feature that assures that these toxins are not active in vertebrates, especially in humans. The expression of these toxins confers protection against crop destruction by insect [37]. These proteins have been commercially produced, targeting the major pests of cotton, tobacco, tomato, potato, corn, maize and rice, notably allowing greater coverage by reaching locations in plants which are inaccessible to foliar sprays [37]. There are numerous strains of Bacillus thuringiensis (Bt), each with different Cry proteins, and more than 60 cry proteins have been identified.

(4) Plant-incorporated-protectants (PIPs)

The adoption of genetically modified (GM) crops has increased dramatically in the last 11 years. Genetically modified (GM) plants possess an insect or pathogen-resistant gene or genes that have been transferred from a different species and so, reduces the destruction of crop by phytophagous arthropod pests [23]. The production of transgenic plants that express insecticidal δ-endotoxins derived from Bacillus thuringiensis (Bt), were first commercialized in the US in 1995. The expression of these toxins confers protection against insect crop destruction [23]. Corn and cotton Bacillus thuringiensis, (Bt) incorporated varieties were introduced in 1995 and a Bt of soy was registered in 2010 [38,39]. Bt incorporated plants have been in use against the following among others; Corn rootworms, Caterpillars and Arbuscular mycorrhizal fungi, [39,40,41,42]. Despite industry claims that PIPs would lessen pesticides dependency, insects have exhibited resistance to the engineered crops [38].

(5) Pheromopesticides

Pheromones are chemical compounds, produced and secreted by animal(s) that influence the behavior and development of other members of the same species. It also has the potent ability to

Advances and Trends in Agricultural Sciences Vol. 1 Bio-pesticidal Properties of Neem (Azadirachta indica)

repel, disrupt mating, or inhibit the growth of several or specific species of insects. Pheromones with this ability are therefore refers to as pheromopesticides. When used in combination with traps, sex pheromones can help to determine what insect pests are present in a crop and what plant protection measures or further actions might be necessary to ensure minimal crop damage. If the attractant is exceptionally effective and the population level is very low, some control can be achieved with pheromone traps or with the "attract and kill" technique.

Generally, however, mating disruption is more effective. Synthetic pheromone that is identical to the natural version is released from numerous sources placed throughout the crop to be protected [31]. The southern pine beetle uses a variety of semi chemicals to mediate mass attack on host pine trees. Two aggregation pheromones, frontalin and trans-verbenol, function in directing other beetles to join in the mass attack of a host tree that is necessary for successful colonization. Once the tree is overcome, no further beetles are needed and two anti-aggregation pheromones, endo- brevicomin and verbenone, are released to divert beetles to other trees [23,31]. The first successful commercial formulation resulted from the discovering of the pink bollworm sex pheromone. In Germany and Switzerland mating disruption has been in use for the control of grape insect pests. It has also been proven effective in grapevine moth, codling moth and European grape moth in the United States [40].

(6) Plants extract

The pest management in agriculture is facing challenge in development of suitable agents to kill insect pests while ensuring the economic and ecological sustainability as majority of the pesticide chemicals are known to cause human and environmental hazards. In the recent past, a variety of new insect control agents have been developed, or are being developed, which may satisfy a variety of insect pest management needs [38,39].

The growing demand for natural products has intensified in the past decades as they are extensively used as biologically active compounds and, are being considered an important alternative strategy for the sustainable insect pest management in agriculture, as they are biodegradable and potentially suitable for use in integrated management programs. Several compounds are present in different plant parts including seeds, fruits, flowers, wood and leaves that acts as natural inhibitors. Magnifera indica is highly rich in polyphenols having antioxidant activity and also glycoside and flavonoids [41]. Sundararaj et al. [42] reported toxic and repellent properties of sugarcane bagasse-based lignin against some stored grain insect pests including Tribolium castaneum. Kumar et al. [43] evaluated the long- term efficacy of the protein enriched flour of pea (Pisum sativum L. var. Bonneville) in its toxicity, progeny reduction and organoleptic properties by combining it with wheat flour and testing the admixture against the red flour beetle, T. castaneum.

3. NEEM PLANT

The use of botanicals is now emerging as one of the most viable means of protecting crop produce and the environment from pollution from chemical pesticides. The most widely used of these botanicals is the neem plant, which is the top on the list of about 2400 plant pesticides in the world [44]. Neem products are effective against more than 350 species of arthropods, 12 species of nematodes, 15 species of fungi, three viruses, and two species of snails and one crustacean species [45,46]. Research has shown that neem extract is effective against nearly 200 species of insects. It is significant that some of these pests are resistant to pesticides, or are inherently difficult to control with conventional pesticides. Among such insects are floral thrips, diamondback moth and several leaf miners. Most neem products belong to the category of medium-to broad-spectrum pesticides, i.e., they are effective over a wide range of pests [47]. According to Jagannathan et al. [44], neem tree extracts has been used against household pests, storage pests and crop pests of field. Neem has been produced as fumigant used as a pesticide and disinfectant in many countries on a commercial basis by farmers and agriculturists. This 100% natural product is nontoxic and environmentally friendly. It assumes more importance in developing countries where millions of deaths are reported every year due to the accidental intake of synthetic pest fumigants.

(i) Neem seed and kernel extract

Advances and Trends in Agricultural Sciences Vol. 1 Bio-pesticidal Properties of Neem (Azadirachta indica)

The active ingredients of the neem plant are located in their maximum amounts in the seed and kernel. The seeds that are used for the preparation of neem kernel extract should be between three and eight months old. When the quantity of Azadirachtin in the seeds is quite high and adequate for efficient pest control [47,48]. Among insects, the Shoot-borer are key forest pests in tropical areas, it belongs to genus Hypsipyla. H. robusta is present in old world tropic while in neotropic region H. grandelle is widely distributed [49]. The two species cause high production of lateral branches as a result of boring into the terminal shoots of young plants [50]. Nim 80 and azatin (neem products) have been shown to produce the insecticidal activity or arrest the development of the pests at certain stages. At low concentration of azatin, the growth rate of both insects was reduced. Increment in concentration led to high mortality rates. Larvae were unable to feed when they were exposed to azatin. It has been shown that azatin acts as direct toxicant instead of inhibiting its growth. On the other hand Nim 80 has showed effectiveness against larval development [51]. To be effective the kernel extract should be milky white in colour and not brownish. The kernel extract is not effective against sucking insects like aphids, white flies and stem borers. In these cases, neem oil spray solution is a better option.

Neem products, Parker oilTM and neemas have been tested for their effectiveness against brown plant hopper. Their mortality rate, food consumption rate and net survival clearly of the insect showed that neem-based products are very effective [52]. Greenhouse evaluation of Azatrol (1.2% Azadiractin A and B), Triple Action Neem Oil (70% neem oil) and Pure Neem Oil at the recommended concentrations aphid colonization reduced by 50-75% after one week of their application as foliar spray. Almost total elimination of aphids was observed following a second application of these formulations seven days after the first application. Results indicate that the neem-based formulations tested were highly effective in suppressing aphid population, but did not act as an efficient repellent at standard application rates. Feeding was suppressed but did not achieve complete inhibition of food intake [53].

(ii) Neem leaf extract

The advantage of using neem leaf extract is that it is available throughout the year. There is no need to boil the extract since boiling reduces the azadirachtin content. Hence the cold extract is more effective. Some farmers prefer to soak the leaves for about one week, but this creates a foul smell [54]. Neem leaves are also used in storage of grains. Neem (leaf and seed) extracts have been found to have insecticidal properties, it is used as foliar spray.

4. MODE AND SPECIFICITY OF ACTION OF NEEM AS BIO-PESTICIDE PRODUCT

(i) Oviposition deterrence

Oviposition deterrence is another way in which neem controls pests. Application of neem formulations have prevented the females from depositing eggs [48]. The effect of neem-based pesticides on the reproductive potential of aphids has been attributed to blocking the neuro-secretory cells by the active ingredient, azadirachtin, which disrupts adult maturation and egg production [54]. Nisbet et al. [55] observed that the reproductive potential of Myzus persicae that fed on diet containing azadirachtin was less than half the Myzus persicae that fed on control diet within the first 26 h, whereas nymph production virtually ceased after 50 h.

(ii) Repellant

The extracts prepared from neem plants have a variety of properties including repellency to pests. According to Shannag et al. [53], the repellent action of Azatrol, Triple Action Neem Oil and Pure Neem Oil is wholly dependent on the concentration that is used. He showed that the three products at higher concentrations were able to repel aphids feeding on sweet pepper plants. Agbo et al. [56] also reported repellent and antifeedant properties of Cyperus articulatus against T. castaneum.

(iii) Antifeedant

Advances and Trends in Agricultural Sciences Vol. 1 Bio-pesticidal Properties of Neem (Azadirachta indica)

The antifeedant and growth inhibitory activities of various crude extracts and purified fractions of the plant were evaluated against economically important polyphagous pest Spodoptera litura [57]. When crops were treated to neem products, anti-peristaltic wave were observed in the alimentary canal which produced an action similar to a vomiting sensation in the insect during feeding. This was attributed to the presence of azadirachtin, salanin and melandriol. Because of this sensation, the insect does not feed on the neem-treated surface. Its ability to swallow was also blocked [48].

5. GROWTH REGULATION

Regulation of the insects’ growth is a very interesting property of neem products which is unique in nature. This is because the products work on juvenile hormones. The insect larva feeds and as it grows, it sheds its old skin (ecdysis or moulting). This process is governed by an enzyme, ecdysone [48]. The degree of abnormality in growth varies with both the growth stage of the insect, and the host plant on which it feeds (53). When the neem components, especially azadirachtin, gains access to the body of the larva, the activity of ecdysone is suppressed and the larva fails to moult, remains in the larval stage and ultimately dies. If the concentration of azadirachtin is not high enough, the larva will die only after it has reached the pupal stage. If the concentration is lower still, the adult emerging from the pupa will be 100% malformed with the formation of chitin (exoskeleton) inhibited and absolutely sterile (Vijayalakshmi et al. 1998).

6. CONCLUSION

The need for steady and safe food supply to the world rising population has led to the exploration of neem tree as a bio-pesticide. With the growing knowledge on the use of bio-pesticides it will gradually replace the conventional chemical pesticides presently in use. One of the problems with the use of chemical pesticides has been their impact on “non-target” species. Often they have been proven to be harmful to various beneficial species in the ecosystem. However, neem extracts are devoid of these effects.

The practice of farmers making their own neem-based products for pest control would reduce their dependence on external inputs for agriculture. It would also reduce their cost of pest control to almost zero, leaving only labour as a potential expenditure item. Pests can also be controlled without the use of toxic chemical pesticides, which will reduce the harm posed to humans and the environment alike. There is wide scope for innovation in developing neem as an efficient bio-pesticide. There is enough information to encourage the use of different neem extracts.

With the increasing trend of using bio fertilizers, insecticides and pesticides, neem should be increasingly cultivated and grown all over the world to get active ingredient-azadirachtin, responsible for stopping the growth cycle of pests. Neem is also assuming a lot of importance in crop management. Considering the fact that neem is not only a cheaper, naturally occurring product and an effective method to control pests and insects, but also has no side effects on plants or other living beings.

COMPETING INTERESTS

Authors have declared that no competing interests exist.

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51. Mancebo F, Hilji L, Mora GA, Salazar R. Biological activity of two neem (Azadirachta indica A. Juss., Meliaceae) products on Hypsipyla grandella (Lepidoptera: Pyralidae) larvae. Crop Protection. 2002;21:107-712. 52. Nathah SS, Choi MY, Paik CH, Seo HY, Kalaivani K. Toxicity and physiological effects of neem pesticide applied to rice on the Nila parvatalugens sta 1, the brown plant hopper. Ecotoxicol Environ Safety. 2009;72:1707-1713. 53. Shannag HK, Capinera JL, Freihat NM. Effects of neem-based insecticides on consumption and utilization of food in larvae of Spodoptera eridania (Lepidoptera: Noctuidae). Journal of Insect Science. 2015;15(1):152. 54. Vimala B, Murugani K, Deecaraman M, Karpagam S, Yalakshmi V, Sujatha K. The toxic effect of neem extract, spinosad and endosulfan on the growth of aphids and its predator. Bioscan. 2010;5(3):383-386. 55. Nisbet AJ, Woodford JAT, Strang RHC. The effects of azadirachtin-treated diets on the feeding behaviour and fecundity of the peach-potato aphid, Myzus persicae. Entomol. Exper. Appl. 1994;71:65–72. 56. Agbo BE, Nta AI, Ajaba MO. A review on the use of neem (Azadirachta indica) as a biopesticide. Journal of Bio-pesticides and Environment. 2015;2(2):58-65. 57. Jeyasankar A, Raja N, Ignacimuthu S. Antifeedant and growth inhibitory activities of Syzygium lineare Wall. (Myrtaceae) against Spodoptera litura Fab. (Lepidoptera: Noctuidae). Cur. Res. J. Biol. Sci. 2010;2:173-177.

Biography of author(s)

Dr. Bassey Etta Agbo Department of Microbiology, University of Calabar, P. M. B. 1115, Calabar, Nigeria

He is a lecturer in the department of Microbiology, Faculty of Biological Sciences, University of Calabar, Calabar, Nigeria. He obtained M.Sc. in Environmental and Public Health Microbiology from the University of Calabar his Ph.D. in Environmental Microbiology at the University of Uyo, Uyo, Nigeria. He has been involved in the teaching of Analytical Microbiology, Environmental Microbiology and Soil Microbiology for almost a decade. He is a researcher, reviewer, editorial board member for renowned international journals and has published widely in both national and international peer-reviewed journals. He is a member of professional bodies including American Society of Microbiology, Nigerian Society of Microbiology, Bio-pesticide Society of Nigeria and Nigerian Institute of Food Science and Technology.

Dr. (Mrs.) Abo Iso Nta Department of Zoology and Environmental Biology, University of Calabar, P. M. B. 1115, Calabar, Nigeria

She is presently the head of Zoology and Environmental Biology Department, Faculty of Biological Sciences, University of Calabar, Calabar, Nigeria. She holds a Ph. D. degree in Entomology from the University of Calabar, Calabar, Nigeria.

Advances and Trends in Agricultural Sciences Vol. 1 Bio-pesticidal Properties of Neem (Azadirachta indica)

Mr. Mathias Okang Ajaba Department of Science Laboratory Technology, University of Calabar, P. M. B. 1115, Calabar, Nigeria

He is currently a Graduate Assistant in the Department of Science Laboratory Technology, University of Calabar, Calabar, Nigeria. He holds B.Sc. degree in Microbiology with certifications in leadership, management, health safety, risk assessment, environmental awareness etc. all attesting to the fact that he is a community oriented person who loves to work in groups to find solutions to problems. He is imaginative in thoughts, honest and hardworking. As an erudite scholar, he has several publications to his credit in both local and international peer review Journals, even at this early stage of his scholarly life. He has presented papers in conferences organized by Nigeria Society of Microbiology. Before he started his current job, he was the Production and Quality Control Manager of Modibbo Adama University of Technology (MAUTECH), Table Water Factory, where he ensured production processes and employee follow both the State and Federal regulations. Also, during his one-year compulsory National Youth Service Corps (NYSC), he worked at the Department of Microbiology, MAUTECH as Teaching Assistant and was involved in academic activities. Again, within this period, he volunteered for Health Safety and Environmental Protection Community Development Service and under his headship of the group; his team initiated health enlightenment campaigns within the community. He has experiences in leadership, volunteerism and teaching. ______© Copyright 2019 The Author(s), Licensee Book Publisher International, This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Chapter 4 Print ISBN: 978-81-934224-3-4, eBook ISBN: 978-93-89246-17-9

Postharvest Heat Treatments to Extend the Shelf Life of Banana (Musa spp.) Fruits

P. K. Dissanayake1*

DOI:10.9734/bpi/atias/v1

ABSTRACT

Bananas are grown in the world mainly for their economic and nutritional value. High perishable nature of banana leads to quality deterioration which distracts consumer and hence high postharvest losses in the market. Climacteric nature of banana make these postharvest losses accelerate by triggering ethylene induced ripening process. Extending banana shelf life could be a considerable commercial benefit for both exporters and retailers. Treatments such as fungicides, heat treatments and low temperature storage are being applied for extending the shelf life of banana. However, nowadays, increased public concern over presence of chemical residues has progressively leads the adoption of heat treatment methods which substitutes as a non-damaging physical treatment for chemical prevention. Heat treatment is one of effective postharvest techniques which have been using as a plant quarantine procedure in other fruits. Indeed, the overall quality of fresh produce treated with optimal hot water temperatures is significantly better than untreated produce, as determined by a sharp reduction in decay incidence and maintenance of several quality traits. Heat treatment can be applied as vapor heat, hot water immersion (hot water dip) of the fruit until the core temperature reaches required effective temperature depending on cultivar. Banana fruit ripening effectively can be delayed by application of hot water treatments such as 40°C for five minutes. These treatments are not negatively effect on fruit taste, brix value around 40°C treatments. Further, more positively suppressed the microbial growth on fruit surface which supportive to the extend shelf life of banana. All findings related to heat treatments on banana suggest that hot water treatment, 40-50°C depending on cultivar, is most suitable for delaying de-greening and hence delaying the ripening during storage at ambient temperature. Food taste and soluble solid content not affected badly by hot water treatments especially up to 40ºC. Microbial growth effectively controlled by hot water treatment over 40°C. As with all this it can be concluded that heat treatments led to increase postharvest life without affecting the food quality of banana.

Keywords: Banana; postharvest life; de-greening; microbial control.

1. BANANA (Musa spp.)

Banana (Musa spp.) is one of the dominant fruits produced in 135 countries and territories and rank among the world's most valuable primary agricultural commodities after citrus and grapes [1]. Banana or plantain plays a vital role in food security and rural development [2]. Indeed, for 600 million people, banana is the main source of daily energy, while for another 400 million people; banana is an important food supplement [3]. Banana ranks fourth in human food after rice, wheat and corn [4]. Bananas are grown in the world mainly for their economic and nutritional value. Global exports of banana, excluding plantain, reached an estimated quantity of 18.1 million tons in 2017, a 6 percent increase compared with 2016. Amidst strong demand in the major markets, export volumes benefited from ample supply growth in the key exporting countries, most notably those located in Latin America [5]. Total annual production of plantain is reportedly 37.2 Tg (million tonnes) [6]. In Liberia, plantain is the most important crop grown by women and the third most important for men, after cacao (Theobroma cacao) and rubber (Hevea brasiliensis) [7]. In Ghana’s humid forest zone, 66 % of ______

1Department of Export Agriculture, Faculty of Agricultural Sciences, Sabaragamuwa University of Sri Lanka, Belihuloya, Sri Lanka. *Corresponding author: E-mail: [email protected];

Advances and Trends in Agricultural Sciences Vol. 1 Postharvest Heat Treatments to Extend the Shelf Life of Banana (Musa spp.) Fruits

households grow plantain, the joint-second most commonly grown food crop nationwide with maize and after cassava [8]. Banana exports by country totaled US$12.4 billion in 2017, up by an average 22.3% for all banana shippers over the five-year period starting in 2013 when bananas shipments were valued at $10.1 billion. The value of global banana exports are appreciated by 14.8% from 2016 to 2017 [9].

Ripened bananas are consumed as dessert fruit. Immature or green bananas (plantain - a type of cooking banana) are consumed in the cooked state and are processed into chips. Bananas provide a good source of energy.

Bananas are harvested at various stages of its maturity depending upon distance to market and the purpose for which it is cultivated, such as culinary, table purpose, etc. Most commonly the fruit is harvested when the ridges on the surface of the skin changed from angular to round i.e., after attainment of the three-fourths full stage [10]. Despite their popularity, bananas have a relatively short shelf life that creates challenges for both producers and consumers. High perishable nature of banana always leads to quality deterioration which distracts consumer and hence high postharvest losses in the market. The banana losses in the market are more significant especially in less developed countries. Climacteric nature of banana make these postharvest losses accelerate by triggering ethylene induced ripening process [11]. Extending banana shelf life could be a considerable commercial benefit for both exporters and retailers. Recently, several technologies have been used alone or combined with relative success. For example, modified atmosphere packaging, hot water treatments and some coatings can be effective in reducing dehydration, delaying color changes, improving appearance and extending shelf life of a diverse group of fresh fruits and vegetables [12, 13, 14].

Bananas are harvested when they are in the "mature green" stage of ripening and treated with ethylene to stimulate ripening before distribution and sale. The fruits generally ripen within 4 to 5 days after ethylene treatment and are then sold primarily at the yellow stage of ripening. After turning yellow, bananas become unsuitable for sale within 1 to 3 days, so finding ways to extend banana's shelf life just 1 to 2 days could enhance their market value.

2. POSTHARVEST TREATMENTS

Short shelf life of banana seriously limits the marketing of the fruit, where extending banana shelf life could be a considerable commercially benefit to both exporters and retailers [15]. The quality of bananas rapidly declines when fully ripened. The ripe banana is soft and delicate with a postharvest shelf life of 5 - 10 days. Generally, the primary factors causing postharvest loss in fruits can be categorized in to mechanical, physiological, pathological or environmental factors [16]. Many storage techniques have been developed to extend the shelf life and prolong freshness of banana for exporting purposes. Cold storage of 13ºC is practiced by developed nations to slow down fruit metabolism and therefore prolong senescence [17]. However, this is costly, and rapid fruit re-warm on the display shelves tend to reduce shelf life [18]. Most research has focused on ways to extend the green life of unripe fruit. Modified atmosphere packaging and ethylene absorbent packaging have been suggested as substitutes for low temperature storage [19], however, these storages are costly as it involves more labour for careful handling to prevent damage to bags and to keep the modified atmosphere conditions. 1-methylcyclopropene (1-MCP) treatment effectively delay peel colour change and fruit softening, and extend shelf life in association with suppression of respiration and C2H4 evolution [20]. Ethylene scrubbing, considered beneficial in storage of climacteric fruits and vegetables [21], may therefore be unnecessary with heated fruit. Use of ethylene oxide and sulphur dioxide is also effective in extending shelf life in Giant Cavendish banana [22]. Treatments such as fungicides, heat treatments and low temperature storage are being applied for extending the shelf life of banana, however nowadays, increased public concern over presence of chemical residues has progressively lead the adoption of heat treatment methods which substitutes as a non-damaging physical treatment for chemical prevention [23,24].

3. USE OF HEAT TREATMENTS

Heat treatment is one of effective postharvest techniques which have been using as a plant quarantine procedure in mango, apple, avocado, and litchi [25]. Indeed, the overall quality of fresh

Advances and Trends in Agricultural Sciences Vol. 1 Postharvest Heat Treatments to Extend the Shelf Life of Banana (Musa spp.) Fruits

produce treated with optimal hot water temperatures is significantly better than untreated produce, as determined by a sharp reduction in decay incidence and maintenance of several quality traits [15,26]. Exposing fruit to high temperatures attenuates some of these processes reduce while enhancing others. This anomalous situation results in heated fruit being more advanced in some ripening characteristics than non-heated fruit while maintaining their quality longer during shelf life at 20ºC [27]. Heat treatment can be applied as vapor heat, hot water immersion of the fruit until the core temperature reaches 47° which takes a number of hours unless radiofrequency is involved [18]. Other treatments, involving hot water dips [29,30] are for a few minutes at different temperatures depending on the fruit type. In addition, there is a short (10-25 s) treatment of hot water rinsing and brushing which may involve temperatures of up to 63°C [25,31]. Hot water is an effective heat transfer medium and, when properly circulated through the load of fruit, quickly establishes a uniform temperature profile [32] However, Record of use of Hot water treatment on banana is very limited compared to other horticultural crops.

4. APPLICATION OF HOT WATER TREATMENTS

In the experiments on effect of hot water treatments on banana [29,30], fruit hands of banana were dipped in hot water for 5 or 10 minutes in the range of hot water temperatures from 30°C to 60°C. In these experiments ten liter (10 L) double distilled water was used in hot water bath to make different hot water temperature regimes. Water was heated until desired temperature and kept the banana to be treated in the water at assigned duration.

5. HOT WATER TREATMENT PLANT FOR BANANA

A hot water treatment plant has been designed and developed for treating banana fruits by Amin and Hossain [33]. It was tested with banana varieties of BARI Kola 1 and Sabri Kola by treating them at 55°C for 5 min. The capacities of the plant is 350 kg h-1 for both the varieties. Break-even point of the hot water treatment plant is 70 h yr-1. Treatment cost of the hot water treatment plant is 0.55 Tk kg-1. The hot water treatment can increase the shelf-life (30%) and reduce the postharvest loss (70%). Hot water treatment plant may be used for treating mango, papaya, guava etc. However, for different fruits the temperature and treatment time should be adjusted for optimum treatment. It is expected that the plant may be profitable for mango, papaya, guava etc.

6. EFFECT ON FRUIT RIPENING AND SURFACE COLOUR

The results of Dissanayake et al. [29] suggested that hot water treatments 35ºC and 40ºC for 5 minutes is most suitable hot water treatments for delaying de-greening and hence delaying the ripening of seeni kesel banana during storage at ambient temperature (Fig. 1 and 2). Further, Fig. 1-B showed that the 40ºC hot water treatment for 10 minutes also can be used for similar effect. Surface colour changes of fruit peel of hot water treated fruits after storage have been assessed for ripening using a colour scores such as, 1= green, 2=colour break, 3=more green than yellow, 4=more yellow than green, 5=yellow with green tip, 6=full yellow, and 7=over ripe [34].

Yanga et al. [35] explained that banana ripening and de-greening is affected by both temperature and ethylene. Without ethylene treatment, fruit remained green when stored at 20°C for 7 d. On the other hand, ethylene treatment turned fruit yellow within 4 d at 20°C. Fruit stored at 30°C remained green regardless of ethylene treatment. The visual color changes were confirmed by instrumental measurements: yellow fruit showed L* > 67, C* > 44 and h◦ < 97; green fruit had L* < 60, C* < 40 and h◦ > 104.

Varit and Songsin [36] explained that Fruit peel colour, expressed as hue angle values, did not change until day 6 regardless of different hot water treatments. Thereafter, hue values of fruit dipped in 45°C water for 5 and 10 min, and untreated fruit sharply declined indicating increased degree of yellowing. In contrast, fruits dipped in 45°C water for 15 min or in 50°C water for 10 min maintained higher hue values and more green colour than the other treatments. Hot water treatment at 50°C for 10 min was more effective maintaining higher hue values and hence delaying yellowing than at 45°C for 15 min. This was evident on day 9 Changes in hue values did not coincide well with those of chlorophyll content which started to decrease on day 6 in all treatments. However, fruit dipped in 50°C

Advances and Trends in Agricultural Sciences Vol. 1 Postharvest Heat Treatments to Extend the Shelf Life of Banana (Musa spp.) Fruits

water for 10 min had much higher chlorophyll content than the other treatments on day 9 coinciding with its hue.

Giri et al. [37] 2016 also investigated same effect on ripening of banana due to hot water treatments. Accordingly, the yellowness index of treated bananas increased slowly compared to control samples. Heat treatment at 45°C for 60 min was found to delay the ripening process and hence increase the shelf-life of bananas by 5 days.

This all disclose that heat treatments are beneficially effect on extending shelf life of banana with appropriate heat treatment according to the variety of banana. However, optimum temperature and duration of treatment application might be vary cultivar to cultivar and need to employ separate investigation.

Fig. 1. Colour scores for banana peel during six days storage after 30, 40 and 50oC hot water treatments for 5 minutes (A) and for 10 minutes (B) Hot water treatments indicated with same English letters in each day are not significantly different at p=0.05 Source: Dissanayake et al. (2015)

Advances and Trends in Agricultural Sciences Vol. 1 Postharvest Heat Treatments to Extend the Shelf Life of Banana (Musa spp.) Fruits

Fig. 2. Colour scores for banana peel during storage after 35, 40, 45 and 50oC hot water treatments for 5 minutes Hot water treatments indicated with same English letters in each day are not significantly different at p=0.05 Source: Dissanayake et al. (2015)

6.1 Total Soluble Solids (% Brix)

Total soluble solid content in banana, remain constant without significant difference among all hot water treatments ranged from 30°C to 55°C [29], which is similar to experiment has been done on other fruit crops [38, 39, 40]. However, 60°C hot water treatment significantly reduces the Brix value of banana [29]. Meanwhile, findings of Kaka et. al. [30] shows increase of Brix Value of banana with increasing hot water treatments which is similar to findings in mango [41]. 60°C hot water dip for 10 minutes results higher brix value 15.60% compare to rest. That might indicate that results could vary with variety of banana. It was observed that the total soluble solids increased with increasing storing days after hot water treatments, initial total soluble solids was observed to be 10.09% which with passage of time increased to 19.55% [30]. Increase in total soluble solids was due to breakdown of starch into soluble sugars [42]. Similar findings have also been reported by Yap et al. [43]. However, these trends are contradictory with compare to sweet orange as total sugars were highest at time zero and decreased with increasing storage duration [44].

6.2 Organoleptic Quality /Taste

Dissanayake et al. [29] showed that there are no significant differences of scores for taste among different hot water treatments on banana compared to the control except 50ºC. There is a trend in reducing scores for taste after 40ºC. Therefore, even though there is no significant differences among taste of hot water treatments (at 45ºC) only up to 40ºC can only be used as hot water treatments to seeni kesel banana to have consumer preferable attributes [29]. This is similar to the results obtained in experiments which has done using Mango (Mangifera indica L.) cultivars where scores for Organopletic qualitities of different hot water treatments were scored to be non-significantly different by panelists [45, 46]. In this way, it is clear that hot water treatments do not negatively affect end- consumer preferences, although, negative effects of Hot water treatments on fruit have been reported in some occasions [47].

7. SUPPRESSION OF MICROBIAL GROWTH

Most postharvest diseases of fruit crops are controlled by fungicides immediately after harvest as a spray or dip application. With the increasing awareness among consumers about fungicide residues

Advances and Trends in Agricultural Sciences Vol. 1 Postharvest Heat Treatments to Extend the Shelf Life of Banana (Musa spp.) Fruits

the use of fungicide are becoming unpopular in the market. So, therefore effective non damaging physical treatment like hot water treatment is highly guaranteed for horticultural products in the market.

In the experiment which has done by Dissanayake et al. [29] shows that the hot water treatment of banana can effectively controlled microbial growth. In that experiment parts of banana peels (1- 2 mm size) were cut-off from randomly selected hot water treated banana fruits (35°C, 40°C, 45°C,50°C and 55°C for 5 minutes and 10 minutes) and placed on potato dextrose agar medium to observe the in vitro growth of microorganisms. The in vitro growth of microorganisms from cultured banana peel was recorded using a scaled ruler (cm) by measuring diameter of the colony every day until day 6.

According to the results (Fig 3) microbial growth of all hot water treated banana was completely suppressed until day 2 of culture. From day 3 onward microbial growths increased significantly but not reached to the level in the control. 40ºC and 45ºC heat treatments suppressed microbial growth in culture media until day 3 of culture period. 50ºC and 55ºC hot water treatments suppressed microbes in greater extent during culture observation period. These suggested that hot water treatments over 40ºC helps to suppress growth of microbes on fruit peel in greater extend compared to the control. It could be inference that the hot water treated banana can be stored suppressing postharvest disease occurring on fruit at least 4 days if treated more than 40ºC hot water. Even though the microbial growth starts later during storage it seems that it could not be vigor enough to cause damages to banana. The use of water dips at 38 to 60ºC for 2 to 60 min has been reported to control in vivo and in vitro spore germination and decay development of postharvest fungi in melons [48], papayas [49], strawberries [50] and tomatoes [51]. Both Couey [52] and Barkai-Golana and Phillips [53] have reviewed the results from these studies and others comprehensively [27]. Further, some scientists [54,55] suggested 50ºC hot water treatment as an optimum temperature for suppressing postharvest fungal diseases in banana varieties. Meanwhile, Giri et al. [37] explained that it is essential to heat the banana at 45°C for at least 45 minutes to reduce the fungal count in banana to an acceptable level.

Hot water treatments and storage period had a significant (p≤0.05) effect on decay incidence of Basari banana fruit (Table 1) (Kaka et al., 2019).

Further, it proved that hot water treatment has the potential to replace chemical fungicides to control crown rot of Banana. Findings of Reyes et al. [42] showed that hot water treatment at 45°C for 20 min reduced crown rot of ‘Santa Catarina Prata’ and ‘Williams’ banana fruits inoculated with Chalara paradoxa spore suspension from 100 to less than 15% and when fruits treated with hot water at 50°C for 20 min, crown rot reduce to <3%.

7.1 Heat Stress and Chilling Injury

Many harvested horticultural commodities are invariably exposed to low temperature in order to retard product respiration and delay ripening and senescence. However, many commodities of tropical fruits such as banana will develop chilling injury if the temperature is too low or if the cold conditions are maintained for too long specially below 13°C. Heat treatments have been found many cases of fruits to delay or prevent the development of chilling injury [23]. In previous researches it was shown that heat pretreatment of banana fruit at 38°C for 3 days before storage at a chilling temperature of 8°C for 12 days prevented increases in visible chilling injury index, electrolyte leakage and malondialdehyde content and also decreases in lightness and chroma, indicating that heat pretreatment could effectively alleviate chilling injury of banana fruit [56]. This has been shown in many commodities to be associated with the prolonged presence of Heat Shock Proteins (HSPs) in the tissue and the protective effect they exert [23, 57, 58, 59, 60]. Findings of He et al. [56] suggested that heat pretreatment enhanced the expression of gene Ma‐sHSPs (small Heat Shock Proteins), which might be involved in heat pretreatment‐induced chilling tolerance of banana fruit.

HSPs increase during a heat stress and generally disappear rapidly when a plant is returned to ambient temperature. Sabahat et al. [61] were the first to show that if a commodity was placed at 2°C

Advances and Trends in Agricultural Sciences Vol. 1 Postharvest Heat Treatments to Extend the Shelf Life of Banana (Musa spp.) Fruits

rather than 20°C following a heat stress the HSPs were not metabolized. Thus, the thermos tolerance induced in a heat stress can give protection against cold stress.

Fig. 3. Diameter of in vitro microbial growth from banana peel after 35, 40, 45, 50 and 55ºC hot water treatments for 5 minutes (A) and for 10 minutes (B) Error bars indicated ±SE of mean at P= 0.05 (n=6). Microbial growth: significant from normal control, ** P<0.001. Microbial growth Indicated with same English letters are not significantly different on each day. Source: Dissanayake et al. (2015)

Table 1. Decay incidence of banana fruit as influenced by different hot water treatments and storage period

Treatment Storage period Mean 0 day 5 day 10 day 15 day Control 0.00 15.36 18.90 27.26 15.38b 40oC 10 min 0.00 13.16 16.19 25.27 13.66c 50oC 10 min 0.00 08.55 10.52 16.43 08.88d 60oC 10 min 0.00 15.23 18.74 29.25 15.81a Mean 0.00d 13.08c 16.09b 24.55a (Source: Kaka et al., 2019)

8. CONCLUSION

All findings related to heat treatments on banana suggest that hot water treatment, 40-50oC depending on cultivar, is most suitable for delaying de-greening and hence delaying the ripening

Advances and Trends in Agricultural Sciences Vol. 1 Postharvest Heat Treatments to Extend the Shelf Life of Banana (Musa spp.) Fruits

during storage at ambient temperature. Food taste and soluble solid content not affected badly by hot water treatments especially up to 40ºC. Microbial growth effectively controlled by hot water treatment over 40ºC. As with all this it can be concluded that heat treatments led to increase postharvest life without affecting the food quality of banana. Further, heat treatment is not only as delaying ripening, but also increase antioxidant levels in banana [62].

COMPETING INTERESTS

Author has declared that no competing interests exist.

REFERENCES

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Advances and Trends in Agricultural Sciences Vol. 1 Postharvest Heat Treatments to Extend the Shelf Life of Banana (Musa spp.) Fruits

19. Scott KJ, McGlasson WB, Roberts EA. Potassium permanganate as an ethylene absorbent in polyethylene bags to delay ripening of bananas during storage. Aust J Exp Agric Anim Husb. 1970;10(43):237–240. 20. Jiang Y, Joyce DC, Macnish AJ. Extension of the shelf life of banana fruit by 1- methylcyclopropene in combination with polyethylene bags. Postharvest Biol Tech. 1999;16(2): 187–193. 21. Wojciechowski J, Blanpied GD, Bartsch JA. A comparison of ethylene removal by means of catalytic combustion and chemical absorption. In: Blankenship SM, editor. Proceedings of 4th National Controlled Atmosphere Research Conference. North Carolina State, University, Raleigh. 1985;363-373. 22. Williams OJ, Raghavan G, Golden KD, Gariépy Y. Postharvest storage of Giant Cavendish bananas using ethylene oxide and sulphur dioxide. J Sci Food Agric. 2003;83(3):180–186. DOI: 10.1002/jsfa.1303 23. Lurie S. Postharvest heat treatments, Postharvest Biol Tech. 1998:14(3);257-269. 24. Paull RE, Chen NJ. Heat treatment and fruit ripening. Postharvest Biol Technol. 2000;21(1):21- 37. 25. Fallik E. Pre-storage hot water treatments (immersion, rinsing and brushing): Review article, Postharvest Biol Tech. 2004;32(2):125–134. 26. Reyes MEQ, Nishijima W, Paull RE. Control of crown rot in ‘San Catarina Prata’ and ‘Williams’ banana with hot water treatments. Postharvest Biol Tech. 1998;14(1):71-75. 27. Klein JD, Lurie S. Heat Treatments for Improved Postharvest Quality of Horticultural Crops. Hort Technology. 1992;2(3):316-320. 28. Tang J, Mitcham E, Wang S, Lurie S. Heat Treatments for Postharvest Pest Control: Theory and Practice. Oxon, UK: CABI International; 2007. 29. Dissanayake PK, Dissanayake MLMC and Wijesekara WMAUM. Effect of Hot Water Treatments on Postharvest Life of Seeni Kesel Banana (Musa spp.cv. Seeni Kesel-Pisang Awak, ABB). Journal of Agriculture and Ecology Research International. 2015;2(4):209-218. 30. Kaka AK, Ibupoto KA, Chattha SH, Soomro SA, Mangio HR, Junejo SA, Soomro AH, Khaskheli SG and Kaka SK. Effect of hot water treatments and storage period on the quality attributes of banana (Musa sp.) fruit. Pure and Applied Biology. 2019;8(1):363-371. Available:http://dx.doi.org/10.19045/bspab.2018.700195 31. Lurie S, Tonutti P. Heat and hypoxia stress and their effects on stored fruits. Stewart Postharvest Review. 2014;3:9. Available:www.stewartpostharvest.com 32. Couey, HM. Heat treatment for control of post-harvest diseases and insect pests of fruits. Horticulture Science. 1989;24(2):198-202. 33. Amin MN and Hossain MM. Development of a hot water treatment plant suitable for banana. Agric Eng Int: CIGR Journal. 2013;15(4):185-193. Available: http://www.cigrjournal.org 34. Sarananda KH, Wijesundara MWMAG. Technology for “embul” banana export.Annals Sri Lanka Dept. Agric. 2006;8:211-217. 35. Yanga X, Song J, Fillmore S, Pangc X, Zhang Z. Effect of high temperature on color, chlorophyll fluorescence and volatile biosynthesis in green-ripe banana fruit. Postharvest Biology and Technology. 2011;62:246–257. 36. Varit S, Songsin P. Effects of hot water treatments on the physiology and quality of ‘Kluai Khai’ banana. nternational Food Research Journal. 2011;18(3):1013-1016. 37. Giri SKR, Singh R, Tripathi MK, More SN. Post-harvest heat treatment of bananas - Effect on shelf life and quality. Journal of Food Safety and Food Quality. 2016;67:132–138. 38. Klein JD, Lurie S. Prestorage heat treatment as a means of improving post storage quality of apples. J Am Soc Hortic Sci. 1990;115(2):255–259. 39. Liu FW. Modification of apple quality by high temperature. J Am Soc Hortic Sci. 1978;103:730– 732. 40. Porritt SW, Lidster PD. The effect of prestorage heating on ripening and senescence of apples during cold storage. J. Am Soc Hortic Sci. 1978;103(4):584–587. 41. Zambrano J, Materano W. Effects of Heat Treatment on Postharvest Quality of Mango Fruits. Tropical Agriculture. 1998;75(4):484-487. DOI: 10.21273/HORTSCI.32.3.434E

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42. Reyes MEQ, Nishijima W, Paull RE Control of crown rot in ‘Santa Catarina Prata’ and ‘Williams’ banana with hot water treatments. Postharvest Biology and Technology. 1998;14:71-75. 43. Yap M, Fernando WMADB, Brennan CS, Jayasena V, Coorey R. The effects of banana ripeness on quality indices for puree production. LWT - Food Sci Technol. 2017;80:10-18. 44. Khan GA, Rab A, Sajid M, Salimullah. Effect of heat and cold treatments on postharvest quality of sweet orange cv. Blood red. Sarhad J. Agric. 2007;23(1):39-44. 45. Anwar R, Malik AU. Hot water treatment affects ripenining quality and storage life of mango (Mangifera indica L.) Pak. J. Agri. Sci., 2007;44(2):304-311. 46. Ram HB, Singh RV, Singh SK, Joshi MC. A note on the effect of Ethrel and hot water dip treatment on the ripening and respiratory activities of mango variety Dashehari. Research notes. Govt. Fruit Preservation Institute, Lucknow, India; 1983. 47. Joyce DC, Hockings PD, Mazzuco RA, Shorter AJ, Brereton IM. Heat treatment injury of mango fruit revealed by nondestructive magnetic resonance imaging. Postharvest Biol. Technol. 1993; 3:305-311. 48. Teite DC, Barkai-Golan lR, Aharoni Y, Copel Z, Davidson H. Toward a practical, postharvest heat treatment for ‘Galia’ melons, Hortic (Amst.).1991;45(3-4):339-344. 49. Couey HM, Linse ES, Natamura AN. Quarantine procedure for Hawaiian papayas using heat and cold treatments. J Econ Entomol. 1984;77:984-988. 50. Couey HM, Follstad MN. Heat pasteurization for control of postharvest decay of fresh strawberries. Phytopathology. 1966;56:1345-1347. 51. Barkai-Golan R. Postharvest heat treatment to control Alternaria tenuis Auct. rot in tomato. Phytopathologia mediterranea. 1973;12:108-111. 52. Couey HM. Heat treatment for control of postharvest diseases and insect pests of fruits. HortScience.1989;24(2):198-202. 53. Barkai-Golan R, Phillips D. Postharvest heat treatment of fresh fruits and vegetables for decay control. Plant Disease. 1991;75(11):1085-1089. 54. Mirshekari A, Ding P, Kadir J, Ghazali HM. Effect of hot water dip treatment on postharvest anthracnose of banana var. Berangan. Afr. J. Agric. Res. 2012;7(1):6-10. 55. De Costa DM, Erabadupitiya HRUT. An integrated method to control postharvest diseases of banana using a member of the Burkholderia cepacia complex. Postharvest Biol Tech. 2005; 36(1):31-39. 56. He LH, Chen JY, Kuang JF, Liu WJ. Expression of three sHSP genes involved in heat pretreatment-inducing chilling tolerance in banana fruit. J Sci Food Agric. 2012;92:1924–1930. 57. Lurie S. Postharvest heat treatments of horticultural crops. Hort Rev. 1998;22:91–122. 58. Zhang JH, Huang WD, Pan QH, Liu Y. Improvement of chilling tolerance and accumulation heat shock proteins in grape berries (Vitis vinifera cv. Jingxiu) by heat pretreatment. Postharvest Biol Technol. 2005;38:80–90. 59. Yi SY, Sun AQ, Sun Y, Yang JY, Zhao CM, Liu J. Differential regulation of tomato plants: analysis of a multiple stress inducible promoter. Plant Sci. 2006;171:398–407. 60. Sevillano L, Mar Sola M, Vargas AM. Induction of small heat-shock proteins in mesocarp of cherimoya fruit (Annona cherimola Mill.) produces chilling tolerance. J Food Biochem. 2010;34: 625–638. 61. Sabehat A, Weiss D, Lurie S. The correlation between heat-shock protein accumulation and persistence and chilling tolerance in tomato fruit. Plant Physiol. 1996;110:531–537. 62. Ummarat N, Matsumoto TK, Wall MM, Seraypheap K. Changes in antioxidants and fruit quality in hot water-treated ‘Hom Thong’ banana fruit during storage, Hortic (Amst.). 2011;130(4):801– 807.

