Morphological and molecular characterisation of plant-parasitic associated with pineapple, roses and tea in Kenya

David Kihoro Sirengo Student number: 01800695

Promoter: Prof. Dr. Wim Bert

Co-promoter: Dr. Laura Cortada

Supervisor: Rolish Singh

A dissertation submitted to Ghent University in partial fulfilment of the requirements for the degree of International Master of Science in Agro- and Environmental Nematology

Academic year: 2019 - 2020

Morphological and molecular characterisation of plant-parasitic nematodes associated with pineapple, roses and tea in Kenya

David Kihoro Sirengo Nematology Research Unit, Department of Biology, Ghent University, K.L. Ledeganckstraat 35, 9000, Ghent, Belgium

Plant-parasitic nematodes in Kenya David Kihoro Sirengo|2020

Preamble

This master thesis is aimed at establishing the diversity and characterization of plant-parasitic nematodes associated with pineapple, cut flowers (roses) and tea from Kiambu county in Kenya. Morphological and molecular characterization of important plant-parasitic nematodes associated with the above-mentioned crops would provide a baseline study for future reference. However, given this thesis was done amid COVID-

19 pandemic, 43 (pineapple 26, roses 35, and tea 9) soil and pumice samples were analysed based on prevalence, density, prominence and identification at genus level. In this study, only two plant- parasitic nematodes from pineapple fields have been characterized through integrative approach. A thorough analysis of both soil and root samples from the sampled geolocated areas in Kiambu, Kenya is needed to provide an overview of all plant-parasitic nematodes in the samples. Having said that and considering the limitations mentioned above, this master thesis has been written using the limited available data and other assignments suggested to fulfil an accepted level of dissertation. The additional off-campus assignments included in this thesis are: 1) resolving taxonomical problem of the cryptic species status of

Scutellonema brachyurus from samples obtained from fingermillet, pineapple, soybean and morphological voucher slides from Rwanda and D2D3 and COI sequence from Tanzania 2) providing an overview of terrestrial nematofauna in Kenya using data from current study and literature search in a joint effort with

Denis Gitonga.

Plant-parasitic nematodes in Kenya David Kihoro Sirengo|2020

Summary - A baseline survey of pineapple, roses and tea fields in Kiambu Kenya was carried out between December 2019 - January 2020 to determine the distribution and abundance of plant-parasitic nematodes and to characterize the prevalent genera using combination of morphological features and molecular analyses. A total of 114 soil and root samples; 60 pineapple, 45 roses and 9 tea samples were collected from Kiambu county, Kenya. Nematodes were extracted from both soil and roots following a standardized protocol and identified at genera and some to species level. In pineapple samples, Helicotylenchus dihystera and Meloidogyne javanica were the two most prevalent parasitic species (100%) followed by Pratylenchus species (36%). Helicotylenchus dihystera was characterized based on its morphology and D2-D3 of 28rDNA sequences similar to GenBank records. The two populations agree with the original description and D2D3. All the four M. javanica populations were identical to each other and a Nad5 gene of mtDNA reference informative polymorphic positions similar to sequence (KU372392) by (Janssen et al., 2016). The top three prevalent genera on roses rhizosphere were Meloidogyne spp. (100%), Xiphinema spp. (54%) and Longidorus spp. (46%). The densities were however relatively low: 9 individuals/100 ml, 5 indiv/100 ml and 4 indiv/100 ml of soil for Meloidogyne, Xiphinema and Longidorus respectively. Plant-parasitic nematode were not detected from tea samples. This results sheds lights on the distribution and diversity of parasitic nematodes on pineapples and roses in Kiambu, Kenya. Additionally, based on samples from fingermillet, soybean, pineapple and potato rhizosphere, our findings revealed all newly generated sequences (D2-D3 and COI) of our S. brachyurus type B populations formed a maximally supported clade with other sequences of African S. brachyurus (type B) and were separated from S. brachyurus type A species which are known to be cryptic species based on molecular data reported by Van den Berg et al. (2016). Morphologically, S. brachyurus type B was found to be different from type A species based on the number of lip region annuli 3-4 vs 4-6 annuli. The species delimitation also revealed taxonomic distinctness of S. brachyurus type B supported by a well significant Rosenberg’s PAB of 1.2E-14 and 1.9E- 8 for D2-D3 and COI respectively, revealing it is a different species from S. brachyurus type A species. Finally, a total of 10 taxa from current study and 116 taxa from literature on Kenyan nematofauna has been revealed. The ten taxa identified were; Criconema spp., Helicotylenchus dihystera, Meloidogyne javanica, Paratylenchus spp., Rotylenchus robustus, macrosoma, Trichodorus spp., Tylenchulus spp, and Scutellonema brachyurus. R. robustus, R. macrosoma and S. brachyurus are new records in Kenya. This checklist is a baseline for future records of new nematofauna identified and described in Kenya.

