ANTIFUNGAL ACTIVITY AND PHYTOCHEMICAL ANALYSIS OF VARIOUS MEDICINAL PLANTS AGAINST PATHOGENIC FUNGI OF TOMATO (Lycopersicon esculentum L.)
BY
SOBIA CHOHAN M.Sc. (Hons.) Agri. (Plant Pathology)
A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree
of
DOCTOR OF PHILOSOPHY
In Plant Pathology
DEPARTMENT OF PLANT PATHOLOGY
FACULTY OF AGRICULTURAL SCIENCES AND TECHNOLOGY
BAHAUDDIN ZAKARIYA UNIVERSITY, MULTAN
2015
1
In the name of Allah, Most gracious, most merciful.
Read! And your Lord is the Most Generous, Who has taught the writing by the pen, Has taught man that which he knew not. (Al-Quran) DECLARATION OF WORK
I hereby declare that contents of the thesis entitled “Antifungal activity and phytochemical analysis of various medicinal plants against pathogenic fungi of tomato (Lycopersicon esculentum L.)” are produce of my own research and no part has been previously submitted for any other degree. Furthermore, work reported in this manuscript does not carry any duplication of research accomplished by other researchers’ except where due reference or acknowledgement is made overtly in the text.
Sobia Chohan
2
The Controller of Examinations Bahauddin Zakariya University, Multan.
It is certify that the contents and form of the thesis submitted by Mrs. Sobia Chohan (2009-ag-191) have been found satisfactory and recommend that it may be processed for evaluation by the External Examiner (s) for the award of the degree.
SUPERVISOR DR. RASHIDA ATIQ Associate Professor (Department of Plant Pathology)
3
Dedicated To my
PARENTS, BROTHERS
And
HUSBAND
ACKNOWLEDGEMENTS
All the acclamations are for The Almighty Allah alone, the most merciful and the most compassionate, Who is the entire source of wisdom and all the knowledge endowed to mankind, Who enabled me to achieve this goal. I offer my heartfelt praise to Holy Prophet Muhammad (PBUH) the most perfect and dignified, glorious one among and of ever borne on the surface of the earth, who is forever model of guidance and knowledge for the humanity.
Ph.D is a sojourn wherein gratification with dissatisfaction, boom with gloom prevails. But, its essence lies what you accomplished in the end and is fuelled by interdependence rather than dependence. Though it is simple to name all the people who lent their support for my endeavor but it is awesome job to gratify them.
I am very appreciative and gratified to my Supervisor, Dr. Rashida Atiq,
Chairperson Department of Plant Pathology, Faculty of Agricultural Sciences &
Technology, BZU, Multan for her valuable suggestions, constructive criticism, guidance and encouragements throughout my work. While working under her, the scrupulous execution of her work has instilled in me a great sense of confidence to carry out my work objectively and successfully.
Undoubtedly, words can't suffice in expressing my sincerest gratitude to
Professor (Retd.) Dr. Sardar Muhammad Mughal - an eminent Plant Virologist/Plant
Pathologist. His cordial behavior, humble attitude, cooperativeness and nobility are 4 some of the exquisite traits which I would certainly like to cherish and immolate as the genuine taught of this charismatic teacher. I could not repay what I achieved today and what I learnt from him. I pay obeisance to this greatest steward of mine to whom I owe all my achievements. The scientific competence in him has rendered my scientific endeavor and zeal more glorious.
I am greatly indebted to Dr. Saeed Akhter, Chairman and Associate Professor of
Department of Food Sciences, FAS & T, BZU, Multan for permitting me to avail the facilities for my work and rendering every sort of encouragement during my tenure. I overwhelmingly acknowledge the services rendered by Tariq Ismail and Amir Ismail,
Lectures of Department of Food Sciences, for providing the spectral data for my compounds.
It gives me immense pleasure to express my deep sense of gratitude and affable thanks are also extended to all my Department colleagues for their constant encouragement and valuable support.
I would like to express my profound gratitude towards my friend Safina Naz,
Assistant Professor of Horticulture.
Working with coordination is more valuable than working in seclusion. I feel fortunate enough to have the company of my brilliant students Mirza Abid Mahmood
(Lecturer, Plant Pathology, MNSUA, Multan), Miss Naila Akram and Syed Atif Hasan for their persistent help and unconditional support. My wholehearted gratitude goes to my laboratory staff for their cooperation and sincere help during lengthy and hard period of my research work.
The author extends sincere thanks to Directorate of External Linkages, Bahauddin
Zakaryia University, Multan for financial supports.
5
The driving forces for accomplishment of my challenge are my beloved parents who extended every care, incessant prayers, moral support and affection not only in this period but during my entire span of life. I pay my sincere obeisance to my cherished parents without whom it was near impossible to fulfill my goal. I could not forget the loving memories of my mother whose hands always rose for my success and my beloved husband whose everlasting enthusiasm bore fruit in the form of this dissertation.
I am indebtly grateful to my brothers, sisters-in-law and my relatives who are the real guiding forces and moral boosters of my triumph. Last but not the least; I cannot overlook my little nephew Umair Bin Saqib for providing energy during my work.
Sobia Chohan
LIST OF CONTENT
6
Sr No . Description Page No.