Advances and Trends in Agricultural Sciences Vol. 1 Postharvest Heat Treatments to Extend the Shelf Life of Banana (Musa spp.) Fruits

Biography of author(s)

Dr. P. K. Dissanayake Department of Export Agriculture, Faculty of Agricultural Sciences, Sabaragamuwa University of Sri Lanka, Belihuloya, Sri Lanka.

He is currently working as a senior lecturer in the Department of Export Agriculture, Faculty of Agricultural sciences, Sabaragamuwa University of Sri Lanka. He is working on chlorophyll degradation on horticultural crops, postharvest life of horticultural commodities and fruit crops diversity concern with underutilized crops. He disclosed in his scientific findings collaboration with Yamguchi University, Japan, that chlorophyll degradation pathway in Japanese bunching onion follows different path instead of well-known pathway. This gives supportive clues to rethink the pathway of chlorophyll degradation in other crops too. Further, he is working on to find the effects of different physical treatments such as heat treatment and light colour spectrum on horticultural commodities for their physio chemical properties. Further, his research interests expand to biodiversity, insect behavior such as bees and fall army worms and biotechnology. ______© Copyright 2019 The Author(s), Licensee Book Publisher International, This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

DISCLAIMER This chapter is an extended version of the article published by the same authors in the following journal with CC BY license. Journal of Agriculture and Ecology Research International, 2(4): 209-218, 2015

Reviewers’ Information (1) Anonymous, Malaysia. (2) Anonymous, Egypt. (3) Anonymous, Thailand.

Chapter 5 Print ISBN: 978-81-934224-3-4, eBook ISBN: 978-93-89246-17-9

Development and Properties of Green Tea with Reduced Caffeine

Kieko Saito1,2* and Yoriyuki Nakamura2

DOI: 10.9734/bpi/atias/v1

ABSTRACT

Caffeine is one of the main components of green tea and has side effects such as sleeplessness. Senior citizens, children, and pregnant woman should avoid tea despite its known beneficial effects. In this study, we developed green tea with reduced caffeine content (low caffeine tea) as a palatable tea that can be offered to everyone. To reduce the tea’s caffeine content, we subjected the plucked tea leaves to a hot-water spray process, and successfully produced a low caffeine tea infusion with 30% the caffeine content. The concentrations of other main components, such as catechins and theanine, in the low caffeine tea infusion did not differ from the control. Further, the physiological function of the tea was assessed; the anti-oxidative activity was investigated using a stable free radical and the anti- lipase activity using an artificial substrate. There were no significant differences between the infusions of low caffeine tea and green tea in anti-oxidative and anti-lipase activities. The results showed that our developed low caffeine tea could be an attractive high quality tea with health benefits for everyone.

Keywords: Camellia sinensis; green tea; reduced caffeine; anti-oxidative activity; anti-lipase activity.

1. INTRODUCTION

Many kinds of tea are produced and consumed worldwide. Tea types, based on processing or harvested leaf development are black (fermented), green (non-fermented) and oolong (semi- fermented). These major tea types differ in how tea is produced and processed according to the different processes of drying and fermentation that determine its chemical composition [1]. One reason for tea’s popularity is that it exhibits various physiological functions, such as improvement of brain function as well as anticancer, anti-obesity, antiallergic and antioxidative activities [2-4]. Green tea (Camellia sinensis (L.) Kuntze) contains catechins (8-20%), caffeine (2-4%) and theanine (1-8%) as the main components, with each component imparting a distinct taste [5]. However, caffeine exhibits some side effects, including sleeplessness. Senior citizens, children, and pregnant woman should avoid tea despite its known beneficial effects. Several kinds of decaffeinated green tea have been produced [6] and some have been commercially available. McKay and Blumberg [7] reported a per capita mean consumption of tea in the world of 120 mL/day. Approximately 76 –78% of the tea produced and consumed is black tea, 20 –22% is green tea and less than 2% is oolong tea [8]. However, these products were not popular with consumers because of their altered taste, attributable to the decrease in main ingredients during the manufacturing process, as well as the high cost. As an effective way to remove caffeine from tea leaves, Tsushida and Murai reported that fresh green tea leaves were steamed with boiling water for a few minutes prior to primary rolling [9]. Hot-water treatment is a simple and economically efficient method to decrease the caffeine content in tea leaves without chemical toxicity. ‘Benifuuki’ and ‘Benihomare’ green teas, which exhibit anti-allergic activity, were soaked in hotwater to reduce the caffeine content, and it was demonstrated that the anti-allergic compound was maintained in the processed tea leaves [10,11]. Thus, hot-water treatment might not decrease the physiological function of tea leaves. The maximum caffeine levels are always limited to 4 mg g-1 for leaf teas and 10 mg g-1 for instant teas [12]. ______

1School of Food and Nutritional Sciences, University of Shizuoka, Shizuoka 422-8526, Japan. 2Tea Science Center, University of Shizuoka, Shizuoka, 422-8526, Japan. *Corresponding author: E-mail: [email protected];

Advances and Trends in Agricultural Sciences Vol. 1 Development and Properties of Green Tea with Reduced Caffeine

In this study, a green tea with reduced caffeine content (low caffeine tea) was manufactured using a hot-water spray process. Further, the main components of the low caffeine tea infusion as well as its anti-oxidative and anti-lipase activities were determined in an effort to elucidate its health benefits.

2. MATERIALS AND METHODS

2.1 Reagent

The reagents used in this experiment were purchased from Sigma-Aldrich (St. Louis, MO, USA), and high performance liquid chromatography (HPLC) grade reagents were used for the HPLC analysis.

2.2 Low Caffeine Tea Manufacturing Process

Fresh tea leaves (Camellia sinensis (L.) Kuntze) were plucked and automatically sprayed with hot water (95°C, 180 seconds) to reduce the caffeine content of tea leaves [13,14]. A tea processing machine with regulated temperature and shower time and possessing high-performance efficiency and stability was used (Terada Co. Ltd. Shizuoka, Japan). After centrifugal dehydration at 3000 rpm for 1 min, the green tea was prepared through a standard manufacturing process.

2.3 Preparation of Tea Leaf Infusions

Three grams of tea leaves (green tea and low caffeine tea) were infused in 100mL of tap water for 0.5, 1, 2 and 6 hours at room temperature. The infusion was centrifuged for 5 min at 3000 rpm and the supernatant was filtered (0.45 μm filter, Millipore, Merck kGaA, Darmstadt, Germany).

2.4 Determination of Caffeine, Catechin and Theanine Contents

To determine the caffeine, catechin and theanine contents, the tea leaf infusions were applied to a reversed-phase high-performance liquid chromatography (Agilent 1100 series HPLC system, Agilent Technologies, Santa Clara, CA, USA) equipped with a reverse phase C18 column (3 μm particle size, 150 x 4.6 mm i.d.; Shiseido, Kyoto, Japan). The HPLC column was maintained at 30°C in an oven. For detection of compounds, 0.1 M NaH2PO4 buffer/acetonitrile was employed at 87:13 for caffeine and catechin, and 87:5 for theanine as the mobile phase at a flow rate of 1.0 ml/min. Individual peaks were identified by comparing their UV-Vis spectral characteristics and retention times with those of commercial standards supplied by Wako Pure Chemicals Industries, Ltd. (Osaka, Japan). Green tea leaves treated without hot water were used as the control.

2.5 Determination of Anti-oxidative Activity

DPPH (2,2-diphenyl-1-picrylhydrazyl, Sigma-Aldrich) as a stable free radical was used to determine the anti-oxidative activity of the tea infusions. A 1.5-ml aliquot of DPPH solution (0.1 mM, in 95% ethanol) was mixed with 100 μL of tea infusion. The mixture was shaken vigorously and left to stand for 20 min at room temperature. The absorbance at 517 nm of the DPPH solution was measured using a spectrophotometer (Bio Spec, Shimadzu, Kyoto, Japan). The radical scavenging activity was measured as a decrease in the absorbance of DPPH, indicating anti-oxidative activity, and was calculated using the following equation:

Scavenging activity (%) = [1- (absorbance of sample/absorbance of control)] × 100

2.6 Inhibition of Lipase Activity

Lipase inhibitory activity was determined in the infusions in order to estimate its anti-obesity effect. 4-methylumbelliferyl oleate (4-MUO) was used as a substrate to measure the pancreatic lipase inhibitory activity. The sample solution (25 μL of 3 h infusion) was added to 50 μL of 0.1 mM 4-MUO solution dissolved in a buffer consisting of 66 mM Tris–HCl (pH 7.4), 7 mM NaCl, 3 mM CaCl2, and 2 mM dimethyl sulfoxide (DMSO). These were mixed in a 96-well microplate, and then 25 μL of lipase solution (50 U/mL) was added to initiate the enzyme reaction. After incubation at 37°C for 60 min, the

Advances and Trends in Agricultural Sciences Vol. 1 Development and Properties of Green Tea with Reduced Caffeine

reaction was stopped with 50 μL of 0.1 mM citric acid, and the amount of 4-methylumbelliferone (4-MU) released by lipase was measured using a fluorometric microplate reader (Varioskan, Fisher Scientific, MA, USA) at λex 355 nm and λem 460 nm.

3. RESULTS

We manufactured a high quality low caffeine tea with health benefits for everyone. First, we determined the caffeine, catechin and theanine contents of the low caffeine tea and green tea (control) infusions at various infusion times (Fig.1). The concentrations of each component in both the low caffeine tea and green tea infusions were increased in an infusion time-dependent manner. The caffeine in the low caffeine tea was infused slowly, and the concentration was extremely low compared to the green tea, i.e., the level was decreased to less than one-third that of green tea at 6 h (Fig. 1A). The caffeine content differed significantly between all of the low caffeine and green tea samples.

The concentrations of catechin and theanine were also increased in an infusion time-dependent manner; moreover, there were no significant differences between the low caffeine tea and green tea, except at the 1 h infusion time (Fig. 1B, C). In other words, the catechin content of the 6 h infusion was very similar between the low caffeine tea and the green tea. Catechins mainly include epicatechin gallate (ECG), epigallocatechin gallate (EGCG), epicatechin (EC), catechin (C), and epigallocatechin (EGC). Among catechins, the most highly infused were EGC, followed by EC, EGCG, C, ECG in both the low caffeine tea and the green tea, and there was no difference in the rank order of catechins between the two groups (Fig. 1B). The analysis of theanine revealed the same trend as for catechins, and there were no significant differences between the low caffeine tea and the green tea at the 0.5, 3 and 6 h infusions (Fig. 1C). The results showed that the low caffeine infusion had reduced caffeine content; however, both catechin and theanine levels, as the main components, were maintained. Next, we determined the physiological function of the low caffeine tea. The 3 h infusion was used as the sample in this experiment, in reference to the result of Fig. 1. Fig. 2 shows the anti-oxidative activity of the low caffeine tea infusion in comparison to the green tea. The stable free radical DPPH was used to determine the radical scavenging activity of the sample. Anti-oxidative activity was indicated by a decrease in DPPH absorbance. Anti-oxidative activity was increased up to 1 h and was maintained at the same level until 6 h; further, the activities of the low caffeine tea and green tea did not significantly differ.

We also determined the anti-obesity function of the low caffeine tea by assessing lipase activity (Table 1). Inhibition of lipase activity did not significantly differ between the low caffeine tea infusions and the green tea infusions.

4. DISCUSSION

As the popularity of green tea has increased recently, caffeine-free green tea options are also being marketed. Taking into account physiological function and taste, we produced a green tea with reduced caffeine content instead of a caffeine-free beverage, and succeeded in reducing the caffeine content by 70%. While caffeine has some side effects, it was reported to enhance the physiological function of catechins through synergistic effects [15-17]. In addition, the combination of L-theanine and caffeine improves brain function in humans [18,19]. It has also been reported that caffeine is necessary for the characteristic taste of tea [20]. Therefore, by reducing the caffeine of green tea instead of completely removing it, the taste and physiological function are maintained, enabling the production of a high quality green tea. The complete removal of caffeine negatively impacts the taste of tea, necessitating the addition of chemicals to improve the quality and taste, and this is a serious issue for tea as a functional food and beverage. We treated fresh tea leaves with a hot water process (95°C, 180 seconds) to produce low caffeine tea; the physiological property of caffeine allows it to be easily eluted by hot water [21]. This is a safe and stable processing method that does not necessitate contamination by chemical substances and resins. From the viewpoint of functionality and taste, it is very important that catechin and theanine levels are maintained as the major components besides caffeine. The total amount of catechins was not reduced in the 6 h infusion compared with the standard green tea beverage, although EGCG, which is the most abundant catechin in tea leaves,

Advances and Trends in Agricultural Sciences Vol. 1 Development and Properties of Green Tea with Reduced Caffeine

was not highly contained in the low caffeine tea infusion. This result is in agreement with a report that, due to their physical properties, EGC is easily dissolved in cold water, while EGCG is difficult to elute [22,21].

A. Caffeine

200 ** Green tea

) Low caffeine tea L 150

m ** **

/ g µ (

100

e n

i ** e f

f 50 a C 0 0.5 1 3 6 B. Catechin Infusion time (h)

0.6

ECG )

L 0.5 EGCG m / EC g 0.4 ** C m

( EGC 0.3 n i h

c 0.2 e t a 0.1 C

0 G L G L G L G L 0.5 1 3 6 Infusion time (h) C. Theanine

) 0.14 Green tea L

m 0.12 Low caffeine tea / g 0.10 * m (

* 0.08

e n i 0.06 n a

e 0.04 h

T 0.02 0.00 0.5 1 3 6 Infusion time (h)

Fig. 1 Quantitative determination of the main components in low caffeine tea and green tea Asterisk (*) indicates statistical significance compared with green tea at the same infusion time. G, green tea; L, low caffeine tea. Each bar shows the mean ± SD (n=3, **p<0.005, *p<0.05).

Green tea (Control) Low caffeine tea ) )

100 % 100 ( %

(

y t y i t i v

i 80 v

80 t i t c c a

g 60 g 60 n i n i g g n n e 40

e 40 v v a a c c s

s 20 l

20 l a a c i c i d

d 0 0 a a R R 0 2 4 6 0 2 4 6 Infusion time (h Infusion time (h)

Fig. 2. Comparison of anti-oxidative activity in green tea and low caffeine tea

Advances and Trends in Agricultural Sciences Vol. 1 Development and Properties of Green Tea with Reduced Caffeine

Table 1. Inhibitory effect of low caffeine tea and green tea on lipase activity

Both catechin and theanine levels were much lower in the low caffeine tea than the green tea only for the 1 h infusion, while no differences were seen for the 3 or 6 h infusions. The manufacturing process might have an effect on the elution of compounds from tea leaves, resulting in the significant difference for the 1 h infusion only. Besides, there appeared to be no differences between the low caffeine tea and the green tea in the contents of catechins and theanine. Moreover, the low caffeine tea exhibited the same level of anti-oxidative activity as the green tea at any infusion time, even with the decrease in EGCG as the most abundant anti-oxidant in tea leaves [23]. EGC, which exhibited relatively high anti-oxidative activity, is easily infused in cold water and might be responsible for the antioxidative activity instead of EGCG.

In regards to the inhibitory effect of low caffeine tea on lipase activity, despite the decrease in caffeine content, the low caffeine tea exhibited the same level of lipase activity as the green tea. The role of caffeine in this function is not clear; however, the lipase inhibitory effect might be enhanced by the synergistic interaction between catechin and theanine.

The low caffeine tea with the high-quality components produced in this study is suitable for consumption by everyone, even those avoiding caffeine, and also exhibits the functions of antioxidative and lipase inhibitory activities.

5. CONCLUSION

We reduced the caffeine content of green tea infusion by 70% to avoid the side effect of caffeine using a hot-water spray process. However, both catechin and theanine levels, as the main components, were maintained. The low caffeine tea exhibited the functions of antioxidative and lipase inhibitory activities at the same level as green tea. We developed more reasonable and high-quality low caffeine tea than ever.

COMPETING INTERESTS

Authors have declared that no competing interests exist.

REFERENCES

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Advances and Trends in Agricultural Sciences Vol. 1 Development and Properties of Green Tea with Reduced Caffeine

5. Horie H, Kohata K. General ingredients of tea. In Health Science of Tea, New Possibility for Physiological Function. Muramatsu K, Oguni I, Isemura M, Sugiyama K, Yamamoto-Maeda M. Eds.; Gakkai Shuppan Center Tokyo, Japan. 2002;26–31. 6. Liang H, Liang Y, Dong J, Lu J, Xu H, Wang H. Decaffeination of fresh green tea leaf (Camellia sinensis) by hot water treatment. Food Chem. 2007;101:1451-1456. 7. McKay DL, Blumberg JB: The role of tea in human health: Anupdate. J Am Coll Nutr. 2002;21: 1–13, 8. Wu CD, Wei GX: Tea as a functional food for oral health.Nutrition. 2002;18:443–444. 9. Tsushida T, Murai T. Selective extraction of caffeine with hot water from intact tea leaves. Nippon Nogei Kagakukaishi. 1985; 59:917-919. 10. Yamamoto MM, Nagai H, Suzuki Y, Moriwaki S, Asai K, Manufacturing Method for low caffeine 'Benifuki', 'Banihomare' green tea remaining the contents of antiallergic compounds. Nippon Shokuhin Kagaku Kogaku Kaishi. 2004;51:435-439. 11. Yamamoto MM, Nagaya H, Mitsumori T, Yamaguchi Y, Horie H, Ema K, et al. A change of chemical components and effect on anti-allergic activity in “Benifuuki” green tea which was produced with low caffeine processing machine. Nippon Shokuhin Kagaku Kogaku Kaishi. 2007;8:109-116. 12. Ye JH, Liang YR, Jin J, Liang HL, Du YY, Lu JL, Lin C. Preparation of partially decaffeinated instant green tea. Journal of Agricultural and Food Chemistry. 2007;55(9):3498-3502. 13. Unno K, Noda S, Kawasaki Y, Yamada H, Morita A, Iguchi K, Nakamura Y. Reduced Stress and Improved Sleep Quality Caused by Green Tea Are Associated with a Reduced Caffeine Content. Nutrients 2017;9: 777. 14. Unno K, Nakamura Y. The Ability of Green Tea with Lowered Caffeine to Reduce Stress and Improve Sleep. Caffeinated and Cocoa Based Beverages 2019;8:209-234. 15. Kajimoto G. On the Antioxidative Components and Antiseptic Components in Tea. Part III. The synergistic action of caffeine to catechin components. Nippon Shokhin Kogyo Gakkaishi 1963;10:11-14.in Japanese） 16. Tanno K, Nonomura H. Antibacterial substances in green tea extract. Nippon Shokuhin Kogyo Gakkaishi. 1974;21:445-449. 17. Zheng G, Sayama K, Okubo T, Juneja LR, Oguni I. Anti-obesity effects of three major components of green tea, catechins, caffeine and theanine, in mice. In vivo. 2004;18:55-62. 18. Giesbrecht T, Rycroft JA, Rowson MJ, de Bruin EA. The combination of L-theanine and caffeine improves cognitive performance and increases. Nutri. Neurosci. 2010;13: 283-290. 19. Foxe JJ, Morie, KP, Laud PJ, Rowson MJ, de Bruin EA, Kelly SP. Assessing the effects of caffeine and theanine on the maintenance of vigilance during a sustained attention task. Neuropharmacol. 2012;62: 2320-2327. 20. Nakagawa M. Correlation of the constituents with the organoleptic evaluation of green tea liquors. Nippon Shokuhin Kagaku Kogaku Kaishi. 1969;16: 252-258. 21. Unno K, Hara A, Nakagawa A, IguchiK, Ohshiro M, Morita A, Nakamura Y. Anti-stress effects of drinking green tea with lowered caffeine and enriched theanine, epigallocatechin and arginine on psychosocial stress induced adrenal hypertrophy in mice. Phytomedicine. 2016;23:1365- 1374. 22. Monobe M, Ema K, Tokuda Y, Maeda-Yamamoto M. Effect on the epigallocatechin gallate/epigallocatechin ratio in a green tea (Camellia sinensis L.) extract of different extraction temperatures and its effect on IgA production in mice. Biosci. Biotechnol. Biochem. 2010;74: 2501–2503. 23. Rice-Evans CA, Miller NJ. Paganga, G. Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radic. Biol. Med. 1996;20:933-956.

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Biography of author(s)

Dr. Kieko Saito School of Food and Nutritional Sciences, University of Shizuoka, Shizuoka 422-8526, Japan and Tea Science Center, University of Shizuoka, Shizuoka, 422-8526, Japan.

She is the Assistant Professor of School of Food and Nutritional Sciences, University of Shizuoka, Shizuoka, Japan. She received her master degree from Graduate School of Agriculture, Nihon University in 1990. After working at RIKEN (Saitama, Japan) and Gerontology Research Center, NIH (USA) as a research associate, she started her career at the University of Shizuoka in 1996. She is in her present position since 2008. During this period, she received her PhD degree based on the thesis of Oxidative stress and Aging in 1991 from Nihon University. Her specialization is in Functional Food and Environmental Science. She joined Tea Science Center of University of Shizuoka in 2014 to assist research related with the tea industry. Her current research interests center on the physiological function of fermented tea and honey from tea flower (Camellia sinensis).

Dr. Yoriyuki Nakamura Tea Science Center, University of Shizuoka, Shizuoka, 422-8526, Japan.

He is the project professor and director of Tea Science Center, University of Shizuoka, Shizuoka, Japan. He graduated from Graduate School of Agriculture, Iwate University in March 1979 and joined the Shizuoka prefectural government in April. Worked at Shizuoka Tea Research Center and Shizuoka Research Institute of Agriculture & Forestry for 36 years. During this period, he received his PhD from Gifu University in 2006 and became the director of Shizuoka Tea Research Center in 2008. He is in his current present position since 2013. He specializes in tea propagation and breeding. He has given the Japanese Society of Tea Science and Technology Award in 1991 and The Society of Tea Science of Japan Award in 2013. He is also an international expert commissioner to evaluate tea quality. ______© Copyright 2019 The Author(s), Licensee Book Publisher International, This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

DISCLAIMER This chapter is an extended version of the article published by the same authors in the following journal with CC BY license. Journal of Experimental Agriculture International, 17(6): 1-6, 2017

Reviewers’ Information (1) Javan Ngeywo, Kenya. (2) Elias Ernesto Aguirre Siancas, Universidad Católica los Ángeles de Chimbote, Perú. (3) Birsa Mihail Lucian, Alexandru Ioan Cuza University of Iasi, Romania.

Chapter 6 Print ISBN: 978-81-934224-3-4, eBook ISBN: 978-93-89246-17-9

Productivity of Some Hausa Potato Accessions (Solenostemon rotundifolius (Poir) J. K. Morton in Jos-Plateau Environment

O. A. T. Namo1* and S. A. Opaleye1

DOI:10.9734/bpi/atias/v1

ABSTRACT

The Hausa potato (Solenostemon rotundifolius (Poir)) J. K. Morton is a tropical, multipurpose crop with different economic values. Its productivity is, however, low in terms of fresh tuber yield in the accessions available for cultivation in Nigeria. Consequently, many farmers are not encouraged to cultivate the crop, thereby limiting its popularity. This study was, therefore, designed to screen different accessions of the Hausa potato for productivity in the Jos-Plateau environment, Nigeria. The nine accessions (Manchok 1, Manchok 2, Bokkos 1, Bokkos 2, Bikka-Baban, Mujir, NRCRI, (White), Tukwak and Langtang) were laid out in a randomized complete block design with five (5) replications. Results indicate that percentage emergence, number of branches per plant, leaf area index (LAI), days to flowering, number of flowers per plant, relative growth rate, net assimilation rate, tuber length, tuber girth, root-top ratio, stand count at harvest, mean tuber weight, dry matter content and fresh tuber yield varied with accessions. Positive correlations were observed between the number of branches and number of flowers and mean tuber weight, root-top ratio and tuber yield, relative growth rate and net assimilation rate, tuber length and harvest index, relative growth rate and harvest index, tuber length and mean tuber weight as well as harvest index. The relative growth rate and net assimilation rate were also positively correlated. Moisture content was negatively correlated with nitrogen free extract. Protein was positively correlated with NFE (0.553*), but negatively correlated with calcium (-0.855**). Ash content and iron were negatively correlated (-0.655*). Total tuber yield was generally low in all the accessions. The positive associations among some growth and yield attributes suggests that these attributes could be used as selection indices in the improvement of the Hausa potato. The crop has the potential to address vitamin C deficiency in children. There is, therefore, the need to intensify research and popularize the production and consumption of the crop. The study also suggests investigation into the source-sink relationship in the Hausa potato.

Keywords: Assessment; accessions; Hausa potato; productivity.

1. INTRODUCTION

The Hausa potato (Solenostemon rotundifolius (Poir) J. K. Morton) is a tropical, multipurpose, minor tuber crop. It has been reported to be one of the best staple tuber crops in terms of its distinctive fragrance, peculiar taste, medicinal, nutritional and economic values. S. rotundifolius is known as Chinese potato, Sudan potato, country potato, Fra Fra potato, Hausa potato, Zulu round potato, innala, fabirama, or pessa [1 and 2]. It is cultivated in the West African countries of Ghana and Nigeria [3]. S. rotundifolius tubers possess elite flavour and taste and have medicinal properties due to the presence of flavonoids that help to lower the cholesterol level of the blood [4, 5 and 6].They also contain enzyme inhibitors [7]. Currently, its genetic resources are disappearing into extinction due to undesirable features such as small tuber size [3], branching of the tubers, lack of balance between the source potential and sink capacity which results in low tuber yield as well as the intense labour required in its production. Yields averaging 5-15 MT/ha have been reported from the crop in Ghana ______

1Cytogenetics and Plant Breeding Unit, Department of Plant Science Technology, University of Jos, P.M.B. 2084, Jos, Plateau State, Nigeria. *Corresponding author: E-mail: [email protected];

Advances and Trends in Agricultural Sciences Vol. 1 Productivity of Some Hausa Potato Accessions (Solenostemon rotundifolius (Poir) J. K. Morton in Jos-Plateau Environment

and Nigeria. The potential yield of the crop could be up to 18-20 MT/ha [8]. Consequently, it is being replaced by more popular root and tuber crops like Irish potato, sweet potato, cassava and yam.

The plant is a small herbaceous, dicotyledonous annual, 15-30 cm high, prostrate or ascending, with a succulent stem and thick leaves. It has an aromatic mint-like smell. Flowers are small and may be white, blue, pink or pale-violet in colour; they are produced on an elongated terminal with distal inflorescence and slender false spikes [9]. It has small dark-brown edible tubers produced at the base of the stem. These flowers are hermaphroditic and the fruits consist of four nutlets which rarely develop. In Africa today, cultivation of this crop is mostly limited to Burkina Faso, Eastern Mali, Northern Ghana and South Africa [10].

The crop is popular in the middle belt region of Nigeria especially in Kaduna, Adamawa, Plateau, Nasarawa and Taraba States where it is known as ‘Beku’, ‘Tumuku’, ‘Hyare’, ‘Nvu’, ‘Gamin’, ‘Ngo’ and ‘Fugi’ [11]. The Hausa potato has the potential of increasing the food bank, solving malnutrition problems, improving food security and increasing yield per unit area of land because of its higher biological efficiency and adaptation to different environments. It also has the potential and prospects for enlarged adoption into other agro-ecological zones in Nigeria, thereby contributing to food security, diversification of the local food base and sustaining livelihood. In Nigeria, 16 such minor root and tuber crops abound, out of about 20 different root and tuber crops cultivated throughout the country [12]. Among these crops are Hausa potato (Solenostemon rotundifolius Poir), is one of the underutilized species, they are important components of subsistence farming systems in their native areas of production; they serve as means of preserving cultural heritage and have a myriad of uses such as food, animal feed, medicines, cosmetics and income generation to rural households [13]. However, farmers growing this crop follow indigenous methods which, coupled with poor agronomic practices and lack of high-yielding varieties result in relatively low yield. The yield can be increased by adopting improved production technologies and cultivars [14]. The objective of this study was to evaluate the productivity of some Hausa potato accessions in the Jos-Plateau environment.

2. MATERIALS AND METHODS

The experiment was conducted between July 2016 and January 2017 at the National Root Crops Research Institute, Kuru in Jos-South Local Government Area of Plateau State (latitude 09°44’N, longitude 08°47’E; altitude1, 293.3 m above sea level). The soil is ferrallitic cambisol developed from volcanic rock [15].

Nine accessions (which were named after their native areas) were obtained from the germplasm collection of the National Root Crops Research Institute (NRCRI), Kuru and from farmers in Bokkos, Langtang, Bikka-Baban, Tukwak, Mujir and Manchok. These include Manchok 1, Manchok 2, Bokkos 1, Bokkos 2, Bikka–Baban, Mujir, NRCRI (white variety), Tukwak and Langtang.

Land preparation, including clearing, ploughing, ridging and plot mapping, was done manually on July 4 and 5, 2016. The net plot size was 3 m x 3 m (9 m2) and the gross plot size was 37 m x 17 m. The accessions were laid out in a randomized complete block design (RCBD) with five replications. One of the replications was used for the growth analysis study.

Fresh and healthy tubers were selected and planted at inter- and intra-row spacing of 1 m and 0.3 m, respectively, giving a total of 33, 333 plants per hectare. Planting was done on July 8, 2016.

The plots were weeded manually at 21 days after planting and earthed up on the same day to avoid the exposure of the tubers to sunlight. Further weeding was done at 45 and 90 days after planting to control weeds. Fertilizer (NPK 15:15:15) was applied at the rate of 200 kg ha1.

2.1 Field Observations and Data Collection

Field observations and data collection were commenced at 15 days after planting (DAP) and continued until harvest.

Advances and Trends in Agricultural Sciences Vol. 1 Productivity of Some Hausa Potato Accessions (Solenostemon rotundifolius (Poir) J. K. Morton in Jos-Plateau Environment

Emergence Rate: This was computed as the ratio of the number of tubers that emerged out of the total number planted and multiplied by 100 as follows:

Percentage emergence = 100% .

Values were subjected to arcsine transformation before the analysis, after which the means were de- transformed.

Number of Branches per Plant: Two plants were sampled from each plot at harvest. The number of primary branches (branches arising from the main stem) was physically counted for each of the plants. The mean number of braches per plant was used for the statistical analysis.

2.2 Growth Analysis Study

Two plants were sampled from each plot for the growth analysis study at 45, 90 and 126 days after planting. Each plant was washed and separated into roots, stems and leaves. All the plant parts were placed in separate labeled envelopes and dried in a moisture-extraction oven at 100°C to obtain constant weight. Leaf area index (LAI), relative growth rate (RGR) and net assimilation rate (NAR) were thereafter computed.

Leaf Area Index (LAI): This was carried out using the leaf-disc method as reported by [16]. The method involves the removal of leaves from the sampled plant from each plot, determination of the total dry weight and of the area/weight relationship of a sample taken from the mass of leaves with a punch of a known diameter. The cross-sectional area of the punch used for this experiment was 1.77 cm2. One hundred discs were taken from each sample and placed in envelopes for drying to constant weight in a moisture-extraction oven at 100°C for 48 hours. The remaining leaves with the punched leaves were placed in separate envelopes and dried at the same temperature and time. Leaf area index was computed using the formula:

LAI = Area of 1 disc x No of discs x Total leaf dry weight / Land area occupied by sampled plant Dry weight of leaf discs

Relative Growth Rate: The relative growth rate was computed on the basis of increase in dry weight of the plant parts over a fixed period, using the formula:

RGR = lnW2 – lnW1 t2 – t1

Where,

W1 and W2 = Total dry weight at times t1 and t2.

Net Assimilation Rate: Net assimilation rate, which is defined as the rate of increase in dry weight per unit leaf area, was computed from the data obtained on dry weights of plants using the method proposed by [17] and reported by [16].

NAR = W – W Loge L – Loge L 2 1 X 2 1 t2 – t1 L2 – L1

Where,

W1 and W2 are the total dry weight of harvested parts at times t1 and t2, respectively. L1 and L2 are the leaf area at t1 and t2. Loge = natural log

Advances and Trends in Agricultural Sciences Vol. 1 Productivity of Some Hausa Potato Accessions (Solenostemon rotundifolius (Poir) J. K. Morton in Jos-Plateau Environment

Days to Flowering: The number of days taken from the date of planting to when the first flower appeared in each plot was recorded as the number of days to flowering.

Mean Number of Flowers per Plant: Five plants were sampled from each plot, the number of flowers of each of which was counted at 90 DAP. The mean number of flowers per plant was used for the statistical analysis.

Root-Top Ratio: At harvest, the shoot (the stem and leaves) and the root (the tubers) were harvested from each of the two plants sampled from each plot and weighed separately. The ratio of the weight of the tubers to that of the shoot was computed as the root-top ratio using the formula:

Root-Top Ratio = Weight of tubers Weight of shoot

Stand Count: This was recorded as the total number of plants in each plot at the time of harvest.

Tuber Length and Tuber Girth: Five tubers were sampled from each plot and measured from the tip to the bottom. The mean tuber length was used for the statistical analysis. The tuber girth was measured as the circumference of the widest portion of the tuber. Five tubers were sampled from each plot and measured using a measuring tape. The mean tuber girth was used for the statistical analysis.

Number of Tubers per plant: The total number of tubers harvested in each plot was divided by the number of plant stands at harvest to obtain the number of tubers per plant.

Mean Tuber Weight: All the tubers harvested from each plot were weighed and the weight was divided by the total number of tubers from the respective plot in order to obtain the mean tuber weight for each accession.

Dry Matter Content: Ten (10) g of fresh tuber sample was taken from the harvested tubers, weighed and dried in a moisture-extraction oven to constant weight at 100°C for 48 hours. The dry matter percentage (DM%) was then computed as follows:

DM% = b x 100 a

Where, a = fresh weight of sample b = dry weight of sample.

Harvest Index: The Harvest Index (HI) was computed at each sampling date of 45, 90 and 120 days after planting as follows:

H.I. = Dry weight of tubers Total dry weight

Total Tuber Yield: All the tubers harvested from each plot were weighed. The weight was converted to the equivalent in tonnes per hectare.

2.3 Data Analysis

Data collected were subjected to the Analysis of Variance (ANOVA) and the F-test was used to test the significance of treatment effects. Means were separated using the Duncan’s new Multiple-Range Test [18]. Growth and yield attributes were correlated using the multiple correlation analysis.

Advances and Trends in Agricultural Sciences Vol. 1 Productivity of Some Hausa Potato Accessions (Solenostemon rotundifolius (Poir) J. K. Morton in Jos-Plateau Environment

3. RESULTS AND DISCUSSION

3.1 Percentage Emergence

Table 1 shows the percentage emergence of some Hausa potato accessions grown in Kuru in 2016. The accession NRCRI (White) had the highest percentage emergence of 87.51%.The accession Mujir had the lowest percentage emergence (30.94%), which did not differ significantly from accessions Bokkos 2 (33.98%) and Langtang (32.39%).

Significant variations observed in percentage emergence might have been caused by differences in the genetic make-up of the different accessions used in this study. The accession NRCRI which recorded the highest percentage emergence had bigger seed tubers than Mujir which had small seed tubers. Rykbost et al. [19] observed that small seed tubers resulted in delayed emergence in all varieties of the Hausa potato.

3.2 Number of Branches per Plant

The highest mean number of branches per plant was observed in the accession Manchok 2 (28.75), followed by Manchok 1 (21.38), Bikka-Baban (17.88), and NRCRI (white) (17.00). The lowest number of branches per plant (6.50) was observed in the accession Langtang (Table 1). Namo [16] also reported variation in the mean number of branches per plant in the sweet potato and noted that the number of branches produced by a plant is primarily a genetic character and that it is influenced by Indole Acetic Acid (IAA) in the plant as well as prevailing environmental conditions. The number of branches contributes to the total dry matter produced by the plant, which in turn may lead to higher tuber yield. However, the high number of branches produced in the Hausa potato, which persist for a long period during the cropping season, may contribute to the generally low tuber yield due to competition for assimilates produced by the photosynthetic source (leaves).

Table 1. Percentage emergence and number of branches per plant of some Hausa potato accessions grown in Kuru in 2016

Accession Percentage Emergence* No of branches per plant Manchok 1 49.80b 21.38ab Manchok 2 50.09b 28.75a Bokkos 1 36.08cd 16.63bc Bokkos 2 33.98d 15.63bc Bikka-Baban 46.72bc 17.88bc Mujir 30.94d 12.25cd NRCRI(White) 87.51a 17.00bc Tukwak 48.83b 16.00bc Langtang 32.39d 6.50d CV (%) 14.20 31.93 Means followed by the same letter(s) within the same column are not significantly different at 5% level of probability (Duncan’s new Multiple-Range Test). *Values were subjected to arcsine transformation before the analysis, and thereafter de-transformed.

3.3 Leaf Area Index (LAI)

Table 2 shows the leaf area index (LAI) of some Hausa potato accessions at different stages of growth in Kuru in 2016. The LAI increased with time up to 90 DAP and thereafter decreased in all but accessions Manchok 2, Bokkos 1 and Bokkos 2. At 45 DAP, the highest LAI value of 0.13 was observed in the accession NRCRI. The lowest LAI value of 0.02 was observed in the accessions Bokkos 1 and Mujir. At 90 DAP, the highest LAI value of 0.44 was observed in the accession NRCRI (White) while the lowest value was observed in accessions Manchok 2, Bokkos1 and Tukwak. At 126 DAP, the highest LAI value of 0.40 was observed in the accession NRCRI (White), while the lowest value (0.11) was observed in the accession Tukwak. LAI increased with time up to 90 DAP and thereafter decreased in all accessions. Deshi et al. [20] reported that LAI in potato increased in all

Advances and Trends in Agricultural Sciences Vol. 1 Productivity of Some Hausa Potato Accessions (Solenostemon rotundifolius (Poir) J. K. Morton in Jos-Plateau Environment

varieties to a peak and then declined due to senescence of leaves and decrease in dry matter production and distribution to the various parts of the plant.

Table 2. Leaf area index of some Hausa potato accessions at 45, 90 and 126 days after planting in Kuru in 2016

Growth Stage ( Days After Planting) Accession 45 90 126 Manchok 1 0.06ab 0.29ab 0.18bcd Manchok 2 0.04ab 0.22b 0.24bc Bokkos 1 0.02b 0.13b 0.26b Bokkos 2 0.06ab 0.25ab 0.40a Bikka-Baban 0.09ab 0.30ab 0.19bcd Mujir 0.02b 0.29ab 0.09d NRCRI (White) 0.13 a 0.44 a 0.40 a Tukwak 0.12ab 0.21b 0.11d Langtang 0.03ab 0.29ab 0.13cd CV (%) 8.45 33.79 19.93 Means followed by the same letter(s) within the same column are not significantly different at 5% level of probability (Duncan’s New Multiple-Range Test)

3.4 Days to Flowering

The highest number of days to flowering was observed in the accession Tukwak (87.00 DAP), which was followed by the accession Mujir (80.25 DAP). The lowest number of days to flowering (71 DAP) was observed in the accession Bokkos 1 (Table 3). The differences in the number of days to flowering indicate that this trait is genotypically controlled [3].

3.5 Number of Flowers per Plant

Table 3 shows the number of flowers per plant of some Hausa potato accessions grown in Kuru in 2016. The number of flowers observed in all the accessions did not differ significantly (P = 0.05). Contrary to this observation, Mwanjah et al. [21] reported that the number of flowers per plant in the Livingstone potato differed significantly with variety.

Table 3. Days to flowering and number of flowers per plant of some Hausa potato accessions grown in Kuru in 2016

Accession Days to Flowering No of flowers per plant Manchok 1 72.00dc 99.90a Manchok 2 75.00cd 70.43a Bokkos 1 70.75e 98.35a Bokkos 2 73.00cde 89.29a Bikka-Baban 75.25c 86.84a Mujir 80.25b 95.24a NRCRI (White) 75.00cd 102.15a Tukwak 87.00a 114.45a Langtang 72.25cde 120.78a CV (%) 2.53 10.13 Means followed by the same letter(s) within the same column are not significantly different at 5% level of probability (Duncan’s New Multiple-Range Test)

3.6 Relative Growth Rate (RGR)

Table 4 shows the relative growth rate (RGR) of some Hausa potato accessions grown in Kuru in 2016. The highest RGR values in all the accessions were observed at 45 days after planting (45 DAP) except in the accessions Bokkos 1 and Mujir, where RGR peaked at 90 DAP.