Keywords – COI, D2-D3, cryptic species, diversity, Kenya, plant-parasitic nematode, pineapple

Agriculture, a principal source of employment and a significant contributor to GDP, remains vital to the

Kenyan economy (World Bank, 2019). Nearly 75% of Kenyans live in rural areas and are actively engaged in the production, processing, and marketing of agricultural products and produce. With an increasing population growth in sub-Saharan Africa (SSA), which is expected to grow even more by 2050 (United

Plant-parasitic nematodes in Kenya David Kihoro Sirengo|2020

Nations, 2017), food security issues need to be addressed and warrants immense increase in crop production through the intensification of cropping systems. However, this will result in the aggravation of pest and disease pressure (Lopes-Caitar et al., 2019). The horticultural industry and cash crops occupy the most prominent position in terms of foreign exchange-earners and source of livelihoods to many Kenyans

(Ongeri, 2014). Over the past decade, Kenya’s horticultural industry has received a great deal of attention due to the rapid and sustained growth of its export to Europe. The sector produces fruits, flowers, vegetables, and herbs, which generates approximately $US 1 billion annually. Currently, the horticultural industry is ranked third after tourism and tea (World Bank, 2019). Pineapple, cut flowers, and tea sub-sectors are among the top export produce. However, these crops are exposed to abiotic and biotic constraints resulting in substantial yield losses. Intensive monoculture of these crops is becoming vulnerable to damage from biotic stressors including plant-parasitic nematodes (PPN), especially root-knot nematodes (RKN,

Meloidogyne sp.) (Jones et al., 2013). Plant-parasitic nematodes are soil-dwelling micro-organisms with over 4100 species described (Decraemer & Hunt, 2006). Being present in all agricultural land, PPN account for 10% yield losses globally (De Waele & Elsen, 2007), ranging from $US 80 billion (Abad et al., 2008) to

$US 157 billion annually (Nicol et al., 2011). Due to their nonspecific symptoms, horticultural damage caused by PPN remains unnoticed and therefore, underestimated (Lopes-Caitar et al., 2019). Research on diagnostic of PPN in SSA is gradually improving as expertise develops and reliable modern techniques becomes available (Coyne et al., 2018). Damage due to PPN, their behaviour, and management options associated with many crops, including pineapple, cut flowers and tea, have received little attention in Kenya and in other SSA countries.

Tea production in Kenya is undertaken by both large-scale and smallholder farmers. According to the tea directorate, there are over half a million registered smallholder tea farmers. Following its favorable climatic conditions and acid soils that foster healthy tea bushes, Kenya ranks first in the export of black tea to United

Kingdom, Egypt, Pakistan, Afghanistan, and the United Arab Emirates (Kibet et al., 2013). According to

Gnanapragasam and Mohotti (2018), several PPN have been recovered from the roots of tea and/or from the rhizosphere of tea plantation globally. However, only a handful have been found to cause economic damage. They include Hemicriconemoides kanayaensis, Meloidogyne sp., Pratylenchus sp., and

Radopholus similis. In Kenya, little research has been conducted concerning PPN of tea.

Plant-parasitic nematodes in Kenya David Kihoro Sirengo|2020

Following tea in export earning is the flower industry which contributes about US$ 1billion annually into the economy (Alessandro et al., 2015). Roses, orchids, carnations, hypericum, lilies, and gladioli are the main flowers grown in different parts of Kenya, including Kiambu where cut flowers produced are mainly for export. Pests and diseases are a considerable challenge in flower production (Rikken, 2011), including PPN

(Handayati & Sihombing, 2017). Globally, Meloidogyne sp. contributes significantly to crop damage and yield reduction (Koenning et al., 2004). In addition, foliar nematodes such as Ditylenchus sp., Aphelenchus sp., and Aphelenchoides sp. cause qualitative damage to cut flowers (Langat et al., 2008). Losses due to

PPN in cut flowers is estimated at 10-20% globally (Agudelo et al., 2006). Preliminary data indicates that

PPN are present in most commercial greenhouses for flower production in SSA (Coyne, unpublished observations). This underpins the need for conducting more studies with regards to PPN associated with flower industries, especially in SSA, given that there is no evidence of important PPN reported.