Acknowledgments i
Contents iv
List of Tables vii
List of Figures ix List of Plates x Abbreviations xi
CHAPTER 1: INTRODUCTION 1
1. THE TOMATO PLANT 1 1.1. Production 1 1.2. Nutritional value 2 2. FUNGAL DISEASES OF TOMATO AND THEIR MANAGEMENT 3
2.1. Damping off and Seed rot 4 2.2. Foliar diseases 4 2.2.1. Early blight 4 2.2.2. Late Blight 6 2.2.3. Anthracnose 7 2.2.4. Wilt and root rot 9
3. IMPLICATIONS OF CHEMICAL CONTROL AND SCOPE OF 11 MEDICINAL PLANTS 4. AIM OF INVESTIGATIONS 13 Objectives 14
CHAPTER 2: REVIEW OF LITERATURE 15
1. INTRODUCTION 15
2. PROCESSING OF PLANTS AND ISOLATION OF ANTIMICROBIAL 15 AGENTS 2.1 Selection of tissue 16 2.2 Choice of Solvents 16 2.3 Choice of extraction procedure 18 3. ASSESSMENT OF ANTIMICROBIAL SUSCEPTIBILITY TEST (AST) 19
3.1 Diffusion test 19 7
3.2 Dilution method 20 4. MAJOR ANTIMICROBIAL PHYTOHEMICALS 21 4.1 Phenolics and Polyphenolics 21 4.1.1. Tannins 23 4.1.2. Flavonoids 24
4.1.3. Coumarins 24 4.1.4. Terpenoids 25 4.2 Alkaloids 25 4.3 Saponins/Steroids 25 5. SECONDARY METABOLITES 26 6. MODE OF ACTION OF ANTIMICROBIAL PHYTOCHEMICALS 27
7. SOME ANTIFUNGAL PROPERTIES OF TARGET BOTANICALS 28 7.1 Garlic (Allium sativum ) 29 7.2 Neem (Azadirachta indica) 30 7.3 Ginger (Zingiber officinale) 31 7.4 Mhendi, Henna (Lawsonia inermis) 31 7.5 Tulsi (Ocimum sanctum, O. basilicum) 32
7.6 Turmeric (Curcuma longa) 32 7.7 Marygold (Tagetes minuta, T. erecta and T. patula) 33 7.8 Eucalyptus (Eucalyptus globules) 33 7.9 Chinaberry (Melia azedarach) 34 7.10 Ashoka tree (Polyalthia longifolia var. Pendula 34 7.11 Periwinkle (Catharanthus roseus) 35
7.12 Mango (Mangifera indica) 35 7.13 Lemon (Citrus limon) 35 7.14 Jambolan, Jaman (Syzgium cumini) 36 7.15 Moringa oleifera 36 8. BENEFICIAL IMPACTS OF ANTIMICROBIAL AGENTS 37
CHAPTER 3: MATERIALS AND METHODS 41
3.1. General methods 41 8
3.2. Sample Collection 42 3.3. Isolation of fungi 42 3.3.1. Isolation from plant tissues 42 3.3.2. Isolation from seed 43 3.4. Identification, maintenance and multiplication of fungi 43
3.5. Pathogenicity tests of fungi 44 3.6. Assessment of antifungal properties of plant extracts 45 3.7. Processing of tissue and Extraction 47 3.8. Phytochemical testing of plant materials 49 3.9. In vitro evaluation of Antifungal activity of plant extracts 50 3.9.1. Poisoned Food Technique 51
3.9.2. Agar well diffusion method 51 3.10. Spore germination inhibition test 52 3.11. In vivo tests of plant extracts and Topsin-M against early blight in 53 greenhouse 3.12. Field evaluation of plant extracts and Topsin-M against early blight 54 3.13. Statistical Analysis 55
CHAPTER 4. RESULTS 56
CHAPTER 5. DISCUSSION 116
CHAPTER 6. SUMMERY 126
CHAPTER 7. REFERENCES 130
APPENDICES 179
LIST OF TABLES
9
Sr. No. Tables Page No.
3.1. Plants used as sources of extract 50 4.1. Frequency % of fungi isolated from tomato seeds using blotter and Agar 57 plate methods 4.2. *Mean Frequency % of fungi isolated from various parts of tomato plant 59 and level of significance
4.3. Qualitative analysis of medicinal plants 63 4.4. Total Phenol Contents in aqueous and methanol extracts of plants 66 4.5. Antifungal effect of aqueous fresh plant extracts at different 72 concentrations against important tomato fungi (Percent growth reduction) 4.6. Antifungal effect of aqueous dry plant extracts at different 76 concentrations against important tomato fungi (Percent growth reduction) 4.7. Antifungal effect of methanol fresh plant extracts at different 86 concentrations against important tomato fungi (Percent growth reduction) 4.8. Antifungal effect of methanol dry plant extracts at different 90 concentrations against important tomato fungi (Percent growth reduction) 4.9. Antifungal activity of aqueous (fresh) plant extracts at different 98 concentrations against selective tomato fungi using agar well method 4.10. Antifungal activity of aqueous (dry) plant extracts at different concentrations 99 against selective tomato fungi using agar well method 4.11. Antifungal activity of methanol (fresh) plant extracts at different 100 concentrations against selective tomato fungi using agar well method 4.12. Antifungal activity of methanol (dry) plant extracts at different 101 concentrations against selective tomato fungi using agar well method 4.13. Efficacy of plant extracts on early blight disease under Greenhouse 110 conditions (2012-13) 4.14. Efficacy of plant extracts on early blight disease under Greenhouse 111 conditions (2013-14) 4.15. Efficacy of plant extracts on early blight disease under Field conditions 113 (2012-13) 4.16. Efficacy of plant extracts on early blight disease under Field conditions 114 (2013-14)
10
LIST OF FIGURES
Figure No. Figures Page No. Figure 4. 1. Gallic Acid calibration curve 66 Figure 4.2. Inhibition percentage (means ± SD) of aqueous extract on spore 104 germination of different fungi
Figure 4.3. Inhibition percentage (means ± SD) of methanol extract on spore 105 germination of different fungi
11
LIST OF PLATES
Plate No. Plates Page No.