Advances and Trends in Agricultural Sciences Vol. 1 Productivity of Some Hausa Potato Accessions (Solenostemon rotundifolius (Poir) J. K. Morton in Jos-Plateau Environment

At 45 DAP, the highest RGR was observed in the accession NRCRI (0.69 gg-1 day-1), which did not differ significantly from those of accessions Manchok 1(0.49 gg-1 day-1), Manchok 2 (0.61 gg-1 day-1), Bokkos 1 (0.46 gg-1 day-1), Bokkos 2 (0.51 gg-1 day-1), Bikka-Baban (0.56 gg-1 day-1) and Tukwak (0.67 gg-1 day-1). The lowest RGR of 0.20 gg-1 day-1 was observed in the accession Mujir.

At 90 DAP, the highest RGR was observed in the accession Bokkos 1 (0.50 gg-1 day-1) while the lowest value was observed in the accession Manchok 2 (0.10 gg-1 day-1). At 126 DAP, the highest RGR value of 0.24 gg-1 day-1was observed in the accession Bokkos 2, while the lowest RGR was observed in the accession Langtang (0.04 gg-1 day-1).

Kuhlase et al. [22] and Vimala and Hariprakash [23] also reported a decrease in relative growth rate over time. In this study, the decrease in RGR over time showed that there was a decrease in total dry matter accumulated and partitioned to various parts of the plants. Usually, more dry matter is partitioned to the tubers at the latter stages of growth compared to the proportion translocated to the leaves and stems.

Table 4. Relative growth rate (gg-1 day-1) (x10-1) at 45, 90 and 126 days after planting (DAP) in some Hausa potato accessions grown in Kuru in 2016

Growth Stage ( Days After Planting) Accession 45 90 126 Manchok 1 0.49ab 0.34ab 0.18ab Manchok 2 0.61ab 0.10c 0.07bc Bokkos 1 0.46ab 0.50 a 0.17ab Bokkos 2 0.51ab 0.32ab 0.24a Bikka-Baban 0.56ab 0.37ab 0.10b Mujir 0.20c 0.31ab 0.12b NRCRI (White) 0.69 a 0.12c 0.15ab Tukwak 0.67ab 0.23bc 0.12b Langtang 0.41bc 0.20bc 0.04c CV (%) 21.65 43.83 44.43 Means followed by the same letter(s) within the same column are not significantly different at 5% level of probability (Duncan’s New Multiple-Range Test)

3.7 Net Assimilation Rate (NAR)

The highest net assimilation rate (NAR) values were observed at 45 days after planting (45 DAP) in all the accessions (Table 5). Thereafter, NAR decreased with time in most of the accessions.

At 45 DAP, the highest NAR of 15.89 gm-2 week-1 was observed in the accession Manchok 2, but this did not differ significantly from accessions Bokkos 1(12.45 gm-2 week-1) and NRCRI (9.15 gm-2 week-1). The lowest NAR value of 5.92 gm-2 week -1 was observed in the accession Bikka- Baban. The highest NAR value at 90 DAP (3.11 gm-2 week-1) was observed in the accession Bokkos 1; the lowest value of 0.10 gm-2 week-1 was observed in the accession Langtang (P = 0.05). At 126 DAP, the NAR values observed in all the accessions did not differ significantly at P = 0.05 (Table 5).

The net assimilation rate varied with genotype and the stage of growth in this study. In most accessions, NAR was higher at the early than at the latter stages of growth. As the cropping season progresses, light interception improves and the rate of dry matter production goes up. But due to mutual shading, photosynthesis no longer exceeds respiration in older leaves, which then cease to be net producers of dry matter [16]. However, in accessions where NAR was higher at the latter stages of growth, there might have been less mutual shading of leaves, due to leaf orientation, which might have resulted in a continuous production of dry matter.

Advances and Trends in Agricultural Sciences Vol. 1 Productivity of Some Hausa Potato Accessions (Solenostemon rotundifolius (Poir) J. K. Morton in Jos-Plateau Environment

Table 5. Net assimilation rate (NAR) (gm-2 week-1) (x10-3) at 45, 90 and 126 days after planting (DAP) in some Hausa potato accessions grown in Kuru in 2016

Growth Stage ( Days After Planting) Accession 45 90 126 Manchok 1 6.73b 1.20ab 0.44a Manchok 2 15.89a 0.42b 0.42a Bokkos 1 12.45ab 3.11a 0.40a Bokkos 2 6.68b 1.08b 0.88a Bikka-Baban 5.92b 1.98ab 0.28a Mujir 7.76b 0.19b 0.74a NRCRI (White) 9.15ab 0.47b 0.43a Tukwak 7.91b 1.60ab 0.54a Langtang 6.43b 0.10b 0.57a CV (%) 36.22 48.01 45.51 Means followed by the same letter(s) within the same column are not significantly different at 5% level of probability (Duncan’s New Multiple-Range Test)

3.8 Stand Count at Harvest

Table 6 shows stand count of some Hausa potato accessions grown in Kuru in 2016. The highest stand count of 25.25 was observed in the accession NRCRI, and this was followed by Manchok 2(17.50), Manchok 1(17.25) and Bikka-Baban (16.50). The lowest number of stand counts at harvest was observed in the accession Langtang (1.75). The differences observed in stand count among the Hausa potato accessions could be attributed to their genotypic differences.

3.9 Root-Top Ratio (RTR)

Table 6 shows the Root-top ratio of some Hausa potato accessions grown in Kuru in 2016.The highest root-top ratio was observed in NRCRI (3.28), followed by accessions Mujir (2.47) and Bokkos 2(2.10). The lowest value of 0.37 was observed in the accession Manchok 2(0.37). Accessions with high root-top ratios are believed to have large sink capacities which enable them to capture assimilates produced by the source (leaves and stems). In other words, more assimilates were partitioned to the tubers in these accessions compared with the leaves and stems [24]. In this study, the accessions NRCRI (White), Mujir and Bokkos 2 with high root-top ratios also had high total fresh tuber yields.

Table 6. Stand count at harvest and root-top ratio of some Hausa potato accessions grown in Kuru in 2016

Accession Stand Count Root-Top Ratio Manchok 1 17.25b 1.20cd Manchok 2 17.50b 0.37e Bokkos 1 10.50c 1.60c Bokkos 2 10.75c 2.10b Bikka-Baban 16.50b 0.96d Mujir 4.00de 2.47b NRCRI (White) 25.25a 3.28a Tukwak 8.50cd 0.90d Langtang 1.75e 1.41c CV (%) 4.43 16.69 Means followed by the same letter(s) within the same column are not significantly different at 5% level of probability (Duncan’s new Multiple-Range Test)

Advances and Trends in Agricultural Sciences Vol. 1 Productivity of Some Hausa Potato Accessions (Solenostemon rotundifolius (Poir) J. K. Morton in Jos-Plateau Environment

3.10 Tuber Length and Tuber Girth

The highest tuber length of 7.08 cm was observed in the accession Tukwak and this was followed by accessions Bokkos I (6.70 cm), NRCRI (White) (6.25 cm), Manchok 2(6.15 cm), Manchok 1(5.70 cm) and Bikka-Baban (5.58 cm).The lowest tuber length was observed in the accession Mujir (4.65 cm).The accessions did not differ significantly (P = 0.05) in tuber length (Table 7). Tuber girth was highest in the accession NRCRI (White) (8.50 cm), but this did not differ significantly from the accession Manchok 2(6.85 cm). All the other accessions were similar in tuber girth.

Namo [16] noted that selection based on the length of tubers was highly desirable for improving sweet potato yield. This could also be true of the Hausa potato. Tuber length and tuber girth varied in all accessions. Ogedengbe et al. [25] reported that variations in tuber length and tuber girth were due to tuber size and the genetic make-up of the plant.

Table 7. Tuber length and tuber girth of some Hausa potato accessions grown in Kuru in 2016

Accession Tuber Length (cm) Tuber Girth (cm) Manchok 1 5.70a 5.80b Manchok 2 6.15a 6.85ab Bokkos 1 6.70a 6.58b Bokkos 2 5.18a 5.13b Bikka –Baban 5.58a 4.95b Mujir 4.65a 6.40b NRCRI (White) 6.25a 8.50a Tukwak 7.08a 6.30b Langtang 4.63a 5.73b CV (%) 26.40 18.85 Means followed by the same letter(s) within the same column are not significantly different at 5% level of probability (Duncan’s New Multiple-Range Test

3.11 Mean Tuber Weight

The highest mean tuber weight of 7.15 g was observed in the accession Bokkos 2, which did not differ significantly from accessions Bokkos 1 (3.99 g), Bikka-Baban (2.66 g), Mujir (3.18 g), NRCRI (White) (2.99 g), Tukwak (2.97 g) and Langtang (4.47 g). The lowest mean tuber weight was observed in the accession Manchok 2(2.12 g) (Table 8). The generally low mean tuber weight in this study confirms the low yield in the Hausa potato. Many tubers are produced per plant, but are small in size due, perhaps, to slow rate of translocation of assimilates from the source to the sink.

3.12 Dry Matter Content

Table 8 shows the dry matter content of some Hausa potato accessions grown in Kuru in 2016. The highest dry matter content of 30.60% was observed in the accession Bikka-Baban, but this did not differ significantly from the other accessions. Dry matter content has been reported to be related to starch content in sweet potato [26]. The dry matter content of 27% and above has been observed to be acceptable to most processors of tubers [26]. Most of the accessions used in this study had dry matter content of 17% and above (Table 8). This implies that only accessions Bikka-Baban, NRCRI (White), Tukwak and Langtang could be used for industrial processing.

3.13 Harvest Index

Table 9 shows the harvest index (HI) of some Hausa potato accessions at different stages of growth. Generally, harvest index increased with time up to 126 DAP in all but accession Mujir. The highest harvest index at 45 DAP was observed in the accession Manchok 2(0.64). The lowest HI was observed in the accession Langtang (0.20). At 90 DAP, the harvest index was statistically similar in all the accessions. At 126 DAP, the highest harvest index was observed in the accession Bokkos 1(0.88) and this was followed by accessions Manchok 1(0.85) and Bikka-Baban (0.83); the accession

Advances and Trends in Agricultural Sciences Vol. 1 Productivity of Some Hausa Potato Accessions (Solenostemon rotundifolius (Poir) J. K. Morton in Jos-Plateau Environment

Mujir had the lowest harvest index of 0.46. Harvest index is valuable determinant of yield as it represents the efficiency of the crop to convert photosynthates to economically valuable products [27 and 28]. Namo [16] observed that harvest index increased with crop age in the sweet potato and that the peak period varied with genotype.

Table 8. Mean tuber weight and dry matter content (%) of some Hausa potato accessions grown in Kuru in 2016

Accession Mean tuber weight (g) Dry matter content (%) Manchok 1 2.15d 17.55 a Manchok 2 2.12e 23.24 a Bokkos 1 3.99b 23.11 a Bokkos 2 7.15 a 21.84 a Bikka-Baban 2.66c 30.62 a Mujir 3.18c 17.40 a NRCRI (White) 2.99c 27.00 a Tukwak 2.97c 25.46 a Langtang 4.47b 25.87a CV (%) 15.30 31.00 Means followed by the same letter(s) within the same column are not significantly different at 5% level of probability (Duncan’s New Multiple-Range Test)

Table 9. Harvest index at 45, 90 and 126 days after planting (DAP) in some Hausa potato accessions grown in Kuru in 2016

Growth Stage ( Days After Planting) Accession 45 90 126 Manchok 1 0.23de 0.51a 0.85ab Manchok 2 0.64 a 0.61a 0.70ab Bokkos 1 0.22de 0.66a 0.88 a Bokkos 2 0.38cd 0.57a 0.76ab Bikka- Baban 0.32cde 0.55a 0.83ab Mujir 0.37cd 0.51a 0.46c NRCRI (White) 0.56ab 0.67a 0.76ab Tukwak 0.43bc 0.64a 0.82ab Langtang 0.20e 0.62a 0.63bc CV (%) 18.29 22.64 12.00 Means followed by the same letter(s) within the same column are not significantly different at 5% level of probability (Duncan’s New Multiple-Range Test)

Table 10. Total tuber yield of some Hausa potato accessions grown in Kuru in 2016

Accession Total Tuber Yield (t ha1) Manchok 1 1.05bc Manchok 2 1.42 b Bokkos 1 1.21 b Bokkos 2 1.42 b Bikka-Baban 0.92bc Mujir 0.12 c NRCRI (White) 3.83 a Tukwak 0.92bc Langtang 0.10 c CV (%) 33.88 Means followed by the same letter(s) within the same column are not significantly different at 5% level of probability (Duncan’s new Multiple-Range Test)

Advances and Trends in Agricultural Sciences Vol. 1 Productivity of Some Hausa Potato Accessions (Solenostemon rotundifolius (Poir) J. K. Morton in Jos-Plateau Environment

Table 11. Correlation matrix of growth and yield attributes of some Hausa potato accessions grown in Kuru in 2016

Parameters NB DTF NF RTR NAR RGR TL TG MTW HI DMC Yield NB 1 -0.065 -0.771** -0.435 0.144 0.160 0.491 0.196 -0.752** 0.392 -0.087 0.299 DTF 1 0.215 -0.088 -0.119 -0.313 0.297 0.109 -0.184 -0.21 0.053 -0.244 NF 1 0.230 -0.039 -0.129 -0.023 0.039 0.276 -0.023 0.095 -0.155 RTR 1 -0.252 -0.096 -0.240 0.485 0.583* -0.299 -0.127 0.560* NAR 1 0.945** 0.586* -0.230 -0.139 0.764** 0.208 0.126 RGR 1 0.424 -0.261 -0.025 0.728** 0.002 0.208 TL 1 0.418 -0.576* 0.704** 0.269 0.432 TG 1 -0.170 -0.086 0.023 0.650* MTW 1 -0.480 -0.176 -0.138 HI 1 0.341 0.430 DMC 1 0.255 YIELD 1 * Significant at 0.05 level of probability ** Significant at 0.01 level of probability Key - NB = Number of branches, DTF = Days to flowering. NF = Number of flowers, RTR= Root Top Ratio, NAR= Net Assimilation Rate, RGR= Relative Growth Rate, TL = Tuber Length, Tuber Girth, MTW =Mean Tuber Weight, HI=Harvest Index, DMC= Dry Matter Content

Advances and Trends in Agricultural Sciences Vol. 1 Productivity of Some Hausa Potato Accessions (Solenostemon rotundifolius (Poir) J. K. Morton in Jos-Plateau Environment

3.14 Total Tuber Yield

Table 10 shows the total tuber yield of some Hausa potato accessions grown in Kuru in 2016. The highest total tuber yield of 3.83 t ha-1 was observed in the accession NRCRI (White).The lowest total tuber yield, which was observed in the accession Langtang (0.10 t ha-1), did not differ significantly from those of accessions Bokkos 1(1.21 t ha-1), Bokkos 2(1.42 t ha-1), Manchok 1(1.05 t ha-1), Manchok 2(1.42 t ha-1) and Bikka-Baban (0.92 t ha-1). Total tuber yield was generally low in all the accessions, contrary to the findings of Enyiukwu et al. [10] and Reddy [29] who reported average tuber yields of between 5 and 15 t ha1. It has been observed that more dry matter could be left in the above-ground portion (leaves and stems) than in the roots at the end of the cropping season, suggesting, perhaps, a lack of balance between the source potential and sink capacity [16]. A further study on the source-sink relationship in the Hausa potato is hereby recommended.

3.15 Correlation Matrix of Growth and Yield Attributes

Table 11 shows the correlation matrix of growth and yield attributes in the Hausa potato. The mean number of branches per plant was negatively and significantly correlated with number of flowers per plant (-0.771**) and the mean tuber weight (-0.752**). The root-top ratio was positively and significantly correlated with the mean tuber weight (0.583*) and total tuber yield (0.560*).

The net assimilation rate (NAR) was positively and significantly correlated with the relative growth rate (0.945**), tuber length (0.586*) and harvest index (0.764**). The relative growth rate (RGR) was positively and significantly correlated with harvest index (0.728**). Tuber length was positively correlated with harvest index (0.704**) but negatively correlated with mean tuber weight (-0.576*). Tuber girth and total tuber yield were positively correlated (0.650*).

Positive correlations were observed between the number of branches and number of flowers and mean tuber weight; root-top ratio and tuber yield; relative growth rate and net assimilation rate; tuber length and harvest index; relative growth rate and harvest index; tuber length and mean tuber weight as well as harvest index. The relative growth rate and net assimilation rate were also positively correlated, as has also been reported by Kuhlase et al. [22]. The results indicate that these growth and yield attributes could make a significant contribution to fresh tuber yield in the Hausa potato, and should, therefore, be further investigated.

4. CONCLUSION

The results of this study show that percentage emergence, number of branches per plant, leaf area index, days to flowering, number of flowers per plant, relative growth rate, net assimilation rate, tuber length, tuber girth, root-top ratio, mean tuber weight, dry matter content and total tuber yield varied with accession. The total tuber yield was generally low in all the accessions. There is a need for further investigation into the source-sink relationship in the Hausa potato. The positive associations among the different growth and yield attributes indicate that there is prospect for improvement in the Hausa potato, using these traits as selection indices.

COMPETING INTERESTS

Authors have declared that no competing interests exist.

REFERENCES

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3. Nanema RK, Traore ER, Bationo P, Zongo J. Morphoagromical characterization of Solenostemon rotundifolius (Poir) J. K. Morton (Lamiaceae) germplasm from Burkina Faso. International Journal of Biological and Chemical Sciences. 2009;3:1100-1113. 4. Horvath T, Linden A Yoshizaki F, Eugster CH, Rüedi P. Abietanes and a novel 20-norabietanoid from Plectranthus cyaneus (Lamiaceae). Helvetica Chimica Acta. 2004;87:2346-2353. 5. Abraham M, Radhakrishnan VV. Assessment and induction of variability in coleus (Solenostemon rotundifolius). Indian Journal of Agricultural Sciences. 2005;75(12): 834-836. 6. Sandhya C, Vijayalakshmi NR. Antioxidant activity of flavonoids from Solenostemon rotundifolius in rats fed normal and high fat diets. Food Research International. 2005;38(6):615- 629. 7. Prathiba S, Nambisan B, Leelamma S. Enzyme inhibitors in tuber crops and their thermal stability. Plant Foods and Human Nutrition. 1995;48(3):247-257. 8. Plant Resources of Tropical Africa (PROTA). Solenostemon rotundifolius Poir (Synonyms: Germania rotundifolius Poir, Plectranthus rotundifolius Sims) database; 2013. PROTA.org/db/w- wpd/exec/db Retrieved November 9. Steentoft M. Flowering Plants in West Africa. Cambridge. Cambridge University Press. 2009; 268. 10. Enyiukwu DN, Awurum AN, Nwaneri JA. Potentials of Hausa Potato (Solenostemon rotundifolius (Poir) J. K. Morton) and management of its tuber rot in Nigeria. Greener Journal of Agronomy, Forestry and Horticulture. 2014;2(2):027 – 037. 11. Olojede AO, Iluebbey P, Dixon AGO. IITA/NRCRI Collaborative Germplasm and Data Collection on Minor Root and Tuber Crops in Nigeria. International Journal of Agriculture and Development (IJARD). 2005;7(2):2006. 12. Asumugha GN, Arene OB. Status of production and utilization of other root crops in Nigeria: Preliminary Investigation. NRCRI Annual Report. 1999;149-152. 13. Olojede AO, Akinpelu AO, Dalyop TY, Lenka D, Nwosu KI, Iluebbey P, Dixon AGO. Exploratory studies on production, utilization and proximate composition of some minor root and tuber crops in Nigeria. In II International Symposium on Underutilized Plant Species: Crops for the Future- Beyond Food Security. 2011; 979:157-164. 14. Akinpelu AO, Olojede AO, Amamgbo EF, Njoku SC. Response of Hausa potato to different NPK 15:15:15 Fertilizer Rates in NRCRI, Umudike, Abia State, Nigeria. Journal of Agriculture and Social Research. 2011; 11(1):22-23. 15. Enwezo WO, Ohiri AC, Opuwaribo EE, Udo JE. Literature Review on Soil Fertility Investigations in Nigeria. Federal Ministry of Agricultural and Natural Resources, Lagos; 1990. 16. Namo OAT. Screening for Source-Sink Potentials in Some Sweet Potato (Ipomoea batatas (L.) Lam.) Lines in Jos – Plateau, Nigeria. Published Ph.D. Thesis, University of Jos, Jos, Nigeria. Published by Lambert Academic Publishing, Omniscriptum GmbH Co. KG, Deutschland, Germany. 2005;240. 17. Gregory FG. Physiological conditions in cucumber houses. 3rd Annual Report of the Experimental Research Station, Nursery and Market Garden Industries Development Soc. Ltd. Chestnut. 1918;19-28. 18. Little TM, Hills FJ. Agricultural Experimentation, Design and Analysis. John Wiley and Sons Ltd., New York, USA. 1977;350. 19. Rykbost KA, Locket KA, Maxwell J. Effect of Seed Piece Size on Performance of Three Potato Varieties. Klamath Experiment Station Report; 1995. Available: http://oregonstate.edu/dept/kbrec/sites/default/files/16_-_95seedpiecesize. 20. Deshi KE, Obasi MO, Odiaka NI, Kalu BA, Ifenkwe OP. Leaf Area Index Values of Potato (Solanum tuberosum L.) stored for Different Periods in Different Kinds of Stores. IOSR Journal of Agriculture and Veterinary Science (IOSR-JAVS). 2015;8(1):09-19. 21. Mwanja YP, Goler EE, Gugu FM. Flowering and Seed-Setting Studies in Livingstone Potato (Plectranthus esculentus N.E. BR.) in Jos-Plateau, Nigeria. International Journal of Plant Breeding and Genetics. 2015; 9: 275-279. 22. Kuhlase LM, Ossom EM, Rhykerd RL. Effect of Plant Population on Morphological and Physiological Parameters of Intercropped Sweet Potato (Ipomoea batatas (L.) Lam.) and Groundnut (Arachis hypogaea L.). Academic Journal of Plant Science. 2009; 2(1):16-24. 23. Vimala B, Hariprakash B. Evaluation of some promising sweet potato clones for early maturity. Electronic Journal of Plant Breeding. 2011;2: 461-465.

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24. Mbwaga Z. Quality and yield stability of orange-fleshed sweet potato (Ipomoea batatas (L.) Lam.) varieties in different agro-ecologies. MScThesis. University of Zambia, Lusaka, Zambia, 2007;76. 25. Ogedengbe SA, Safwan I, Ajala BA. Effects of Seed Tuber Size and NPK Fertilizer on Some Yield Components of Coleus Potato (Solenostemon rotundifolius (Poir) J.K. Morton). International Journal of Agriculture and Rural Development. 2015;18(2):2240-2245. 26. Nwankwo IIM Afuape SO. Evaluation of High Altitude Orange-Fleshed Sweet potato (Ipomoea batatas) Genotypes for Adaptability and Yield in Lowland Rainforest Ecology of Umudike, South-eastern Nigeria. IOSR Journal of Agriculture and Veterinary Science. 2013; 5(6):77-81. 27. Wnuk A, Gorny AG, Bocianowski J, Kozak M (2013). Visualizing harvest index in Crops. Communications in Biometry and Crop Science. 2013;8(2):48–59. 28. Masango S. Water use efficiency of orange-fleshed sweet potato. M.Sc. Thesis University of Pretoria, South Africa. 2014;112. Academic Publication. Available http://www.repository.up.ac.za. [Date assessed 2nd Feb. 2017]. 29. Reddy, PP. Plant Protection in Tropical Root and Tuber Crops. Springer International Publishers, India. 2015;336.

Biography of author(s)

Ms Seun Abimbola Opaleye Cytogenetics and Plant Breeding Unit, Department of Plant Science Technology, University of Jos, P.M.B. 2084, Jos, Plateau State, Nigeria.

She is from Oyo State, south-western Nigeria. She was born in 1979, She had her first and second degrees in Ladoke Akintola University of Technology, Ogbomosho, Oyo State and University of Jos, Jos, Nigeria. She worked as Agricultural Science teacher in Government Day Secondary School, Dakko in Taraba State, and as a trainee under the student Industrial Work Experience (SIWES) programme, she worked in the International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria. She has co-authored two peer-reviewed research articles in international journals. She is a member of the Genetics Society of Nigeria (GSN). Her interests include Research and Development, crop production and teaching.

Dr. Timothy Otsnjugu Aku Namo Cytogenetics and Plant Breeding Unit, Department of Plant Science Technology, University of Jos, P.M.B. 2084, Jos, Plateau State, Nigeria.

He is from Nasarawa State, north-central Nigeria. He is a Professor of genetics and plant breeding in the Department of Plant Science and Biotechnology, University of Jos, Jos, Nigeria. His research areas include crop breeding, agronomy and germplasm collection and conservation. His main interests in crops are maize, peanut, Bambara groundnut, sweet potato, Hausa potato, Livingstone potato and the Polynesian arrowroot. He is particularly concerned about neglected (orphan) crops, especially root and tuber crops cultivated in Nigeria. He has received several awards and prizes, including Nasarawa State and the Federal Republic of Nigeria scholarships for post-graduate studies, Leadership Gold Award for Excellence by the African

Advances and Trends in Agricultural Sciences Vol. 1 Productivity of Some Hausa Potato Accessions (Solenostemon rotundifolius (Poir) J. K. Morton in Jos-Plateau Environment

Age International Magazine, Commendation for distinguished contribution to researches in crop science by the Canadian Centre of Science and Education, invitation to propose a Special Issue by SciencePG, Best Lecturer/Researcher Award by the Botanical Society of Nigeria; Fellow, Directorate of Crop Science, International Agency for Standards and Ratings. He has supervised sixteen (16) M.Sc theses and thirty (30) undergraduate projects. He is currently supervising two (2) PhD projects. He has published his research findings in thirty-nine (39) peer-reviewed scientific journals and Conference Proceedings. He has presented many research papers in local and international conferences. He is the Editor-in-Chief of the Nigerian Journal of Botany; he is also serving as Editor/Reviewer in a number of national and international scientific journals. He has recorded some citations in Google scholar. ______© Copyright 2019 The Author(s), Licensee Book Publisher International, This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Reviewers’ Information (1) Ahmed Karmaoui, Morocco. (2) Raúl Leonel Grijalva Contreras, Instituto Nacional de Investigaciones Forestales, México. (3) Anonymous, Bidhan Chandra Krishi Viswavidyalaya, India.

Chapter 7 Print ISBN: 978-81-934224-3-4, eBook ISBN: 978-93-89246-17-9

Roots of Hydroponically Grown Tea (Camellia sinensis) Plants as a Source of a Unique Amino Acid, Theanine

Kieko Saito1,2* and Yoriyuki Nakamura2

DOI:10.9734/bpi/atias/v1

ABSTRACT

The beneficial effects of green tea are well documented. However, most research has reported the effects of green tea brewed solely from leaves or leaf extracts. We focused on tea roots and developed a hydroponic system to explore the effect on roots that biosynthesize one of the rarest functional amino acids, theanine. The level of theanine in tea roots was much higher than in leaves, which was analyzed using HPLC. Moreover, a higher level of theanine was detected in white rootlets than in lignified roots. Thus, tea roots cultured hydroponically in a controlled environment might be considered a natural drug containing theanine, which could lead to synergistic effects with other ingredients of the root. This novel medicinal material from the roots demonstrates a significant medical function for tea that extends beyond its leaves.

Keywords: Tea; Camellia sinensis; theanine; roots; hydroponics.

1. INTRODUCTION

Green tea (Camellia sinensis) leaves are used to make a well-known beverage with beneficial effects on health, and the functions of the main leaf components have been widely studied [1]. Theanine (γ-ethylamide-L-glutamic acid), one of the rarest amino acids and an ingredient of green tea (also found in Camellia genus, C. assamica, C. taliensis, C. irrawadiensis, C. furfuracea), and has not been found in any other plantand has only been found in one mushroom, Xerocomus badius [2,3]. Recently, the biosynthesis of theanine in two species belonging to the genus Schima (S. wallichii and S. mertensiana) was also investigated [4]. The current research has shown that theanine has psychoactive properties, because it is readily absorbed and permeates the blood-brain barrier to function in the brain [5-9], leading to reduced mental and physical stress, improved cognition, and boosting of mood in a manner that is synergistic to caffeine [10-12]. Thus, tea leaves containing theanine, which can exhibit preventive or ameliorating effects on brain dysfunction, have begun to attract attention in our aging and stressed society. Though theanine is synthesized from glutamic acid and ethylamine by γ-L-glutamylethylamide ligase in the roots, and accumulates in leaves through stems [13], the roots have not been extensively studied. Detailed quantitative analysis of roots cultivated in soil is complicated by the presence of a lignified taproot with very fine lateral roots that are intricately shaped. In addition, it appears that lignified taproots contain less theanine than leaves. We therefore employed a modified hydroponic culture system to examine whether the roots of tea plants could be used as a potential source of theanine. We analyzed the root theanine content and assessed the potential application of tea roots as a medicine for improving human physiological function.

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Advances and Trends in Agricultural Sciences Vol. 1 Roots of Hydroponically Grown Tea (Camellia sinensis) Plants as a Source of a Unique Amino Acid, Theanine

2. MATERIALS AND METHODS

2.1 Hydroponic Culture of Tea Plants

In this experiment, we used tea plant (Camellia sinensis var. Yabukita) cuttings that had been grown in soil until roots were established for approximately 1-2 months in order to conveniently obtain young plants with roots (Fig. 1B). The plants with fresh roots were moved to plastic pots and cultured in a nutrient solution with continuous aeration under controlled conditions in a Biotron incubator (Nihonika, Japan) [14]. Day/night temperatures were kept at 25/18°C, photosynthetic photon flux density (PPFD) at the plants was 40.0 μmol m-2 s-1 during the 12 h day period, and the relative humidity was about 60%. The nutrient solution was changed once a week. The roots were shaded and cultured for several months to supply materials for this experiment.

2.2 Determination of Theanine

To determine the concentration of theanine, actively growing white roots were washed with distilled water, dried in a drying oven at 50°C overnight, homogenized with three times the volume of 3% sulfosalicylic acid solution using an ultrasonic homogenizer, and then centrifuged at 12,000 g for 10 min. The concentration of the amino acids in the filtered supernatant was analyzed using an L-8500 automatic amino acid analyzer (Hitachi Co. Ltd., Tokyo, Japan).

3. RESULTS AND DISCUSSION

We employed hydroponics to allow quantitation of the content of theanine in the roots of tea. Fig. 1A shows the appearance of a representative plant cultured hydroponically for one month after transplanting from soil, and thenthe plants were grown for six months to obtain a large amount of fine whiteroots (Fig. 1B). The yield of roots of the tea plant produced depends entirely on the growth (data not shown).

Tea roots cultivated hydroponically were ideally suited for the analysis and biosynthesis of theanine; the white rootlets contained 12 g theanine per 100 g dry weight of roots, a value three times higher than that of lignified taproots cultivated hydroponically (Table 1); for comparison, the typical theanine content of leaves from plants cultivated in soil is about 1-2 g/100 g.

The various biosynthesized substances obtained by hydroponic cultivation (e.g. saccharides, flavonoids) were present at lower amounts than in plants cultivated in soil due to the effect of PPFD on photosynthesis in leaves (data not shown). In the presence of sunshine or other light, theanine is converted to other compounds, such as catechins, so high PPFD inhibits the accumulation of theanine in leaves [13,14]. In addition, only a trace amount of theanine was detected in roots cultivated in soil, indicating that roots cultivated in soil are not a suitable source of theanine. However, hydroponically cultivated tea roots could contain higher amounts of theanine. In addition, the composition and amount of amino acids contained in the roots are different from those in leaves [15], suggesting that tea root might be a medicine or remedy effective in treating a disease or part of the body.

Generally, high-quality green tea is cultured in the shade so that it will accumulate theanine, which has a pleasant flavor; shade inhibits the decomposition of theanine. However, this procedure leads to only 2% theanine in dried leaves, which is inefficient for collection of theanine and is not industrially practical. Accordingly, a chemical means of synthesis was developed as a method for industrial production of theanine in large quantities [16]. However, the yield of this organic synthesis is low, and the operation is complicated by the need for separation and purification of theanine from a mixture of unreacted materials and byproducts. In addition, recently a synthetic method of theanine using bacteria was developed, which has now become an important source of theanine [17-20]. However, the product obtained by this method is not the genuine theanine from Camellia genus. In this study, our findings suggested that hydroponic culture could be employed as an alternative method to obtain large amounts of theanine, albeit not in high purity. However, tea roots may offer a new type of drug

Advances and Trends in Agricultural Sciences Vol. 1 Roots of Hydroponically Grown Tea (Camellia sinensis) Plants as a Source of a Unique Amino Acid, Theanine

based not only on the function of theanine but also possible synergy with other tea root components, which might offer benefits as a Chinese herbal medicine.

Consequently, hydroponics makes it possible to control environmental conditions during growth of tea plants. We have already succeeded in rooting cuttings of tea plants in a nutrient solution only. Therefore, it is likely that this approach to cultivation will facilitate the extraction of theanine from the roots.

Recent demand for theanine has increased due to its use as a food additive for enhancing flavor and as a supplement for supporting human health, especially mental health [5-9]. Unno demonstrated that theanine exhibit the stress-reducing function in humans and animals [21-24].

Indeed, we propose that the roots of tea plants, which, may attenuate brain dysfunction.

Further study using animals will likely reveal the effects of tea roots on the brain and other organs [25]. Tea roots hydroponically cultivated, which include phytochemicals might be a novel material for our health.

Fig. 1. Tea roots cultivated hydroponically (A) Tea plant one month after transplanting from soil (B) Actively growing tips of the roots after six months

Table 1. Concentration of theanine produced by different cultivation systems

4. CONCLUSION

We determined high amounts of theanine from tea roots, especially fine white roots, which was hydroponically cultivated under a controlled environment, and suggested tea roots.

ACKNOWLEDGEMENTS

We appreciate Hamada K, Kan T, Takahashi N, and Fukazawa N for technical assistance.

Advances and Trends in Agricultural Sciences Vol. 1 Roots of Hydroponically Grown Tea (Camellia sinensis) Plants as a Source of a Unique Amino Acid, Theanine

COMPETING INTERESTS

Authors have declared that no competing interests exist.

REFERENCES

1. Eto H, Tomita I, Shinmura J, Isemura M, Hara M, Yokogoshi H, Yamamoto M, Editors. Health science of tea (Cha no Kinou); 2013. No-Bun-Kyo, Tokyo (in Japanese) 2. Casimir J, Jadot J, Renard M. Separation and characterization of N- ethyl-g-glutamine in Xerocomus badius (Boletus ladius). Biochim. Biophys. Acta. 1960;39:462–468. 3. Wei-Wei D, Shinjiro O, Hiroshi A. Distribution and biosynthesis of theanine in Theaceae plants. Plant Phys. Biochem. 2010;47:70-72. 4. Hiroshi A. Occurrence, biosynthesis and metabolism of theanine (γ-Glutamyl-L-ethylamide) in plants: A comprehensive review; 2015. Available:https://doi.org/10.1177/1934578X1501000525 5. Yokogoshi H, Kobayashi M, Mochizuki M. Terashima. Effect of theanine, gamma glutamylethylamide, on brain monoamines and striatal dopamine release in conscious rats. Neurochem Res. 2002;23:667–73. 6. Juneja LR, Chu DC, Okubo T, Nagato Y, Yokogoshi H. L-theanine—a unique amino acid of green tea and its relaxation effect in humans. Trends in Food Sci. & Tech. 1999;10:199-204. 7. Kimura K, Ozeki M, Juneja LR, Ohira H. L-Theanine reduces psychological and physiological stress responses. Biol. Psychol. 2007;74:39–45. 8. Unno K, Fujitani K, Takamori N, Takabayashi F, Maeda K, Miyazaki H, Tanida N, Iguchi K, Shimoi K, Hoshino M. Theanine intake improves the shortened lifespan, cognitive dysfunction and behavioural depression that are induced by chronic psychosocial stress in mice. Free Radic. Res. 2011;45:966–974. 9. Vuong QV, Bowyer MC, Roach PD. L-Theanine: properties, synthesis and isolation from tea. J. Sci. Food Agric. 2011;91:1931–1939. 10. Kimura K, Ozeki M, Juneja L, Ohira H. L-Theanine reduces psychological and physiological stress responses. Biol Psychol. 2007;74:39–45. 11. Park SK, Jung IC, Lee WK. Lee YS, Park HK, Go HJ. A combination of green tea extract and L- theanine improves memory and attention in subjects with mild cognitive impairment: A double- blind placebo-controlled study. J Med Food. 2011;14:334–343. 12. Haskell CF, Kennedy DO, Milne AL, Wesnes KA, Scholey AB. The effects of Ltheanine, caffeine and their combination on cognition and mood. Biol Psychol. 2008;77:113–122. 13. Konishi S, Takahashi E. Metabolism of theanine in tea seedlings and transport of the metabolites. Nippon Dojouhiryougaku Zasshi. 1969;40:479-84. (In Japanese) 14. Saito K, Ikeda M. The function of roots of tea plant (Camellia sinensis) cultured by a novel form of hydroponics and soil acidification. Am J Plant Sci. 2012;3:646-48. 15. Ohta K, Yoshida A, Harada K. HPLC analysis of free aminoacids and caffeine in green tea cultivated by hydroponics. Nippon Nogeikagaku Kaishi. 1995;69:1331-1339. 16. Kimura R, Miura T. Influence of alkylamides of glutamic acid and related compounds on the central nervous system. II. 1) Syntheses of amides of glutamic acid and related compounds, and their effects on the central nervous system. Chem Pharm Bull. 1971;19:1301-307. 17. Kawagishi H, Sugiyama K. Facile and large-scale synthesis of L-Theanine. Biosci Biotechnol Biochem. 1992;56:689. 18. Suzuki H, Izuka S, Miyakawa N, Kumagai H. Enzymatic production of theanine, an “umami” component of tea, from glutamine and ethylamine with bacterial γ-glutamyltranspeptidase. Enzym. Microbial. Technol. 2002;31:884-889. 19. Mu W, Zhang T, Jiang B. An overview of biological production of L-theanine. Biotechnol. Advan. 2015;33:335-342. 20. Bindal S, Gupta R. L-Theanine synthesis using γ-Glutamyl transpeptidase from Bacillus licheniformis ER-15. J. Agric. Food Chem. 2014;62:9151-9159. 21. Unno K, Fujitani K, Takamori N, Takabayashi F, Maeda K, Miyazaki H, Tanida N, Iguchi K, Shimoi K, Hoshino M. Theanine intake improves the shortened lifespan, cognitive dysfunction and behavioural depression that are induced by chronic psychosocial stress in mice. Free Radic. Res. 2011;45:966–974.

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22. Unno K, Iguchi N, Tanida N, Fujitani K, Takamori N, Yamamoto H, Ishii N, Nagano H, Nagashima T, Hara A, Shimoi K, Hoshino M. Ingestion of theanine, an amino acid in tea, suppresses psychosocial stress in mice. Exp. Physiol. 2013;98:290–303. 23. Unno K, Tanida H, Ishii N, Yamamoto H, Iguchi K, Hoshino M, Takeda A, Ozawa H, Ohkubo T, Juneja LR, Yamada H. Anti-stress effect of theanine on students during pharmacy practice: Positive correlation among salivary α-amylase activity, trait anxiety and subjective stress. Pharmacol. Biochem. Behav. 2013;111:128–135. 24. Unno K, Furushima D, Hamamoto S, Iguchi K, Yamada H, Morita A, Horie H, Nakamura Y. Stress-reducing function of matcha green tea in animal experiments and clinical trials nutrients. 2018;10:1468. 25. Saito K, Ikeda M, Hideki Kametani H. Theanine in the tea roots attenuates memory deficits in the aged rats. Free Radical Biology and Medicine. 2011;51(Suppl.):S58.

Biography of author(s)

Dr. Kieko Saito School of Food and Nutritional Sciences, University of Shizuoka, Shizuoka 422-8526, Japan and Tea Science Center, University of Shizuoka, Shizuoka, 422-8526, Japan

She is the Assistant Professor of School of Food and Nutritional Sciences, University of Shizuoka, Shizuoka, Japan. She received her master degree from Graduate School of Agriculture, Nihon University in 1990. After working at RIKEN (Saitama, Japan) and Gerontology Research Center, NIH (USA) as a research associate, she started her career at the University of Shizuoka in 1996. She is in her present position since 2008. She received her PhD based on the thesis of Oxidative stress and Aging in 1991 from Nihon University. Her specialization is in Functional Food and Environmental Science. She joined Tea Science Center of University of Shizuoka in 2014 to assist research related with the tea industry. Her current research interests center on the physiological function of fermented tea and honey from tea flower (Camellia sinensis). ______© Copyright 2019 The Author(s), Licensee Book Publisher International, This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Reviewers’ Information (1) Anonymous, Nigeria. (2) Anonymous, UK.