Pineapple is the second most important tropical fruit in the world after banana (Muimba-Kankolongo,

2018) and contains components such as malic acid and bromelin enzyme among others, as an alternative form of supplementary nutrition for improved health (Hossain, 2016). Pineapple contributes undoubtedly to the Kenyan economy and puts it on the globe among the top five exporters (Cheruiyot & Tarus, 2016). The crop is cultivated by large scale and smallholder farmers, of which 90% of the plantation is dominated by large scale producers such as Delmonte and Kakuzi limited companies (Hossain, 2016). However, a majority of smallholder farmers are turning into pineapple production for home consumption and commercial purposes (Koech et al., 2013). Due to the perennial nature and monocropping of pineapple in commercial production systems, there have been increased incidences of pests and diseases; among these PPN have proven to be problematic (Bartholomew et al., 2002). According to Guerout (1975), PPN account for up to

40% yield losses globally. Actual losses may even be higher considering that there is limited data available from many countries where nematological expertise is lacking. Sipes & Chinnasri (2018) reported over 100 species of nematodes associated to pineapples worldwide, yet the diversity and impact of PPNs in these crops remain poorly studied. Meloidogyne javanica, M. incognita, Pratylenchus brachyurus and Rotylenchus reniformis have been documented as economically damaging species (Sipes & Chinnasri, 2018). Several

PPN have been recorded from commercial pineapple production in Kenya, M. javanica and M. incognita

Plant-parasitic nematodes in Kenya David Kihoro Sirengo|2020 being the most important ones (Kiriga et al., 2018). Besides, unpublished record (Olajide et al., 2017) reported the presence of M. enterolobii in a commercial pineapple field.

Prompt and reliable diagnostic of nematodes are of paramount importance for the development of effective strategies for monitoring and managing potential targets. Traditionally, morphological features play a crucial role in helping to differentiate, identify, and classify nematodes (Troccoli et al., 2008). The use of non-overlapping morphological traits in classical provides immediate results and ease in quantitative evaluations (Carneiro et al., 2017). However, nematode identification using morphology needs trained and experienced personnel. For example, tropical Meloidogyne sp. identification using morphology is often problematic due to difficulty in distinguishing key diagnostic features; therefore identification is conducted at the genus level only or specimens are classified either as M. javanica or M. incognita (Coyne et al., 2018). It is also problematic given the phenotypic plasticity among populations and absence of clear taxonomic characteristics for cryptic species (Derycke et al., 2010a). On these grounds, molecular characterisation is a vital complement to morphological identification. Molecular barcoding is rapid, robust and can be used routinely.

In the light of these views, the aim of this work was to study morphological and molecular characterisation of plant-parasitic nematodes associated with pineapple, roses and tea in Kenya. To achieve this, the following objectives were defined:

a) To determine the distribution and density of plant-parasitic nematodes in pineapple, cut flowers

(roses) and tea growing areas in Kiambu county, Kenya.

b) To characterise Kenyan population of Helicotylenchus and Meloidogyne species associated with

pineapple.

c) To resolve taxonomical challenges, such as the cryptic species status of Scutellonema brachyurus.

(see addendum 1: Taxonomic challenge of cryptic species status of Scutellonema brachyurus

(Nematoda: ))

d) To provide an updated checklist of terrestrial nematodes in Kenya. (see addendum 2: An overview

of terrestrial nematodes in Kenya)

Plant-parasitic nematodes in Kenya David Kihoro Sirengo|2020

Materials and methods

SAMPLE COLLECTION AND NEMATODE EXTRACTION

Both smallholder and commercial farms were sampled from pineapple (60 soil and root samples), roses

(45 soil and root samples) and tea (9 soil samples) between December 2019 and January 2020 in Kiambu county, Kenya (Figure 3). Samples collected from each farm were geolocated using a Garmin GPS device.

Samples collection was organized in two phases: in the first phase, soil and root samples were collected randomly using a zigzag pattern, while in the second phase samples were further taken around the rhizosphere of individual plants. For roses, additional information on the application of nematicides (Velum

Prime) to the plants was recorded.