Plate 4.1 Colony growth on PDA medium of isolated fungi and their reproductive 60 structures
Plate 4.2 Bioefficacy of neem and garlic extract on mycelial growth of A. solani 80 Plate 4.3 94 Bioefficacy of methanol extracts of neem and garlic on mycelial growth of A. solani Plate 4.4 Effect of neem leaf extract on tomato plant in greenhouse 112
12
Plate 4.5 Effect of neem and garlic extract on tomato plants in field 115
ABBREVIATIONS
A Absorbance
α Alpha β Beta ANOVA Analysis of variance BZU Bahauddin Zakaryia University oC Degree centigrade cm Centimeter DAP Days after planting
DAT Days after transplanting 13 f. sp forma specialis
FAO Food and Agriculture Organization g Gravity, gram GAE Gallic acid equivalent h Hour ha Hectare
I.U International unit kg Kilogram
L liter
L.S.D Least significant difference M, m Meter mg Milligram ml Milliliter min Minute mm millimeter nm Nanometer (1 x 109 meter)
NZ No zone
NUV Near ultra violet
P Probability
PARC Pakistan Agricultural Research Council PDA Potato dextrose agar
PDI Percent disease index
Percent Percentage, % ppm Parts per million Pv Pathovar ® Registered rpm Revolutions per minute
TPC Total phenol contents t/ha Tons per hectare µm Micro meter (1 x 10-6 meter)
UV Ultra violet v/w Volume per weight
14
1. INTRODUCTION
1. THE TOMATO PLANT
Tomato, also called as “golden apple” or “love apple” belongs to nightshade family
‘Solanaceae’ which comprises of over 1000 species of flowering plants including economically important crops such as tomato, potato, eggplant, chili and tobacco. Tomato was initially placed in the largest and diverse genus Solanum by Linnaeus in 1753 and the species was designated as Solanum lycopersicum L. Later on in 1768, it was moved by
Phillip Miller to the genus Lycopersicon and named as Lycopersicon esculentum Mill. on the basis of morphological features of pollens of tomato and other members (Taylor,
1986). Recently on the basis of genetic identity, tomato is taxonomically called as
Lycopersicon lycopersicum (L.) Karst. with a synonym as Lycopersicon esculentum
(Peralta and Spooner, 2001).
The genus Lycopersicon of the Solanaceae family includes both cultivated tomatoes and their wild relatives (Peralta et al., 2005) that are native to Peru and Galapagos
Islands (Smith, 1994). So far approximately 7500 tomato cultivars have been identified producing tomatoes of various colors, shapes and sizes (Sims, 1980). Tomatoes were initially cultivated as ornamentals and thought to be poisonous (Gentilcore, 2010).“Cherry tomato” found outside the South America is perhaps the ancestor of modern cultivars
(Rick, 1979). Tomato is supposed to have early domestication in Mexico from where it was introduced into Europe during 16th century, to Asia in 18th century and in Indo-Pak subcontinent about 200 years ago (McCue, 1952).
1.1. Production
15
Tomato (Lycopersicon lycopersicum (L.) Karst./(Lycopersicon esculentum Mill.) is most extensively grown and widely consumed vegetable crop after potato in the world.
Tomato cultivars are divided as determinate and indeterminate types depending on their growth habits; the former type is suitable for production of uniform ripened fruits whereas the indeterminate cultivars are appropriate for continuous production of fruit throughout the year (Jones, 2008). Low cost, ease of tomato production, short-duration and high economic returns has attracted the growers to produce the crop throughout the year even in places with warmer climates (Naika et al., 2005). Tomato production and consumption since 1993 have almost doubled in the world (Van de vooren et al., 1986). Due to its assorted fruits, it is considered as an important cash and industrial crop in many countries including United States, China, Turkey, India and Egypt. According to world’s statistics,
China is the leading tomato producing country in the world with annual production of about 34 million tones. World production of tomato is estimated at 145 million tones annually over an area of 4.36 million ha, yielding on average basis up to 336 thousand kg/ha. Diversified ecological and climatic conditions and improved agro techniques in
Pakistan favor the production of quality tomatoes throughout the year, presently covering an area of 58.1 thousand hectares with an annual production of about 574 thousand tones.
Punjab alone shares or contributes 86.3 thousand tones from an area of 6.5 thousand ha giving an average yield of 13.1 thousand tones/ha (GOP, 2013). Traditionally, tomatoes are grown under open field conditions, but as a result of increasing demands and consumption the crop is also raised in tunnels, greenhouses, kitchen gardens and some hydroponics where facilities exists.
1.2. Nutritional value
Tomato is widely consumed vegetable crop due to its savory fruit, flavor and nutritive values in a variety of forms such as salads, soups, ketchup, puree and in delicious 16 cuisines. Among the 29 major fruits and vegetables, tomatoes rank first in the relative contribution to human nutrition (Saltueit, 2003). Tomato fruit is rich in minerals, antioxidants and vitamins (A and C), beta-carotene and potassium (Hobson and Davies,
1971). Presence of a natural antioxidant-lycopene and low calorie food make it highly suitable and beneficial for human consumption. A fresh ripe tomato contains; 900 (I.U) of vitamin A; 233mg vitamin C; 0.6mg thiamine; 0.4mg Riboflavin; 0.7mg Niacin; 13mg Ca;
27mg P; 0.5mg Fe; 3mg Na and 244mg K (Nonnecke, 1989). Red tomatoes contain high levels of carotenoids and lycopene which act as powerful antioxidant known to reduce the risks of cancers and cardiovascular diseases associated with type 2 diabetes (Dorgan et al.,
1998; Shidfar et al., 2011). According to Fernandez-Ruiz et al; (2002), lycopene contents can be increased by about five times in cultivated tomato through breeding with their wild relatives. Fresh ripened fruit are good appetizers, help in wound healing because of antibiotic properties (Conn and Stumpy, 1970), and the extract have more profound effects on urinary acidity than of orange juice (Saywell and Lane, 1933).