Chapter 8 Print ISBN: 978-81-934224-3-4, eBook ISBN: 978-93-89246-17-9

Genetic Variability of Sugarcane Clones as Affected by Major Endemic Diseases in Ferké, Northern Ivory Coast

Yavo M. Béhou1,2 and Crépin B. Péné1*

DOI:10.9734/bpi/atias/v1

ABSTRACT

Background: Sugarcane is a major commercial crop grown in tropical and subtropical areas of the world, including West and Central Africa. Across this region, smut, leaf scald and pokkah boeng are considered as endemic diseases, the first two being economically important. Aims: The overall objective of study was to contribute to sugarcane yield improvement in Ivory Coast. The specific objective was to evaluate the diversity of susceptible sugarcane genotypes mainly in first ratoon crop to three major endemic diseases under natural infection, namely leaf scald, smut and pokkah boeng. Methodology: The study was carried out over 2 seasons (2016-18) as plant and first ratoon cane at Ferké 1 experimental station under full covering sprinkler irrigation in northern Ivory Coast. Treatments were composed of 863 sugarcane genotypes split into 39 families planted at single row density. Planting was done per genotype in rows of 3 m long depending on families, without replication and compared to the check variety SP70/1006. That check was replicated every five rows to ease comparison with the clones. Phytosanitary observations regarding the three endemic diseases made at the age of five months were subjected to a series of multivariate analyses. Results: The study showed that most relevant diseases determining the diversity of susceptible sugarcane genotypes were, in descending order, pokkah boeng, smut and leaf scald. Increase in clone infestations on first ratoon cane compared with plant cane was observed regarding the three endemic diseases but more importantly for smut by 51%. The dendrogram deduced from cluster analysis showed that infected genotypes were split into six groups with same families belonging often to different clusters so that no family investigated specifically susceptible or resistant to any disease was determined. In other words, each family investigated was composed of disease-free as well as susceptible genotypes in proportions varying from one family to another. Conclusions: All families investigated were relevant to maintain the diversity required for the breeding process under way. Examples of recommended families were the following: disease-free (F02, F03, F04, F05, F06); resistant (F01, F06, F07, F08, F09); moderately resistant (F10, F11, F12, F13, F14). It came out from the study that most relevant diseases determining the diversity of susceptible sugarcane genotypes were, in descending order, pokkah boeng, smut and leaf scald. Increase in clone infestations on first ratoon cane compared with plant cane was observed regarding the three endemic diseases but more importantly for smut by 51%. Each family investigated was composed of disease-free as well as susceptible genotypes in a certain proportion which varied from one family to another. Cluster 5 was the most prolific of infected genotypes with 286 clones (33%) split into 36 families (92%) whereas clusters 2, 3 and 4 were the least prolific ones, with 42, 52 and 56 infected genotypes split into 14, 23 and 21 families, respectively. Clusters 1 and 2 were much more associated with genotypes susceptible to smut and pokkah boeng but also with genotypes moderately susceptible to pokkah boeng. Clusters 3 and 4 were associated with genotypes susceptible or highly susceptible to leaf scald. In contrast, clusters 0, 5 and 6 were related to disease-free, resistant or

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1Department of Research and Development, SucafCI/SOMDIAA, 22 Rue Des Carrossiers Treichville Zone 3, 01 P.O.Box 1967 Abidjan 01, Côte d’Ivoire. 2UMRI: Agricultural Sciences and Engineering, EDP/National Polytechnic Institute (INPHB), P.O.Box 1313 Yamoussoukro, Côte d’Ivoire. *Corresponding author: E-mail: [email protected];

Advances and Trends in Agricultural Sciences Vol. 1 Genetic Variability of Sugarcane Clones as Affected by Major Endemic Diseases in Ferké, Northern Ivory Coast

moderately resistant genotypes and which crosses or families would, therefore, be recommended for Ferké agro-ecology. Examples of such families were the following: disease-free (F02, F03, F04, F05, F06); resistant (F01, F06, F07, F08, F09); moderately resistant (F10, F11, F12, F13, F14).

Keywords: Leaf scald; smut; pokkah boeng; susceptibility; resistance; agro-ecology; multivariate analysis.

1. INTRODUCTION

Sugarcane is a major commercial crop grown in tropical and subtropical regions of the world, including West and Central Africa. Sugarcane is a tall-growing monocotyledonous perennial grass that is cultivated in the tropical and subtropical regions of the world, primarily for its ability to store high concentrations of sucrose, or sugar, in the stem [1]. During the last 100 years, many sugarcane producing countries such as Mauritius, Reunion Island, Java-Indonesia, New Guinea, India and South Africa, have experienced epidemics of various diseases like red rot, smut, wilt, rust leaf scald gummosis and yellow leaf [2-4]. The damage caused during each epidemic would vary depending on the nature of disease and spread of affected varieties. Number of sugarcane varieties were replaced because of their breakdown to new diseases or to new pathogenic strains. Many biotic and abiotic stresses affected the sugarcane production and are known to be one of the oldest cultivated plants in the world. Improving sugarcane production will greatly help in economic prosperity of the farmers and others associated with sugarcane cultivation. Large numbers of sugarcane pathogens have been recorded all over the world [5]. Propagation of sugarcane through vegetative cuttings enhances spread of diseases through planting materials. Primary transmission of diseases by seed canes causes a serious threat to sugarcane growth and yields. Therefore, disease resistant varieties play a key role in controlling numerous biotic constraints in sugarcane and several varieties were developed to manage diseases in the past. In parallel, different agronomic practices and physical methods like hot water therapy are being effectively used to control diseases transmission in sugarcane. More recently, propagation of sugarcane through tissue culture is being used in some advanced countries to produce virus, phytoplasma and bacteria disease-free planting materials. Sugarcane diseases, such as smut, white leaf, and wilt/top rot/Pokkah Boeng, are critical limitations of production, causing serious losses in yield and quality among susceptible cultivars [6]. Use of disease resistant or tolerant varieties along with healthy seed nursery programs would form the basis to successfully manage diseases in sugarcane. Across West and Central Africa, smut, leaf scald and pokkah boeng used to be considered as endemic diseases in sugarcane (Fig. 1). The first two (smut and leaf scald) were revealed economically important compared to pokkah boeng. Still, severe symptoms of pokkah boeng with top-rotting damages were often observed on highly susceptible cane genotypes which needed to be identified and eliminated in the crop breeding process. That’s why the three diseases used to be considered as one of the key criteria in variety selection of sugarcane carried out in Ivory Coast [7-8]. Pokkah boeng is a fungus caused by Fusarium moliniforme. Injury varies from slight chlorosis and splitting of the base of young unfolding leaves to top rotting, which may kill the growing point. While common in certain susceptible varieties during warm and rainy weather, it is seldom of commercial importance. Leaf scald, caused by a bacterium (Xanthomonas albilineans), is considered among sugarcane major diseases and therefore of commercial importance. It is primarily a vascular disease with streaks produced on cane leaves. Sometimes, instead of definite stripes, the entire shoot is chlorotic to nearly white. Diseased plants have a characteristic stunted appearance and the terminal whorl of leaves curves inward at the tips, which are often dried or withered [9]. Sprouting of the lateral buds beginning at the base of stalk is characteristic and may occur when there is no apparent injury to the top. In the acute stage, some shoots or the entire stool may suddenly wilt and die [10]. Several symptoms are expressed in Leaf scald, varying in severity from a single, white, narrow, sharply defined leaf stripe to death of shoots or entire plants. A common symptom in mature diseased cane is the abnormal formation of side shoots on stalks, and basal side shoots tend to develop more rapidly than those higher up, whereas the opposite occurs in healthy stalks [11]. The disease causes marked reductions in growth, tillering and ratooning ability of susceptible varieties. It is highly infectious and spreads through infected seed cane, knife cuts and probably by other means of physical contact [12]. However, aerial transmission and epiphytic survival have also been reported for this pathogen [13-15]. Smut, caused by a fungus namely Ustilago scitaminea or Sporisorium scitamineum is characterized by the production from the growing point of a long whiplike shoot [16-17]. Smut teliospores are scattered when the membrane covering this shoot bursts and carried by wind or rain/irrigation water

Advances and Trends in Agricultural Sciences Vol. 1 Genetic Variability of Sugarcane Clones as Affected by Major Endemic Diseases in Ferké, Northern Ivory Coast

[18]. Infection takes place through seed pieces and through axillary buds of the growing plant. Germination of buds from infected cuttings may be seriously reduced, the plant is stunted and ratooning ability is weakened [19]. Sugarcane streak mosaic disease caused by SCSMV was first recognized on Zuénoula and Ferké 2 sugar estates in Ivory Coast in 2017 and 2018 respectively (Fig. 2). It is also a disease of great economic importance in sugarcane only recognised in the late 1990s [20-21]. It was also first recognised in Indonesia in 2005 on the island of Java [22-24] where average yield losses of 20% were reported [25]. Its symptoms are similar to those of Sugarcane mosaic disease (caused by SCMV), nitrogen deficiency, drought or susceptibility to herbicides. That is why its pathogen detection is important through accurate diagnostic tools like ELISA – Enzyme Linked Immunosorbent Assay or RT-PCR – Reverse Transcriptase Polymerase Chain Reaction [26]. Managing these endemic diseases through variety selection in the targeted African region is of crucial importance regarding the context of growing interest for early stage crop improvement. That breeding program is based on cross hybridization and selection of genotypes at early stages where numerous plant materials investigated are often highly susceptible [27]. Having knowledge of crosses responsible for susceptible or highly susceptible genotypes would help in the choice of parental material for location-oriented hybridizations.

The aim of the study was to characterize the genetic variability of susceptible sugarcane clones mainly in first ratoon crop to three endemic diseases at one-row screening stage, namely smut, leaf scald and Pokkah Boeng.

2. MATERIALS AND METHODS

2.1 Site Characteristics

The study was carried out at Ferké 1 experimental station in northern Ivory Coast (9°20’ – 9°60’ N, 5°22’ – 5°40’ W, 325 m). Prevailing climate was tropical dry with two seasons: one was dry which occurs from early November to April and the other, wet, from May to late October. The dry season was marked by a northern and warm trade wind (Harmattan) taking place from mid-November to late January. Rainfall pattern is unimodal and centered over August and September which totalize almost half of annual average rainfall (1 200 mm) with an average daily temperature of 27°C, maximum and minimum values yielding 32.5 and 21.0°C, respectively. Irrigation water requirements for sugarcane growth and yield performance ranged to about 650-700 mm/yr [28-30]. Main soil units (ferralsol or hydromorphic type) were characterized by shallow to moderate depths (30-80 cm) with sand-clay as predominant soil texture where the experiment was located.

2.2 Sugarcane Crop Material

The crop material investigated which comprised 863 sugarcane genotypes, was grown over two years as plant crop and first ratoon at one-row screening stage. All clones were planted with stem cuttings following families and compared to a check commercial variety (SP70-1006) which was moderately susceptible to smut and resistant to leaf scald and pokkah boeng. Genotypes derived from the second generation of sugarcane hybrid seeds were provided in November 2015 by Reunion Island sugarcane breeding center (eRcane). They resulted from bi-parental crosses of commercial or elite varieties of diversified origins (Reunion, Brazil, Australia, Sudan, Florida, Colombia, South Africa, etc.).

2.3 Experimental Design

The experimental design used at one-row screening stage was an incomplete block design comprising 863 clones, each planted in a single-row plot of 3 m long without replication apart from the check variety. That one was replicated many times (173) every 5 rows of clones subjected to visual screening. Clones split into 39 families (or crosses) as well as the check variety were planted separately in single row plots with 1.5 m of row spacing (4.5 m²/plot) in November 2016 following 11 blocks of 7 m wide and 30 m long with 3 m spacing. Families were not repeated except for the check variety. The number of clones per family varied from 2 (F31 or F32) up to 161 (F07). Within each block, there was a 1 m spacing between adjacent clones to allow distinction of individual clones during disease ratings, growth vigor evaluation and selection. To prevent edge effects the field trial

Advances and Trends in Agricultural Sciences Vol. 1 Genetic Variability of Sugarcane Clones as Affected by Major Endemic Diseases in Ferké, Northern Ivory Coast

was surrounded by a buffer zone of 3 m wide and 30 m long planted with a commercial variety (R579) rather resistant to the three diseases.

2.4 Epidemic Disease Observations

Symptoms, based on natural infection of smut, leaf scald and pokkah boeng as epidemic diseases with high pressure in West and Central African agro-ecology, were observed on sugarcane genotypes at the age of five months. Disease ratings were based on percentage of cane shoots or stools infected by fungus or leaf scald. The susceptibility scale of smut was provided by Rao, et al [31] as follows: Free of symptom (0%); Resistant (0.1 - 5%); Moderately resistant (5.1 - 15%); Moderately susceptible (15.1 – 30%); Susceptible (> 30%).

That of pokkah boeng was provided by Gulya, et al. [32], Karuppaiyan et al [33] as follows: Free of symptom (0%); Resistant (0.1 - 1%); Moderately resistant (1.1 - 10%); Moderately susceptible (10.1 – 25%); Susceptible (25.1 - 50%); Very susceptible (50.1 – 100%).

The leaf scald incidence scale as described by Rott, et al. [11] is the following: Free of symptom (0 %); Susceptible (0.1 - 10%); Very susceptible (11 - 100%).

2.5 Statistical Analysis

Data processing was conducted using Excel 2013, Statistica 7.1 and R 2.2 software packages which was based on clone phenotypic traits observed in the experiment. To do so, data were firstly recorded as a database and processed on Excel following a dynamic crossed table. Percentage of disease infestations and qualitative assessment of infestations (ratings) were used in data processing. A series of 3 multivariate analyses using R software, i.e. principal component analysis (PCA), cluster analysis (CA) and corresponding factor analysis (CFA), were made. The data were computed in application of Mahalanobis D² statistics among all possible combinations of genotypes grouped into different clusters following canonical root method described by Rao [34].

3. RESULTS

3.1 Estimates of Disease Free or Infected Clones in Plant and First Ratoon Cane

Phytosanitary profiles of test families in terms of number of healthy or infected clones regarding the three endemic diseases whose symptoms were observed is shown in Table 1. All families were infected by at least one disease, except for family F32 which was composed of 2 clones. Over both plant cane and first ratoon, the number of non-infected families decreased from nine (23%) to two (5%) and one (2.5%), respectively for leaf scald, pokkah boeng and smut. Therefore, the most infectious disease across the experiment was, in descending order, smut, pokkah boeng and leaf scald. As shown in Fig. 1, number of disease-free genotypes in plant cane decreased significantly (P=0.05) from leaf scald to smut and pokkah boeng with, respectively, 98, 86 and 80%. Number of naturally infected genotypes increased significantly (P=0.05) in first ratoon cane for the three diseases compared to plant cane, with 13.4, 65 and 39.5%, respectively, for leaf scald, smut and pokkah boeng. Smut was the most infectious disease in first ratoon cane whereas leaf scald was the least infectious one with, respectively, 50 and 12% increase compared to that of plant cane.

3.2 Clone Susceptibility to Endemic Diseases in First Ratoon

The number of disease free genotypes in first ratoon cane was significantly high (P=0.05) regarding leaf scald and pokkah boeng with, respectively, 87 and 70% as opposed to low (35%) for smut. About 50 and 28% of cane genotypes were resistant or moderately resistant to smut and pokkah boeng, respectively (Fig. 4). In contrast, 13.4, 16.5 and 0.1% of genotypes were susceptible or highly susceptible to leaf scald, smut and pokkah boeng, respectively. This shows the economic importance of leaf scald and smut.

Advances and Trends in Agricultural Sciences Vol. 1 Genetic Variability of Sugarcane Clones as Affected by Major Endemic Diseases in Ferké, Northern Ivory Coast

Shoot infected by Pokkah Shoots infected by Leaf Scald Shoots infected by Smut Boeng (Fusarium moliniforme) (Xanthomonas albilineans) (Ustilago scitaminea)

Fig. 1. Symptoms of three sugarcane endemic diseases investigated three months after harvest (first ratoon) in Ferké, Ivory Coast

Leaf of variety R570: 100% of disease Leaves of variety R579: Disease severity scale following incidence with severity level 2 observed Putra et al [24]: from asymptomatic (level 0) to highly in R3-005 commercial field. infested (level 4).

Fig. 2. Symptoms of Sugarcane Streak Mosaic disease (caused by SCSMV) observed four months after planting in Ferké, Ivory Coast SCSM disease was first recognised on Ferké sugarcane plantations in December, 2018

Fig. 3. Percentage of disease free and naturally infected clones depending on endemic disease observed in plant cane and first ratoon in Ferké 1, Ivory Coast

Advances and Trends in Agricultural Sciences Vol. 1 Genetic Variability of Sugarcane Clones as Affected by Major Endemic Diseases in Ferké, Northern Ivory Coast

Table 1. Phytosanitary profiles under natural infection of sugarcane genotypes in plant cane (R0) and first ratoon (R1) at one-row stage in Ferké (Ivory Coast)

Families Parents Leaf scald Smut Pokkah boeng Total Heathy Infected Heathy Infected Heathy Infected Female Male R0 R1 R0 R1 R0 R1 R0 R1 R0 R1 R0 R1 F01 R98/4009 R95/4065 14 12 1 3 10 0 5 15 10 3 5 12 15 F02 N42 R96/6422 4 4 0 0 4 3 0 1 3 2 1 2 4 F03 R92/2401 R97/6375 42 33 0 9 39 16 3 26 32 29 10 13 42 F04 NCo 310 R99/6153 28 27 0 1 28 13 0 15 24 19 4 9 28 F05 R03/4018 (e) R04/8052 17 17 0 0 17 8 0 9 16 15 1 2 17 F06 R01/0277 R95/2100 27 23 0 4 26 14 1 13 21 13 6 14 27 F07 RB83/5486 (e) R575 160 137 1 24 142 44 19 117 134 127 27 34 161 F08 R81/0833 SP70/1143 12 8 2 6 13 2 1 12 13 12 1 2 14 F09 N14 (e) R585 45 45 1 1 41 11 5 35 36 30 10 16 46 F10 H32/8560 R585 52 41 3 14 30 6 25 49 39 40 16 15 55 F11 R03/4018 (e) N14 39 36 2 5 26 9 15 32 36 35 5 6 41 F12 R584 R99/6153 19 17 0 2 17 13 2 6 17 16 2 3 19 F13 M1042/86 PR83/1248 17 17 0 0 15 10 2 7 14 13 3 4 17 F14 R98/0814 R585 13 13 1 1 13 6 1 8 13 11 1 3 14 F15 R96/2569 R585 13 12 1 2 14 7 0 7 8 12 6 2 14 F16 H72/8597 (e) R585 24 22 2 4 19 10 7 16 19 19 7 7 26 F17 R582 R585 20 18 0 2 18 6 2 14 14 16 6 4 20 F18 R83/0444 N14 5 4 0 1 5 1 0 4 2 5 3 0 5 F19 R575 N6 4 2 0 2 4 1 0 3 4 4 0 0 4 F20 R575 CP81/1384 6 6 0 0 6 4 0 2 6 4 0 2 6 F21 R93/2351 R99/6254 17 13 0 4 15 9 2 8 15 13 2 4 17 F22 R90/2992 R97/2332 3 2 0 1 3 1 0 2 3 2 0 1 3 F23 R579 R92/0804 6 5 0 1 6 4 0 2 6 4 0 2 6 F24 R91/4188 R00/2460 10 10 0 0 8 0 2 10 6 8 4 2 10 F25 R94/6113 R93/6769 16 4 0 12 15 5 1 11 15 11 1 5 16 F26 R92/6545 R93/6683 11 8 0 3 11 6 0 5 5 5 6 6 11 F27 R96/2569 R97/2332 2 2 0 0 1 1 1 1 2 1 0 1 2 F28 R582 R570 35 30 0 5 34 20 1 15 31 25 4 10 35 F29 R01/2072 VMC71/238 7 6 0 1 6 3 1 4 5 7 2 0 7

Advances and Trends in Agricultural Sciences Vol. 1 Genetic Variability of Sugarcane Clones as Affected by Major Endemic Diseases in Ferké, Northern Ivory Coast

Families Parents Leaf scald Smut Pokkah boeng Total Heathy Infected Heathy Infected Heathy Infected Female Male R0 R1 R0 R1 R0 R1 R0 R1 R0 R1 R0 R1 F30 R93/0136 R00/2460 14 13 0 1 13 4 1 10 10 12 4 2 14 F31 R89/2042 R97/2332 10 9 0 1 9 6 1 4 7 10 3 0 10 F32 R11/7003 N27 2 2 0 0 2 2 0 0 2 2 0 0 2 F33 R93/0136 SP80/3280 2 1 0 1 1 0 1 2 2 1 0 1 2 F34 R579 R94/6447 12 11 0 1 12 6 0 6 10 2 2 10 12 F35 R98/6095 HoCP85/845 10 10 0 0 10 2 0 8 8 8 2 2 10 F36 TC9 R95/4065 33 33 0 0 32 19 1 14 26 21 7 12 33 F37 R00/4009 R95/4053 43 41 0 2 32 9 11 34 31 17 12 26 43 F38 R98/4009 R98/4001 28 28 0 0 25 16 3 12 20 18 8 10 28 F39 RB83/5054 R97/2335 26 25 1 2 19 4 8 23 24 16 3 11 27 Total 848 747 15 116 741 301 122 562 689 608 174 255 863

Leaf scald Smut Pokkah Boeng 13.33 5.79% 1.62% 0.12% % 20.86 10.54 34.88 % % 0.12% %

6.95% 19.70 %

86.56 29.08 70.45 % % % Disease-free Disease-free Disease-free Resistant Resistant Susceptible Moderately resistant Moderately resistant Moderately susceptible Moderately susceptible Highly susceptible Susceptible Susceptible

Fig. 4. Distribution of sugarcane genotypes in first ratoon crop following their susceptibility to each of the three endemic diseases observed under natural infection at one-row screening stage in Ferké 1, Ivory Coast

Advances and Trends in Agricultural Sciences Vol. 1 Genetic Variability of Sugarcane Clones as Affected by Major Endemic Diseases in Ferké, Northern Ivory Coast

3.3 Clone Susceptibility in First Ratoon Depending on Endemic Diseases Observed

In first ratoon cane, all genotypes studied were split into eight different groups depending on endemic disease infestations observed (Table 2). Group G0 was composed of 209 disease-free sugarcane genotypes (24%) split into 34 families out of 39 (87%). Groups G1, G2 and G3 were composed of genotypes infected only by leaf scald, smut and pokkah boeng, respectively. Smut was the most infectious disease with a rate of 37% corresponding to 319 genotypes split into 34 families out of 39 (87%). Leaf scald was the least infectious one with a rate of 3% involving 25 genotypes split into 15 families. Infestation rate of pokkah boeng alone gave 6.5% corresponding to 56 genotypes split into 22 families. Group 7 was composed of genotypes infected by the three endemic diseases with a rate of 3% which corresponded to 25 clones split into 15 families out of 39 (38.5%).

Groups G1, G4, G5 and G7 were associated with genotypes susceptible or highly susceptible to leaf scald whereas G0, G2, G3 were associated with disease-free genotypes (Fig. 5). Group G6 was associated with genotypes resistant, moderately resistant or moderately susceptible to pokkah boeng and smut.

) a

i Leaf S_H.sus. t r e

n Leaf S_Sus. I ' d

5 G1 5 , G54 2 3

( G7

2 8 6 3 3 ,

: e u l a v Smut_D-free Pok_D-free n

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2 G6

n Pok_Res. o i Pok_M.sus.

s Pok_Sus. n e m i D

Cluster 0 Disease type Dimension 1; Eigen value : ,37638 (36,38 % of Inertia)

Fig. 5. Projection of groups of sugarcane genotypes in 1-2 factor plane following correspondence factor analysis (CFA)

3.4 Cluster Analysis of Naturally Infected Cane Genotypes

The dendrogram deduced from cluster analysis showed that infected genotypes were split into six groups (Fig. 6, Table 3) with same families belonging often to different clusters so that no family specifically susceptible or resistant to any disease investigated was determined. In other words, each family investigated was composed of disease-free as well as susceptible genotypes in a certain

Advances and Trends in Agricultural Sciences Vol. 1 Genetic Variability of Sugarcane Clones as Affected by Major Endemic Diseases in Ferké, Northern Ivory Coast

proportion which varied from one family to another. Therefore, all families investigated were relevant to maintain the diversity required for the breeding process under way.

Table 2. Grouping of sugarcane clones following their susceptibility or not to endemic diseases observed under natural infection at one-row stage in first ratoon, Ferké 1 (Ivory Coast)

Groups Characteristics Number Families represented (Number of clones) G0 Disease free 209 F02 (2), F03 (12), F04 (10), F05 (7), F06 (9), F07 (34), clones F08 (1), F09 (8), F10 (2), F11 (7), F12 (9), F13 (10), F14 (6), F15 (6), F16 (7), F17 (5), F18 (1), F20 (3), F21 (3), F22 (1), F23 (3), F25 (2), F26 (3), F27 (1), F28 (13), F29 (2), F30 (3), F31 (5), F32 (2), F35 (1), F36 (12), F37 (5), F38 (11), F39 (3) G1 Infected clones 25 F03 (2), F07 (6), F08 (1), F10 (1), F11 (2), F12 (2), F15 (1), only by leaf scald F19 (1), F21 (3), F25 (1), F26 (1), F28 (1), F29 (1), F31 (1), F39 (1) G2 Infected clones 319 F01 (1), F03 (11), F04 (9), F05 (8), F06 (4), F07 (75), F08 (6), only by smut F09 (21), F10 (27), F11 (24), F12 (5), F13 (3), F14 (5), F15 (4), F16 (10), F17 (9), F18 (3), F19 (2), F20 (1), F21 (7), F23 (1), F24 (8), F25 (1), F28 (9), F29 (4), F30 (8), F31 (4), F33 (1), F34 (2), F35 (7), F36 (9), F37 (12), F38 (7), F39 (11) G3 Infected clones 56 F02 (1), F03 (1), F04 (3), F05 (1), F06 (3), F07 (2), F09 (3), only by pokkah F10 (3), F12 (2), F16 (1), F17 (1), F20 (1), F21 (3), F25 (1), boeng F26 (2), F28 (4), F30 (1), F34 (6), F35 (1), F36 (7), F37 (4), F38 (5) G4 Infected clones 55 F01 (2), F03 (4), F07 (12), F08 (4), F09 (1), F10 (10), F11 (2), by leaf scald and F15 (1), F16 (2), F17 (2), F18 (1), F19 (1), F22 (1), F25 (7), smut F26 (1), F28 (2), F30 (1), F39 (1) G5 Infected clones 11 F03 (1), F06 (2), F07 (2), F16 (2), F23 (1), F25 (1), F28 (2) by leaf scald and pokkah boeng G6 Infected clones 163 F01 (11), F02 (1), F03 (9), F04 (5), F05 (1), F06 (7), F07 (26), by smut and F08 (1), F09 (13), F10 (9), F11 (5), F12 (1), F13 (4), F14 (2), pokkah boeng F15 (2), F16 (4), F17 (3), F20 (1), F22 (1), F23 (1), F24 (2), F26 (3), F27 (1), F28 (4), F30 (1), F34 (3), F35 (1), F36 (5), F37 (20), F38 (5), F39 (11) G7 Infected clones by 25 F01 (1), F03 (2), F04 (1), F06 (2), F07 (4), F08 (1), F10 (3), the three endemic F11 (1), F14 (1), F21 (1), F25 (3), F26 (1), F33 (1), F34 (1), diseases F37 (2)

Mahalanobis square distance between clusters displayed in Table 4 shows that they were significantly different from one another (P<0.001). Cluster C5, as the most prolific of infected genotypes, was composed of 286 clones (33%) split into 36 families over 39 (92%). It was followed by clusters C6 and C1 with 125 and 93 infected genotypes split into 29 and 28 families, respectively. Clusters C2, C3 and C4 were the least prolific, with 42, 52 and 56 infected genotypes split into 14, 23 and 21 families, respectively. Extremely high values of coefficient of variation obtained were in line with the high diversity in clone susceptibility or resistance to disease observed with clusters determined (Table 5). It is particularly the case for clusters 1 and 2, cluster 3 and 4 all six clusters regarding, respectively, leaf scald, smut and pokkah boeng.

3.5 Susceptibility of Clusters to Endemic Diseases

Clusters C1 and C2 were much more associated with genotypes susceptible to smut and pokkah boeng but also with genotypes moderately susceptible to pokkah boeng (Fig. 7). Clusters C3 and C4 were rather associated with genotypes susceptible or highly susceptible to leaf scald. In contrast, clusters C0, C5 and C6 were related to disease-free, resistant or moderately resistant genotypes.

Advances and Trends in Agricultural Sciences Vol. 1 Genetic Variability of Sugarcane Clones as Affected by Major Endemic Diseases in Ferké, Northern Ivory Coast

4. DISCUSSION

4.1 Diversity of both Resistant and Susceptible Clones

The diversity of disease-free clones and that of susceptible clones to smut came from 34 families, not necessarily the same, over a total number of 39 families investigated (87%). This sounds relatively high genetically in both cases. Also, 25 clones (3%) from 15 families (38.5%) were susceptible to the three endemic diseases, which shows their relative genetic diversity. Similarly, clones infected by smut, pokkah boeng and leaf scald came from, respectively, 34, 22 and 15 families, which indicates the genetic diversity of diseased clones. Even clones infected by leaf scald, as the least infectious disease, were relatively diversified genetically, too. Therefore, the disease susceptibility observed was not only prolific within families (about 3% with smut as the most infectious disease) but also not specific to a limited number of families or crosses (more than 10 crosses at least, i.e. 25%). This denotes the complexity of sugarcane breeding in search for resistant or tolerant parental varieties through their progenies while maintaining high genetic diversity for effective selection programs. Similar findings were reported on sugarcane brown rust in Florida [35].

F17_L08 F07_L41 F09_L06 F05_L08 F09_L19 C1 F16_L03 F10_L03 F21_L10 F10_L05 F05_L11 C2 F10_L24 F07_L98 F14_L05 F37_L37 F07_L87 F03_L03 C3 F06_L12 F23_L06 F25_L05 F25_L08 F25_L12 F11_L07 C4 F21_L04 F07_L69 F01_L02 F07_L06 F07_L34 F16_L07 F08_L03 F37_L22 F07_L19 F10_L27 F07_L17 F07_L129 F09_L24 F39_L27 F37_L31 C5 F10_L34 F37_L29 F28_L21 F35_L03 F05_L10 F13_L14 F36_L07 F07_L60 F07_L15 F07_L09 F21_L01 F03_L34 F06_L10 F35_L05 F34_L05 F09_L36 F37_L06 F15_L09 F07_L110 F07_L28 F07_L121 F09_L02 F10_L35 C6 F03_L10 F39_L10 F23_L03 F17_L12 F04_L02 F10_L19 0 20 40 60 80 100 120

(Dlien/Dmax)*100

Fig. 6. Dendrogram deduced from cluster analysis regarding 654 naturally diseased cane genotypes in first ratoon split into six different clusters

4.2 Breeding for High Yields and Disease Resistance

It is evident from this study that breeding for disease resistance is complicated by the frequent emergence of new pathogenic variants. These tend to overpower the resistant varieties, as witnessed from withdrawal of former ruling varieties from commercial cultivation [18].

However, growing genetically resistant varieties is the most cost effective and appropriate means for managing pest and diseases in sugarcane. Therefore, introgression of resistance genes into productive varieties is a key component of sugarcane breeding strategies. In this study, most of the

Advances and Trends in Agricultural Sciences Vol. 1 Genetic Variability of Sugarcane Clones as Affected by Major Endemic Diseases in Ferké, Northern Ivory Coast

resistant genotypes to smut came from families such as F02, F03, F04, F05, F06, F01, F06, F07, F08 and F09. They were morphologically characterized by well protected buds with scale leaves as opposed to that of highly susceptible clones to smut from families related to clusters C1 and C2 such as F35, F36, F37, F38 and F39. These findings were in line with the fact that sugarcane smut resistance mechanism is characterized into bud resistance (infection resistance) and inner tissue resistance (colonisation resistance) [36-38]. It was observed by Singh and Budharaja [39] that hyphae will not penetrate cells of the scale leaves. Hence buds tightly enclosed with the scale leaves have a better chance of escaping infection. On this basis, Waller [40] hypothesized that varietal resistance was determined by bud morphological characteristics. Structural characterization of sugarcane buds could provide clues for classification of test clones according to its smut resistance. Da Gloria et al [41] established an association between the bud structural characteristics and the cultivar resistance. Presence of outer scales was hypothesized to provide protection against bud invasion of the smut pathogen.

Table 3. Families composing different clusters of sugarcane genotypes in first ratoon determined by multivariate analysis

Clusters Total of Families represented (Number of clones) clones C0 209 F02 (2), F03 (12), F04 (10), F05 (7), F06 (9), F07 (34), F08 (1), F09 (8), (disease F10 (2), F11 (7), F12 (9), F13 (10), F14 (6), F15 (6), F16 (7), F17 (5), F18 (1), free) F20 (3), F21 (3), F22 (1), F23 (3), F25 (2), F26 (3), F27 (1), F28 (13), F29 (2), F30 (3), F31 (5), F32 (2), F35 (1), F36 (12), F37 (5), F38 (11), F39 (3) C1 93 F01 (4), F04 (3), F05 (2), F06 (2), F07 (17), F08 (2), F09 (9), F10 (11), F11 (5), F12 (1), F13 (1), F15 (3), F16 (3), F17 (2), F18 (1), F19 (1), F20 (2), F21 (1), F24 (1), F25 (1), F26 (1), F27 (1), F28 (1), F29 (1), F30 (3), F35 (2), F37 (7), F39 (5) C2 42 F05 (1), F07 (10), F09 (1), F10 (5), F11 (4), F14 (1), F16 (4), F24 (1), F29 (1), F31 (1), F36 (2), F37 (6), F38 (3), F39 (2) C3 52 F01 (1), F03 (7), F04 (1), F06 (4), F07 (9), F08 (2), F09 (1), F10 (4), F11 (2), F12 (2), F15 (1), F16 (3), F17 (1), F18 (1), F19 (1), F21 (3), F23 (1), F25 (1), F26 (1), F28 (3), F29 (1), F33 (1), F39 (1) C4 56 F01 (1), F03 (2), F07 (14), F08 (4), F10 (7), FF11 (3), F14 (1), F15 (1), FF17 (1), F19 (1), F21 (1), F22 (1), F25 (10), F26 (1), F28 (2), F30 (1), F31 (1), F34 (1), F37 (2), F39 (1) C5 286 F01 (4), F02 (2), F03 (15), F04 (11), F05 (5), F06 (9), F07 (57), F08 (3), F09 (20), F10 (11), F11 (6), F12 (6), F13 (4), F14 (3), F15 (2), F16 (6), F17 (7), F18 (1), F19 (1), F20 (1), F21 (7), F23 (1), F24 (5), F25 (2), F26 (5), F28 (15), F29 (2), F30 (4), F31 (2), F33 (1), F34 (11), F35 (5), F36 (15), F37 (16), F38 (12), F39 (9) C6 125 F01 (5), F03 (6), F04 (3), F05 (2), F06 (3), F07 (20), F08 (2), F09 (7), F10 (15), F11 (14), F12 (1), F13 (2), F14 (3), F15 (1), F16 (3), F17 (4), F18 (1), F21 (2), F22 (1), F23 (1), F24 (3), F28 (1), F30 (3), F31 (1), F35 (2), F36 (4), F37 (7), F38 (2), F39 (6)

Table 4. Mahalanobis square distance (bellow diagonal) between clusters taken 2 by 2 and Fisher values (above diagonal) regarding the first ratoon crop

C1 C2 C3 C4 C5 C6

C1 - F = 300,3497 F = 255,6737 F = 787,0107 F = 349,0343 F = 114,117 C2 31,23867 - F = 770,4648 F = 1114,496 F = 1085,876 F = 685,3198 C3 23,06915 99,7911 - F = 212,3803 F = 122,3954 F = 128,7563 C4 67,75786 139,7433 23,70339 - F = 930,1317 F = 777,3201 C5 14,96656 89,2283 8,37098 59,7696 - F = 53,39004 C6 6,43988 65,6016 10,55097 60,4845 1,84710 - P < 0.001 for all values

Advances and Trends in Agricultural Sciences Vol. 1 Genetic Variability of Sugarcane Clones as Affected by Major Endemic Diseases in Ferké, Northern Ivory Coast

Table 5. Means of cluster genotypes infected by each of the three endemic diseases observed at five months of age at one-row screening stage in first ratoon cane, Ferké 1 (Ivory Coast)

Clusters Rate of disease infestation (%) Leaf scald Smut Pokkah boeng Mean CV (%) Mean CV (%) Mean CV (%) C1 (n = 93) 1.33 a 362.8 22,65 a 20.0 1.76 a 297.9 C2 (n = 42) 0.48 b 648,1 52,21 b 30.4 1.69 b 215.3 C3 (n = 52) 15.76 c 17.6 2,39 c 129.5 0.70 c 158.4 C4 (n = 56) 42.16 d 41.0 6,74 d 126.9 0.86 d 239.1 C5 (n = 286) 0.00 - 2,20 e 77.8 1.31 e 188.9 C6 (n = 125) 0.00 - 9,42 f 27.1 0.87 f 204.8 Letters a, b… f: means followed by different letters in the same column are significantly different (P<0.001) after Mahalanobis square distance statistics deduced from cluster analysis

Smut_D-frCe0e ) a i t r

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Smut_RPePosCko._6k_DR-ferse.e %

0 Smut_M.res. 2 5

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2 Pok_M.res.

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3 5 8 0 3

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Cluster 0 Disease ty pe Dimension 1; Eigen v alue : ,33521 (24,46 % d'Inertia) Fig. 7. Projection of clusters determined by cluster analysis and susceptibility to endemic diseases in 1-2 factor plane following correspondence factor analysis in first ratoon cane at one-row stage, Ferké 1 (Ivory Coast)

4.3 Increase in Disease Infection with Age in Sugarcane

Increase in clone infections in first ratoon cane compared with that of plant cane was observed regarding the three endemic diseases but more importantly for smut by 51%. This was similar to observations made by several authors like Bailey [42] in South Africa, Croft, et al. [43] in Indonesia, Whittle and Irawan [44], Sundar, et al. [44] in India and Zhao et al [35] in Florida (USA). Increase in infection of susceptible sugarcane varieties with age is the result of increasing pressure of natural infection from contaminated soil by the crop itself or nearby infected fields with age through whip shelting teliospores, as far as smut is concerned [45]. That’s why hot-water and fungicide treatment of seed cane for nurseries and roguing of nursery plantations are key recommendations to reduce disease infections in commercial fields [46-48]. Currently, the use of pre-sprouting seedlings with a phytosanitary certificate and seedlings from micro-propagation methods are alternatives to prevent smut and other diseases affecting sugarcane cultivation [45]. Therefore, plant canes which derives from disease-free planting material are prone to be much less infected compared to ratoon crops, as far as susceptible genotypes or cultivars in favourable environments are concerned.

Advances and Trends in Agricultural Sciences Vol. 1 Genetic Variability of Sugarcane Clones as Affected by Major Endemic Diseases in Ferké, Northern Ivory Coast

5. CONCLUSION

It came out from the study that most relevant diseases determining the diversity of susceptible sugarcane genotypes were, in descending order, pokkah boeng, smut and leaf scald. Increase in clone infestations on first ratoon cane compared with plant cane was observed regarding the three endemic diseases but more importantly for smut by 51%. Each family investigated was composed of disease-free as well as susceptible genotypes in a certain proportion which varied from one family to another. Therefore, all families investigated were relevant to maintain the diversity required for the breeding process underway. Cluster 5 was the most prolific of infected genotypes with 286 clones (33%) split into 36 families (92%) whereas clusters 2, 3 and 4 were the least prolific ones, with 42, 52 and 56 infected genotypes split into 14, 23 and 21 families, respectively. Clusters 1 and 2 were much more associated with genotypes susceptible to smut and pokkah boeng but also with genotypes moderately susceptible to pokkah boeng. Clusters 3 and 4 were associated with genotypes susceptible or highly susceptible to leaf scald. In contrast, clusters 0, 5 and 6 were related to disease- free, resistant or moderately resistant genotypes and which crosses or families would, therefore, be recommended for Ferké agro-ecology. Examples of such families were the following: disease-free (F02, F03, F04, F05, F06); resistant (F01, F06, F07, F08, F09); moderately resistant (F10, F11, F12, F13, F14).

COMPETING INTERESTS

Authors have declared that no competing interests exist.

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Advances and Trends in Agricultural Sciences Vol. 1 Genetic Variability of Sugarcane Clones as Affected by Major Endemic Diseases in Ferké, Northern Ivory Coast

12. Royer M, Pieretti I, Cociancich S. Recent progress in understanding three major bacterial diseases in sugarcane: Gumming, leaf scald and ratoon stunting. In: Achieving sustainable cultivation of sugarcane.: Breeding, pests and diseases. P Rott (eds). 2018;2:311-36. 13. Autrey LJC, Saumtally S, Dookun A, Sullivan S, Dhayan S. Aerial transmission of the leaf scald pathogen, Xanthomonas albilineans (Ashby) Dowwson. Proceed. Intern. Soc. Sugar cane Technol. 1992;21:508-26. 14. Daugrois JH, Dumont V, Champoiseau P, Costel L, Boisne-Noc R, Rott P. Aerial contamination of sugarcane in Guadeloupe by two strains of Xanthomonas albilineans. Europ. J. Plant Pathol. 2003;109:445-58. 15. Champoiseau P, Rott P, Daugrois JH, 2009. Epiphytic population of Xanthomonas albilineans and subsequent sugarcane stalk infection are linked to rainfall in Guadeloupe. Plant Dis. 2003;93:339-46. 16. Piepenbring M, Stoll M, Oberwinkler F. The generic position of Ustilago maydis, Ustilago scitaminea and Ustilago esculenta (Ustilaginales). Mycological Progress 1. 2002;71-80. 17. Que Y, Xu L, Wu Q, Ling H, Liu Y, Zhang Y, Guo J, Su Y, Chen J, et al. Genome sequencing of Sporisorium sctamineum provides insights into the pathogenic mechanisms of sugarcane smut. BMC Genomics. 2014;15:1-19. 18. Sundar AR, Barnabas EL, Malathi P, Viswanathan R. A mini-review on smut disease of sugarcane caused by Sporisorium scitamineum. In: Mworia JK (eds). Botany. In Tech, Rijeka, Croatia. 2012;107-28. Available:www.intechopen.com 19. Peters LP, Carvalho G, Vilhena MB, Creste S, Azevodo Ra, Monteiro-Vitorello CB. Functional analysis of oxidative burst in sugarcane smut-resistant and smut-susceptible genotypes. Planta. 2017;245:749-64. 20. Hall JS, Adams B, Parsons TJ, Frech R, Lane LC, Jensen SG. Molecular cloning, sequencing and phylogenetic relationships of a new potyvirus: sugarcane streak mosaic virus, and a re- evaluation of classification of the Potyviridae. Mol. Phylogen. Evol. 1998;10:323-32. 21. Hema M, Joseph J, Gopinath K, Sreenivasulu P, Savithri HS. Molecula characterization and interviral relationships of flexuous virus causing mosaic disease of sugarcane (Saccharum officinarum L.) in india. Arch. Virol. 1999;144:479-90. 22. Kristini A, Irawan, Sasongko D. Mosaic booming. Gula Indonesia. 2006;30:36-8. 23. Magarey RC, Kritini A, Sallam N, Achadian E, Samson PR, Goebel FR, Thompson NP, McGuire PJ, Lonie KJ. Preparations to enhance Australia’s biosecurity: part 1- review of IPM for moth borers and sugarcane streak mosaic in the Javan sugarcane industry. Proc. Aust. Soc. Sugar Cane Technol. 2012;34:8. 24. Putra LK, Kristini A, Achadian EM, Damyanti TA. Sugarcane streak mosaic virus in Indonesia: distribution, characterization, yield losses and management approaches. Sugar Tech. 2014; 16(4):392-99. 25. Magarey RC, Kristini A, Achadian E, Thompson N, Wilson E, Reynolds M, Sallam N, Goebel FR, Putra L. Sugarcane streak mosaic: Researching a relatively new disease in Indonesia. Proc. Aust. Soc. Sugar Cane Technol. 2018;40:257-66. 26. Addy HS, Nurmalasari, Wahyudi AHS, Sholeh, Anugrah C, Iriyanto FES, Darmanto W, Sugiharto B. Detection and response of sugarcane against the infection of sugarcane Mosaic Virus in Indonesia. Agron. 2017;7:50. DOI:10.3390/agronomy70330050. 27. Béhou YYM, Péné CB, Zézé A. Genetic Diversity of First Sugarcane Varieties of Reunion- Ivorian Origin Preselected in Ferké, Ivory Coast. J. Experim. Agric. Intern. 2018;23(3):1-15. 28. Péné CB, N’Diaye S and Konan NC. Sprinkler irrigation and soil tillage practices in sugarcane plantations as influenced by soil texture and water storage in northern Ivory Coast. J. Life Sci. 2013;7(3):293-301. 29. Konan EA, Péné CB, Dick E. Main factors determining the yield of sugarcane plantations on Ferralsols in Ferké 2 sugar complex, Northern Ivory Coast. J. Emerg. Trends Engineer. Appl. Sci. JETEAS. 2017a;8(6):244-56. 30. Konan EA, Péné CB, Dick E. Caractérisation agro-climatique du périmètre sucrier de Ferké 2 au Nord de la Côte d’Ivoire. J Appl. Biosci. 2017b;116:11532-45.