The samples were kept in plastic ziplock bags, labelled and put into cooler boxes for transit into the laboratory at the international centre for insect physiology and ecology (icipe) for processing. For each sample, a representative volume of 100 ml of soil and 5 g of roots were extracted using the modified

Baermann method (Coyne et al., 2018). Nematode suspensions were concentrated into 3 ml volume before dividing them into two equal portions that were subsequently transferred into a 1.5 ml Eppendorf tubes. One portion was fixed with DESS solution (DMSO-EDTA salt saturated solution) and the other half contained living nematodes. All samples were well labeled, wrapped with Parafilm®, and including the remaining soil samples for systematics (300 ml each) and ten samples from commercial pineapple fields, transported to

Nematology Research Unit at Ghent, Belgium. Each sample from commercial pineapple field (200 ml) was extracted using the modified Baermann tray method (Whitehead and Hemming, 1965) in Belgium.

Plant-parasitic nematodes in Kenya David Kihoro Sirengo|2020

Fig. 1. Above and below ground symptoms observed in pineapple from sampling field.

Fig. 2. Sampled locations in Kiambu county, Kenya.

Plant-parasitic nematodes in Kenya David Kihoro Sirengo|2020

DENSITY, PREVALENCE AND PROMINENCE OF PLANT-PARASITIC NEMATODES FROM

PINEAPPLE AND FLOWER (ROSES) SOIL SAMPLES

The nematode suspensions were counted using a 10x10 square counting dish under the stereomicroscope to determine the density (nematodes/100 ml of soil) and identify nematodes to genus level. Nematode density was determined by counting the two diagonals of the counting dish using a stereomicroscope and was presented based on mean density calculated as the number of individuals of nematode species in a sample divided by number of samples positive for that nematode species. Plant- parasitic nematode prevalence was given as the number of samples having a particular nematode species divided by the total samples examined multiplied by 100 (Boag, 1993). Prominence was determined as mean density multiplied by the square root of prevalence divided by 10.

MORPHOLOGICAL CHARACTERISATION

Morphological and morphometric analyses were conducted based on live and fixed nematodes. The PPN from each sample were hand-picked into a drop of clean water in an embryo dish. Samples in the embryo dish were killed and fixed by adding a few drops of 4% PFA (paraformaldehyde fixative), buffered in PBS

(phosphate buffer saline), microwaved at 700 W for 3 sec and left overnight at 4˚C. In addition, a suspension of Scutellonema from finger millet sample into embryo dish were killed and fixed by adding a few drops of

Trump’s fixative (2% paraformaldehyde, 2.5% glutaraldehyde) in 0.1M Sorenson buffer (Sodium phosphate buffer, pH 7.5). The embryo dish was heated in the microwave 700 W for 5 secs, left for an hour at room temperature and at 4˚C for 24 h allowing maximum penetration of the fixative. A few drops of De Grisse 2 solution (5% glycerine in ethanol) were added into the embryo dish five times ensuring a 1h 30 min interval followed by the addition of a few drops of De Grisse 3 solution (50% glycerine in ethanol) at the end of the day. Fixed samples were left in the incubator overnight. As per Seinhorst protocol (Seinhorst, 1959), nematodes were gradually transferred to anhydrous glycerin for permanent slides and mounted on glass slides for light microscopy study aided by an Olympus BX51 DIC microscope (Olympus Optical) equipped with an Olympus camera. Measurements were made based on light microscopy pictures using ImageJ

(Schneider et al., 2012).

Plant-parasitic nematodes in Kenya David Kihoro Sirengo|2020

MOLECULAR CHARACTERISATION

Live nematodes were each hand-picked into a drop of distilled water and mounted on temporary slides for the assessment of the key morphological features and for morphometrics by photographing individual specimens, as mentioned above. Following the protocol of Singh et al. (2018), each specimen processed for DNA extraction was cut and transferred into a PCR tube with 20 µl of worm lysis buffer (50 mM KCl, 10 mM Tris at pH 8.3, 2.5 mM MgCl2, 0.45% NP 40 (Tergitol Sigma), 0.45% Tween-20), and incubated at

−20°C for 10 min. A 1 μl proteinase K (1.2 mgml−1) was added to each PCR tube and incubated at 65°C (1 h) and 95°C (10 min), with a final centrifugation step of the mixture at 14000 g for 1 minute. The vouchered species were processed to sequence informative regions including the large subunit ribosomal RNA (28S), small subunit ribosomal RNA (18S), Cytochrome c oxidase I (COX1) and NADH dehydrogenase subunit 5

(NAD5), for RKN) were amplified and sequenced to determine species identity and their barcodes.

PCR amplification was executed using TopTaq polymerase (Qiagen, Germany) into a 23 µl PCR reaction mix with 2 µl of extracted genomic DNA. The primer sets 18A (5’- AAA GAT TAA GCC ATG CAT G -3’) and

26R (5’- CAT TCT TGG CAA ATG CTT TCG -3