2. FUNGAL DISEASES OF TOMATO AND THEIR MANAGEMENT
Tomato is vulnerable to the attack of different pathogens at all stages of its growth and development such as fungi, bacteria, nematodes, viruses and viroids which spread through seed, soil, water, air and vector. Jones et al; (1991) have listed 24 fungal, 7 bacterial, 7 nematodes, 10 viral and 3 viroid diseases on tomato, but the diseases incited by the fungi are predominantly occurring (Persley, 1993), and cause serious diseases, reduce yield by 90-100% and deteriorate nutritional value and quality of fruits (Dellavalle et al., 2011). Apart from post-harvest decays, main fungal diseases of tomato encountered in Pakistan are: damping off, seed rot, early and late blights, leaf spots, anthracnose, wilts and root rot. These diseases are briefly reviewed and discussed with respect to biology, 17 morphology and taxonomy of the causal pathogens on one hand, and the symtomatology, epidemiology and management on the other.
2.1. Damping off and Seed rot
These diseases are generally caused by a complex of seed and soil borne fungi as species of Pythium, Rhizoctonia, Macrophomina, Fusarium, and Sclerotinia as well as air borne Alternaria, Colletotrichum, Cladosporium, Rhizopus, Curvularia, and Aspergillus.
These fungi are quite active under wet conditions and cause rotting and damping off in seedlings and retard germination process, affect establishment and stand in the field. The surviving seedlings develop necrosis, softening and stem flaccidity and finally wilting of plants. Infected tomato seeds do not develop any typical or conspicuous symptoms on seed surface. However, seed-borne can easily spread from one place to another and serve as an initial source of inoculum potential (Nishikawa et al., 2006). Seed treatment with Captan or other seed dressing chemicals is a conventional practice to eliminate and exclude seedborne pathogens (Dwivedi and Pathak 1981; Messiaen 1992). Dipping of tomato seed and seedlings in the extracts of some medicinal plants is reported to be highly effective.
2.2. Foliar diseases
Two important foliar diseases which are historically important and pose threat to tomato production are: early blight and late blight (Fry et al., 1993; Fakir 2001; Ketelaar and Kumar 2002; Reni and Roeland 2006; Than et al., 2008). The incidence of early blight in Pakistan range between 49-91% and under severe epiphytotic conditions causes colossal losses in production (Azam and Shah, 2003).
2.2.1. Early blight
Early blight incited by Alternaria solani (Ell. and Mart.) Jones & Grout is a common disease of tomato and potato. It is placed in Phylum Ascomycota, class Hyphomycetes, order Pleosporales and family Pleosporacae. On the basis of its phylogenetic analysis,
18 cosmopolitan nature and morphological characters, the pathogen belongs to a large genus
Alternaria which comprises of non catenated spores with long beak (Varma et al., 2006;
Simmons 2007). The mycelium of A. solani is light brown, septate with branched hyphae and bear short conidiophores measuring 50-90µm. Pale golden or olivaceous brown conidia are melanized cells, muriform, formed either singly or in chains with 9-11 transverse and 1-5 longitudinal septa and measure 150-300 x 15-19µm in size ((Neergaard
1945; Ellis 1971; Rotem 1998). The isolates of Alternaria consist of various races which can be differentiated on the basis of color, growth pattern, sporulation and size of conidia and number of septa, but sporulation is greatly influenced by temperature, light and nutrition. Different culture media and plant extracts have been tested which favored best sporulation, storage, viability, and sub culturing (Dhingra and Sinclair 1985; De Avila et al., 2000; Rodrigues et al., 2010). Color variation or colony pigmentation of A. solani depends upon media and range from initially white, yellow, but later turning brown, black or brownish to greenish black.
The disease is characterized by the appearance of small brown lesions or spots on lower or older leaves which enlarge and develop concentric rings in a bull’s eye pattern in the center of the diseased area surrounded by yellow hallo. Lesions also develop on stems and fruits (Agrios, 2005). Due to necrotrophic nature of the pathogen, entire crop is blighted under favorable weather conditions, mainly the high temperature and humidity, resulting in complete defoliation (Datar and Mayee 1981; Peralta et al., 2005). A. solani is a soil and air-borne pathogen and overwinters in crop residues or wild members of the
Solanaceae family. The fungus reproduces asexually by means of multicellular conidia in spring season and spreads by rain splashes or wind. Conidia directly penetrate the plant tissues or enter through wounds usually present on older leaves close to the soil.
19
Early blight is generally controlled with 3-4 fungicidal applications made at 15-day intervals during the growth and development of crop. Initially, inorganic copper fungicides such as Bordeaux mixture, Cupravit, Cobox and Copper oxychloride were used (Datar and
Mayee 1981; Sinha and Prasad 1991; Singh et al., 2001), and then organic or synthetic chemicals as Mancozeb, Dithane M-45, Captan and Polyram were introduced which gave satisfactory results (Devanathan and Ramanujam, 1995). Most of the fungicidal formulations were effective, protectant but hardly eradicant or curative, and restricted the development of early blight by 40-61% and increased the yield accordingly. Leaf extracts of neem, tulsi and garlic etc were shown to possess antifungal activity against the pathogen. Other methods of disease control as cultural, biological and varietal resistance have little value.
2.2.2. Late Blight
It is a very serious and destructive disease of potato and tomato and is caused by the fungus Phytophthora infestans (Mont.) de Bary. It has been extensively studied by the scientists in the world for about 150 years and thus associated with the historical development of plant pathology (Robin and Choen, 2004). The disease was responsible for Irish potato famine in 1840’s which culminated into death of millions of people and the survivals were forced to migrate to America and other countries (Govers and
Latijnhouwers, 2004).