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31. Rao GP, Triphathi DNP, Upadhaya UC, Singh RDR, Singh RR. CoSe-91423 and CoSe-93232: New promising red rot and smut resistant sugarcane varieties for Eastern Utterpardesh. Ind. Sugar J. 1996;261-63. 32. Gulya TJ, Martinson CA, Loesch PJJr. Evaluation of inoculation techniques and rating dates for Fusarium ear rot of opaque-2 maize. Phytopatol. 1980;70(1):116-8. 33. Karuppaiyan R, Ram B, Ramdiya S, Ali M, Meena MR. The incidence of pokkah boeng in indigenous and exotic sugarcane (Saccharum officinarum) clones. Ind. J. Agricul. Sci. 2015; 85(4):596-601. 34. Rao CR. Advanced statistical methods in biometric research. New York, John Wiley & Sons (eds); 1952. 35. Zhao D, Davidson RW, Baltazar M, Comstock JC, McCord P, Sood S. Screening for sugarcane brown rust in the first clonal stage of the Canal Point sugarcane breeding program. Agronomy 2015;5:341-62. 36. Dean JL. The effect of wounding and high pressure spray inoculation on the smut reactions of sugarcane clones. Phytopathol. 1982;72:1023-25. 37. Sabalpara AN, Vishnav MU. Screening of sugarcane clones/varieties for resistance to smut caused by Ustilago scitaminea Syd. Ind. Sugar J. 2002;12(7):507-09. 38. Khan HMWA, Chattha AA, Munir M, Zia A. Evaluation of resistance in sugarcane promising lines against whip smut. Pak. J. Phytopathol. 2009;21(1):92-3. 39. Singh K, Budhraja TR. The role of bud scales as barriers against smut infection. Proc. Bien. Conf. Sugarcane Res. Dev. 1964;5:687-90. 40. Waller JM. Sugarcane smut (Sporisorium scitaminea) in Kenya. II. Infection and resistance. Trans. British Mycol. Soc. 1970;54:405-14. 41. Da Gloria BA, Albernas MC, Amorim L. Structural characteristics of buds of sugarcane cultivars with different levels for resistance in smut. J. Plant Dis. Protec. 1995;102(5):502-08. 42. Bailey RA. Possibilities for control of sugarcane smut (Ustilago scitaminea) with fungicides. In: Proceed. South Afr. Sugarc. Technol. Assoc. 1979;137-42. 43. Croft BJ, Irawan, Berding N. Screening Australian sugarcane clones for smut reaction in Indonesia: Initial results. In: Proc. Austr. Sugarc. Technol. 2000;22:170-77. 44. Whittle PJL, Irawan. Pokkah boeng: A guide to sugarcane diseases. In: Rott P, Bailey RA, Comstock JC, Croft BJ, Saumtally AS (eds). Montpellier, CIRAD/ISSCT. 2000;136-40. 45. Monteiro Vitorello CB, Carvalho Schaker PD, Benevenuto J, de Sousa, N, de Silva T, de Almeida SS. Progress in understanding fungal diseases affecting sugarcane: Smut. In: Achieving sustainable cultivation of sugarcane. Vol 2: Breeding, pests and diseases, P Rott (eds). 2018;221-43. 46. Gupta SC. Control of diseases by ho water treatment of sugarcane seed material. Indian Sugar Crops. J. 1978;5:28-9. 47. Bhuiyan SA, Croft BJ, Tucker GR. New method of controlling sugarcane smut using Flutriafol fungicide. Plant Dis. 2015;99:1367-73. 48. Rott P, Comstock JC. Sugarcane smut disease. Institute of Food and Agricultural Sciences. University of Florida, Gainesville, Florida (USA). 2015;1-2.

Biography of author(s)

Yavo Yanick Michaël Béhou Department of Research and Development, SucafCI/SOMDIAA, 22 Rue Des Carrossiers Treichville Zone 3, 01 P.O.Box 1967 Abidjan 01, Ivory Coast, Côte d’Ivoire and UMRI: Agricultural Sciences and Engineering, EDP/National Polytechnic Institute (INPHB), P.O.Box 1313 Yamoussoukro, Ivory Coast, Côte d’Ivoire.

Advances and Trends in Agricultural Sciences Vol. 1 Genetic Variability of Sugarcane Clones as Affected by Major Endemic Diseases in Ferké, Northern Ivory Coast

He is a junior ivorian scientist born in May 17, 1992 in Man, western Ivory Coast. He is an agronomist, graduated from the University College of Agronomy which is part of the National Polytechnic Institute Félix HOUPHOUET-BOIGNY (INP-HB) of Yamoussoukro (Ivory Coast), since 2016. He has been working as a PhD candidate in Agronomy and Agricultural Engineering at the INP-HB Doctoral University School since 2017-18. His PhD topic deals with varietal selection in sugarcane based on true seeds, under the supervision of Prof Dr. Crépin B. PÉNÉ in the R&D Department of SUCAF-CI/SOMDIAA Company (Ferké 1 & 2 Sugar Estates, northern Ivory Coast).

Crépin B. Péné Department of Research and Development, SucafCI/SOMDIAA, 22 Rue Des Carrossiers Treichville Zone 3, 01 P.O.Box 1967 Abidjan 01, Ivory Coast, Côte d’Ivoire.

He is currently holding the position of Director of Research in the field of Irrigation/Agricultural Engineering basically in sugarcane. Since February 2008, he has been elected as a Member of The Ivorian Academy of Sciences and Arts and Cultures. In the frame of his PhD-graduations (State PhD and University PhD, Abidjan/Ivory Coast), he has gained some valuable on-the-job training experience at three international research institutions, i.e.: the Institute of Soils and Water, Volcani Center (Israel) : Irrigation and Soil Management (oct - dec 1990); the IAEA Seibersdorf Laboratory, Vienna (Austria): Use of Nuclear Techniques in Irrigation and Soil Fertility Management (May - July 1993) and the International Institute of Land Reclamation and Improvement (ILRI), Wageningen (The Netherlands): Drainage Design and Management (Aug – Nov. 1994). He has been involved in some research work dealing with various tropical crops, namely cereals (maize, sorghum, per-millet and rice), vegetables (tomato, pepper), legumes (soybean, groundnut) as well as palm oil. He has been acting as Head of the R&D Department of SUCAF-CI/SOMDIAA sugar industry since October 2006 where his duty was to increase significantly cane yields through variety improvement as well as to develop cost effective agricultural practices regarding the management of Irrigation, soil tillage, soil fertility and weeds. He acted as sugarcane research team leader at the Ivorian Agricultural Research Centre (CNRA and IDESSA) where he conducted applied research work in irrigation but also to coordinate their research and development efforts regarding various disciplines. He had been involved as principal investigator in numerous R&D Projects as SODESUCRE, SUCAF-CI, SUCRIVOIRE, IAEA-FAO Research Coordinated Program on the Use of Nuclear Techniques to increase Irrigation as well as Fertilizer Use Efficiency etc. He also has as much as 30 publications records issued in several international scientific journals, such as: J Experim. Agric. Intern., J Agric. Crop Res., Mod Conc. Dev. Agron., Afr. J Plant Sci., Am. J. Biosci. Bioengin., J of Life Sci., J of Agric. Sci. Technol., Sécheresse, Cahiers Agric., Agron. Afr., J. Appl. Biosci., Sci. & Nature, Europ. J. of Biosci. About 50 technical notes were also issued in Technical Journals from their research efforts. As a scientist, he took part in numerous international congresses held in foreign countries for oral or poster presentations, like Netherlands, South Korea, Brazil, USA, South Africa, Australia, Thailand, Canada, Romania, Morocco, France, Reunion Island, Cameroon, and Austria. ______© Copyright 2019 The Author(s), Licensee Book Publisher International, This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Reviewers’ Information (1) Zeynel Dalkiliç, Turkey. (2) Wawan Sulistiono, Institute for Agricultural of Technology of North Maluku. Indonesia. (3) Kagoda Frank, Uganda.

Chapter 9 Print ISBN: 978-81-934224-3-4, eBook ISBN: 978-93-89246-17-9

Riparian Buffer Strip Width Design in Semiarid Watershed Brazilian

Victor Casimiro Piscoya1*, Vijay P. Singh2, Jose Ramon Barros Cantalice3, Sergio Monthezuma Santoianni Guerra3, Moacyr Cunha Filho4, Cristina dos Santos Ribeiro3, Renisson Neponuceno de Araújo Filho5 and Edja Lillian Pacheco da Luz3

DOI:10.9734/bpi/atias/v1

ABSTRACT

The use of riparian areas as water quality management tools, primarily derived from the studies of agricultural watersheds, where low phosphorus and large nitrate reductions in the suspended sediment are observed. A riparian strip performs many key functions, such as nutrient uptake, trapping of sediment or pesticides. Therefore, a number of different forms of protection strips have been applied in the field according to relief, steepness and location for use. Studies assessing technologies to design riparian strips using plant covers, based on sediment yield in river basins, are required for environmental protection. The removal of semi-shrubby, native vegetation in the Brazilian semiarid region, has contributed to the degradation of semiarid basins. The aim of this study was to design a riparian strip for the Jacu River in the semiarid region of Pernambuco as a function of sediment yield. Experiments were conducted during the years 2008-2011 in the Jacu River basin at Serra Talhada, Pernambuco State, Brazil. The sediment yield in the Jacu River channel was obtained by measuring suspended and background solid discharge. The riparian strip width estimated in the riparian areas of the Jacu River basin was 15 m. It was concluded that for this study, the sediment yield time and observation of hydrological data were important factors for determining the riparian strip width with greater security.

Keywords: Riparian vegetation; degradation; erosion; soil conservation.

1. INTRODUCTION

Studies for assessing technologies to design riparian strips using plant covers, based on the sediment yield in river basins, are needed for environmental protection. Some forms of riparian strips are agricultural landscape features that have arisen as a result of an environmental law or are areas that are difficult to work [1]. Riparian buffers perform many key functions, such as the ability to trap nutrients, sediment, or pesticides transported from upslope areas. This evidence has contributed to a number of different forms of buffer strips being applied according to the localized landform, slope, and field use. In addition to their primary role of trapping nutrients and sediment, riparian buffers can provide multiple benefits in terms of biodiversity and water regulation [1]. Some authors reported that large permanent vegetation strips produce an effective reduction of pesticides carried by runoff, because of the ability of vegetation to slow runoff and promote water infiltration and adsorb

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1Department of Rural Technology-Environmental Engineering, Rural Federal of Pernambuco University (UFRPE), Recife-PE, Brazil. 2Department of Biological and Agricultural Engineering and Zachry Department of Civil Engineering, Texas A&M University, College Station, Texas 77843-2117, USA. 3Department of Environmental Engineering, Rural Federal of Pernambuco University (UFRPE), Recife-PE, Brazil. 4Department of Informatics and Statistics, Rural Federal of Pernambuco University (UFRPE), Recife-PE, Brazil. 5Department of Forestry Engineering, Federal University of Tocantins (UFT), Tocantins, Brazil. *Corresponding author: E-mail: [email protected];

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herbicides, these strips can improve the quality of water and produce additional environmental benefits when used with other soil management practices [2].

The use of riparian areas as water quality management tools, primarily derived from the studies of agricultural watersheds, where low phosphorus and large nitrate reductions in the suspended sediment are observed [3] and [4], in addition to influencing the processes of direct flow generation due to rain, peak flood attenuation, runoff power dissipation due to the roughness of banks, heat water balance, stability of banks and ravines, nutrient cycling, and sediment control [5]. Georgakakos et al. [6] observed the development of new runoff source areas and short-circuiting of the riparian buffer as well as repeated presence of cows in the fenced-out area, all of which may diminish the potential effectiveness of this practice.

Riparian buffer zone is an ecotone located between human-disturbed lands and wetlands, lakes, or rivers. The main functions of the riparian buffer zone is to protect wetland ecosystem through flooding control, water protection, soil conservation, habitat provision for wild species diversity, and the influence they have on ecosystem processes in wetlands [7]. A riparian strip performs many key functions, such as nutrient uptake, trapping of sediment or pesticides. Therefore, a number of different forms of protection strips have been applied in the field according to relief, steepness and location for use. In addition to capturing nutrients and sediments, riparian strips can provide multiple benefits in terms of biodiversity and water regulation. However, the current practice conceptualizes landscape components interacting with a range of goods and ecosystem services [8]. Vegetation strips or buffer strips represent a soil conservation technique of low potential for reducing the transport of pesticides by runoff from adjacent agricultural areas to water bodies [9].

Studies have shown that the buffer function of a riparian strip is complex because not all factors can be fully controlled and the final destination of chemicals is closely related not only to their chemical properties but also with the characteristics of events, such as rain, harvest and plant cover conditions [10]. Much has been currently discussed in riparian areas forming part of plant systems essential to environmental balance, representing, therefore, a central concern for sustainable rural development.

Despite their undeniable environmental importance, riparian forests have been eradicated in many parts of Brazil. There are a few major studies on a micro experimental watershed which provide limited information for the development of hydrological and sedimentological criteria that can be utilized to determine the minimum riparian strip width in a riparian zone in order to ensure the protection of waterways [11]. On the other hand, the Brazilian Forest Code of 1965 only presents strict limits of width for vegetation strips on watercourses and does not define scientific criteria for determining the width of riparian areas. In Brazil, the conversion of forests to pastures, agricultural land and/or urban areas is one of the main causes of impairment on water bodies. Protected areas are often created as a conservation strategy to halt the deforestation and start the recovery process in ecologically relevant areas. The limits of these Conservation Units are often defined following the patterns of terrestrial vegetation.

Accordingly, we must review our methodologies to design, understand and evaluate riparian buffer strips to maximize their potential benefits, which should be according to local needs, pressures and landscape. It is necessary to consider the vulnerability of a number of watershed areas to prioritize resources for the design of effective riparian strips. This may involve evaluating vulnerable ecological function areas and intensive farming areas [1].

Studies are needed, especially in areas with steep slopes and subjected to intensive land use that will help determine the width of vegetation strips for the restoration of riparian vegetation using dense vegetation cover of variable width that is able to retain soil losses [12]. There is a critical need for field assessment to validate models of erosion simulation and production of sediment in river watersheds with the objective of determining riparian strip widths [13]. The objective of this study, therefore, was to design a riparian buffer strip for the Jacu River in the semiarid region of Pernambuco, Brazil as a function of sediment yield and test it using field data.

Advances and Trends in Agricultural Sciences Vol. 1 Riparian Buffer Strip Width Design in Semiarid Watershed Brazilian

2. MATERIALS AND METHODS

2.1 Location and Characteristics of the Experimental Area

The study was conducted in the Jacu River basin located within the municipal boundary between cities of Serra Talhada and Floresta, forming part of the São Pedro River watershed and part of the Pajeú River watershed, both in the state of Pernambuco, with an area of 2.10 km2. The watershed is limited by the coordinates latitude -8º07'55” S between -8º09'07” S and longitude -38º23'20” W between -38º24'14” W.

The characteristic climate is Bwh type - tropical climate called semiarid, warm and dry, with summer- autumn rains, according to the Köppen classification, with average annual rainfall of 484.06 mm yr-1 for the period from 1992 to 2007 and average annual temperature above 25°C with summer rains that delay to fall and extend to April [14].

In the study area, there is a predominance of Entisol Fluvent [15], with limitation regarding the water storage capacity due to the higher percentage of sand in these soils, especially in those the coarse sand predominates over thin sand.

2.2 Physical and Water Parameters of the Jacu River Watershed

The physical and water parameters of the watershed were obtained from the Shutlle Radar Topography Mission image processing, SC.24-X-A (1: 250,000), as shown in Table 1.

Table 1. Physical and water parameters of the Jacu River semiarid basin

Parameters Value Area 2.10 km2 Perimeter 6.50 km Basin length 2.00 km Form factor 0.0497 Length of main channel 2.66 km Vector distance from the main channel 1.85 km Number of channels in the basin 34 channels Basin order Third order Number of 1st order channels 26 channels Number of 2nd order channels 7 channels Number of 3rd order channels 1 channel Total length of channels 11.06 km Average length of channels 0.43 km Drainage density 1.32 km/km2 Hydrographic density 12.38 channels /km2 Compactness coefficient 1.26 Highest basin altitude 638.9 m Lowest basin altitude 422.4 m Altimetry amplitude of the basin 216.5 m Steepness of the main channel 17.26 m/km Concentration time 0.984 hour

2.3 Vegetation and Use of the Jacu River Semiarid Watershed

For preparing the vegetation chart for the Jacu River basin, photo interpretation techniques were used over a GEOCOVER image [16], corresponds to a mosaic of Landsat images that were orthorectified and processed with a high quality standard (Geo Cover Technical Guide) adopted for the

Advances and Trends in Agricultural Sciences Vol. 1 Riparian Buffer Strip Width Design in Semiarid Watershed Brazilian

georeferencing of other orbital products suggested by [17,18]. In the case of the Jacu River watershed, the chart was based on the individualization of vegetation type units (shrub semi-shrubby and upland agriculture) and used according to their tonal and textural characteristics with prior field verification to validate the results obtained in the initial stage. The values obtained as a result of quantification of the vegetation chart and use in the Jacu River watershed is shown in Table 2.

Table 2. Distribution of vegetation classes and use in the Jacu river semiarid basin

Interval Area (m2) Soil use (%) Shipht cultivation upland 364.611 17.24 Shrubby 258.248 12.21 Semi-shrubby 1.492.411 70.55 Total 2.115.270 100.00

Table 3. Soil physical characterization at depths of 0-20 cm, 20-40 cm, 40-60 cm, 60-80 cm in the Jacu River basin

Depth Ds Dp Sand Silt Clay Total porosity Texture g cm-3 ------g kg-1 ------% 0 -20 1,14 2,59 72,94 18,61 8,46 55,9 Sandy clay loam soil 20-40 1,20 2,50 66,48 23,65 9,87 52,0 Sandy clay loam soil 40-60 1,13 2,66 72,88 17,39 9,74 57,5 Sandy clay loam soil 60-80 1,17 2,63 75,33 15,76 8,91 55,5 Sandy clay loam soil Ds = soil density; Dp = particle density

Table 4. Chemical characterization of soil in the Jacu river semiarid watershed collected at depths of 0-20 cm, 20-40 cm, 40-60 cm, 60-80 cm

Sorption complex Depth (cm) 0 - 20 20 - 40 40 - 60 60 - 80 Exchangeable cations 2+ -1 Ca (cmolc kg ) 0.52 0.57 0.50 0.67 2+ -1 Mg (cmolc kg ) 0.35 0.31 0.35 0.35 + -1 Na (cmolc kg ) 0.27 0.25 0.58 0.38 + -1 K (cmolc kg ) 0.16 0.57 0.50 1.35 -1 SB (cmolc kg ) 1.30 1.70 1.93 2.75 PST (%) 13.09 10.63 21.89 14.97 CTC (%) 2.0 2.11 2.42 3.05 Soluble cations pHes 7.30 7.20 7.05 7.02 CE(dS m-1) 1.41 2.07 4.28 6.70 2+ -1 Ca (mmolc L ) 0.85 1.02 1.71 1.72 2+ -1 Mg (mmolc L ) 0.35 0.45 0.90 0.99 + -1 Na (mmolc L ) 8.13 15.23 45.31 77.91 -1 K+(mmolc L ) 1.10 1.41 2.54 3.09 - -1 Cl (mmolc L ) 7.60 10.91 11.69 10.60 -1 0,5 RAS (mmolc L ) 10.19 16.18 32.45 54.84

2.4 Soil Physical and Chemical Characteristics

For characteristics physical Table 3 and chemical characteristics Table 4, samples were collected at depths of 0-20 cm, 20-40 cm, 40-60 cm, and 60-80 cm; air dried; crushed; homogenized; and passed through a 2 mm mesh. The particle size distribution was determined using the test tube method, and the particle density was obtained using the volumetric flask method and volumetric moisture [19]. The

Advances and Trends in Agricultural Sciences Vol. 1 Riparian Buffer Strip Width Design in Semiarid Watershed Brazilian

soil density was determined by the volumetric cylinder method [20]. The total porosity was obtained following the expression:

Ds Pt= 1- . 100 (1) Dp

-3 where Pt = the total porosity expressed in percentage (%); Ds = the soil density (g cm ), and Dp = the particle density (g cm-3).

For the chemical characteristics of soil samples were determined, soluble cations and exchangeable cations [21], as presented in Table 4.

2.5 Determination of the Jacu River Riparian Strip Width

The Jacu River riparian strip width was determined using the equation proposed by [22] and [12], which is appropriate for designing the width of vegetation strips in riparian areas to protect streams from high sediment and attached nutrient loss from hillslopes in areas of intensive land use on sloping ground. We argued that the filter strip should be a dense ground vegetation cover. The strip should be of variable width designed for the incoming discharges and sediment loads. The equation, which depends on the slope area and annual soil loss, can be expressed as:

2 1 Aa  H w=Y+ - b b (2) gHg ls 2tan where a = the riparian area (ha); A = the sediment yield of the basin (t ha-1); B = the deposited sediment weight (t); G = the sediment storage capacity of the vegetation (t); w = the width of the permanent vegetation strip (m); I = the strip length (m), ls = deposit width (m), Y = the additional width -3 required for sediment accumulation (m), s = the mean deposit density (tm ), g = the height of sediment deposit across the strip (m), Hg =the plant height (m) Hb = height of the sediment deposit across the strip (m) or sediment deposited in front of the vegetable strip (m), tan = slope angle tangent, and w = the riparian strip width (m). The weight of sediment accumulated in the vegetation strip B is the deposit volume (m3) multiplied by the average deposit density (tm-3). The sediment storage capacity of the vegetation strip is G (t).

The extent of sediment deposition over the riparian strip is much smaller than the total length of riparian area (I), due to the convergence of flow in preferential flow paths which can be expressed as a flow convergence factor (C) defined as:

l C= (3) ls where I = the strip length (m), and Is = the deposit width (m).

Substituting equation (3) into equation (2), the result is

2 1 cAa  H w=Y+ - s b (4) gHg l 2tan

Equation (4) was used in the calculation [12] recommend y = the additional width of 2 m for moderately erodible soils and 5 m for very erodible soils.

3. RESULTS AND DISCUSSION

3.1 The Jacu River Basin Characteri-zation

The Jacu River semiarid basin has an area of 2.10 km2, which covers the entire area drained by the river system [23]. The basin has a form factor of 0.0497 and a drainage density of 1.32 km / km2

Advances and Trends in Agricultural Sciences Vol. 1 Riparian Buffer Strip Width Design in Semiarid Watershed Brazilian

[which shows that the basin has an average drainage] (Table 3). The density can vary from 0.5 km / km2 for basins with poor drainage to 3.5 km / km2 or more for well-drained watersheds [24]. The roughness value obtained for the basin was 0.2 due to its sparse vegetation cover, with small areas of shrubby arboreal caatinga (moderately uncovered) with upland crops in the rest of the area and extensive breeding of small animals.

3.2 Design of the Jacu River Riparian Strips

The sediment yield (Y) was calculated by the solid discharge obtained in the exudate according to the procedure of [25], from the sum of the sample collections of suspended sediment with the use of one of the sediment samplers (US DH-48), with the collection of bottom sediment. For both the suspended sediment sampling and the bottom sediment sampling, the Equal Width (IIL) method was used.

The sediment yield (Y) ranged from 0.45 to 1.72 t y-1 and was considered to have low values for 4 years. The respective amounts of suspended sediment concentration (SSC) ranged from 874 to 376 mg L-1, which was considered high for a small watershed and for low values of the discharged liquid [26]. The sediment concentration values of watercourses in semiarid regions exhibited different behaviors when compared with events in a humid climate. In these regions, vegetation is limited by the production of sediment, while in the dry regions vegetation does not promote an efficient coverage of soil, thus allowing the generation of large volumes of sediment being carried by runoff coming to waterways [27].

Table 5 shows the riparian strip widths designed, following the method of [22] and [12], for the Jacu River according to the sediment yield in the period from 2008 and 2011. For design, the sediment deposition area was 30 m for calculating the convergence factor (C), the soil density and density of particles were obtained from the flow depth of 0 to 20 cm (Table 3), the average plant height was 1 m. and the sediment deposit height was 0.1 m.

Table 5. Width dimension (ω) of riparian vegetation strips in the Jacu River as a function of sediment yield (Y) [according to Karssies & Prosser (1999; 2001)]

Year Y W – Riparian strip width (t ha-1) (m) 2008 1.722 15.35 2009 1.568 14.40 2010 0.152 5.71 2011 0.451 7.54 Y = sediment yield in Jacu River basin in 2008 to 2011 (Piscoya, 2012)

It was observed that the dimensioning of riparian strip width for the Jacu River watershed was correlated to the sediment yield values that inherited the variability associated with the distribution pattern of precipitation and local runoff. However, this variation in riparian strip width values is not a disadvantage, since the values that should be considered for the Jacu River watershed are the highest.

Although the values of 15 meters for the riparian strip width were indicated, based on the sediment yield determined for the period 2008-2011 and the interaction of hydrological, physical, soil, climate and vegetation factors, these are not yet final. The 4-year period is possibly not enough to capture the whole range of variability of the semiarid region, requiring the observation of hydrological data and production of larger sediments in order to have a more conclusive riparian strip value for the Jacu River [12] presented widths of 26 m. for all erosion rates in the range 40 t ha-1.

Inácio [28] also applied the methodology of [12] in experimental plots and obtained riparian strip widths ranging from 5 to 10 m, respectively, for slopes between 4 and 38% for watercourses in southern Bahia.

Advances and Trends in Agricultural Sciences Vol. 1 Riparian Buffer Strip Width Design in Semiarid Watershed Brazilian

Researches such as [29] speculated that the vegetation strips in riparian areas in the form of dense strips of grass or even of shrubby vegetation are considered effective in controlling sediment transport; however, many of these strips have been established in experimental plots, and rarely have been evaluated at the river basin scale, which is important in assessing the real impact of these strips on the control of siltation and water quality of rivers. Thus, [29] evaluated the effectiveness of these strips applied in isolation in river basins and suggested that for use in river basins, vegetation strips should be accompanied by other practices to reduce sediment loads in rivers and the gross erosion on slopes in order to maintain their efficiency.

4. CONCLUSION

The determination of the Jacu River riparian strip width using the Karssies Prosser method, based on sediment yield, seems to be promising for the conditions of a small river basin.

The value of riparian strip width considered in this study for the riparian areas of the Jacu River basin is 15 meters.

The sediment yield time and observation of hydrological data are important factors for the determination of riparian strip width with greater security.

COMPETING INTERESTS

Authors have declared that no competing interests exist.

REFERENCES

1. Stutter MI, Chardon WJ, Kronvang B. Riparian buffer strips as a multifunctional management tool in agricultural landscapes: Journal of Environmental Quality. 2012;41:297-303. 2. Otto S, Vianello M, Infantino A, Zanin G, Di Guardo A. Effect of a full-grown vegetative filter strip on herbicide runoff: Maintaining of filter capacity over time. Chemosphere. 2008;71:74-82. 3. Likens GE, Bormann FH, Pierce RS, Eaton JS, Johnson NM. Biogeochemistry of a forested ecosystem, Ed. Springer-Verlag. New York; 1977. 4. Peterson BJ, Wollheim WM, Mulholland PJ, Webster JR, Meyer JL, Tank JL, et al. Control of nitrogen export from watersheds by headwater streams. Science. 2001;292:86-90. 5. Zakia MJB. Identification and characterization of the riparian zone in an experimental microwatershed: Implications for river watershed management and forest restoration. Brazil; 1988. 6. Georgakakos CB, Morris CK, Walter MT. Challenges and opportunities with on-farm research: Total and soluble reactive stream phosphorus before and after implementation of a cattle- exclusion, riparian buffer. Frontiers in Environmental Science. 2018;6:71. 7. Ma M. Riparian buffer zone for wetlands. The Wetland Book: I: Structure and Function, Management, and Methods. 2018;149-156. 8. Costanza R, Wilson MA, Troy A, Voinov A, Liu S. The value of the world’s ecosystem services and natural capital. Nature. 1997;1:253–260. 9. Borin M, Bigon E, Zanin G, Fava L. Performance of a narrow buffer strip in abating agricultural pollutants in the shallow subsurface water flux. Environment Pollution. 2004;131:313–321. 10. Lacas JG, Voltz M, Gouy V, Carluer N, Gril JJ. Using grassed strips to limit pesticide transfer to surface water: A review. Agronomy for Sustainable Development. 2005;25:253-266. 11. Lima WP, Zakia MJB. Hydrology of riparian woods. Riparian woods matas in: Conservation and Recovery. Fapesp. Brazil; 2000. 12. Karssies L, Prosser I. Designing filter strips to trap sediment and attached nutrient, River and Riparian land management technical guideline, Number 1, Australia; 2001. 13. Flanagan DC, Foster GR, Neibling WH, Burt JP. Simplied equations for fillter strip design. Transactions of the American Society of Agricultural Engineers. 1989;32:2001-2007.

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14. Mascarenhas JC, Beltrão B, Miranda J, Souza Junior LC, Galvão M, Pereira S. Design of sources underground water supply. Brazil; 2005. 15. Brazilian Agricultural Research Corporation. Brazilian. System of soil classification. Brazil; 2013. 16. Tavares JB, Santos DR, HayaKawa EH, Prado BR, Martins VA, Antunes MAH. Evaluation of geo cover data from field data collected with GPS receivers. 2009;14:1889-1896. 17. Lima MIC. Introduction to radargeological interpretation. Brazil; 1975. 18. Florenzano TG. Satellite images for environmental studies. Brazil; 2002. 19. Brazilian Agricultural Research Corporation. Manual of soil analysis methods. Brazil; 1997. 20. Klute A. Water retention: Laboratory methods, in: Klute A. (Ed.), Methods of soil analysis. American Society of Agronomy, Madison. 1985;635-62. 21. Brazilian Agricultural Research Corporation. Manual of chemical analyzes of soils, plants and fertilizers. Brazil; 2009. 22. Karssies L, Prosser I. Guidelines for riparian filter strips for Queensland irrigators. Canberra; 1999. 23. Alcântara EH, Amorim AJ. Morphometric analysis of a coastal hydrographic watershed: A case study. Brazil. 2005;14:70-77. 24. Villela SM, Mattos A. Applied hydrology. Brazil; 1975. 25. United States Geological Survey-USGS. Techniques of water resources investigations. EUA; 1973. 26. Piscoya VC. Management in the Jacu river watershed: Sediment production, ciliary vegetation sizing and water salinity in underground watershed. Brazil; 2012. 27. Vanoni VA. Sedimentation engineering: American society of civil engineers, manuals and reports on engineering practice. EUA; 1975. 28. Inácio ESB, Cantalice JRB, Araújo QR, Nacif PGF. Quantification of erosion in agroforestry system and pasture in Southern Bahia. Brazil. 2005;18:238-244. 29. Verstraeten G, Poesen J, Gillijns K, Govers G. The use of riparian vegetated filter strips to reduce river sediment loads: An overestimated control measure. Hydrological Processes. EUA. 2006;4259-4267.

Biography of author(s)

Mr. Victor Piscoya Department of Rural Technology-Environmental Engineering, Rural Federal of Pernambuco University (UFRPE), Recife-PE, Brazil.

He holds a PhD in Soil Science from the Federal Rural University of Pernambuco in Recife - Pernambuco, Brazil, where he is a professor of Agroforestry Systems. He is currently the author of the project "Riparian Buffer Strip Width Design in Brazilian Watershed" and develops projects related to soil fertility in agroecological plantations, quantification of carbon and nitrogen stocks in agroforestry systems, soil water infiltration in semi-arid environments. His experience in the areas of Agronomic Engineering, Environmental Engineering and Forestry Engineering, with emphasis on Soil and Water Management and Conservation, Agroforestry Systems, Soil and Water Conservation Engineering, Wood Harvest and Transport, Forest Technology, mainly acting in the following subjects: semi-arid watersheds, sediment production, and hydrology relations, Caatinga vegetation, irrigation water quality and agroforestry systems. The author holds a postdoctoral degree from TEXAS A & M UNIVERSITY in College Station, Texas (USA) with CAPES SUPPORT -and a MSc in Forestry Engineering from the Federal University of Paraná. and Graduation in Forestry Engineering from the National University of the Peruvian Amazon.

Advances and Trends in Agricultural Sciences Vol. 1 Riparian Buffer Strip Width Design in Semiarid Watershed Brazilian

Professor V. P. Singh Department of Biological and Agricultural Engineering and Zachry Department of Civil Engineering, Texas A&M University, College Station, Texas 77843-2117, USA.

He is a Distinguished Professor, a Regents Professor, and Caroline and William N. Lehrer Distinguished Chair in Water Engineering at Texas A&M University. He received his B.S., M.S., Ph.D. and D.Sc. degrees in engineering. He is a registered professional engineer, a registered professional hydrologist, and an Honorary diplomate of ASCE-AAWRE. He has published extensively in the area of hydrology and water resources. For seminal contributions he has received more than 92 national and international awards, as well as three honorary doctorates. He is a member of 11 international science/engineering academies. He has served as President of the American Institute of Hydrology (AIH), Chair of Watershed Council of American Society of Civil Engineers, and is currently President-Elect of American Academy of Water Resources Engineers. He has served/serves as editor-in-chief of three journals and two book series and serves on editorial boards of more than 25 journals and three book series. ______© Copyright 2019 The Author(s), Licensee Book Publisher International, This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Reviewers’ Information (1) Shubha Mundodu, BMS College for Women, India. (2) Rebecca Yegon, University of Embu, Kenya. (3) R. K. Mathukia, Junagadh Agricultural University, India. (4) Hesbon Otieno, South Eastern Kenya University, Kenya.

Chapter 10 Print ISBN: 978-81-934224-3-4, eBook ISBN: 978-93-89246-17-9

Phenotypic Plasticity: The Best Approach for Stress Selection

Ciro Maia1*, Paulo Mafra de Almeida Costa1, Cleverson de Freitas Almeida1, Luiz Alexandre Peternelli2 and Márcio Henrique Pereira Barbosa1

DOI:10.9734/bpi/atias/v1

ABSTRACT

The abiotic stresses are the main factors associated to low productivity, since they are related to the soil and the conditions of the environmental adversities. These are difficult to control factors and when severely affects production dramatically. The expand agricultural production to new crop areas in the tropical regions is an important strategy to supply the huge demand for food and renewable energy sources. However toxic aluminum (Al) present in tropical soils is a limiting factor for agricultural production. The objective of this study was to identify Al-tolerant and Al-sensitive sugarcane genotypes, based on phenotypic plasticity. Eleven sugarcane genotypes were evaluated under non- stress and Al-stress conditions. Genetic variability regarding Al tolerance was observed among the sugarcane genotypes by phenotypic plasticity. Al-stress caused a reduction in the primary root length and in the shoot dry weight, but an increase in the lateral root length. There was a difference between the genotypes related to Al accumulation in the roots and shoot, suggesting the existence of distinct tolerance mechanisms. Based on phenotypic plasticity genotypes can be classified as tolerant or sensitive to Al. The phenotypic plasticity is a simple form of analysis; however, it has great information about the behavior of the genotype. We characterized genotypes associated with Al-stress. The characterization of contrasting genotypes will be important for breeding programs involving sugarcane yield in regions subjected to stress.

Keywords: Abiotic stress; root system; Saccharum spp.; selection.

1. Al IS AN IMPORTANT ABIOTIC STRESS IN TROPICAL REGIONS

The biological significance of stress can be defined as a significant deviation from the ideal conditions in which plants are grown, preventing them from fully expressing their genetic potential for growth, development and reproduction. It can also be understood as an external factor that exerts influence on the plant.

In general, we can divide environmental stresses into two large groups, abiotic and biotic stresses. Abiotic stresses are related to nutritional factors, Al toxicity, salinity, heat, irradiation and others. Biotic stresses are associated with organisms such as insects, diseases, viruses, nematodes, weeds, among others.

Plants may be exposed to stress and depending on that intensity of exposure, may be reversible to plants. The plants receiving a reversible stress condition can recover and return to produce dry matter at the original rates, but below the ideal condition [1].

The stress characteristics (severity, duration, exposure time, combined stresses) and plant characteristics (organ or tissue affected, stage of development, genotype) influence the response of

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1Department of Crop Science, Federal University of Viçosa, Minas Gerais, Brazil. 2Department of Statistics, Federal University of Viçosa, Minas Gerais, Brazil. *Corresponding author: E-mail: [email protected];

Advances and Trends in Agricultural Sciences Vol. 1 Phenotypic Plasticity: The Best Approach for Stress Selection

plants to stress. Consequently, a different combination of conditions can cause different plant responses to the same type of stress [2].

Plants generally use three strategies to deal with stress: (i) specialization - the genotype is adapted to a specific environment; (ii) generalization - the genotype presents moderate aptitude for most environments and (iii) phenotypic plasticity - environmental signals interact with the genotype and stimulate the production of alternative phenotypes [3].

In practice, phenotypic plasticity can be measured by the production difference in the two cultivation conditions, that is, in the normal condition and in the stress condition. The low phenotypic plasticity has high relation with the productive stability, therefore more tolerant to the stress. Tolerant genotypes, which have low phenotypic plasticity, or generalists, are more recommended for conditions of constant exposure to stress or marginal regions. However, genotypes considered as efficient have high phenotypic plasticity when compared with tolerant genotypes [3].

In this chapter we will analysis the phenotypic plasticity of sugarcane genotypes under abiotic stress conditions, more specifically in Al stress. Thus, we can better investigate how breeders can select genotypes for inclusion in new crossing blocks to develop genotypes tolerant and / or efficient to abiotic stress.

Sugarcane (Saccharum spp.) crops occupy approximately 23 million hectares in more than 100 tropical and subtropical countries. Currently, sugarcane and its derivatives represent the second largest source of primary energy in the Brazilian energy matrix, and Brazil is responsible for the production of more than half of all globally commercialized sugar. In Brazil, sugarcane has been cultivated for nearly 500 years and is utilized for sugar and ethanol fuel production, being Brazil the major sugarcane producer in the world, with 719.1 million tonnes produced in 2010, around 43% of the world production for this year, that was approximately 1.69 X 103 million tones [4] Due to predicted population growth and increasing worldwide demand for renewable energy sources, there is a need to expand agricultural production to new crop areas in the tropical regions, including areas considered less appropriate for agriculture, i.e. with low fertility, low pH and aluminum (Al) toxicity [5].

Al-stress damages primarily the radicular system, with several secondary effects such as low water and nutrient absorption, and reduction of plant growth and development [6]. Growth impairment has been observed in the radicular system of sugarcane [7], corn [8], sorghum [9], rice [10], barley [11], and wheat [12].

The Al stress tolerance is associated with the ability to maintain cellular division and elongation, and the viability of meristematic tissues even under stress conditions [13]. Plants present two main mechanisms of resistance against toxic Al. With Al exclusion, they can prevent toxic Al from entering plant tissues through exudation of organic acids by the root tip and the consequent complexation of Al into non-toxic forms. It is well-known that root apex is the critical region of Al toxicity. Many plants secrete organic acid (malate, citrate, and oxalate) from the root tip in response to Al stress, which prevents trivalent Al ion from entering root tip cells [14]. In addition, plants possess tolerance mechanisms like Al detoxification inside the cells by means of complexation with organic compounds [15,16,17]. Genotypes that exhibit any of these mechanisms are Al-tolerant, if not exhibit are considered Al-sensitive. However, distinct crops show different tolerance levels. Higher tolerance is also observed in cereals crops, with rice being more tolerant to Al-stress, followed by corn, soybean, sorghum, and wheat [8]. In sugarcane, Saccharum officinarum is more tolerate to Al than Saccharum spontaneum [18].

Plants have developed several mechanisms to circumvent the lack of certain resources under stress conditions, among which is phenotypic plasticity, i.e., the ability to express an alternate phenotype under environmental stimuli to endure an adverse situation [19]. Phenotypic plasticity, is defined as the ability of a genotype (individual) to express different phenotypes according to its environment [20]. A number of methods exist to quantify phenotypic plasticity with the use of various indices such as the trait mean, the trait variation coefficient, the trait reaction norm, and the trait extreme values and phenotypic distances [21]. From the agronomic point of view, it could be reflected as the yield

Advances and Trends in Agricultural Sciences Vol. 1 Phenotypic Plasticity: The Best Approach for Stress Selection

difference in contrasting environments. In plant breeding, such approach is crucial for the selection of genotypes since phenotypic plasticity has a strong correlation with stability, that is, low phenotypic plasticity implies high production stability [22,23].

Studies on Al-stress in sugarcane based on phenotypic plasticity are still scarce, especially on the selection of contrasting genotypes for use in breeding programs. Therefore, the present study will analyses a study of case, and identify Al-tolerant or Al-sensitive sugarcane genotypes based on phenotypic plasticity in Al-stress.