P. infestans is a hemibiotrophic oomycete pathogen and belongs to a diverse group of eukaryotic microorganisms (Margulis and Schwartz, 1988), placed in Peronosporales order, family Pythiaceae and genus Phytophthora. According to Baldauf et al; (2000), its molecular characteristics and biochemical analysis indicate that oomycetes are more closely related to heterokonts (stramenopiles) and diatoms (brown algae) rather than to true fungi.
20
Microscopic sporangia produced by the fungus are asexual and sexual, hyaline, pear-shaped measuring 20-40µm in size and formed on coenocytic branches called sporangiophores. The swelling at the point of attachment of sporangia to sporangiophores constitutes a distinct feature of P. infestans that helps in its identification (Alexopoulos et al., 1996). Bouwmeester et al; (2009) stated that P. infestans has both biotrophic and necrotrophic phases in its life cycle. Vega-Sánchez et al; (2000) and Smart et al; (2003) observed extended period of biotrophy in tomato leaves, might be due to compatible interaction with tomato-specialized isolates. The disease is characterized by the development of pale green water soaked lesions on leaves which later turn brown and necrotic. Under moist and humid weather, whitish mycelial growth comprising of sporangiophores and sporangia appear on the underside of the leaves. Oily brown lesions may develop on petioles and stem which later turn black causing the entire plant to die.
Olive colored shiny lesions are also produced on infected fruits followed by complete defoliation of unprotected crop within 10-14 days, resulting into serious yield losses
(Govers, 2005). The fungus survives as mycelium in plant debris during asexual cycle but oospores (A1 and A2 mating types) in the sexual cycle can live in the absence of host
(Ristaino, 2002). Due to short life cycle of disease, fungus spreads in cool nights and warm days with heavy dew. Foggy and rainy weather favor the infection, disease development and production of sporangia (Majid et al., 1992; Stevenson 1997).
Few inorganic and systemic fungicides as Ridomil and thiophenate methyl may reduce late blight by 77% if applied at proper time. Disease forecast models are available indicating start of spraying when relative humidity is 90% and environmental temperature is 18oC. Disease resistance has been identified in Solanum andegenum but not in tomato.
Neem formulations were found to be antifungal against the pathogen (Rashid et al., 2004;
Naz et al., 2006; Yeni et al., 2010).
21
2.2.3. Anthracnose
The disease is very common, widespread and occasionally destructive and is caused by several species of Colletotrichum, mainly Colletotrichum acutatum (Živković et al., 2010), C. coccodes (Wallr.) Hughes (Yonghao, 2013), C. dematium (Pers. ex Fr.)
Grove (Dillard 1989; LeBoeuf et al., 2008) and C. gloeosporioides (Penz) Penz.& Sacc.
Taxonomically, the species are positioned in phylum Ascomycota, class Sordariomycetes, order Glomerellales, family Glomerellaceae and Genus Colletotrichum. Morphologically,
Colletotrichum species are identified on the basis of vegetative, reproductive characters and colony color. The morphology varies considerably between species because different isolates have overlapping ranges of conidial and colony characteristics (Sutton 1980, 1992;
Than et al., 2008). Živković et al; (2010) described morphology of C. acutatum which comprises of branched, hyaline and septate mycelium. Conidia are 1-2 celled, aseptate, hyaline fusiform and cylindrical with obtuse apex and tapering at bases, measuring 12.3-
14.7 × 4.6-5.3µm (Smith and Black, 1990), and 12.5-20 × 3-5μm (Gunnell and Gubler,
1992). Initially its colonies are white or creamy and then become grey or dark grey on
PDA. Black acervuli can be seen around the culture. Appressoria when formed are simple, clavate to ovate, smooth and light to dark colored. The anthracnose is a serious disease of ripe and unripe fruits of tomato (Yonghao, 2013). The causal fungus sporulates and survives on infected fruit in the form of sclerotia which developed on sunken, water soaked lesions that later become black in color. These sclerotia can survive in soil for up to three years.
Fungicide sensitivity among six isolates of C. gloeosporioides (Cg1-Cg7) were tested by Kumar et al; (2007) using Propiconazole hexaconazole, Thiophenate-methyl,
Carbendazim, Mancozeb and Copper oxychloride by the poisoned food technique. Isolates were highly sensitive to mancozeb and copper oxychloride except Cg3 which was 22 reasonably resistant to thiophenate-methyl. Carbendazim was the most effective against all isolates. Intana et al; (2007) reported that Benomyl was most useful in controlling chili anthracnose. Duby et al; (2000), Sangoyomi et al; (2009) and Amadi et al; (2010) reported that aqueous extracts of Ocimum contained biologically active antifungal compounds against Colletotrichum species.
2.2.4. Wilt and root rot
Tomato wilt caused by Fusarium species is a serious problems limiting tomato production in the world and inflicting heavy economic losses (Booth 1971; Hafiz 1986;
Baysal et al., 2009). Two discrete forms of the pathogen have been recognized; Fusarium oxysporum f. sp. lycopersici (FOL) (W. C. Snyder & H. N. Hans) causing vascular wilt, and F. oxysporum f. sp. radicis-lycopersici (FORL) (W. R. Jarvis & Shoemaker) causing crown and root rot. However, morphological characteristics of all FOL and FORL isolates are similar to that of F. oxysporum (Summerell et al., 2003). Fusarium species constitute a most diversified and complex genus which belongs to phylum Ascomycota, class
Sordariomycetes, order Hypocreales, family Nectriaceae. According to Leisle et al;
(2001), morphological characters are the most important criteria to identify Fusarium species. Three types of spores (microconidia, macroconidia and chlamydospores) are produced in Fusarium species (Nelson et al., 1983). Abundant microconidia are hyaline, aseptate, ellipsoidal to straight, 1-2 celled measuring 5-12 x 2.3-3.5µm whereas macroconidia are fusiform, sickle-shaped, slightly curved, mostly with pedicillate basal cell, hyaline, having 3-5 septation and two to many celled measuring 23-54 x 3-4.5µm.