2. ABIOTIC STRESS EXPERIMENT TO MEASURE THE PHENOTYPIC PLASTICITY

Eleven sugarcane genotypes were evaluated: RB966928, RB867515, RB937570, RB957610, RB93509, RB92579, RB008041, SP801842, SP813250, RB935744, and RB928064. These genotypes consisted of cultivars and clones occupying an extensive crop area in Brazil and/or are used as parental plants in the main breeding programs. The experimental design consisted of randomized complete block design with three replications, in factorial scheme. Factor 1: 11 genotypes and factor 2: Al conditions (Non-stress – with free of Al-stress, with 0% Al saturation, and the other one, Al-stress – subjected to Al-stress with 53% Al saturation [24]).

Plants were harvested 90 days after transplantation. The shoot was separated from the radicular system and dried in a forced-air incubator at 70°C for 72 h in order to determine the shoot dry weight (SDW, g). The radicular system was evaluated by image analysis using the software WinRHIZO Pro 2009 a (Basic, Reg, Pro & Arabidopsis for Root Measurement) coupled to an Epson Perfection V700/V75 scanner equipped with an extra light and a resolution of 400 dpi. The length of the radicular system was divided into diameter classes (d) for the lateral roots (LRL, d≤0.5 mm) and the primary roots (PRL, d>0.5 mm). The roots were dried in an incubator with forced ventilation at 70°C for 72 h, after which Al contents in the root (ALR, dag kg-1) and in the shoot (ALS, dag.kg-1) [10]. For more details see [25].

3. HOW PHENOTYPIC PLASTICITY CAN USED TO DEFINE GENOTYPES

3.1 Experimental Analyses and Genotype Performance

Significant differences were observed between the genotype averages for all traits, suggesting the existence of genetic variability among the genotypes. Al concentration in the soil had a significant effect on all traits, except for primary root length and Al contents in the shoot, indicating that the experimental conditions were adequate for the evaluation of Al-stress in sugarcane. The GxA interaction was significant for all traits with the exception of shoot weight (data not shown), suggesting that the genotypes responded differently to the environmental variations, that is, to Al-stress.

3.2 Shoot Weight

Under non-stress conditions, the average shoot weight was 96 g ± 18.03 g. The cultivars RB937570 and RB92579 presented the highest (117 g), and RB966928 the lowest (69 g), value. Under Al-stress, the average was 53 g ± 11.36 g, with RB867515 being the genotype that produced the most shoot weight (73 g). In contrast, the genotype RB957610 produced only 38 g. These results reveal great genetic variability among the genotypes (Fig. 1).

Al-stress caused an average reduction of 44% (28–60%) in shoot weight, consequently, will affecting the genotypes productive performance. Sugarcane cultivated in greenhouse trails under drought and soil acidity, showed a decrease of 71.8% and 58.9% in the growth of leaves and culms, respectively. However, in soil with sufficient water availability, increasing soil acidity resulted in a less drastic reduction of only 11% [26]. Ecco et al. [27] with the aim of to study the interaction between water deficit and soil acidity in sugarcane, two genotypes were evaluated in greenhouse with Al stress and the combinations of drought and Al stress. The author observed a 23% reduction in biomass production under Al-stress and 69% under drought stress combined with Al toxicity.

Advances and Trends in Agricultural Sciences Vol. 1 Phenotypic Plasticity: The Best Approach for Stress Selection

The genotype RB966928 presented, under both cultivation conditions, an under-average shoot weight. Nevertheless, it showed less variation in biomass production between the different environments. On the other hand, the genotype RB928064 had an above-average performance under non-stress conditions and below average under Al-stress, showing the highest production amplitude between the different environments (Fig. 1). Plants have developed distinct strategies to deal with stress, among which is phenotypic plasticity, which is associated with productivity stability [22].

Genotypes with low phenotypic plasticity, that is, less productivity variation in different environments, present high stability and can be characterized as tolerant [19]. So, based on phenotypic plasticity and considering the plant shoot like the photosynthetic machinery, in the present study, the genotypes RB966928, RB867515, RB008041 and RB935744 can be regarded as tolerant. In contrast, the genotypes RB937570, RB92579, and RB928064 are considered sensitive to Al, and the remaining present intermediate tolerance. These results reveal that it is possible to identity tolerant genotypes based on phenotypic plasticity, enabling the use of natural variability in the breeding of the characteristic. This is the first work to study such strategy as a tool to evaluate the tolerance of sugarcane genotypes in Al stress.

3.3 Morphology of the Radicular System

For the variable primary root length (PRL), no significant differences were found between the Al-stress and the non-stress conditions, with averages of 3.40 ± 1.02 m and 3.70 ± 0.41 m, respectively (Fig. 2a). The wider dispersion around the average shows a great effect of stress on this variable and the different responses of each genotype to this condition. Under stress, the genotypes usually show distinct responses, resulting in larger variability of the affected characteristic since each genotype shows a distinct potential response to stress [28]. The genotype RB935744 showed the smallest phenotypic plasticity, that is, the greatest stability for the production of primary roots, while the greatest phenotypic plasticity was found for the genotype SP80-1842. Only the genotypes RB867515, RB957610 and RB928064 produced more primary roots when subjected to stress conditions than under non-stress conditions. This result suggests that these genotypes probably respond to stress using mechanisms different from those used by the other genotypes, producing more primary roots or increasing the root diameter in lieu of lateral root formation.

Regarding the variable LRL, there was an opposite effect. Al-stress caused an increase in the LRL from 15.2 ± 2.6 m to 19.3 ± 5.18 m (Fig. 2b). All genotypes produced more lateral roots under stress conditions, with the exception of RB008041 and SP80-1842. The genotype SP80-1842 showed a drastic decrease in LRL under stress. RB957610 and RB008041 presented the greatest and the smallest phenotypic plasticity, respectively. When subjected to stress, the genotypes RB008041 and SP80-1842 produced approximately half the amount of lateral roots when compared to RB867515 and RB957610.

These results show that the sugarcane genotypes responded to Al-stress by modifying their radicular system, exhibiting phenotypic plasticity (i.e., the expression of alternative phenotypes under environmental stimuli). Under non-stress conditions, the radicular system consisted, on average, of 80% lateral roots and 20% primary roots. When subjected to Al-stress, there was an increase in the production of lateral roots (85%) in lieu of primary roots (15%). The genotype RB937570 produced less primary roots (25% under non-stress conditions and 12% under Al-stress) and more lateral roots (75% under non-stress conditions and 88% when subjected to Al-stress). RB928064 showed an opposite behavior, producing more primary roots (17% under non-stress conditions and 21% when subjected to stress) and less lateral roots (83% without stress and 79% under stress). The first symptom of Al toxicity is the rapid inhibition of root growth, especially under conditions of drought stress or restricted P availability. Al toxicity results in low absorption of water and mineral nutrients due to a decrease in the relative surface of the radicular system [6].

3.4 Aluminum Contents is Related to Al-Stress Tolerance Mechanisms

The average ALR and ALS were 1.33 dag.kg-1 and 0.017 dag.kg-1, respectively, varying between 0.78 and 2.39 dag.kg-1 for ALR and 0.009 and 0.027 dag.kg-1 for ALS (Fig. 3). Higher Al contents in the radicular system were expected since most of the absorbed element remains in the roots, and a small

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portion may be translocated to the leaves [6,7]. Some of the Al effects on the photosynthetic process are apparently a consequence of the toxic effects expressed initially in the roots [13]. A few studies show that Al affects the absorption and/or transport of mineral nutrients to the leaves [29], resulting in low rates of liquid CO2 assimilation and reduced biomass accumulation [30].

The genotype RB92579 presented 97.3% of the Al in the radicular system and 2.7% in the shoot. In contrast, RB93509 had 99.4% and 0.6% of the Al in the roots and in the shoot, respectively (Fig. 3). RB957610, RB92579, and RB928064 showed the lowest Al contents in the plant (0.820, 0.808, and 0.792 dag.kg-1, respectively), while RB008041 and SP80-1842 had the highest Al contents (2.402 and 2.045 dag.kg-1, respectively). These results suggest that different mechanisms of tolerance to Al exist in sugarcane.

Plants can express tolerance to toxic Al using two main mechanisms: (i) the exclusion of Al and (ii) tolerance of Al [15,16,17]. Exclusion of Al is associated with the exudation of organic acids by the radicular tip in the presence of activated Al, avoiding the toxic Al before its penetration in the plant. Exudation may occur through the overexpression of genes encoding enzymes involved in the synthesis of organic acids. The mechanisms of tolerance are associated with cellular detoxification of Al.

Exclusion can occur in different ways. The Al-carboxylate complex is not translocated into the roots or through the cellular membranes. The amount of activated carboxylated Al released depends on the Al activity in the rhizosphere, indicating that stress conditions are responsible for activating this mechanism [15]. In the presence of Al, wheat [31] and oat [32] exudate malate; corn [33], oat [32], rice [34], sorghum [35], and soybean [36] exudate citrate, while corn exudates also oxalate [37]. However, little is known about the organic acids exudated by sugarcane. Trejo-Tellez [38] reported that the overexpression of the enzyme pyruvate phosphate dikinase in tobacco roots causes an increase in the exudation of organic acid anions, with a strong reduction in Al accumulation in the plant. This observation suggests that the genotypes that least absorbed Al, such as RB957610, RB92579, and RB928064, probably use the mechanism of Al exclusion. However, further studies are needed in order to fully elucidate how this process takes place in sugarcane.

Fig. 1. Averages of the shoot dry weight (SDW in grams), and phenotypic plasticity (PP, in grams) of 11 sugarcane genotypes evaluated under non-stress and Al-stress conditions. The horizontal lines indicate the general averages under non-stress (dotted) and Al-stress (dashed) conditions. Tukey’s honestly significant difference (HSD) under Al-stress was 53.6 g (P = .01) and 44.2 g (P = .05), and under non-stress conditions was 106.5 g (P = .01) and 87.9 g (P = .05) [25]

Advances and Trends in Agricultural Sciences Vol. 1 Phenotypic Plasticity: The Best Approach for Stress Selection

Fig. 2. Average primary root length (PRL, m), lateral root length (LRL, m), and phenotypic plasticity (PP, m) of 11 sugarcane genotypes evaluated in non-stress and Al-stress environments. The horizontal lines indicate the general averages under stress (dotted) or non- stress (dashed) conditions. (a) Tukey’s HSD under stress conditions was 3.2 m (P = .01) and 2.6 m (P = .05), and under non-stress conditions was 2.8 m (P = .01) and 2.3 m (P = .05); (b) Tukey’s HSD under stress was 17.2 m (P = .01) and 14.2 m (P = .05), and under non-stress conditions was 13.2 m (P = .01) and 10.9 m (P = .05) [25]

In the tolerance mechanism, Al enters the cytoplasm, and, once inside the cell, a detoxification process takes place with Al complexation with organic compounds [17]. Several compounds can form stable complexes with Al within the cell, including organic acids such as citrate, oxalate, malate, and proteins [16]. Free Al+3 or Al complexed with chelating agents can be translocated into the cellular vacuole, where they are stored without causing toxicity [6]. Tolerance to acid soils with high toxic Al

Advances and Trends in Agricultural Sciences Vol. 1 Phenotypic Plasticity: The Best Approach for Stress Selection

concentration involves a complex interaction that is controlled by many genes and transcription factors [15]. This mechanism is associated with plant growth even in the presence of Al, that is, Al in its inactive form. Thus, the genotypes RB867515, SP81-3250, and RB935744, which presented elevated Al contents in the plant (1.4, 1.1, and 1,2 dag.k-1 Al, respectively), were able to produce a fair amount of shoot (73, 67, and 67 g SDW, respectively).

Fig. 3. Average Al contents in the root (ALR, in dag.kg-1) and in the shoot (ALS, in dag.kg-1) and the ratio between them (expressed in percentage of the average) of 11 sugarcane genotypes evaluated under Al-stress. Tukey’s HSD was 1.16 dag.kg-1 (P = .01) and 0.96 dag.kg-1 (P = .05) for ALR, and 0.018 dag.kg-1 (P = .01) and 0.015 dag.kg-1 (P = .05) for ALS [25]

The characterization of genotypes, and knowledge of the relationship between the traits involved in tolerance to Al-stress, constitute the initial step in the breeding for tolerance against abiotic stresses. From that point, breeders have the challenge to plan breeding strategies in order to increase production under stress conditions. Parentoni et al. [39] suggested that, for corn, a satisfactory selection criterion to increase the efficiency of P utilization should include grain production under P stress and the evaluation of P contents in the grain under conditions of high P. Mundim et al. [40] concluded that, for popcorn, the selection performed in environments with contrasting P conditions should be performed in each of these environments, via direct or indirect selection. An attempt to select genotypes subjected to low fertilization hinders the optimal expression of many desired traits, especially those associated with productivity and quality [41]. The identification of sugarcane genotypes tolerant to Al must consider traits of the roots and the shoots, as well as a possible correlation with productivity at advanced stages of plant development.

4. FINAL CONSIDERATIONS

The genotypes were characterized and the relationships between some of the features involved in Al tolerance were elucidated. The identification of sugarcane genotypes tolerant to Al should consider root properties as well as the phenotypic plasticity.

The present study revealed genetic variability between sugarcane genotypes’ tolerance to Al by the phenotypic plasticity approach. Al-stress caused a reduction in the SDW and PRL, as well as an increase in the LRL.

Advances and Trends in Agricultural Sciences Vol. 1 Phenotypic Plasticity: The Best Approach for Stress Selection

The genotypes RB867515, SP81-3250, and RB935744, even presenting high contents of Al in the plant, still produced a fair amount of shoot. Based on the phenotypic plasticity, that is, the ability of a genotype to produce an alternative phenotype under environmental stimuli in order to circumvent adverse conditions, RB966928, RB867515, RB008041, and RB935744 were classified as tolerant. On the other hand, the genotypes RB937570, RB92579, and RB928064 were considered as sensitive to Al.

Therefore, considering the current history in terms of the increasingly frequent occurrence of climate anomalies, added to the uncertainties of scenarios published periodically in recent years, a greater commitment to research related to abiotic stresses is necessary.

ACKNOWLEDGEMENTS

Authors would like to thank Fapemig, Capes, CNPq and the Sugarcane Genetic Breeding Program (Ridesa/UFV) for financial support.

COMPETING INTERESTS

Authors have declared that no competing interests exist.

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Advances and Trends in Agricultural Sciences Vol. 1 Phenotypic Plasticity: The Best Approach for Stress Selection

14. Nakamura A, Kitching RL, Cao M, Creedy TJ, Fayle TM, Freiberg M, Malhi Y. Forests and their canopies: Achievements and horizons in canopy science. Trends in Ecology & Evolution. 2017; 32(6): 438-451. 15. Inostroza-Blancheteau C, Rengel Z, Alberdi M, Mora ML, Aquea F, Arce-Johnson P, et al. Molecular and physiological strategies to increase aluminum. Molecular Biology Reports. 2012; 39:2069-2079. 16. Kochian LV. Cellular mechanism of aluminum toxicity and resistance in plants. Annual Review of Plant Physiology. 1995;46:237-260. 17. Simões CC, Melo JO, Magalhaes JV, Guimarães CT. Genetic and molecular mechanisms of aluminum tolerance in plants. Genetics and Molecular Research. 2012;11:1949-1957. 18. Landell MGA. Behavior of sugarcane (Saccharum spp) against aluminum levels in nutrient solution. Jaboticabal: Unesp; Portuguese;1989. 19. Maia C, DoVale JC, Fritsche-Neto R, Cavatte PC, Miranda GV. The difference between breeding for nutrient use efficiency and for nutrient stress tolerance. Crop Breeding and Applied Biotechnology. 2011;11:270-275. 20. Przybylo R, Sheldon BC, Merilä J. “Climatic effects on breeding and morphology: Evidence for phenotypic plasticity,” Journal of Animal Ecology. 2000;69(3):395–403, 21. Valladares D, Sanchez-Gomez, Zavala MA. “Quantitative estimation of phenotypic plasticity: bridging the gap between the evolutionary concept and its ecological applications,” Journal of Ecology. 2006;94(6):1103–1116. 22. Bradshaw AD. Unravelling phenotypic plasticity – why should we bother? New Phytologist. 2006;170:644-648. 23. Cavatte PC, Martins SCV, Morais LE, Silva PEM, DaMatta FM. The Physiology of Abiotic Stresses. In Fritsche-Neto, Borem (Ed.) Plant breeding for abiotic stress tolerance. Berlin: Springer-Verlag; 2012. 24. Sobral AF, Guimarães VOS. Relation between Aluminium toxicity and sugarcane (Saccharum spp.). Pesquisa Agropecuária Brasileira. 1992;27:287-292. 25. Maia C, Almeida CF, Costa PMA, Melo-Junior JAG, Silveira G, Peternelli LA, Barbosa MHP, Bhering LL. Phenotypic Plasticity of Sugarcane Genotypes under Aluminum Stress. Journal of Experimental Agriculture International. 2018;22:1-11. 26. Carlin SM, Santos DMM. Physiological indicators of the interaction between water deficit and soil acidity in sugarcane. Pesquisa Agropecuária Brasileira. 2009; 44:1106-1113 27. Ecco M, Santiago EF, Lima PR. Biometric answers in young plants of cane sugar under the water stress and the aluminum. Comunicata Scientiae. 2014;5:59-67. 28. Da Silveira G, Costa PMA, Kist V, Almeida CF, Baffa DCF, Barbosa MHP. Genetic variation affecting agronomic traits in sugarcane in response to high and low phosphorus availability. Agronomy Journal. 2014;106:2296-2304. 29. Giannakoula A, Moustakas M, Mylona P, Papadakis I, Yupsanis T. Aluminum tolerance in maize is correlated with increased levels of mineral nutrients, carbohydrates and proline and decreased levels of lipid peroxidation and Al accumulation. Journal of Plant Physiology. 2008; 165:385-396. 30. Jiang HX, Chen LS, Zheng JG, Han S, Tang N, Smith BR. Aluminum-induced effects on photosystem II photochemistry in citrus leaves assessed by the chlorophyll a fluorescence transient. Tree Physiology. 2008;28:1863-1871. 31. Papernik LA, Kochian LV. Possible involvement of Al-induced electrical signals in Al tolerance in wheat. Plant Physiology. 1997;115:657-667. 32. Zheng S, Ma J, Matsumoto H. Continuous secretion of organic acids is related to aluminum resistance during relatively long-term exposure to aluminum stress. Physiologia Plantarum. 1998;103:209-214. 33. Piñeros MA, Magalhaes, JV, Carvalho Alves, VM, Kochian LV. The physiology and biophysics of an aluminum tolerance mechanism based on root citrate exudation in maize. Plant Physiology. 2002;129:1194-1206. 34. Ishikawa S, Wagatsuma T, Sasaki R, Ofei-Manu P. Comparison of the amount of citric and malic acids in Al media of seven plant species and two cultivars each in five plant species. Soil Science and Plant Nutrition. 2000;46:751-758.

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35. Magalhaes J. Molecular genetics and physiological investigations of aluminum tolerance in sorghum (Sorghum bicolor L Moench). Ithaca: Cornell University; 2002. 36. Silva IR, Smyth TJ, Raper CD, Carter TE, Rufty T. Differential aluminum tolerance in soybean: an evaluation of the role of organic acids. Physiologia Plantarum. 2001;112:200-210. 37. Kidd PS, Llugany M, Poschenrieder C, Gunse B. The role of root exudates in aluminium resistance and silicon-induced amelioration of aluminium toxicity in three varieties of maize (Zea mays L). Journal of Experimental Botany. 2001;52:1339-1352. 38. Trejo-Téllez LI, Stenzel R, Gómez-Merino FC, Schmitt JM. Transgenic tobacco plants overexpressing pyruvate phosphate dikinase increase exudation of organic acids and decrease accumulation of aluminum in the roots. Plant Soil. 2010;326:187-198. 39. Parentoni SN, Souza CL Jr, Carvalho Alves VM, Gama EEG, Coelho AM, Oliveira AC, et al. Inheritance and breeding strategies for phosphorus efficiency in tropical maize (Zea mays L). Maydica. 2010;55:1-15. 40. Mundim GB, Viana JMS, Maia C. Early evaluation of popcorn inbred lines for phosphorus use efficiency. Plant Breeding. 2013;132:613-619. 41. Hawkesford MJ. Improving nutrient use efficiency in crops. Chichester: John Wiley & Sons Ltd; 2012.

Biography of author(s)

Ciro Maia Department of Crop Science, Federal University of Viçosa, Minas Gerais, Brazil.

Doctor Scientia in Genetics and Plant Breeding by UFV (2014), Master in Genetics and Plant Breeding - UFV (2009). Agronomist from Federal University of Viçosa-UFV (2007). Professional experience: Currently soybean breeder and manager of SEEDCORP HO research station in Primavera do Leste-MT (2018-current). Substitute Professor at UFV-Campus Florestal (2017); Senior Associate Researcher - Soybean Breeding in Corteva in Sorriso-MT (2013-16); Associate Researcher - Maize Breeding, in Corteva in Sorriso-MT (2009-10). Experience in coordination and development of research projects. Orientation of undergraduate and graduate students. Experience in the routines of soy and corn breeding. Project leadership with integration of molecular markers SSR and SNP in plant breeding and plant breeding for abiotic stress.

Paulo Mafra de Almeida Costa Department of Crop Science, Federal University of Viçosa, Minas Gerais, Brazil.

Agronomist (2009), Master (2011) and Doctor in Genetics and Plant Breeding (2015) by the Federal University of Viçosa. Professor of Basic, Technical and Technological Education of the Federal Institute of Santa Catarina - Campus Concordia, Statistical Area. It acts in the Genetic Plant Breeding, with emphasis on: Sugarcane Breeding, Biometry and Abiotic Stress. He acts in Statistics with emphasis in: Data analysis, Experimentation, Genomic Statistics and R. environment.

Cleverson de Freitas Almeida Department of Crop Science, Federal University of Viçosa, Minas Gerais, Brazil.

PhD student and master in genetics and breeding by the Federal University of Viçosa (UFV), agronomist engineer by the same institution. He has experience in the plant breeding of sugarcane and pumpkin crops, acting in the lines of research in quantitative genetics, genetic diversity, phenotyping aiming at tolerance to abiotic stresses and the use of microsatellite molecular markers. Currently, his research involves the study of the inheritance of pumpkin (Cucurbita moschata Dush.) seeds oil and fatty acid profile, development of calibration curve Near infrared reflectance (NIR) for these characteristics and the implementation of intrapopulational recurrent intrapopulation selection aiming at the launching of an open pollinated variety of reduced growth habit and with higher yield of functional seed oil. It has articles published in topics related to biofortification, evaluation of genotypes aiming at tolerance to aluminum stress and phosphorus deficiency, microsatellite molecular markers and genetic diversity.

Luiz Alexandre Peternelli Department of Statistics, Federal University of Viçosa, Minas Gerais, Brazil.

Full Professor of the Statistics Department of the Federal University of Viçosa and Research Productivity Scholar at CNPq - Level 1C, with experience in Mixed Models, experimental designs, stochastic simulation, sugarcane, plant breeding, genomic selection, predictive models for NIR data, and Statistical Learning.

Advances and Trends in Agricultural Sciences Vol. 1 Phenotypic Plasticity: The Best Approach for Stress Selection

Márcio Henrique Pereira Barbosa Department of Crop Science, Federal University of Viçosa, Minas Gerais, Brazil.

He holds a bachelor's degree in agronomy from the Federal University of Lavras (1989), a master's degree in Agronomy (Phytotechnology) from the Federal University of Lavras (1992) and a PhD in Agronomy from Universidade Federal de Lavras (1996). He is currently a full professor at the Federal University of Viçosa and coordinator of the Sugarcane Genetic Improvement Program that integrates the Inter-University Network for the Development of the Sugar-Energy Sector-RIDESA. He has experience in the field of agronomy with emphasis on Plant Breeding and works mainly in the following subjects: sugarcane, genetic improvement, quantitative genetics. ______© Copyright 2019 The Author(s), Licensee Book Publisher International, This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Reviewers’ Information (1) Nebi Bilir, Suleyman Demirel University, Turkey. (2) S. A. C. N. Perera, University of Peradeniya, Sri Lanka. (3) Aruna Rai, University of Mumbai, India.

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Chapter 11 Print ISBN: 978-81-934224-3-4, eBook ISBN: 978-93-89246-17-9

Abundance and Incidence of Zucchini (Cucurbita pepo L) Flies in the Korhogo Department of Northern Côte d’Ivoire and Pest Control Methods Used by Farmers

Yalamoussa Tuo1*, Klana Kone2, Michel Laurince Yapo1 and Herve Kouakou Koua2

DOI:10.9734/bpi/atias/v1

ABSTRACT

To improve the production of zucchini in Côte d'Ivoire and particularly in to Korhogo Department, a study was carried out at the Peleforo Gon Coulibaly University research site and at four farmers’ sites during dry and rainy seasons. On each plot, the number of healthy and attacked fruits was evaluated, based on a random sample of 100 fruits. The attacked fruits were transported to the laboratory and incubated to determine the causative agents. The methods and pesticides used to control insect pests by farmers were listed. During the rainy season, 86.06% of the fruit was attacked by flies while 13.94% remained healthy. In the dry season, for a total of 9,617 controlled fruits, 7,439 (77.35%) were healthy and 22.65% were attacked. Four insects species emerged from infested fruit. They were Bactrocera cucurbitae, Dacus ciliatus, Dacus bivittatus, belonging to the family of Tephritidae, and Scaeva pyrastri belonging to Syrphidae. The method used by farmers to control pests was not effective. In conclusion, flies represent the limiting factor of zucchini production during the rainy season in to Korhogo Department. At the end of this research, it emerges that the main zucchini pests in the Korhogo Department are Bactrocera cucurbitae, Dacus ciliatus, and Dacus bivittatus of the family Tephritidae. The species Scaeva pyrastri of the family of Syrphidae is not responsible for the loss of fruit; it comes only secondarily after the attack of Tephritidae. Pesticides used by growers in the control of pests are not indicated. Because of the strong presence of flies in the rainy season on zucchini and the damage they cause, the production of zucchini during this season may not be profitable.

Keywords: Zucchini; fly; attacked fruits; dry season and rainy season.

1. INTRODUCTION

Zucchini is an herbaceous plant of the family Cucurbitaceae. It is a vegetable plant, grown mainly for its fruits [1]. Zucchini contains protein, amino acids, minerals, vitamins, and fatty acids. The seeds and leaves of this vegetable are used in the treatment of many uro-genital diseases [2] this vegetable is therefore very important for health. The production cycle of zucchini is, on average, 45 days. The harvest index of immature fruit vegetables, including zucchini, cucumber, eggplant, or green beans, is based principally on size and color, depending upon market needs. For zucchini produced in Spain and consumed in Europe, the fruits have an average length of about 20 cm, just before hardening and darkening of fruit peel, and before undesirable seed development [3]. It could be an excellent substitute for eggplant (4 months) and cabbage (3 months) in the diet. The cultivation of zucchini could thus contribute to improving the living conditions of people. According to the 2014 estimates of ______

1Unité de Formation et de Recherche (UFR) des Sciences Biologiques, Département de Biologie Animale, Université Peleforo Gon Coulibaly, BP 1328 Korhogo, Côte d’Ivoire. 2Unité de Formation et de Recherche (UFR) Biosciences, Département de Zoologie, Biologie Animale et Ecologie, Université Felix Houphouet-Boigny de Cocody, 22 Bp 1611 Abidjan 22, Abidjan,Côte d’Ivoire. *Corresponding author: E-mail: [email protected];

United Nations Food and Agriculture Organization (FAO), zucchini production in Côte d'Ivoire was 19,296 tons. However, production is limited by several constraints, including insects, pests, and particularly fruit flies [4]. The pressure of fruit flies causes damage ranging from 30 to 100% depending on the species of cucurbits and the season [5]. Zucchini is pollinated by wild bees [6] and honey bee A. mellifera [7]. In addition, for Delaplane and Mayer [8], the increase in the number of bee visits on cucurbit flowers generally leads to an increase in production. Using their oviposter, females generally lay their eggs in tender fruits. The sting area is marked by the presence of a brown resin deposit. At hatching, the larvae feed on the pulp, causing the fruit to rot [5,9]. The sting marks depreciate the market value of the fruit and the activity of the larvae renders them unfit for consumption. One of the most damaging plant physiological disorders in cucurbits is squash silverleaf (SSL) disorder. SSL is associated with the feeding of immature whiteflies, Bemisia argentifolii Bellows and Perring, and is characterized by silvering of the adaxial leaf surface and blanching of fruit [10]. Seeing the extent of the damage, it would be important to know the species of zucchini flies in Côte d'Ivoire, evaluate their impact on production, and study the methods used by the producers to control the insects. These different activities will allow the establishment of an appropriate control method.

2. MATERIALS AND METHODS

2.1 Study Site

The present study was carried out in the sub-prefecture of Korhogo, located between 8°26 and 10º27 N, and 5º17 and 6º19 W, 600 km from Abidjan in the north of the Côte-d'Ivoire. This locality belongs to the Sudano-Sahelian dry tropical climate regime in which the rhythm of the seasons is regulated by the displacement of the Intertropical Front [11]. This climate is characterized by two seasons.

The rainy season extends from May to October with a maximum of precipitation in September. The dry season lasts from November to April and is characterized by the harmattan that settles from December to February. During the year 2016, an average rainfall of 1324.7 mm was recorded, and the annual average temperature varies between 24.6°C and 30.2°C [12]. This research was done on 5 sites, including an experimental plot housed in the botanical garden of Peleforo Gon Coulibaly University and four (4) others distributed throughout the sub-prefecture. These sites (Promafolo, Lataha, Kassirimé and Takali) were chosen for their high production of zucchini.

2.2 Methodology

Vegetable equipment was made up by the varieties of zucchini most produced in the north of Côte d’Ivoire, in particular Aurore F1 and Color F1.

2.2.1 Experimental device

The experimental device was a block of Fisher, completely randomized with two (2) blocks and five (5) repetitions. Each variety of zucchini was represented by a block, with two (2) objects (T0=untreated plots, T1=plots treated with approved synthetic insecticide). The zucchini varieties used were Aurore F1 and Color F1.

2.2.2 Abundance of zucchini flies

Fruits with bite marks were systematically harvested during the various surveys. These fruits were brought back to the laboratory in plastic bags and were incubated. The fruits were placed on wet sand, which facilitated the metamorphosis. Once a week, this sand was washed and sieved to collect pupae of the week. These pupae were then placed on moistened blotting paper in boxes and were monitored. After emergence, young flies were kept in 70% alcohol.

2.2.3 Incidence of flies on production and pest control methods

Following the method used by [13] in 2014, each plot was visited once a week from flowering to fruiting. During these visits, the number of healthy fruits and the number attacked by the flies was

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Advances and Trends in Agricultural Sciences Vol. 1 Abundance and Incidence of Zucchini (Cucurbita pepo L) Flies in the Korhogo Department of Northern Côte d’Ivoire and Pest Control Methods Used by Farmers estimated. The attack of the flies on the fruits is characterized by the presence of exudates and the shape of the fruit at the point of bite. In addition, farmers were questioned. The questionnaire submitted to the farmers requested information about the fighting methods, the kind of pesticide, and the efficiency of pesticide.

2.2.4 Identification of flies

Harvested flies were identified using the binocular loupe based on morphological characteristics. Identification keys of [14] and [15] were used.

2.2.5 Statistical analyses

The analyses were performed using Statistica software (version 7.1). Single-factor variances (ANOVA, p<0.05) were performed. Homogeneous averages were pooled using the Newman-Keuls test. Thus, the abundance of species, the rate of attacked fruits, and the impact of insecticides have been evaluated. The four sites of the study were compared by an Ascending Hierarchical Classification (ACH) based on fly populations.

3. RESULTS AND DISCUSSION

3.1 Abundance and Incidence of Flies

In the Korhogo Department the fruits of zucchini were attacked by B. cucurbitae, D. ciliatus, D. bivittatus of the family Tephritidae, and Scaeva pyrastri of the family Syrphidae (Fig. 1). The averages of the different fly species differed significantly at α=0.05, p=0.000001 and ddl=44. The Newman- Keuls test showed that B. cucurbitae, with a total of 109 specimens, was the most abundant. In addition, no significant difference was observed between the means of D. ciliatus (4.75), D. bivittatus (2.66), and S. pyrastri (2.00) (Fig. 2). These results are similar to those obtained by [16,17,18] on the island of Reunion. According to these authors, Bactrocera cucurbitae, Dacus ciliatus, and Dacus demmerezi were the most devastating flies of Cucurbitaceae on this island. According to the results of these authors, the three species of Tephritidae identified are responsible for fruit rot.

This result is similar to those obtained by [5]. According to this author, female fruit flies lay their eggs in the fruit. At hatching, the larvae feed on the fruit pulp, thus causing the decomposition of the fruit. At the experimental plot level, 86.06% of the fruits were attacked in the rainy season while 13.94% were healthy. In the dry season, 75.48% of the fruits were healthy versus 24.52% of attacked fruits. Regarding the farming environment, 77.35% of the fruits were healthy and 22.65% were attacked. For all four sites surveyed in the dry season, those of Kassirimé and Promafolo were the most infested by the flies with 26.70% and 26.22%, respectively, of fruits being attacked. The site of Takali, with 20.82% of fruit destroyed, was a little less attacked. With an attack rate of 17.26%, fruits from Lataha were the least infested. Based on the fruit infestation rate, the Ascending Hierarchical Classification revealed two groups: The one consisting of the Kassirimé and Promafolo sites, which have the highest infestation rates, opposed to the group formed by the sites of Lataha and Takali with low infestation (Fig. 3). Ultimately, these results suggest that the incidence of flies on fruit depend on the season and the production site.

According to [5], losses related to flies could be between 90% and the total production, depending on the site, the season and the species of Cucurbitaceae. These high losses may explain the low production of zucchini during the rainy season. Indeed, during this study, very few producers of zucchini were counted. The main reason is the poor performance of yield during this season.

3.2 Characteristics of the Flies Harvested

No specimens of S. pyrastri emerged from the attacked fruits that were in a poor state of field degradation. Those specimens hatched fruits whose decomposition was already well advanced in the field. As regards three species of Tephritidae, they were found only in the boxes containing stung fruits whose decomposition was in the very early stages. The decomposition of zucchini fruits is not

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Advances and Trends in Agricultural Sciences Vol. 1 Abundance and Incidence of Zucchini (Cucurbita pepo L) Flies in the Korhogo Department of Northern Côte d’Ivoire and Pest Control Methods Used by Farmers directly due to S. pyrastri. The action of this fly is secondary. In fact, during this study, the fly only emerged from fruit that was in a state of advanced decomposition in the field. This species only lays its eggs on the zucchini when it is in process of decomposition. This observation is consistent with the results of [13]. According to these authors, some zucchini flies are saprophagous.

a) Bactrocera cucurbitae b) Dacus bivittatus

c) Dacus ciliatus d) Scaeva pyrastri

Fig. 1. Zucchini flies species

100.00% a 90.00%

80.00%

70.00% s e i c e

p 60.00% s

f o

n 50.00% o i t r

o 40.00% p o r p 30.00%

20.00%

10.00% b b b 0.00% B. cucurbitae D. ciliatus D. bivittatus S. psytri Species

Fig. 2. Proportion of fly species recovered from infested zucchini fruits

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Fig. 3. Classification of collection sites based on fly infestation rate

Table 1. Insecticides used by zucchini producers

Commercial name Active substance Plant approved Lambda 25 EC Lambda-cytrine Food crops Cypercot 336EC Cyperméthrine 36 g/L Coton, Tropist P336 EC Cyperméthrine 36 g/L Coton Conquest C 88 cyperméthrine 72 g/L Coton K.optimal Lambda-cyhalotrine 15 g/L Market gardening Doni 672 EC Cypermetrine 36 g/L Coton Blast 52 EC Lambda-cyhalotrine 36 g/L Coton Polytrine 336 EC Cyperméthrine 36 g/L Coton

3.3 Pest-Control Methods

The investigation revealed that farmers used chemical methods to control pests in zucchini production. Eight (8) types of insecticides were used. These are Lambda 25 EC, Cypercot 336EC, Tropist P336 EC, Conquest C 88, K. Optimal, Doni 672 EC, Blast 52 EC, Polytrine 336 E (Table 1). Among all the pesticides used, 6 (96, 38%) are advised in cotton cultivation, while 2 (3, 62%) are authorized on food crops in Côte d’Ivoire. Therefore, despite the use of these pesticides, insect damage is important. In the farmers’ environment, nearly ¼ of the production is lost in the dry season. In addition, due to high parasite pressure and bad phytosanitary practices, very few farmers produce zucchini in the rainy season. In conclusion, the chemical method is not effective in controlling zucchini flies. The inefficiency of the chemical method observed during our investigation could be explained by the fact that zucchini fruits serve only as a substrate for spawning. According to several authors [5] and [17], flies spend more time on refuge plants than on Cucurbitaceae. Females come to zucchini plantations during the flowering period and fruiting only for egg-laying. According to Deguine et al. [19], the selection behavior of the plant by flies involves a system of exchange of information between the plant and the phytophagous insect. Perceived information can be of a physical nature (visual stimuli) but is mainly of a chemical nature (volatile compounds emitted by the plant). In addition, the ineffectiveness of insecticides could be related to the way of life of Tephritidaae. In fact, at hatching, larvae develop inside the fruit and pupation occurs in the soil [14]. All of these constraints could be the cause of the inefficiency of the chemical method. The investigation revealed that the pesticides used in zucchini cultivation are not indicated for this plant. This could be explained by the high cost of pesticides approved in market gardening compared to those of cotton. Due to the production of cotton

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Advances and Trends in Agricultural Sciences Vol. 1 Abundance and Incidence of Zucchini (Cucurbita pepo L) Flies in the Korhogo Department of Northern Côte d’Ivoire and Pest Control Methods Used by Farmers in this area, cotton pesticides are available on the market at low cost. Indeed, cotton companies offer insecticides to cotton growers at the beginning of the season. Moreover, when farmers need money, producers offer pesticides at low prices. In addition, according to the survey, the majority of farmers are unaware of the risks associated with unconventional use of pesticides.

These results are similar to those obtained by Doumbia and Kwodjo [20].

According to these authors, market gardeners in the city of Abidjan and suburbs (Dabou and Anyama) use unapproved pesticides. The phytosanitary practices that are appropriate would be linked to the total ignorance of the producers. According to a study conducted in Abidjan, 73.4% of producers are not aware of the risks of contamination due to their behavior versus 8.3% who claim to know their share of responsibility in the contamination of market garden produce [21]. The majority of market gardeners cannot read. Consequently, they cannot make use of the instructions of good use written on the ambalages of the pesticides. Similar results have been obtained by [22]. According to these authors, the inappropriate use of pesticides is due to the inability of market gardeners to implement the instructions for use on packaging.

4. CONCLUSION

At the end of this research, it emerges that the main zucchini pests in the Korhogo Department are Bactrocera cucurbitae, Dacus ciliatus, and Dacus bivittatus of the family Tephritidae. The species Scaeva pyrastri of the family of Syrphidae is not responsible for the loss of fruit; it comes only secondarily after the attack of Tephritidae. Pesticides used by growers in the control of pests are not indicated. Because of the strong presence of flies in the rainy season on zucchini and the damage they cause, the production of zucchini during this season may not be profitable.

COMPETING INTERESTS

Authors have declared that no competing interests exist.

REFERENCES

1. Klana Koné, Yalamoussa Tuo, Michel Laurince Yapo, Fokin Soro, Drissa Traoré, Kouakou Hervé Koua. Main insect pests of zucchini (Cucurbita pepo L), in the dry season and impact on production in Northern Côte d'Ivoire. Journal of Entomology and Zoology Studies. 2019;7(1): 523-527. 2. European Medicines Agency. Assessment report on Cucurbita pepo L. semen, EMA/HMPC/136022/2010 Committee on Herbal Medicinal Products (HMPC). 2012;44. Available:www.ema.europa.eu/.../document...assessment_report/.../WC5001 3. Megías Z, Martínez C, Manzano S, García A, del Mar Rebolloso-Fuentes M, Garrido D, Jamilena M. Individual shrink wrapping of zucchini fruit improves postharvest chilling tolerance associated with a reduction in ethylene production and oxidative stress metabolites. PloS one. 2015;10(7):e0133058. 4. Bharathi TE, Sathiyanandam VKR, David PMM. Attractiveness of some food baits to the melon fruit fly, Bactrocera cucurbitae (Coq.) (Diptera: Tephritidae). International Journal of Tropical Insect Science. 2004;24:125–134. 5. Dhillon MK, Singh R, Naresh JS, Sharma HC. The melon fruit fly, Bactrocera cucurbitae: A review of its biology and management. Journal of Insect Science. 2005;5(40):16. Available:https://doi.org/10.1093/jis/5.1.40 6. Enriquez E, Ayala R, Gonzalez VH, Farfán JN. Alpha and beta diversity of bees and their pollination role on Cucurbita pepo L. (Cucurbitaceae) in the Guatemalan cloud forest. Pan- Pacific Entomologist. 2015;91(3):211222. 7. Walters SA, Taylor BH. Effect of honey bee pollination on pumpkin fruit and seed yield. Hortscience. 2006;41:370-373.