Chlaymydospores, if present, are hyaline, smooth or rough walled, intercalary or terminally attached to conidiophores. The fungal colonies are fast growing with initially white aerial mycelium and then turn reddish or purple color.
23
Both forms of Fusarium causing tomato wilt are soil-borne (Nash and Synder 1962;
Sangalang et al., 1995). The pathogen after coming in contact with tomato roots moves through water conducting tissues thus blocks the vascular vessels, which is the main reason for wilting. The infected plants show drooping and yellowing of lower leaves followed by wilting, necrosis and death of plants under warm weather (Booth, 1971). Upon longitudinal sectioning of the wilted stem, dark brown discolorations of vascular vessels are observed. The fungus survives in soil as chlamydospores (Synder and Hansen, 1940), although it is present as mycelium in soil debris. High temperature and warm moist soils favor disease development (Goss, 1936), soil temperature above 30oC stimulates root infection but seed infection is favored by low temperature up to 14oC. The soils of Pakistan are generally rich in Fusarium species which have a wide host range. Because of lack of fungistatis in the soil, wilt diseases are of common occurrence with varying degrees of severity (Hafiz, 1986).
Verticillium wilt caused by Verticillium albo-atrum (Reinke & Berthold.) has emerged as an important disease in tomato. The disease can be easily identified by the symptoms, plant wilts on one side but other side looks healthy. Initially, marginal and interveinal chlorosis with characteristic V-shaped lesions developed on lower leaflets. The pathogen is soil borne and attacks wide range of host plants (Pegg and Brady, 2002). V. albo-atrum typically has large conidiophores with one septate, single, larger conidia mainly 3.5-8 x 2-3µm and survive as dark thickened resting mycelium and hyphal knots
(Smith, 1965). Carbendazim, Curzate M-8, Ridomil-gold and Metyram are reported to be the best chemical for inhibiting radial growth of F. oxysporum and P.infestans and increasing the yield by 24% (Song et al., 2004; Anju et al., 2007). The synergistic effect of various fungicides has been reported when Carbendazim was mixed with Mancozeb,
Zinab and Carbofuran (Latha et al., 2000; Gangawane and Kamble, 2001). Soil-borne
24 pathogens are difficult to control by the chemical treatments which are generally expensive. There are variable and conflicting reports on the efficacy of aqueous and methanol extracts of medicinal plants except neem, melia, garlic and eucalyptus which were partially effective against Fusarium species.
3. IMPLICATIONS OF CONTROL MEASURES AND SCOPE OF MEDICINAL
PLANTS AGAINST TOMATO DISEASES
As far cultural methods are concerned, Han et al; (2000) summarized that approaches such as selection of tomato variety, planting site, timely irrigation, weeding, crop rotation and adequate fertilization are beneficial to maintain health and vigor of plant but are time consuming and non specific. In the green houses with continuous cropping pattern, Hanafi (2003) recommended to plant more than one tomato cultivar to escape attack of early blight disease. Jones and Woltz (1981) achieved effective control of vascular wilt of tomato (F. oxysporum f. sp. lycopersici) by changing soil pH up to 6.5-7 and fumigation of infected soil. Macro conidia of Fusarium wilt were destroyed with additional fumigation of glass house (Weststeijn, 1973). Tomato cultivars possessing good and substantial disease resistance are required but these are not easily available in every case. Shtienberg et al; (1995) observed that resistant cultivars strengthened the time period of fungicidal sprays. Certain biological methods as use of beneficial microorganisms
(Fravel et al., 1999) and microbial products are considered as alternative but nonconventional approaches (Elad and Zimand 1993; Fravel et al., 2005; Segarra et al.,
2009). Biocontrol organisms are reported to reduce the damage caused by fungi in fruits and vegetables (Elliott et al., 2009), but only limited work is available in case of tomato diseases. Biocontrol agents include Trichoderma harzianum, Pseudomonas chlororaphis and Gliocladium virens (Cuevas et al., 2005; Abdullah et al., 2008; Srinivasan et al., 2009).
However, mechanisms involved in biological control including competition for nutrients,
25 induced resistance, mycoparasitism and secretion of bioactive compounds still need to be investigated for specific tomato diseases (De Curtis et al., 2010).
Chemical control is very common, traditional and effective approach to protect the crops from the ravages of plant diseases. The worldwide use of agrochemicals at present has exceeded about 2.5 million tones per annum. At least 150 different fungicides of different groups have been formulated, introduced and used in agriculture (Brent and
Hollomon, 2007). However, it has been realized that use of chemicals is unsafe and unfriendly. Continuous, indiscriminate and haphazard use of pesticides has created many problems (Malkhan et al., 2012). One of the main implications is that the pesticides deteriorate the environment, disturb natural process and ecosystem, contaminate food and feed and affect human and animal health (Eilenberg et al., 2001; Mancini et al., 2008).
Indiscriminate use of chemicals enhances development of resistance in the pathogens and production of highly virulent strains (Osman and Al-Rehiayani 2003; Pasche et al., 2004).
According to Reddy (1986), approximately 72 fungal species have already developed resistance to 62 synthetic chemicals world over. The synthetic chemicals are also source of carcinogenicity, hormonal imbalance and high levels of residues (Dubey et al., 2007;
Kumar et al., 2007). Therefore, it is extremely necessary to develop alternatives for disease management and to reduce dependence on synthetic chemicals (Eilenberg et al., 2001;
Cuthbertson and Murchie 2005; Riaz et al., 2010). There is also increasing public concern over the environmental pollution and level of pesticide residues on vegetables including tomato. The scientists have identified several medicinal plants and their extracts as alternatives which are inexpensive, non-toxic, biodegradable, ecofriendly and fit very well in the integrated disease management (Hashim and Devi 2003; Okigbo and Nmeka 2005;
Okigbo and Omodemiro 2006).