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8. Delaplane KS, Mayer DF. Crop pollination by bees. CABI Publishing, Oxon, United Kingdom; 2000. 9. Ganie SA, Khan ZH, Ahangar RA, Bhat HA, Barkat H. Population dynamics, distribution, and species diversity of fruit flies on cucurbits in Kashmir valley, India. Journal of Insect Science. 2012;13(65):1-7. Available:https://doi.org/10.1673/031.013.6501 10. Frank DL, Liburd OE. Effects of living and synthetic mulch on the population dynamics of whiteflies and aphids, their associated natural enemies, and insect-transmitted plant diseases in zucchini. Environmental Entomology. 2005;34(4):857-865. 11. Jourda JP, Saley BM, Djagoua EV, Kouamé KJ, Biémi J, Razack M. Utilisation des données ETM+ de Landsat et d’un SIG pour l’évaluation du potentiel en eau souterraine dans le milieu fissuré précambrien de la région de Korhogo (Nord de la Côte d’Ivoire): Approche par analyse multicritère et test de validation. Télédétection, Google Scholar. 2005;5(4):339-357. 12. SODEXAM 2017. Etat du climat de l’année 2016 en Côte d’Ivoire. 12 p Côte-d Ivoire consulté le 10/12/2017. Available:www.acmad-au.org/wpcontent/uploads/.../stateofclimate in2016 13. Mokam DG, Djiéto-Lordon C, Bilong CFB. Patterns of species richness and diversity of insects associated cucurbit fruits in the southern part of Cameroon. Journal of Insect Science. 2014;14(1):9. Available:https://doi.org/10.1093/jisesa/ieu110 14. White IM, Elson-Harris MM. Fruit flies of economic significance: Their identification and bionomics. CAB/ACIAR, London, United Kingdom; 1992. Available:https://trove.nla.gov.au/version/43034159.xii,601p.: ill.; 24cm 15. White IM. Taxonomy of the dacina (Diptera: Tephritidae) of Africa and the Middle East (Hatfield, South Africa). Entomological Society of Southern Africa. African Entomology Memoir, 0373- 4242; no. 2 BOOK [vi]. 2006;156. 16. Vayssières JF, Carel Y, Coubes M, Duyck PF. Development of immature stages and comparative demography of two cucurbit-attacking fruit flies in reunion island: Bactrocera cucurbitae and Dacus ciliatus (Diptera Tephritidae). Environ. Entomol. 2008;37(2):307-314. Available:https://doi.org/10.1603/0046-225X(2008)37[307:DOISAC]2.0.CO;2 17. Bonnet E. Interactions entre les mouches des Cucurbitaceae (Diptera: Tephritidae), une plante hôte (courgette) et une plante piège (maïs) disposée en bandes et patchs intra-parcellaires à La Réunion. Mémoire de Master professionnel « Biodiversité des Écosystèmes Tropicaux » Spécialité « Biodiversité des Écosystèmes Cultivés » Université de La Réunion Année 2009 / 2010. 18. Boyer E. Etude des composés volatils des cucurbitaceae susceptibles d’attirer les femelles de mouches de fruits (Diptera: Tephritidae). Memoire de stage master 2 mention « sciences du vivant » specialite « valorisation des ressources naturelles » Universite de la Reunion . UFR des Sciences et Technologies. 2012;84. 19. Deguine JP, Lavigne A, Atiama M. Dynamiques des populations de Mouches des légumes durant l'hiver austral à La Réunion. Cah Agric. 2012;21:395-403. 20. Doumbia M, Kwodjo KE. Pratiques d’utilisation et de gestion des pesticides par les maraîchers en Côte d’Ivoire: Cas de la ville d’Abidjan et deux de ses banlieues (Dabou et Anyama). Journal of Applied Biosciences. 2009;18:992–1002. Available:http://www.biosciences.elewa.org/ 21. Wognin AS, Ouffoué SK, Assemand EF, Tano K, Koffi-Nevry R. Perception des risques sanitaires dans le maraîchage à Abidjan, Côte d’Ivoire. Int. J. Biol. Chem. Sci. 2013;7(5):1829- 1837. DOI: 10.4314/ijbcs.v7i5.4 22. Tuo Y, Dougba DN, Michel Laurince Yapo ML, Koua KH. Screening of phytosanitary practices in vegetable growth activities northern of Côte D’Ivoire. International Journal of Recent Scientific Research. 2017;8(6):17396-17402. DOI: 10.24327/IJRSR

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Biography of author(s)

Yalamoussa Tuo Unité de Formation et de Recherche (UFR) des Sciences Biologiques, Département de Biologie Animale, Université Peleforo Gon Coulibaly, BP 1328 Korhogo, Côte d’Ivoire.

He is Ivorian, Holder of a doctorate from Félix Houphouet-Boigny University in Côte d'Ivoire he’s actually Senior Lecturer at Peleforo Gon Coulibaly University of Côte d'Ivoire. President of the Korhogo Local section of Entomological Society of Côte d'Ivoire, he is the author of 20 publications in the field of agricultural entomology. Research thematic: Insect Biology-Pollinating Insects-Pest Insects-Beekeeping.

Klana Kone Unité de Formation et de Recherche (UFR) Biosciences, Département de Zoologie, Biologie Animale et Ecologie, Université Felix Houphouet-Boigny de Cocody, 22 Bp 1611 Abidjan 22, Abidjan,Côte d’Ivoire.

He is Ivorian, PhD student in agricultural entomology at the Félix Houphouet-Boigny University in Côte d'Ivoire. Holder of a Master's degree in agricultural entomology, he is doing his thesis on the entomofauna of zucchini in the department of Korhogo. He has already published four articles from this thesis on zucchini flies, insect pests and pollinators of this crop. He is also the author of articles on insect pests of other crops such as cabbage, millet, squash and pistachio.

Michel Laurince Yapo Unité de Formation et de Recherche (UFR) des Sciences Biologiques, Département de Biologie Animale, Université Peleforo Gon Coulibaly, BP 1328 Korhogo, Côte d’Ivoire.

He is Ivorian, Holder of a doctorate from the University of Cocody, Senior Lecturer at Peleforo Gon Coulibaly University of Côte d'Ivoire. He is the author of 21 publications in the field of General entomology, Aquatic Entomology, Insect Biodiversity and Tropical Ecology.

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Herve Kouakou Koua Unité de Formation et de Recherche (UFR) Biosciences, Département de Zoologie, Biologie Animale et Ecologie, Université Felix Houphouet-Boigny de Cocody, 22 Bp 1611 Abidjan 22, Abidjan,Côte d’Ivoire.

He is Ivorian full professor of entomology in faculty of Biosciences in University Felix Houphouet-Boigny of Côte d'Ivoire. He is the author of 48 publications in the field of agricultural entomology. His expertise research areas include Bio-Ecology and Management of Insect-Spatial-Temporal Insect Control and Insect Control-Physiology of Insect's Digestion. Fulbright Alumni University of Florida (USA) 2014 at Tropical Research and Education Center (TREC, Homestead, FL), he has supervised 3 PhD thesis and 12 Masters. Member of the Ivorian Agricultural Sciences Association, and the African Entomologists Association he is also the President of the Entomological Society of Côte d'Ivoire. ______© Copyright 2019 The Author(s), Licensee Book Publisher International, This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Reviewers’ Information (1) Hamit Ayberk, Istanbul University, Turkey. (2) Hanife Genç, Canakkale Onsekiz Mart University, Turkey. (3) Jian-Hong Liu, Southwest Forest University, China. (4) M. Indar Pramudi, Lambung Mangkurat University, Indonesia.

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Chapter 12 Print ISBN: 978-81-934224-3-4, eBook ISBN: 978-93-89246-17-9

Soluble Bases and CEC Variation across Undisturbed and Disturbed Coastal Forests in Tanzania

Elly Josephat Ligate1* and Can Chen2

DOI:10.9734/bpi/atias/v1

ABSTRACT

Understanding of different levels of soil calcium (Ca), magnesium (Mg), potassium (K), sodium (Na), cation exchange capacity (CEC), and percentage base saturation (BS), is important in the management of forest ecosystems. However, there is limited documentation on the status of these elements in the undisturbed forest sites (CFS) crop-agriculture (ADS) and livestock grazing (DGS) disturbances in the tropical coastal forests. This chapter attempts to address this deficit by presenting soil fertility based on exchangeable bases’ status and variations across undisturbed forest sites (used as a control), crop-agriculture and livestock disturbed sites in the coastal zone of Tanzania. The chapter aims to add knowledge on the management of tropical coastal forests. Indeed, this chapter shows that crop-agriculture and livestock grazing disturb soil chemical properties in tropical coastal forests. Therefore, it is essential to protect undisturbed forest while putting more efforts to restore the disturbed sites for sustainable forest management along the coastal areas.

Keywords: Soluble bases; cation exchange capacity; coastal forests; forest ecosystem; Tanzania.

1. INTRODUCTION

Forest disturbances due to human pressures and poor management systems affect forest structure and ecosystems [1,2,3]. Human induced forest disturbances and degradation affect the structure of forest ecosystems at large [4]. Indeed, human activities contribute to forest biodiversity decline or loss. The main activities contributing to forest loss, especially in the tropics include clearing land for crop-agriculture, pole cutting, charcoal burning, timber harvesting, and settlements [5,6]. Human disturbances reduce the capacity of forest to regenerate, function, and offer various ecological services [7,8,9]. However, documentation shows that some degree of disturbances are actually beneficial, as they contribute to the increase of biodiversity and nutrient circulation. Beneficial disturbances are thus considered important for long term sustainability and productivity of most ecosystems on earth. Certainly, disturbances are important in the modification of forest structures (i.e., stand parameters and species diversity), thus helping forests to undergo successional stages and maintain values. Unfortunately, in many cases these structures are affected by natural and human activities under varied environmental conditions [4].

Encroachment through human activities threatens the coastal forests, which cover an area of about 800 km2 along the coastal zone of Tanzania [8]. These activities alter the distribution and structure of the forests. Changes in spatial and temporal patterns, and the subsequent regeneration capacity put forest management efforts in jeopardy [10,11]. Yet, studies on how coastal forests, such as those comprising the study area, respond to crop-agriculture and livestock grazing disturbances are not available.

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1Department of Biosciences, Solomon Mahlangu College of Science and Education, Sokoine University of Agriculture, Morogoro, Tanzania. 2College of Forestry, Fujian Agriculture and Forestry University, Fuzhou, P.R. China. *Corresponding author: E-mail: [email protected];

Advances and Trends in Agricultural Sciences Vol. 1 Soluble Bases and CEC Variation across Undisturbed and Disturbed Coastal Forests in Tanzania

Human disturbance by activities such as plowing and logging greatly affect soil properties [12]. Forest disturbances for example, strongly affect soil characteristics mainly soil volume, chemistry and texture. Impacts consequences are soil degradation, soil erosion and the destruction of species, biomass and biodiversity [13]. Forest disturbances start to affect species composition, which in turns affects soils nutrients [14]. The impacts of vegetation destruction in soils nutrients pools is that, different plant species have different nutrient requirements and returns to soils [12]. Disturbances in forest affect the ecological relationship between forest vegetation and forest soils [15,16]. Therefore, in this chapter soil disturbances is defined as any physical, biological, or chemical alteration of the soil caused by forestry operations [17].

Human activities especially those involving clearance of forest vegetation pose soil to erosion, loss of organic matter and other necessary elements that are useful for vegetation growth. For example, a study by [12] shows that soil nitrogen of different ecosystems is concentrated mostly at the top 10 cm depth hence any effects on this layer would affect soil nutrients in these ecosystems. This effect is supported in [12] that soil nutrients such as phosphorus differences in different soil horizons may result from change of biological and geochemical processes at different depths after disturbances. However, in the same study, soil potassium was slightly higher in the disturbed than primary intact forest sites. Therefore, the findings in this work supports many existing literatures that disturbances on vegetation component of the ecosystems affects soil fertility. In this chapter we tried to present the variation of soil fertility across undisturbed, crop- agriculture and livestock grazing sites. Crop- agriculture and livestock grazing are used in this piece of work because these activities largely contribute to disturb coastal forest ecosystems in Tanzania [18].

The existing studies have documented on the impacts of land cover change and carbon storage [19,20,21]. Studies on soil organic carbon conducted by [22], Nitrous Oxide and Methane by [23] and plant diversity in [24] and [25]. Although a study by [26] investigated soil fertility on different land uses, documentation on the comparative differences of soluble bases and CEC across forest sites subjected to different land uses along the tropical coastal forests including those found in Tanzania is lacking. This lack of information is a challenge on the management of coastal forests in the tropics.

Inadequate information about soil soluble bases and CEC puts forests management in risk because the knowledge about the existence of forest resources is not enough to address the entire reciprocal function of soil properties and the interplays between vegetation and soils soluble bases in the ecosystems [27,28]. A chapter about soluble bases status and variation is important in the tropical coastal forests because these forests face pressure from human activities mainly crop-agriculture and livestock grazing [18]. Information generated in this chapter is crucial in contributing on the effective management and protection of tropical coastal forest ecosystems [29].

1.1 Crop-agriculture and Forest Disturbances

Coastal ecosystems especially forests are overexploited because of unsustainable use of resources as well as pressure from the growing agricultural activities [1]. Clear tree felling from intensive agriculture is associated with timber removal, and with major disturbances by using powered machinery contributes to the opening of larger sites for crop production [2] making coastal ecosystems vulnerable to disturbances. The detrimental effects of agricultural practices is deforestation, which in turn affects soils in ways such as erosion, desertification, salinization, compaction, lowering soil structure quality and loss of soil fertility [3]. Deforestation usually led to land degradation and ecological imbalance especially when clearing and burning are accompanied in deforestation methods in preparation of lands that are used for crop production [3]. Crop-agricultural activities disturb forests soils and cause high scale severity in soil and vegetation properties [4,30]. It is obvious that the ongoing agricultural practices of clearing land for crop production and improved pasture management by using uncontrolled fire accelerate the problem of forest disturbances [5].

1.2 Livestock Grazing and Forest Disturbances

Livestock grazing disturbances in forests is a concern in management because the life of every kind of human beings and civilization all over the world show well connections between these activities and

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forest ecosystems [6,7,8]. Livestock grazing affects species composition, ecosystem function, and socioeconomic value of forests [9]. Literature show that livestock induced disturbances might be among the major factors constraining regeneration and recruitment of species in terrestrial ecosystems [31].

The physical structure of plant communities is often changed by grazing. Defoliation by grazing herbivores alter plant height and canopy cover, and change species composition to include structurally different types of plants [32]. Defoliation can promote shoot growth and enhance light levels, soil moisture, and nutrient availability [31,10]. However, grazing animals can decrease flower and seed production directly by consuming reproductive structures, or indirectly by stressing the plant and reducing energy available to develop seeds [11]. Grazing animals can also disperse seeds by transporting them in their coats (fur, fleece, or hair), feet, or digestive tracts [11]. For some plant species, grazing may facilitate seed germination by trampling seed into the soil. Trampling and pawing disturb the soil and in some cases completely destroy soil crusts [32,12]. Trampling may also change the structure of plant communities by breaking and beating down vegetation [32]. In addition, the effect of trampling is compaction of soils, which damages plant roots, causing them to be concentrated near the soil surface [13].

Reduced vegetative cover and disturbed soil surfaces results into increased wind and water erosion [14]. The hoof-action of large grazing animals can incorporate plant materials into soils and increase organic matter. Grazers enhance mineral availability by increasing nutrient cycling within patches [18]. Also, the organic components of feces and urine from grazing animals can build soil organic matter reserves [6]. These organic components results into soils having increased water-holding capacity, increased water-infiltration rates, and improved structural stability, which can decrease soil loss by wind and water erosion [16]. Certainly, these changes may prevent plants from acquiring sufficient resources for vigorous growth [12].

2. LOCATION AND BIOPHYSICAL CHARACTERISTIC OF THE STUDY AREA

2.1 Location

This chapter presented the soil fertility information based on the study that was conducted in the coastal ecosystems located along the Coastal Zone of Tanzania. This zone stretches within 850km from the boarder of Tanzania and Kenya in in the north, and Tanzania and Mozambique in the south. This ecological area is rich in biodiversity as it has about 190 forest species, of which 92 are endemic [17]. However, as in many other tropical forests, farming (crop-agriculture), livestock grazing, timber harvesting, and charcoal production threaten these forests. As a result, these forests are disappearing at an alarming pace [18]. Because of the human activities, tropical coastal forests located in the coastal zone of Tanzania have lost about 69% of their primary vegetation [17]. If not abetted, further degradation will continue to threaten about 1500/300,000 (i.e., 0.5%) of global vascular plants found in this zone [19]. Nevertheless, crop-agriculture and livestock grazing continue to be the main human activities accelerating the rate of coastal forests degradation in Tanzania [20].

The coastal zone was purposely chosen because is among the areas with the leading forest cover loss in Tanzania particularly between 2000 and 2016 (Fig. 1 a & b). Specifically, the chapter presents information, which were obtained from the forestland cover and land use classifications for Uzigua Forest Reserve (UFR) found in Bagamoyo and Chalinze Districts, Pwani Region in the Coastal Zone of Tanzania Mainland.

The UFR has a coverage area of 24,730 ha [21]. This forest was purposely selected to represent other forest ecosystems along the coastal zone, which have been encroached mainly for crop- agriculture and livestock grazing. Certainly, this forest is within 100 km from the coast of Indian Ocean and thus considered among the tropical coastal forests in Tanzania [22]. The forest is under the Central Government that is represented by the Forest and Bee-keeping division of the United Republic of Tanzania, Ministry of Natural Resources and Tourism [21].

The UFR is supposed to be completely restricted from human use, serving for catchment and biodiversity conservation [21]. Unfortunately, due to poor protection and surrounding settlements, the

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entire forest is affected by human based activities such as harvesting trees for fuel-wood, fodder, grazing pressure and encroachments for agriculture. These activities have significantly affected this forest. Yet, this forest reserve is among a few remaining tropical coastal forests in Tanzania. Therefore, addressing the status of coastal forests contributed to generate useful information for management. This contribution is crucial in dealing with ecological management challenges emanating from crop- agriculture and livestock grazing pressures. Actually, the chapter highlights the variation on the status of soil fertility using cation exchange capacity differences to gain an understanding on how-crop agriculture and livestock grazing threaten the soils which harbors diverse plant species [23,24]. Consequently, understanding the variation of nutrients on soils affected by crop- agriculture and livestock grazing is very crucial in management of coastal forests.

2.2 Climate, Soils and Vegetation

The coastal zone of Tanzania mainland receives annual average rainfall of 917.23 mm where by the peak periods of rainfall are in January to April and November to December. Uzigua forest reserve is located in the tropical and sub-humid area with 700 mm to 1000 mm rainfall. October to May is a wet season while June to September is dry. The annual minimum temperature is 22.4°C while the maximum temperature is 31.7°C [25]. The soils are well-drained, red sand clay, loamy with brown friable top soils covered by more or less decomposed litter. The area is undulating with continuous hills with altitude ranging from 400 to 600 meters above sea level (masl) [26]. However, the current climate change and variability along the coast greatly influence temperature, rainfall, and the distribution pattern of plant species in these tropical coastal forests, and therefore the composition of the forest fragments at large [19].

a. b. Fig. 1. a. The coastal zone of Tanzania, b. A section of a disturbed area in the coastal zone of Tanzania

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3. HUMAN DISTURBANCES ON THE COASTAL FORESTS ECOSYSTEMS

Deforestation due to human pressures and poor forest management systems affects forest structure and ecosystems [27,28,29]. Forest disturbances and degradation affect the structure of forest ecosystems at large [33]. Human activities contribute to forest biodiversity decline or loss [34]. The main activities contributing to forest loss, especially in the tropics, include clearing land for crop- agriculture, pole cutting, charcoal burning, timber harvesting, and settlements [35,36,37,29]. Human disturbances reduce the capacity of forest to regenerate, function, and offer various ecological services [38,39]. However, documentation shows that some degree of disturbances are actually beneficial, as they contribute to the increase of biodiversity and nutrient circulation. These disturbances are thus considered important for long term sustainability and productivity of most ecosystems on earth [40,41]. Definitely, disturbances are important in the modification of forest structures (i.e., stand parameters and species diversity), thus helping forests to undergo successional stages and maintain values. Unfortunately, in many cases these structures are affected by natural and human activities under varied environmental conditions [33]. The impacts of disturbances are not only observed on vegetation but also on soil nutrients including calcium, magnesium, potassium and sodium hence cation exchange capacity along the coastal zones of many tropical ecosystems.

3.1 Status of Soluble Bases on the Coastal Ecosystem of Tanzania

3.1.1 Disturbances and soluble bases

An understanding of different levels of soil calcium, magnesium, potassium, and sodium, is important in the management of forest ecosystems [42,43,44], because cation exchange capacity highly influence vegetation growth in forest ecosystems [45]. Despite the importance of these elements, little is understood about their patterns and variability in tropical coastal forest ecosystems particularly on crop-agriculture and livestock grazed land uses [46]. Disturbances on the tropical coastal forests affect soluble bases [47]. As a result, many of the tropical forests are characterized by limited soluble bases [48,49]. The variation of nutrients exists between different ecosystems because of processes such as pedogenesis variability of parent rock materials and land uses [43,44,50]. While cutting down of native vegetation to convert forestland into farms counts as one of the processes that add soil nutrients, yet this addition is considered a temporal return of mineral nutrients in soil stock [51]. Thus, any conversion of natural vegetation into crop or grazing lands contributes to alter some soil nutrients. The depletion of nutrients is severe especially when fertilizers are not used as one of the corrective measures [51]. Unfortunately, crop-agriculture in the coastal forest reserves is practiced without additional of fertilizers. Therefore, this piece of work tries to establish that forest disturbances brought by human activities or processes affect vegetation, which in turn influence nutrients biogeochemistry through variation in the quantity and chemistry of plant litter [52]. The processes of nutrients depletion begin with impacts of disturbances on litter accumulation, thus lowering the capacity of forest ecosystems to slow soil erosion and mineral nutrients leaching (the most factors for soluble nutrients loss in the tropics) [47,50].

Activities that cause land cover change for example those associated with deforestation cause soluble bases depletion and extinction of some plant species in the tropics hence limiting the development of forest ecosystems [53,54]. Because of the roles played by soluble bases in controlling soil acidity and plant community welfare, an understanding about soluble elements quantities and variation is crucial in forest management [55].

3.1.2 Variations of soluble bases across land uses

The tested hypotheses in this chapter shows that there is significant variation of soluble bases, cation exchanges capacity and base saturation across forests sites subjected into different management practices (Tables 1, 2 3 and 4). This variation supports the findings by other researchers that spatial nutrients variations are contributed by land use management options [56]. From the findings and reviewed literature, we establish that soluble bases vary because of different land uses and management supporting the documentation by [57].

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Across the study sites, agriculture and grazed sites have lost soluble bases, the conditions, which in this work is associated with loss of vegetation (Lehmann et al. 2003). The effects of land use and management systems on soil fertility and chemical properties presented in this chapter is in agreement with some observations made by [45] and [56]. The evidence that intact soil sites harbor higher bases than disturbed sites has been clearly observed in Ca, Mg and CEC where by these bases and CEC were high in CFS than in ADS and DGS unlike the K, Na and BS.

The variation shows that disturbances affect soluble bases differently across land uses. The significant differences of Ca and Mg in CFS and, DGS and ADS is a good indicator of impacts of disturbances on these two major soluble bases in the tropical coastal forests. The interpretation is that Ca and Mg highly get lost in disturbed than in the intact sites [51].

Low amount of Ca and Mg in ADS than in DGS shows that converting land into crop- land and grazing land use makes soil vulnerable to soil erosion and leaching and uptakes by crops [54,58,59]. Low amount of Ca and Mg in ADS and DGS partially shows that human activities in these land uses disturb nutrients through conversion of forests into other land uses. Indeed, [49], support low quantities of soluble bases in disturbed soils by indicating that soluble bases in disturbed (cropped and grazed sites) have declined. The loss is contributed by vegetation loss, whereby loss of vegetation influences soil chemical properties by manipulating the distribution and concentrations of soluble bases unlike in the intact forest sites where trees and other vegetation contribute to increase exchangeable bases in the soils [52,45].

This chapter establishes that low base elements in disturbed sites is partially explained by loss of vegetation or the removal of soil elements from the soil by crop harvests or livestock grazing and leaching [59]. A combination of these three factors (i.e. clearing vegetation, crop harvests and grazing) affects the status of nutrients in the coastal forests; in turn, these factors affect forests ecosystems because of the interdependence between above and below ground forests ecosystem components. For example, variations of Ca, Mg, K and Na between CFS and the disturbed sites indicate that conversion of forests in other land uses results into release of nutrients locked in vegetation mainly in the form of woody [49].

The wooden locked nutrients are released into soils and animals where they are temporarily stored before getting lost [51]. Therefore, crop-agriculture and grazing disturb vegetation and litter hence soil nutrients in the tropics. Higher quantity of soluble bases in intact sites is a good indicator that undisturbed sites maintain nutrients circulation than the disturbed ones agreeing the findings of [60].

Indeed, the variation across soluble bases in response to disturbances shows that nutrients loss is not uniform throughout all soluble bases. For example, across the study sites, K and Na were low in all land uses compared to Ca and Mg. Low K and Na is the condition reported in the tropical forests because of the origin of the soils, high rainfall and high temperatures effects [61]. These environmental factors when combined with crop-agriculture and livestock grazing pressure affect more K and Na in the tropics than other soluble bases [61]. It shows that human activities accelerate the loss of K in the tropics, in turn low K affects carbohydrate and protein formation in forests trees [62]. In this view, human activities cause K deficiency in forest ecosystems partly threatening the productivity of these forests [63,62].

Calcium had higher correlation with almost all other soluble especially in CFS. This correlation indicates that intact forest sites have the capacity to retain nutrients than disturbed sites in agreement with [51]. Soluble bases such as Ca and Mg showed a positive and strong correlation across all the land uses except in ADS. The negatively correlated Ca and Mg in ADS is in line with [55].

The interplays of nutrients because of disturbances is used to indicate that certain activities accelerated loss of some nutrients. For example, the negative correlation of Ca and Mg in ADS than in any other land uses is useful to show that there are more declines in Mg than Ca in the disturbed forests sites supporting the findings in [47]. The main reason for high loss of Mg than Ca is that, the former base is vulnerable to leaching than the latter in disturbed sites [64]. Because crop-agriculture and livestock grazing contribute to disturb forests sites by affecting vegetation and accelerating soil

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erosion and leaching, it is recognized that crop agriculture and livestock grazing contribute to loss of Mg than Ca through leaching [52,55]. The positive and negative correlation findings on soluble bases in the intact forests and disturbed sites are also reported in [65]. Therefore, there is no uniformity in nutrients trends and dynamics other than variation across forests sites when exposed to different land use.

Table 1. Soluble bases variation across land uses

LU Ca Mg K Na mean p mean p mean p mean p CFS vs. ADS 3.75 ± 0.99 <.001 0.80 ± 0.17 <.001 0.03 ± 0.06 <.680 0.01 ± 0.01 <.240 CFS vs. DGS 3.11 ± 1.07 <.001 5.87 ± 0.42 <.001 0.55 ± 0.09 <.001 0.31 ± 0.04 <.001 ADS vs. DGS 0.63 ± 0.58 <.280 6.67 ± 0.39 <.001 0.52 ± 0.09 <.001 0.31 ± 0.04 <.001 Where: p = p-value

Table 2. The variation of CEC and BS across CFS, ADS and DGS

Land use CEC BS mean p-value Mean p-value CFS vs. ADS 2.61 ± 0.84 < .030 10.29± 3.74 < .010 CFS vs. DGS 13.74 ± 1.59 < .001 5.86 ± 2.67 < .030 ADS vs. DGS 16.36 ± 2.19 < .001 36.03± 5.26 < .400

Table 3. Paired soluble bases correlation between across land uses

LU Ca Mg K Na r p-value r p r p r p CFS vs. ADS 0.373 <.010 0.135 <.365 0.247 <.094 0.042 <.780 CFS vs. DGS 0.074 <.623 0.320 <.028 0.074 <.622 0.421 <.003 ADS vs. DGS 0.288 <.050 0.463 <.001 0.051 <.734 0.075 <.616 Where: r = Correlation value, p = p-value

Table 4. Paired sample correlation of CEC and BS across land uses

LU CEC BS r p r p CFS vs. ADS 0.279 <.058 0.538 <.000 CFS vs. DGS 0.079 <.596 0.082 <.584 ADS vs. DGS 0.613 <.000 0.263 <.001 Where: r = Correlation value, p = p-value

3.1.3 Soluble bases, CEC and BS vs. elevation levels

Soil fertility varies with elevation variations. The variation is real as indicated by differences on levels of Mg in ADS, Ca, in ADS, CEC in ADS, and Na in CFS and BS in DGS (see Table 5). These variations show that Mg and Ca were low at high elevation (350 to 600m) across the study area meaning that agricultural activities that were carried out at high elevations posed some potential risk for soluble bases depletion [55]. The low variations of nutrients in CFS against elevation could be associated with less leaching on nutrients in the CFS across different elevations. The variation of nutrients in DGS against elevation compared to other land uses shows non-significant values. This little variation partially explains that, grazed land contains some vegetation especially woods, which contribute to recycle soil nutrients, and partially returning the nutrients through animal feces [66,67,49].

Although DGS had less variation of nutrients across the elevation, it is established that low nutrients availability at high elevation (350 to 600 m) contributed to limit vegetation growth, which upon grazing pressure it resulted into loss of soil nutrients more than the lower bottoms. This limited supply of

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nutrients in-turn promotes nutrients insufficiency for wood production and thus livestock grazing continues to be among the factors affecting soluble bases in tropical coastal forests [54,57].

Table 5. Correlation of Ca, Mg, K, Na, CEC and BS with elevation

LU and Ca Mg K Na CEC BS Elevation r p r p r p r p R p r p Elevation 1 0.250 0.04 0.794 0.09 0.539 0.14 0.365 0.08 0.615 0.05 0.750 and CFS Elevation 0.18 0.222 0.25 0.095 0.02 0.987 0.08 0.589 0.15 0.329 0.01 0.972 and ADS Elevation 0.04 0.775 0.03 0.863 0.02 0.890 0.03 0.851 0.05 0.727 0.12 0.418 and DGS Where: r = Correlation value, p = p-value

3.1.4 Soluble bases CEC, BS and UFR sustainability

Although in this chapter we lacked baseline quantities of soluble bases, CEC and BS to make a comparison of whether the variation and quantities are sufficient or not to sustain UFR, still the current variation used to establish soluble bases in the coastal forests. The available data on Ca and Mg, CEC and BS are useful in predicating sustainability of coastal forests relationships because these factors largely control forest ecosystems by affecting the distribution of plants in forests [55]. Higher amount of Ca and Mg (for example) in CFS is a good indication that the uptake and recycling of these nutrients by trees and other vegetation is not in excess than the amount lost by leaching in disturbed soils [55]. Again, high amount of Ca and Mg in CFS is a good indicator that CFS health is promising because these two soluble bases are important in natural sustainability of forest ecosystems.

In order that coastal forests maintain the forest capacity to retain nutrients, protecting the remnant of these forests and recovering disturbed sites is an important worldwide approach [68,24,8]. It is essential to implement the common strategies used locally and globally for examples applying approaches such as excluding human settlements, crop-agriculture, and livestock grazing [69,70,71,72]. These efforts aim to allow the regeneration of trees and other vegetation to return soil nutrients since tropical forests have a pronounced power of self-maintenance through regeneration [73].

It is important to protect vegetation in intact forests sites and restore disturbed sites to rejuvenate the lost nutrients and prevent further degradation of forests ecosystems. Protection and restoration must aim in improving the amount of soluble bases because these elements largely govern soil acidity and, consequently plant species composition [55]. Improvement on the composition of species in turn affects forest soils nutrients [55]. Protection and restoration efforts might contribute into providing the function of reducing runoff, soil and nutrient loss and improvement of nutrients circulation in coastal tropical forests [65]. Improvements on forest ecosystems should not necessarily need addition of base elements, rather it can be done by avoiding further disturbances, protecting the intact sites and restoring disturbed sites by taking the advantage of natural forest capacities to recycle nutrients [73].

4. CONCLUSIONS

The chapter concludes by showing that there is significant spatial chemical attributes variation of calcium, magnesium potassium as well as sodium, cation exchange capacity and base saturation across closed forest, crop agriculture and livestock grazing disturbances. These elements were significantly different among sites. From chemical variations of soluble bases as representative of soil chemical properties, it shows that disturbed forest sites have low nutrients than undisturbed sites. These variations indicate that soils in the disturbed sites at high elevation ranging between 350 to 600m are well conserved for essential nutrients to maximize forest vegetation growth and development along the ecological gradients. Some variations are recorded and expected to change because the effects of disturbances can be short or long-term occurring over decades or centuries. The variation across forest sites shows that forest disturbances affects an integral relationship

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between vegetation and soils. This relationship is vital because soil gives vital support such as provision of moisture, nutrient and anchorage to vegetation while vegetation provides protective cover and nutrient maintenance. Certainly, the chapter shows that disturbances can eliminate some species and species composition while some disturbances bring changes in succession pathways, which are beneficial to maintain energy flow, nutrient cycling, species, genetic and structural diversity. Therefore, it is suggested that further studies should be carried out to identify soils, correlation of soil elements in the tropics in different land uses. These studies should aim to establish a trend of nutrients at risk because of human activities mainly crop-agriculture and livestock grazing, and suggest possible remedies for sustainable coastal forests across different regions and landscapes in Tanzania and elsewhere globally.

COMPETING INTERESTS

Authors have declared that no competing interests exist.

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Biography of author(s)

Elly Josephat Ligate Department of Biosciences, Solomon Mahlangu College of Science and Education, Sokoine University of Agriculture, Morogoro, Tanzania.

Dr. Elly Josephat Ligate is a lecturer, researcher and consultant working in the Department of Biosciences, Solomon Mahlangu Campus of Science and Education at Sokoine University of Agriculture (SUA), Morogoro Tanzania. Elly holds a Bachelor of Science SUA. He holds a Master of Science in Natural Resources Assessment and Management from the University of Dar es Salaam-Tanzania. Indeed, Elly holds a Doctor of Philosophy (Ph.D) in Ecology offered by Fujian Agriculture and Forest University, Fuzhou, Fujian China. Elly has worked as an academician at higher learning institutions for over 12 years by teaching Ecology, Botany, and Environmental Health and EcosystemS Restorations. He has researched and published in a wide range of areas through an inter-disciplinary approach to research and educational training. Elly has published articles in the areas of Agriculture, Environmental Management, Carbon Stocking, Ecosystem Services and Management, Land Cover and Use Changes, and Coastal Forests Ecosystem Management. His key fields of research and consultancy interest include: Ecosystems Disturbances and Restorations, Environment and Climate Change, Sustainable Agriculture, Natural Resources Management.

Can Chen College of Forestry, Fujian Agriculture and Forestry University, Fuzhou, P.R. China.

Can Chen, (Ph.D.) Associate Professor of Fujian Agriculture and Forestry University Forestry College in China, a master tutor, director of the Department of ecological environment. He is a visiting scholar in the Department of Natural Resources

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Management, Iowa State University, USA and a visiting professor in the Science Forest Centre at University of British Columbia in Canada. He has been engaged in the research of coastal forest environment and urban ecological forestry for a long time. He is now a core member of the National Chinese Fir Research Center of the Ministry of Forestry, the Southern Forest Resources and Environmental Engineering Technology Research Center of Fujian Province (Provincial Key Laboratory), the Key Laboratory of Forest Ecology Management and Process of Fujian Province, the Forest Ecology Research Institute and the Eucalyptus Research Center. ______© Copyright 2019 The Author(s), Licensee Book Publisher International, This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

DISCLAIMER This chapter is an extended version of the article published by the same authors in the following journal with CC BY license. Asian Journal of Environment & Ecology, 6(2): 1-12, 2018.

Reviewers’ Information (1) Fabio Aprile, Western of Pará Federal University, Brazil. (2) Muhammad Farhan, Government College University, Pakistan.

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Chapter 13 Print ISBN: 978-81-934224-3-4, eBook ISBN: 978-93-89246-17-9

Surface Water Nitrogen Load Due to Food Production-Supply System in South Asian Megacities: A Model-based Estimation

Syeda Jesmin Haque1,2*, Shin-ichi Onodera1 and Yuta Shimizu1

DOI: 10.9734/bpi/atias/v1

ABSTRACT

Food production and supply system contributed more than 90% of the nitrogen originated in south Asian megacities that pollute the surface water. Five megacities of three South Asian countries were considered in this study. These countries are developing and their population is increasing tremendously. All the five megacities are very densely urbanized. A numerical model has been used to calculate the anthropogenic nitrogen load on the environment. FAO statistics on fertilizer consumption and food balance data sheet has been used to calculate the nitrogen load. Human waste plays the vital role in nitrogen production of south Asian megacities. So, in these contexts the nitrogen load for all the study areas extremely harmful for environment and ever increasing population also increased the load of nitrogen on surface water produced from human waste which also very awful for the environment. So, a proper sewage treatment facility is compulsory for all the study areas. Four findings has been identified are; (1) for all three countries, rice and wheat production-supply produce the maximum amount of nitrogen. (2) Though the amount of nitrogen due to fertilizer input more or less same among the countries but amount of produced nitrogen due to human waste is huge in Bangladesh. (3) Moreover, in city scale, the amount of nitrogen due to fertilizer input is maximum in Delhi city and negligible in Kolkata due to an insignificant amount of farmland. (4) Interestingly, the maximum amount of nitrogen load in surface water is in Kolkata city due to human waste but Mumbai and Dhaka shows a medium amount of nitrogen load. This can give the estimation for city wise untreated nitrate content and this is necessary for the capacity development of existing sewerage treatment plant as well as the establishment of new plants.

Keywords: Nitrogen load; South Asia; megacities; numerical model; water pollution.

1. INTRODUCTION

South Asian countries are the densest areas over the world [1]. Population growth rate also increased over the years [2]. Increased population requires high agricultural production. For the world as a whole, per-hectare output of cereals, which account for more than half the food people eat if the grain fed to livestock is factored in, had risen by the late 1990s to 3.0 metric tons, which was double the average yield in the early 1960 [3]. High yielding variety with excessive use of pesticides and fertilizer enhance the agricultural production and meet the excessive demands. Overuse of fertilizer and pesticides affect the environment. Sometimes alter the ecological balance and chemical cycles. In many developing countries there are many fertilizer management technologies suited to regional agricultural and socio-cultural structures, such as crop management knowledge models [4], N fertilizer models [5], leaf color charts (LCC) [6], and soil–plant analysis development (SPAD) [7], but adoption rates are very low. Fertilizer contains nitrogen which also affects the nitrogen cycle in both micro and macro level. Eutrophication followed with excessive nitrogen use. This nitrogen pollution transmitted to the surface water and finally seepage to groundwater. To quantify the effects of anthropogenic

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1Graduate School of Integrated Arts and Science, Hiroshima University, Japan. 2Geological Survey of Bangladesh, Room#415, 153 Pioneer Road, Segunbagicha, Dhaka-1000, Bangladesh. *Corresponding author: E-mail: [email protected];

Advances and Trends in Agricultural Sciences Vol. 1 Surface Water Nitrogen Load Due to Food Production-Supply System in South Asian Megacities: A Model-based Estimation

nitrogen load, this research has been modeled at country and city scales. In South Asia, currently the main origin of anthropogenic nitrogen is Food production and supply [8, 9, 10, 11, 12]. Future scenarios based on past trends coincide with assumptions on increased animal protein consumption, while population increase alone cannot explain the projected demand. If this scenario happens, it will imply massive losses of reactive N to the environment, with eutrophication, loss of biodiversity, air pollution via higher NOx and NH3 emissions, water pollution, soil acidification, and emission of N2O [13].

The Rapid urban growth of the megacities deteriorates water quality simultaneously [14, 15, 16]. Pollutants increase over the years due to urbanization which affects the pollution load to the surface and groundwater [17]. Discharge of domestic and industrial effluent wastes, leakage from water tanks, marine dumping, radioactive waste and atmospheric deposition are major causes of water pollution. Heavy metals that disposed off and industrial waste can accumulate in lakes and river, proving harmful to humans and animals [18]. Toxins in industrial waste are the major cause of immune suppression, reproductive failure and acute poisoning. According to United Nations, the probable economic cost of environmental deterioration due to water contamination is very rigorous in the South Asian region in terms of restoring the quality of life and installing controls [19]. In the context of the South Asian region, specifically in Bangladesh, India and Pakistan, nitrogen pollution has turn into more severe and critical near the urban areas due to high pollution loads discharged from urban activities. In this study, the authors try to evaluate the nitrogen load to surface water caused by farming and livestock production, food trade and human waste to clarify nitrogen flow and its effects on water quality for the year 2005 in 3 countries and their five megacities in South Asia.

2. DATA AND METHODS

2.1 Nitrogen Flow Model

This model calculates the flow of nitrogen due to food production and supply system (Fig 1). By following Shindo et al. (2003) [12] the nitrogen flow model has been used for the calculation of this research. In this model, magnitude of nitrogen flow estimated from the published statistics on fertilizer consumption and crop production and from food balance sheets for three South Asian countries and also for five megacities. The data were collected from the FAO (2005) [20]. Table 1 show the food list which is considered in the calculation. In the table, the protein contents and conversion factors from protein to nitrogen follows the “Standard Tables of Food Composition in Japan” [21].

Fig. 1. Nitrogen flow in food production- supply system and environmental load Source: [12]

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Advances and Trends in Agricultural Sciences Vol. 1 Surface Water Nitrogen Load Due to Food Production-Supply System in South Asian Megacities: A Model-based Estimation

In FAO food balance data [20], utilization and food supply are classified in to different items like, “production”, “imports”, “stock changes”, “exports”, “feed”, “seed”, “processing”, “domestic supply quantity”, “other uses”, and “food”. Shindo et al. (2003) described that the net food supply was calculated as “production”+ “imports”+ “stock changes”- “exports”. The authors assumed that “seed” and “other uses” were applied to farmland, “feed” was eaten by livestock and “processing” and “food” were used by humans. They also assumed that all crop residues, meat and fish are unused for food and feed were returned to farmlands. Nitrogen is taken out of farmlands by crop harvesting, and remaining nitrogen was considered to discharge from farmland to the environment (NLf). All nitrogen was considered to discharge from humans to the environment in human waste, food waste and food processing industrial waste (NLw). Some of These wastes can be treated through the sewerage system or septic tank. In Asia, only 35% of urban wastewater is properly treated by sewerage system and a large part of the waste directly discharged into the water without any treatment [22]. So, that value has been assumed for five megacities water treatment rate. Land use data with a spatial resolution of 1km*1km [23] was used to classify the farmland, forest and also calculate the total country and city area. The calculated values have been displayed by graph and map.

Table 1. Foods used in the nitrogen cycle model, their protein contents, and conversion factors from protein to nitrogen

Foods Protein content (g/100g) Factor for converting protein to N Cereals Wheat 10.6 5.77 Rice 6.8 5.95 Barley 8.0 5.83 Maize 8.6 6.25 Rye 12.7 5.83 Oats 13.7 5.83 Millet 10.6 6.25 Sorghum 10.3 6.25 Other cereals 12.0 6.25 Starchy roots Cassava 1.6 6.25 Potatoes 1.6 6.25 Sweet potatoes 1.2 6.25 Yams 2.0 6.25 Other roots 3.0 6.25 Legumes Pulses general 22.0 6.25 Soyabeans 34.2 5.71 Oil crops excluding soyabeans 18.5 5.31 Vegetables 1.5 6.25 Fruits 0.7 6.25 Meat 18.1 6.25 Milk 3.0 6.38 Eggs 12.3 6.25 Fish and seafood 18.7 6.25 Source: [12]

3. RESULTS AND DISCUSSION

3.1 Country wise Nitrogen Budget

Fig. 2, 3 and 4 show the calculated nitrogen from the FAO Stat food supply data sheet in three South Asian countries. In Bangladesh the highest amount of nitrogen provided by rice production. Wheat, and fish, seafood also provided some nitrogen (Fig 2).