26
Historically, several plants or their parts have been used for the treatments of diseases in man, animals and plants, in the form of extracts, powder, decoction and infusions (Nostro et al., 2000). Recently, several antibacterial or antimicrobial proteins or peptides with low mammalian toxicity have been identified from the green plants
(Subramanyam and Roesli, 2000; Huynh et al., 2001; Jamil et al., 2007). Antifungal properties of extract from several plants have been reported against Alternaria and
Fusarium species (Khalil, 2001; Anand and Bhaskaran 2009; Jabeen and Javaid 2010;
Kanwal et al., 2010; Wang et al., 2004; Khair and Wafaa 2007). It is now well established that higher plants are reservoir of natural drugs, compounds, metabolites and oils which posses antimicrobial properties (Banso and Olutimayin, 2001; Siva et al., 2008; Amadi et al., 2010). A meager work is available on the identification and isolation of such compounds from the medicinal plants against fungal diseases of tomato in Pakistan which now constitute the important subject.
4. AIM OF INVESTIGATIONS
Tomato is an important vegetable crop and constitute essential component of our daily diet throughout the year (Raja and Khokhar, 1993). In spite of favorable environmental and ecological conditions for tomato production in Pakistan, we face certain challenges and crises such as low and unstable production, irregular supplies, post harvest losses, increased consumption and high pricing sometimes out of reach of the common man. Heavy losses also occur in the field due to many diseases particularly caused by the fungal pathogens. Control of fungal diseases through chemicals is problematic and creates numerous difficulties relating to environmental pollution, food contamination and hazards to human health. This trend is now changing through the use of botanicals which have better advantages over chemical control (Jabeen and Javaid 2010; Bajwa and Ahmad
27
2012). It was, therefore, desirable to monitor fungal diseases of tomato in a production area, identify fungal pathogens and characterize bioactive and antifungal compounds as alternatives of agrochemicals. The objectives of present investigations were as follows:
Objectives
Qualitative and quantitative monitoring of different fungal diseases of tomato in a
production area.
Partial characterization of fungal pathogens isolated from different parts of tomato
plant (seeds, leaves, fruits and roots) and establishment of their pathogenicity.
Selection of botanical plants, their phytochemical analysis and biological and
antifungal interaction with the target pathogens.
In vitro evaluation of plant extracts or different formulations (aqueous, dry powder,
methanol extracts) on mycelial growth, spore germination or sporulation.
In vivo efficacy of promising antifungal plant extracts against early blight of
tomato under greenhouse and field conditions.
28
2. REVIEW OF LITERATURE
1. INTRODUCTION
Fungal plant diseases are traditionally controlled by cultural, genetically and chemical means. The chemical methods involve the application of organic, inorganic and synthetic fungicides which are cost effective and their continuous, haphazard and indiscriminate use create many environmental and toxicological problems (Bianchi et al.,
1997; Malkhan et al., 2012; Bhagwat and Datar 2013). Therefore, present approaches by the plant scientists aim at exploring potentials of extracts and compounds from higher plants as alternatives of chemicals, being cheaper, safe, nontoxic, quickly degradable and environment friendly. From the earliest known civilization plants have served mankind as sources of food, feed, fiber, drugs, additives, binders and antimicrobial agents against human infections (Siddiqui et al., 2004; Falodun et al., 2006; Gurjar et al., 2012). Muto et al; (2005), Jabeen and Javaid (2010), Kanwal et al; (2010) and Riaz et al; (2010) reported that the crude extracts and compounds isolated or purified from medicinal plants are highly and economically effective in the management of number of plant diseases.
2. PROCESSING OF PLANTS AND ISOLATION OF ANTIMICROBIAL
AGENTS
Many scientific steps and series of operations are involved in the processing of plants for the isolation of antifungal substances, their identification, assessment and evaluation. The
29 determination of biologically active compounds from medicinal plants depends on many factors as age of tissue, identification of plant part to be used, cleaning and storage of material, type of solvent, extraction procedures and antimicrobial assays (Rizvi et al.,
1999; Singh et al., 2001). Extraction medium as water, buffers, antioxidants, organic solvents and homogenizing tools should be clean (Rice, 1995).
2.1. Selection of tissue
An excellent source of antimicrobial agents is natural products obtained from the
vascular plants and microorganisms. As far as selection of tissue is concerned, source
plant should be biologically actively growing, easily manageable and available in
vicinity, free from decays or infections, and could be easily stored under suitable
condition.
2.2. Choice of Solvents
The extraction of naturally active compounds depends on the selection of proper
solvent. The ideal ratio of solvent-to-sample is 10:1 (v/w) solvent to dry weight (Green,
2004). Water is a general solvent and used to extract plant products with antimicrobial
action, but water soluble phenolics or other compounds act only as antioxidants and water
soluble flavonoids have no antimicrobial importance, although conventional healers use
mostly water (Parekh et al., 2005). Plant extracts with organic solvents, however, provide
far more reliable antimicrobial activity than water extracts. Ncube et al; (2008) and Gurjar
et al; (2012) recommended that a good solvent should be nontoxic, evaporates quickly at
low heat, aids in rapid absorption of extracts, maintains preservative integrity and has
incapability to cause dissociation or precipitation of extract.