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Fig. 2. Amount of nitrogen content due to food supply in Bangladesh (calculated from FAO data)

In India wheat, rice and oil crops provided a large amount of nitrogen. Other food like beans, soya beans, milk-excluding butter and fish, seafood also generate a large amount of nitrogen (Fig. 3).

Fig. 3. Amount of nitrogen content due to food supply in India (calculated from FAO data)

In Pakistan, wheat, rice and beans, oil crops, soya beans, milk-excluding butter are the main contributor to nitrogen production (Fig. 4). For all country, food production sector contributes very high nitrogen compared to other sectors. The stock variation shows some negative value for some foods for every country.

Fig. 5 displays the calculated nitrogen budget due to the food supply, fertilizer input and human waste of three South Asian countries. Among the three countries, Bangladesh shows very high nitrogen load per square km compared to other two countries. These may be due to the small country area and large population burden since fertilizer input shows more or less similar in each country but a large

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amount of human waste in Bangladesh. These wastes discharged into the surface water and pollute the surface as well as groundwater. Calculated residues input for all the country shows negative result so we assume the result is zero.

Fig. 4. Amount of nitrogen content due to food supply in Pakistan (calculated from FAO data)

Fig. 5. Nitrogen budget due to agriculture and food supply in three south Asian countries

3.2 Spatial Distribution of Nitrogen

After calculating the country scale nitrogen load the model is used for calculating the five megacities nitrogen load. The three studied country and their five megacities are displaying in Fig 6. Circle of the Fig.6 displays the amount of nitrogen due to fertilizer input. Surface water of Delhi city receives the maximum amount of nitrogen due to fertilizer input. In Kolkata city total amount of farmland area is negligible so the amount of fertilizer input also negligible and the calculated nitrogen load displays negative value. The second large value of nitrogen load display in Karachi city, Dhaka and Mumbai city display less than one ton of nitrogen per km square area.

3.3 Nitrogen Load to Megacities and Concentrations in Surface Water

Among the five megacities, Kolkata city shows a large amount of nitrogen due to human waste. These may be due to the small area and very vast amount of population growth (Fig. 7). This nitrogen directly discharged into the surface water, so the amount of nitrogen per sq. km for all the cities

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excluding Karachi is excessive. In total nitrogen, all the country shows a very high amount of nitrogen due to human waste. Because all the five cities are megacity and their population also more than ten million so human wastes are increasing gradually. These excessive human wastes increase the nitrogen load in the water. These scenarios will turn in to more severe because of the increasing rate of population.

Fig. 6. Nitrogen load (tN km-2) due to fertilizer input in five megacities (0 indicate negligible amount of farmland)

Sewerage treatment facilities can treat the sewerage of less than 25% of the city dwellers of Dhaka city [24]. Dhaka situated by the river Buriganga is one of the most polluted rivers in Bangladesh. Industrial untreated effluents, natural and human activities change the water quality properties. Dhaka also surrounded by three other rivers (i.e. Balu, Turag and Shitalakhya). Water quality of these surrounded rivers also deteriorated since the last couple of decades. Most of the industries have no effluent treatment plant and these industries release effluents without any treatment and a huge volume of toxic wastes released into Buriganga river day and night [25]. Dhaka city produces about 3500 to 4000 m tons of solid wastes per day now [24]. Pollutant release amount increases over the years.

The Yamuna is the major rivers of Delhi and has a social, economic and religious consequence for vast sections of the population. Domestic and industrial sewage generated within the Delhi is the main source of pollution of the river Yamuna during its course through the city. A number of fecal coliforms severely increased since last ten years. In March 2004, the estimated sewage generation was about 719 MGD but Government of Delhi provided sewage treatment capacity of only 512 MGD whereas actual sewage being treated was only 335 MGD. This amount represented only 47 percent of the total estimated sewage production [26]. The balance of 384 MGD discharges into the river without any treatment.

Hooghly river of Kolkata is used for different purposes from livelihood to industrial pollutants release and transportation. But BOD and DO found satisfactory that means the water is good for aquatic life. Besides, fecal coliform concentration found high in value (approximately 750000 MPN/100ml) which indicate that direct consumption this water without any treatment is unsafe for human [27]. All the

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open wells of wetlands are hydraulically connected with surface water that is biogenically polluted [27,28].

Fig. 7. Nitrogen load to the environment due to food supply, farmland and human excretion in five south Asian megacities

Drinking water of Karachi city is provided from Indus river at Kotri. Total nitrogen (Nitrate+ Nitrite) content found in the acceptable limit (3.078 mg/l) and not exceeds the standard of EPA (10mg/l) [29]. Besides, total nitrogen concentration found increasing trend in the coastal area and adjacent area and river water (Malir, Lyari river) due to urbanization. The Lyari river is the main transporter to an estimated amount of 909.218 million liters of raw sewage to the Arabian Sea [30].

4. CONCLUSION

In the entire region of South Asia, about 95% of the anthropogenic nitrogen input was due to agriculture and food supply. Nitrogen content can give a clear idea of nitrate pollution in surface water. In this study, nitrogen budget are calculated on the country basis. Besides, treated sewerage is excluded to calculate nitrogen in case of the city.

Four findings has been identified are; (1) for all three countries, rice and wheat production-supply produce the maximum amount of nitrogen. (2) Though the amount of nitrogen due to fertilizer input more or less same among the countries but amount of produced nitrogen due to human waste is huge in Bangladesh. (3) Moreover, in city scale, the amount of nitrogen due to fertilizer input is maximum in Delhi city and negligible in Kolkata due to an insignificant amount of farmland. (4) Interestingly, the maximum amount of nitrogen load in surface water is in Kolkata city due to human waste but Mumbai and Dhaka shows a medium amount of nitrogen load. This can give the estimation for city wise untreated nitrate content and this is necessary for the capacity development of existing sewerage treatment plant as well as the establishment of new plants.

From this model, it can give a good idea about the current status of nitrogen of three south Asian developing countries and their important megacities. It also can realize the effect on the environment due to this nitrogen load.

5. LIMITATION OF THE MODEL

Deficiency of accurate sewage treatment data is the main bottlenecks of this study. Some industry has own sewerage treatment plant and some has absent. Besides, the government also installed some that not capable for all. So, all the treatment facilities are not inventoried properly.

Because of the total city area, farmland and forest area calculated from land use data with the spatial resolution of 1 km* 1 km [23] so it may vary with actual area.

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This study considers only food supply production and agricultural data, energy production is not included. As energy production has no prominent share for nitrogen production in South Asia. Even though, the inclusion of energy production data may give the more accurate budget. Nitrogen load to catchments and concentrations in river water also not included in this calculation.

6. FUTURE SCOPE

Energy production may include in the future model with other promising sectors. Data sources and sectors might be identifying for proper calculation. Future projection and its effect on the environment also be a new arena for this kind of study.

COMPETING INTERESTS

Authors have declared that no competing interests exist.

REFERENCES

1. UNFPA. Urbanization: A Majority in Cities; 2007. Available:http://www.unfpa.org/pds/urbanization.htm Cited 5 April, 2011 2. UN. World Population Prospects: The 2008 Revision and World Urbanization Prospects: The 2009 Revision, Population Division of the Department of Economic and Social Affairs of the United Nations Secretariat. United Nations; 2010. Available:http://esa.un.org/wup2009/unup 3. Southgate D. Population growth, increases in agricultural production and trends in food prices. The Electronic Journal of Sustainable Development. 2009;1(3):41. 4. Paustian M, Theuvsen L. Adoption of Precision Agriculture technologies by German crop Farmers. Precis. Agric. 2017;18:701. 5. Yan D, Zhu Y, Wang S, Cao W. A quantitative knowledge-based model for designing suitable growth dynamics in rice. Plant Prod. Sci. 2006;9:93–105. 6. Zhu Y, Cao W, Dai T, Tian Y, Yao X. A knowledge model system for wheat production management. Pedosphere. 2007;17:172–181. 7. Dobermann A, Cassman KG. Plant nutrient management for enhance productivity in intensive grain production systems of the United States and Asia. Plant Soil. 2002;247:153–175. 8. Van Aardenne JA, Carmichael GR, Levy II H, Streets D, Hordijk L. Anthropogenic NOx emissions in Asia in the period 1990-2020. Atmos. Environ. 1999;33:633-646. 9. Galloway JN. Nitrogen Mobilization in Asia. Nutr. Cycl. Agroecosyst. 2000;57:1-12. 10. Streets DG, Waldhoff ST. Present and future emissions of air pollutants in China: SO2, NOx, and CO. Atmos. Environ. 2000;34:363-374. 11. Zheng X, Fu C, Xu X, Yan X, Huang Y, Han S, Hu F, Chen G. The Asian nitrogen cycle case study. AMBIO. 2002;31:79-87. 12. Shindo J, Okamoto K, Kawashima H. A Model –based Estimation of Nitrogen Flow in the Food Production- Supply System and its Environmental Effects in East Asia. Ecological Modelling, 2003;169:197-212. 13. Liu J, Ma K, Ciais P, Polasky S. Reducing human nitrogen use for food production. Scientific reports. 2016; 6:30104. 14. Foster SSD. The interdependence of groundwater and urbanisation in rapidly developing cities. Urban Water. 2001;3:185–192. 15. Morris BL, Lawrence AR, Foster SSD. Sustainable groundwater management for fast-growing cities: mission achievable or mission impossible? In: Chilton, P.J. (ed.) Groundwater in the Urban Environment. Vol. 1: Problems, processes and management. Proceedings of the XXVII Congress of the International Association of Hydrogeologists; Nottingham, UK. 1997;55–66. 16. Onodera S. Subsurface Pollution in Asian Megacities Groundwater and Subsurface Environments. 2011;Part III:159-184, DOI: 10.1007/978-4-431-53904-9_9 . 17. Karn SK, Harada H. Surface water pollution in three urban territories of Nepal, India, and Bangladesh. Environmental management. 2001;28(4):483-96. DOI:10.1007/s002670010238.

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18. Mehtab Haseena, Muhammad Faheem Malik, Asma Javed, Sidra Arshad, Nayab Asif, Sharon Zulfiqar and Jaweria Hanif. Water pollution and human health. Environmental Risk Assessment and Remediation. 2017;1(3). 19. UN. Sources and nature of water quality problems in Asia and the pacific; 1998 (United Nations). 20. FAO. Food and Agriculture Organization of the United Nations, Viale delle Terme di Caracalla, 00153 Rome, Italy; 2005. Available:http://faostat.fao.org. 21. Resources Council, Science and Technology Agency. Standard Tables of Food Composition in Japan, 5th ed. (revised). Printing Bureau, Ministry of Finance, Japan. 2000;589 (in Japanese) 22. WHO. Global Water Supply and Sanitation Assessment 2000 Report. World Health Organization, Geneva; 2002. Available: http://www.who.int/water_sanitation_health/Globassessment/GlasspdfTOC.htm. 23. USGS. The USGS Land Cover Institute (LCI); 2000. Available: http://landcover.usgs.gov/glcc/download.php, Global land cover map 2000 24. Ahmad MK, Islam S, Rahman S, Haque MR, Islam MM. Heavy Metals in Water, Sediment and Some Fishes of Buriganga River, Bangladesh. 2010;4(2):321-332. Available: http://www.sid.ir/en/VEWSSID/J_pdf/108220100216. 25. Islam MM, Akhtar MK, Masud MS. Prediction of environmental flow to improve the water quality in the river Buriganga. Proceedings of the 17th IASTED International Conference on Modelling and Simulation, Montreal, QC, Canada; 2006. 26. CAGI. Report on Government of NCT of Delhi of 2005, Measures to Control Water Pollution in River Yamuna in Delhi; 2005. Available: www.cag.gov.in/html/cag_reports/delhi/.../civilvolII_yamu_rev.pdf 27. WBPCB. Annual Report 2007-08, West Bengal Pollution Control Board. West Bengal Pollution Control Board; 2008. Available:http://www.wbpcb.gov.in/html/annualreps/ar0708/chapter_10.pdf. 28. KMC. Ground Water Information Booklet, Kolkata Municipal Corporation, West Bengal; 2007. 29. GEMSTAT. Global water quality database, The United Nations Global Environment Monitoring System (GEMS) Water Programme; 2010. Available:http://www.gemstat.org/. Data downloaded in December, 25, 2010 30. Burt N. Environmental Assessment and Protection of Karachi Harbor; 1997.

Biography of author(s)

Syeda Jesmin Haque Graduate School of Integrated Arts and Science, Hiroshima University, Japan and Assistant Director, Geological Survey of Bangladesh, Room#415, 153 Pioneer Road, Segunbagicha, Dhaka-1000, Bangladesh.

She serves for Geological Survey of Bangladesh (GSB) as Assistant Director. After her graduation from Department of Geology, University of Dhaka, she joined this organization and till now she serves the country as a geologist. She obtained her masters degree from hydrogeology and environmental geology stream from Dhaka University too and did a unique water quality assessment and hydrogeological characterization on third aquifer of Dhaka city. She did her second masters in Engineering Geology from University Kebangsaan, Malaysia (National University of Malaysia). There also she did an exceptional research on hydrogeology and hydro-geo-chemical comparison of Dhaka and three cities of Malaysia. She successfully completed a post graduation diploma from Hiroshima University, Japan as a Visiting Researcher. At that time she did an outstanding review work on five Asian megacities water quality, urbanization effect on water quality and trend analysis. In her career she also did some distinctive palynological and paleontological research to identify the paleoenvironment of the country and climate change. She also did a distance course on Systematic Paleontology from Missouri University of Science and Technology, USA. She has published more than thirteen articles, posters and conference proceedings in peer reviewed

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journals. She also authored a book, titled “Hydrogeology of the lower Dupi Tila Aquifer of Dhaka City Bangladesh, The hydrogeological and hydrochemical characterization”.

Prof. Dr. Shin-ichi Onodera Graduate School of Integrated Arts and Sciences, Hiroshima University, Japan.

He is a Professor with doctorate in hydrology, with strong background in groundwater hydrology and in material transport especially for nitrogen and phosphorus in a catchment scale. Experienced in numbers of managing projects from conception to completion as principal investigator.

Dr. Yuta Shimizu Graduate School of Integrated Arts and Science, Hiroshima University, Japan.

He is a hydrologist who works on spatiotemporal changes in nutrient transportation and geographical information science. He is well experienced on distributed hydrological modeling on nutrient cycle analysis, nutrient behavior in reservoir, nitrogen contamination in river and groundwater. ______© Copyright 2019 The Author(s), Licensee Book Publisher International, This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Chapter 14 Print ISBN: 978-81-934224-3-4, eBook ISBN: 978-93-89246-17-9

Nutrient Solution: Agronomic Characteristics and Quality of Strawberry Fruits Cultivated in Substrate

Dalva Paulus1* and Anderson Santin1

DOI:10.9734/bpi/atias/v1

ABSTRACT

Aims: This study determined a nutritive solution and evaluated the performance in the development, production and quality of strawberry cultivated in the substrate. Study Design: The treatments were commercial and recommended nutritional solutions for strawberry using the methods of Castelane and Araújo (C.A.), Furlani and Fernandes Junior (F.F.J.) and the proposed solution with seven replicates. Place and Duration of Study: The experiment was carried out in the experimental area of the Federal Technological University of Paraná, Brazil, in the period between May and December 2014. Methodology: Agronomic variables such as yield, number of fruits, nutrient content, physiological indicators, physical and chemical characteristics of fruits were analysed. Results: The proposed nutrient solution resulted in larger masses of fresh and dry matter (225.4 g plant-1 and 27.5 g plant-1), number of fruits (40.1) and fresh fruit mass (750.4 g plant-¹), in relation to the other evaluated solutions. The proposed solution resulted in better physical and chemical characteristics such as soluble solids, reducing and total sugars, anthocyanins, flavonoids, phenolic compounds and ascorbic acid and the strawberry fruits presented an attractive colour and met the quality standards for the consumer. The highest levels of nitrogen (33.7 g kg-1), phosphorus (9.3 g kg- 1), and potassium (28.2 g kg-1) in the leaf tissue were found in the proposed solution and contributed to productivity and fruit quality gains of a strawberry. Conclusion: These results provide a nutrient base and can be adapted to other cultivars in different locations. In this study, the proposed nutrient solution contributed to productivity gains, fruit quality and comes as an option of adequate nutrient content for the strawberry, with ionic balance, without excess nutrients. These results provide a nutrient base and can be adapted to other cultivars in different locations.

Keywords: Fragaria x ananassa Duch; nutrients; colour; physical and chemical characteristics.

1. INTRODUCTION

The strawberry (Fragaria x ananassa Duch) is one of the fruits most appreciated by consumers in different regions of the world, highlighting its color, aroma, flavor and versatility in cooking and gastronomy. For this reason, strawberries are in great demand both in the natura and industrial processings [1]. U.S. consumption of strawberries has increased significantly during the past two decades, from 2 lb per capita in 1980 to 8 lb in 2013 [2]. Consumption is expected to increase as a result of increased awareness of the health benefits associated with berry consumption, year-round availability made possible through domestic production and protected berry culture, increased imports, and improved cultivars [3].

Strawberry fruits with better physicochemical characteristics guarantee acceptance by the consumer market and increase yield in processing and industrialization. Ripening is a biochemical process in fruits, in which physical and chemical characteristics including dramatic bioactive compounds production, such as reducing sugars, organic acids, ascorbic acid, anthocyanin, and ellagic acid for

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1Department of Agronomy, Federal University of Technology - Paraná, Paraná - 85660-000, Brazil. *Corresponding author: E-mail: [email protected];

Advances and Trends in Agricultural Sciences Vol. 1 Nutrient Solution: Agronomic Characteristics and Quality of Strawberry Fruits Cultivated in Substrate

each fruit stages at maturity. In general, strawberry fruits [4]. In this sense, the nutritional solution concentration, together with the use of processing techniques have been important factors taken into account to improve the productivity and physicochemical properties of the fruit [5].

Strawberry cultivation in the substrate is a production technique used in several regions of Brazil and around the world, allowing to obtain high production and greater ergonomics in crop management [6]. The main problem faced by producers in this production system is with regard to composition and management of the concentration of the nutrient solution. The cultivation of strawberries in semi- hydroponic systems has been growing annually. In these systems, the use of substrates with ideal physical characteristics for the development of the plants is important; an ideal substrate should, for example, provide mechanical support for the roots, a balanced and stable porosity in order to provide sufficient air and water for root metabolic processes, and a good water retention capacity in order to avoid stress due to water deficit or hypoxia [7].

In the literature Paranjpe et al. [8], reported that concentrations of nutrient solution with values of electrical conductivity (EC) between 1.4 and 1.8 dS m-1 and up to 2.0 dS m-1 [9] are proposed to obtain quality and productivity of strawberry fruits, but the great difficulty with most nutrient solutions is to adjust the amount of nutrients for substrate cultivation.

In this sense, the need arises for studies with nutritive solutions with the determination of ionic balance of nutrients and their relationship with yield and quality of strawberry fruits in substrate cultivation. In this study we determined a nutrient solution for strawberry and evaluated the agronomic characteristics and fruit quality. The results provide nutrient content information extracted by the plants with the proposed solution, production data and physiological indicators of fruit quality, which contribute to meet the demands of the consumer market and make the production system more sustainable.

2. MATERIALS AND METHODS

2.1 Plant Material and Growing Conditions

The experiment was carried out in the experimental area of the Federal University of Technology - Paraná, Brazil (25º42'52 "S, 53º03'94" W, 530 m altitude), in the period between May and December 2014, covered with a 150-micron plastic film.

The seedlings of the cultivar Camino Real were purchased from a suitable nurseryman of varietal quality, from Maxxi Mudas®, from Patagonia, Argentina. These were transplanted in plastic pots with a capacity of 8 L in dimensions 24 × 23 cm, placed in lines, on the soil of the protected environment, filled with sand of medium granulometry, being transplanted one plant per pot, distributed with a density of eight plants per square meter.

The replenishment of nutrients was carried out daily by means of a drip irrigation system, with drippers of the brand netafim®, with a spacing of 0.20 m and a flow of 3.2 L hour-1, with a dripper per vessel, thus maintaining the sand in the field capacity. The total fertigated volume was 535.7 mm and the total irrigation time was 47.5 hours for all treatments.

The meteorological data (temperature, relative air humidity and solar radiation) were obtained every 15 minutes using Akso® brand AK 172 dataloggers installed in meteorological shelters, located in the center of the protected environment.

The fertilisers used to compose the evaluated nutrient solutions were potassium nitrate (KNO3), calcium nitrate Ca(NO3)2, monoammonium phosphate (NH4H2PO4) and magnesium sulfate (MgSO4). For micronutrients the amount of 25 g per 1000 L of water of the commercial product Conmicros Standard® was used in all the nutrient solutions, which presented the concentrations of B (2.0%), CuEDTA (2.0%), FeEDTA (7.9%), MnEDTA (2.0%), Mo (0.4%) and ZnEDTA (0.8%).

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The nutrient solutions after addition of the nutrients presented the following values of electrical conductivity and pH 2.0 and 6.0 mS cm-1 for the commercial solution (F.F.J.) [10] 1.7 and 6.2 mS cm-1 for the commercial solution (C.A.) [11], and 1.8 and 5.8 mS cm-1 for the proposed solution.

In relation to the management of nutrient solutions, the fertigations were done daily, and at each application of the fertirrigation, a new solution for each treatment was prepared. Also, twice-a-week irrigations were carried out only with water to avoid salinisation of the substrate. Electrical conductivity and pH were measured with conductivity and portable HI 98130 Hanna® brand portable pH meters each time the solution was prepared. pH values between 5 and 6, and electrical conductivity greater than 1.5 mS cm-1 were maintained during the experiment [12].

2.2 Treatments and Experimental Design

The treatments were commercial and recommended nutritional solutions for strawberry using the methods (C.A.) [11], (F.F.J.) [10] and proposed solution, with seven replicates. The amounts of nutrients used for each solution are shown in Table 1. The calculation of the proposed solution was based on ionic nutrient balance [13].

2.3 Evaluated Parameters

The content of macronutrients and micronutrients in leaf tissue was determined and four leaves per plant were completely expanded in the flowering period [14].

During the full flowering period (120 days after transplanting [DAT]) and at 190 DAT, measurements of the relative index of total chlorophyll in the abaxial and adaxial parts of the last two expanded leaves of each plant were performed at 11:00 AM using the chlorophyllometer model Clorofilog Falker® brand. The fresh matter mass of fruits and number of fruits per plant was determined by adding all the harvests during the evaluated period (fifteen harvests).

The average mass of fruits was obtained by dividing the fresh matter mass of fruits by the number of fruits per plant. The fruits were harvested when they presented more than 75% of the epidermis with pink colouration [15].

The fruit colour was determined in 10 fruits randomly selected from each nutrient solution, using a digital colourimeter (Minolta model, Cr 200 b), where the values of luminosity ("L") were determined, ranging from light to dark. The value 100 corresponds to white colour and value 0 (zero), the black colour, and component “c” which expresses chroma degree of the fruits, where, by the proposed classification, more colourful fruits present smaller values and less colourful fruits present higher values [16].

The soluble solids (SS) content was obtained by direct reading in Hanna® bench refractometer model HI 96801, using the homogenized pulp and filtered at room temperature, obtaining the values in degrees (Brix). The determination of the titratable acidity (T.A.) was by titration with 0.1N NaOH until it reaches pH 8.1. The ratio (SS/TA) was determined by dividing the soluble solids content by the titratable acidity.

Total sugar concentrations were determined by the method described by Dubois et al. [17] those of reducing sugars were obtained by the method described by Miller [18].

The quantification of total phenolic compounds (mg gallic acid 100 g pulp-1) was carried out according to the spectrophotometric method of Follin-Ciocauteau, proposed by Woisky and Salatino [19]. The ascorbic acid content (Vitamin C) was determined by standard titration method of AOAC modified by Benassi and Antunes [20]. Vitamin C content was calculated based on titration values of a standard solution of ascorbic acid and the results expressed in mg of 10 g of ascorbic acid 100 g pulp-1.

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Table 1. Quantities of nutrients used in the preparation of nutrient solutions for strawberry cultivation on the substrate

Nutrients Commercial solution Commercial solution Proposed (mg L-1) C.A. F.F.J. solution - N-NO3 124.6 116.2 166.9 - N-NH4 5.6 5.6 31.92 - P - H2PO4 46.5 49.6 78.12 K+ 195.0 234.0 182.13 Ca++ 124.0 104.0 68.0 Mg++ 24.60 36.0 13.7 -- S-SO4 43.20 48.0 16.6

In the quantification of anthocyanins and flavonoids, the procedure described by Mazaro et al. [21] was used. All the physicochemical analyses were determined in a single crop, at 150 DAT, which corresponded to the peak of production. A composite sample of 100 fruits per treatment was used for all the analyses, taking seven subsamples of approximately 50 g each.

At 190 DAT, which corresponded to end of the experiment, the mass of fresh matter in a precision scale (0.001 g) of all the plants of the experiment was determined. After the plants were placed to dry in a forced circulation air oven at 65°C until reaching constant mass to determine the mass of dry matter.

2.4 Statistical Analysis

The data of experiment were submitted for analysis of variance (Test F), when the F test was significant the means were compared by Tukey's test (P=0.05), using “SAS Studio” [22].

3. RESULTS AND DISCUSSION

During the conduction of experiment, the average temperature, relative humidity and average daily radiation were 19.2°C, 75% and 949.7 kJ m-2. The temperature conditions during the experiment were found to be within the ranges suitable for the crop. Temperatures that range between 18°C and 24°C are considered adequate for the development of the crop [23].

The electrical conductivity (EC) in the solutions tested ranged from 1.5 to 2.1 dS m-1, with an average of 1.8 dS m-1. The mean conductivity is at the upper limit of the recommended range of 1.4 to 1.8 dS m-1 [10]. The pH variations of the solutions were between 5.0 and 7.0, with an average of 6.2. pH ranges between 5.5 and 6.5 are most indicated for the culture [10].

It was observed that the EC in the evaluated solutions improved fruit quality by increasing the solids content and sugars. It was found that evaluated solutions were within the recommended pH range for strawberry.

Nutrient solutions significantly influenced nutrient content in leaf tissue. The highest levels of nitrogen (33.7 g kg-1), phosphorus (9.3 g kg-1) and potassium (28.2 g kg-1) in the leaf tissue were found in the proposed solution (Table 2). The other macronutrients did not differ significantly. For micronutrients, there were significant differences for the boron content in F.F.J. solution and higher iron and manganese contents in the proposed solution.

The macronutrients, in descending order, nitrogen (N), potassium (K), calcium (Ca), phosphorus (P), magnesium (Mg), and sulphur (S) were the nutrients extracted in greater quantity by the strawberry. The following ranges are recommended: N, 15-25 g kg-1; P, 2-4 g kg-1; K, 20-40 g kg-1; Ca, 10-25 g kg- 1; Mg, 6-10 g kg-1; and S, 1-5 g kg-1. For boron (B), iron (Fe), manganese (Mn), copper (Cu) and zinc (Zn) are 35-100, 50-300, 30-300, 5-20, and 20-50 mg kg-1, respectively [24]. The contents found in the foliar tissue for the studied solutions are superior to those suitable for N and P, within the

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recommended range for K, Ca, Mg, B, Fe, Mn, Zn, and Cu. No visual symptoms of nutritional deficiency were observed in the strawberry plants during the experiment.

The proposed solution resulted in the highest relative indices of total chlorophyll in the flowering phase and at the end of the crop cycle (Table 3). There was also a decrease in the relative index of total chlorophyll in final phase of the cycle in all evaluated solutions.

The mass of the fresh and dry matter presented significant differences, being largest accumulation of fresh (225.4 g plant-1) and dry (27.5 g plant-1) mass obtained in the proposed solution (Table 3). The fresh mass of the proposed solution was 6.74% higher than the Castelane and Araújo commercial solution.

The highest relative chlorophyll index in the proposed solution is justified by higher nitrogen content present in the leaf tissue. The content of chlorophyll in the leaf is used to predict the nutritional level of N in plants, due to the fact that the amount of this pigment correlates positively with N content in the plant [25]. This relationship is attributed mainly to the fact that 50% to 70% of total N of the leaves is integral with enzymes, which are associated with chloroplasts [26].

The decrease of relative index of chlorophyll in the final phase of crop cycle can be explained by the advancing age of the leaf because in this phase, there is a decline of photosynthetic capacity. The photosynthetic efficiency is linked to the amount of chlorophyll and consequently, to the growth phase of the plant [27].

The results of mass of the fresh and dry matter obtained with the proposed solution may be related to higher nutrient intake, especially the nitrogen present in the foliar tissue of the proposed solution (Table 2). Plant development, productivity and strawberry fruit quality are strongly influenced by nitrogen fertilization [28].

Moreover, the increase of K in the plant causes an increase in the production of photoassimilates and consequently, a greater mobilization of leaf N in the synthesis of macromolecules, which in turn are used in vegetative growth and fruit production [29].

The number of fruits per plant, average fruit mass, and fresh fruit mass was influenced by evaluated treatments, obtaining best results in the proposed solution (Table 4). There were gains of 6.1% and 7.94% in the fresh fruit mass in relation to commercial solutions F.F.J. and C.A, which can be attributed to ionic balance of the proposed solution, which met the nutritional demand of strawberry with nutrient amounts without excesses or deficiencies, contributing to fruit quality and sustainable management of fertilizers in agriculture.

For the luminosity (L) of the epidermis, the nutrient solutions evaluated did not present significant influence (Table 4). The values of luminosity of the evaluated solutions were below the value 29.24 and according to Conti et al. [16] indicate dark colour. The dark colour of strawberry fruits in the evaluated solutions is a desirable characteristic for both industry and consumers because dark red fruits are more attractive in the eyes of consumers.

For the colour component or chroma value of the epidermis, the proposed solution presented darker and more colourful fruits. The “C” component expresses the colour of fruits, where values less than 24.92 have more colour of the epidermis, values between 24.92 and 36.08 have intermediate colour, and values above 36.08 have less colourful fruits [16]. It is of great importance that the external aspect of the fruit in commercialisation is mainly in natura, the proposed solution resulted in fruits being more attractive for commercialization.

The superiority in the number of fruits and fresh fruit mass in proposed solution can be attributed to the ionic balance of the solution, which favoured the absorption of some ions, such as potassium (28.2 g kg-1) (Table 2), which improved productivity and fruit quality [30]. The increase in mean mass of fruits, influenced by potassium present in the proposed solution, can be attributed to the important role that this nutrient plays in the translocation of photoassimilates from leaves to the fruits and the role it exerts in cell extension [29].

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Table 2. Nutrient content in leaf tissue of fertigated strawberry with different nutrient solutions

Nutrients N P K Ca Mg S B Cu Fe Mn Zn g kg-1 g kg-1 g kg-1 g kg-1 g kg-1 g kg-1 mg kg-1 mg kg-1 mg kg-1 mg kg-1 mg kg-1 Sol. C.A. 31.1 b* 7.8 b 21.8 b 11.1 ns 6.9 ns 1.1 ns 100.5 b 5.3 ns 82.6 b 194.0 b 30.0 ns Sol. F.F.J. 30.4 b 8.0 b 23.4 b 10.9 6.8 1.2 103.0 a 5.7 81.7 b 216.5 b 29.5 P. solution 33.7 a 9.3 a 28.2 a 10.5 7.0 1.2 98.4 b 5.5 86.5 a 222.0 a 31.6 Mean 31.7 8.4 24.5 10.8 6.9 1.2 100.6 5.5 83.6 210.8 30.4 C.V. (%) 3.9 9.2 3.3 3.1 5.6 4.3 1.2 2.3 3.5 2.6 2.2 *Means followed by the same letter in the column do not differ significantly by Tukey test, at P=0.05; ns: no significant; C.V.: Coefficient of variance

Table 3. Relative index (I.R.) of total chlorophyll phases full flowering and the end, masses of fresh and dry matter of shoot (M.F. and M.S.) of fertigated strawberry plants with different nutrient solutions

Solutions Full Flowering End of cycle M.F. M.S. I. R. of total chlorophyll I. R. of total chlorophyll (g planta-1) (g planta-1) Sol. C. A. 54.4 b* 50.2 b 210.2 b 21.0 b Sol. F. F.J. 57.1 b 52.9 b 208.7 b 20.7 b P. solution 62.2 a 59.4 a 225.4 a 27.5 a Mean 57.9 54.17 215.0 23.1 C.V. (%) 9.3 8.4 20.4 22.0 *Means followed by the same letter in the column do not differ significantly by Tukey test, at P=0.05; C.V.: Coefficient of variance

Table 4. Number of fruits plant-1 (N.F.P.), mean fruit mass (M.F.M), fresh fruit mass (F.F.M.), luminosity of the epidermis, the colour of the epidermis (Chroma) of fertigated strawberry with nutritive solutions

Solution N.F.P. M.F.M. F.F.M. Luminosity Chroma (g) (g plant-1) Sol. C.A. 30.3 b* 12.5 b 690.8 b 28.7 ns 35.1 a Sol. F.F.J. 32.5 b 13.3 b 704.6 b 27.8 34.93 a P. solution 40.1 a 15.7 a 750.4 a 26.0 32.10 b Mean 34.3 13.83 715.3 27.8 34.0 C.V. (%) 22.4 13.7 27.8 3.81 3.87 *Means followed by the same letter in the column do not differ significantly by Tukey test, at P=0.05; ns: no significant; C.V.: Coefficient of variance

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The number of fruits found in the proposed solution was 33.9% higher than that observed by Vignolo et al. [31], where 26.5 fruits per plant with the cultivar Camino Real in cultivation were carried out in the soil. The fresh fruit mass transformed into yield results in 60 t ha-1, which is higher than the yield obtained from 9.07 and 10.55 t ha-1, with the same cultivar in conventional and organic systems, respectively [32].

The proposed solution resulted in more colourful fruits, with a higher content of soluble solids and of total and reducing sugars, possibly due to the higher content of potassium. Potassium is one of the nutrients most used by strawberry, considered the “element of quality” in plant nutrition, to improve physical-chemical characteristics and to increase production [14].

The SS/TA were not influenced by the evaluated nutrient solutions, the average value being 0.90 g.100 g pulp-1 and 9.33, respectively (Table 5). It was verified that the proposed nutrient solution resulted in a higher content of soluble solids and concentration of total and reducing sugars, anthocyanins, flavonoids, phenolic compounds and ascorbic acid (Table 5).

The soluble solids content in the proposed solution was 25.3% higher than the results obtained by Andriolo et al. [30] of 6.65 °Brix. The minimum values of soluble solids should be higher than 7.0 °Brix, guaranteeing acceptable taste [15], all nutritional solutions presented values above 7.0 °Brix, considered acceptable for consumers.

Table 5. Soluble solids (S.S.) (°Brix), total sugars (T.S.) (mg.g-1 fresh fruit mass), reducing sugars (R.S.) (mg.g-1 fresh fruit mass), flavonoids (F.) (mg.100 g-1 fresh fruit mass), anthocyanins (A.) (mg.100 g-1 fresh fruit mass), phenolic compounds (P.C.) (mg of galic acid 100 g-1 fresh fruit mass) and ascorbic acid (A.A.) (mg.100 g-1 pulp) of fertigated strawberry fruits with different nutrient solutions

Solution S.S. T.S. R.S. F. A. P.C. A.A. Sol. C. A. 8.0 b* 8.7 b 1.1 b 3.3 b 34.0 b 75.1 b 45.1 b Sol. F. F. J. 8.2 b 9.0 b 1.3 b 3.1 b 35.0 b 76.4 b 47.0 b P. solution 8.9 a 10.1 a 1.9 a 4.0 a 40.4 a 80.6 a 52.5 a Mean 8.4 9.3 1.4 3.5 36.1 77.4 48.2 C.V. (%) 8.1 7.7 19.3 17.9 20.2 10.3 12.7 *Means followed by the same letter in the column do not differ significantly by Tukey test, at P=0.05; C.V.: Coefficient of variance

In the relationship between sugar content and acidity (SS/TA) there was no statistically significant difference between the evaluated solutions, with a mean value of 9.33. This value meets the minimum relationship patterns for strawberry fruits of 8.75 [15]. The strawberry fruits of the cultivar Camino Real presented an adequate SS/TA ratio, with a degree of maturation and fruit quality. The SS/TA ratio is an important parameter to determine fruit maturation, and fruit taste evaluation, as well as an indicator of fruit palatability, being directly linked to the preference and acceptance of the fruits by the consumer [33].

Commercially, the colour of the fruits can be influenced by the anthocyanins, which contributes greatly to quality evaluation, since the consumers correlate between the colour and total quality of specific products [34]. The anthocyanin content in the proposed solution was higher than that reported in the literature (20.93 mg 100 g-1 fresh fruit mass) by Calvete et al. [35] with the same cultivar, on different commercial substrates. According to Clifford [36] the anthocyanin levels may present variations related to climatic factors, seasonality, the degree of maturation, nutrition and type of cultivar.

The results of the present study indicate that fruits of the proposed solution presented an attractive colour and fruits with higher concentrations of anthocyanins, allowing greater benefits to the consumer due to the antioxidant effect.

As the anthocyanins content may be a criterion of choice at the time of feeding, due to the health benefits [35], the consumer will be eating higher anthocyanin content when consuming strawberries of

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the Camino real proposed solution. For humans, the intake of foods rich in anthocyanins, such as red fruits, is related to health benefits, as these components have high antioxidant and antitumor activity, as well as acting as an anti-inflammatory and preventing the formation of edemas [37].

The phenolic compounds and ascorbic acid contents presented significant differences for the evaluated solutions. The proposed solution resulted in an increase in phenolic compounds and ascorbic acid. In the literature, it is reported that potassium fertilisation exerts a beneficial effect on vitamin C levels [38].

Furthermore, the phenolic contents found in this study are lower than those verified by Pineli et al. [39] with the same cultivar (174.3 mg 100 g-1 pulp). Phenolic compounds are significantly influenced by the genetic factors of the cultivar [40]. In addition, the "open" culture system provides a higher content of phenolic compounds than the protected environment system [41].

Another factor that possibly influenced the content of phenolic compounds was the temperature. It is known that the synthesis of phenolic substances is favoured by the milder temperatures, especially the nocturnal ones and also the temperature variation from day to night, affects pigment deposition [42]. The average temperature of 19.2°C favoured the deposition of phenolic compounds, anthocyanins and flavonoids in fruits.

Potassium exerts influence on phenolic content, as it is related to photosynthesis and to biosynthesis of starch and proteins. With the increase of K doses in the plant, the production of photosynthates increases, which may increase the targeting of excess carbon fixed to the pathway of shikimic acid, which is the pathway for the formation of phenolic compounds, which may increase the concentration of phenolics in the plant [43].

The ascorbic acid (Vitamin C) in strawberry may vary according to the cultivar, stage of ripening and fertilization. It is one of the most important nutritional components in fruits and human food and its content can be used as an index of food quality [15]. In addition to mineral nutrition, the intensity of solar radiation (949.7 kJ m-2) associated with the time of year (summer) contributed to the increase in ascorbic acid content. The intensity and duration of fruit exposure to sunrays during growth influence the amount of ascorbic acid formed is synthesized from sugars supplied by photosynthesis, which increases with the highest incidence of radiation [42].

The phenolic compounds and ascorbic acid contents presented significant differences for the evaluated solutions. The proposed solution resulted in an increase in phenolic compounds and ascorbic acid. In the literature it is reported that potassium fertilization exerts a beneficial effect on vitamin C levels [38].

4. CONCLUSION

In this study, the proposed nutrient solution contributed to productivity gains, fruit quality and comes as an option of adequate nutrient content for the strawberry, with ionic balance, without excess nutrients. These results provide a nutrient base and can be adapted to other cultivars in different locations.

ACKNOWLEDGEMENTS

The authors thank the company Maxxi mudas ® for the donation of the seedlings to carry out the research.

COMPETING INTERESTS

Authors have declared that no competing interests exist.

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Biography of author(s)

Dalva Paulus Department of Agronomy, Federal University of Technology - Paraná, Paraná - 85660-000, Brazil.

She is a professor of the Agronomy Course at the Federal University of Technology, Paraná, Brazil. She graduated in Agronomy from the Federal University of Santa Maria, Rio Grande do Sul, Brazil. She obtained a master's degree from the same university in the area of vegetable production in 2001. She holds a doctorate degree from the University of São Paulo, Luiz de Queiroz College of Agriculture. She is part of the Master in Agroecosystems of the Graduate Program of the Federal University of Technology, Paraná, Brazil. She has published several articles in national and international periodicals. Editor and author of several book chapters, such as the book Sustainable Agricultural Management Techniques. She guided undergraduate students of the Agronomy course, master's degree and doctorate. She held several lectures in the area of hydroponic crops and protected crops. Organized events and field days, such as Hydroponics Workshops. Currently, she is dedicated to studies related to the reuse of wastewater in seedlings and greenery crops. Effects of colors of shading meshes on the quality of strawberries. Her achievement and recognitions are as follows: Agricultural Engineer and PhD in Plant Production presented the scientific community and the general public with the book chapter: Nutrient Solution for Production and Quality of Strawberry Grown in Substrate.

Anderson Santin Department of Agronomy, Federal University of Technology - Paraná, Paraná - 85660-000, Brazil.

He is an agronomic engineer from the Federal University of Technology, Paraná, Brazil. He completed his Master's Degree in Agronomy/Phytotechny from the State University of Western Paraná Campus, Marechal Cândido Rondon (Unioeste), Brazil, with the Dissertation "Physiological indicators and productivity of strawberry cultivated under mulchings and spatial arrangements in field crop". He achieved his PhD degree in Agronomy / Phytotechny from the State University of Western Paraná Campus Marechal Cândido Rondon (Unioeste), Brazil, with the thesis "Production and quality of strawberry fruit cultivated on plastic soil covers". ______© Copyright 2019 The Author(s), Licensee Book Publisher International, This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Reviewers’ Information (1) Arun Kumar Mahawar, SKN College of Agriculture, Sri Karan Narendra Agriculture University, India. (2) Ehlinaz Torun Kayabaşi, Arslanbey Vocational School, University of Kocaeli, Turkey.

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