Initial test of plants for antimicrobial activities normally begins by using the
rudimentary extractions followed by various organic solvent extraction methods. Almost
all the known antimicrobial compounds from plants are saturated or aromatic organic
30 compounds, and mostly obtained through initial extraction. Methanol, ethanol and water are the most regularly used solvents to prelude investigations of antimicrobial activity in plants. Chloroform, dicholro-methane, hexane and acetone are other solvents. Some workers use a mixture of solvents which offers paramount solvent systems for extraction
(Erdemgil et al., 2004; Gurjar et al., 2012). Cowan (1999) examined a variety of organic solvents for extractants for their capability and efficiency to solubilize antibacterials from plants and ranked them as methylene dichloride > methanol > ethanol > water (Erdemgil et al., 2004; Gurjar et al., 2012). Amadioha (2000) reported that the mycelial growth of
Pyricularia oryzae was drastically reduced by the use of water and ethanol extracts of leaves than oil extracts from A. indica seeds. The disc diffusion assay revealed that ethanolic extract of C. zeylanicum was effective against Penicillium sp. and chloroform fraction was partially useful against Phytophthora infestans. In general, methanol is reported to be effective than ethanol, chloroform, benzene and petroleum against most of the fungi and plant tissues. lvanovska et al; (1996), Tura and Robards (2002) and Alkhail
(2005) preferred ethanol extraction for phytochemicals and other compounds as tannins, polyphenols, polyacetylenes, flavonol, sterols and alkaloids which are better soluble in ethanol. Zafar et al; (2002) recommended chloroform for better extraction. Different host plants and large number of fungal species were tested by the above workers and antifungal activities were equally reported. Hassanein et al; (2008) observed that growth of F. oxysporum at 20% concentration with ethyl acetate and ethanol extracts of neem leaves was completely inhibited than A. solani. The leaf extracts prepared in ethyl acetate compared with ethanol showed that the inhibition increased steadily with the extract concentration, both absolutely reduced the radial growth of F. oxysporum. Solvent extraction with petroleum ether, benzene, chloroform, methanol and ethanol of twelve plants indicated their antifungal activity against the fungus. Methanol extract of the
31
Curcuma rhizome exhibited good fungicidal activity. Govindachar et al; (1999) found that hexane extract of Azadirachta indica leaves and its chromatographic fractions had antifungal action against the deuteromyceteous fungi, i.e. Colletotrichum lindemuthianum and F. oxysporum. Siva et al; (2008) obtained 98% inhibition with acetone extract of A. indica but less in other cases. Chloroform fraction was partially useful against P. infestans. Methanol extract of the Curcuma rhizome exhibited good fungicidal activity.
2.3. Choice of extraction procedure
Extraction methods frequently rely on solvent used, length of extraction period, temperature, pH of the solvent, solvent-to-sample ratio and particle size of the plant tissues. The fundamental standard is to crush the plant material (dry or wet) in a fine shape which increases the surface area. Eloff (1998) recorded that five minute extractions of extremely fine particles of 10µm diameter gave higher quantities of compounds than values obtained with less finely ground material after 24 hours in a shaking machine. The extraction method that has been generally used by researchers is homogenization of healthy plant tissues in solvent. Fresh and healthy plant parts (wet or dried) are crushed in a blender to fine particles, dissolved in a certain quantity of solvent and stirred dynamically for 5 - 10 minutes or left for 24 hours after which the extract is filtered. The filtrate is then dried under reduced pressure and to determine the concentration, redissolved in the solvent. Some researchers use centrifuge (20,000 g for 30 min approximately). Many workers prefer use and application of crude extracts for the control of diseases, but others recommend pure forms of allelochemicals which facilitate scientist for analysis, variable or comparative activity and quantitative results (Singh et al., 2001;
Tura and Robards 2002; Shafique et al., 2011). Darout et al; (2000) recommend homogenization of plant tissue in the presence of extraction medium in warring and
32
electric blenders instead of crushing in a pestle and mortar. Under these conditions,
properties of ethanol and chloroform extracts of 10 plants were evaluated against five
pathogenic fungi of anthracnose disease (Mogle, 2011).
3. ASSESSMENT OF ANTIMICROBIAL ACTIVITY
Different methods are used for the evaluation of antimicrobial activity. Antimicrobial
Susceptibility Test (AST) is standard tests and classified into diffusion and dilution
methods. Diffusion tests include poison food technique, agar well method, disk diffusion
and bioautography (Pelttari et al., 2002; Khan et al., 2009), while dilution method
comprises of agar dilution and broth dilution assay (Okusa et al., 2007).
3.1. Diffusion test
Screening of extracts from different botanicals against the test pathogens is usually done by the poisoned food technique (Nene and Thapliyal 2000; Bhagwat and Datar 2013).
Plant extracts at different concentrations are incorporated in the medium plates followed by inoculation with the test pathogens and recoding their growth and development. Sasode et al; (2012) used this and evaluated in vitro effectiveness of neem, eucalyptus, datura, mint, tulsi and lantana against Alternaria brassicae, and fungitoxicity was assessed at different concentrations from 10 to 100%; completely suppression of the mycelial growth was found at 40% strength. Aqueous and organic solvents extracts of neem and some other hosts were highly effective against Aspergillus niger, Fusarium oxysporum, Rhizopus stolonifer and Geotrichum candidium from tomato (Yeni et al., 2010). Antifungal activity of flavonoids from mango leaf extracts against five fungal species (Penicillium citrii,
Aspergillus fumigates, A.niger, Macrophomina phaseolina and Alternaria alternata) revealed best effectiveness at 1000 ppm (Kanwal et al., 2010).
33
Antifungal potential of aqueous and ethanolic extracts of garlic, ginger, chili and
Cinnamom was established against 276 fungal isolates associated with post-harvest storage rots of fruits (Birhanu et al., 2014). Similarly, Sahejpal et al; (2009) through disc diffusion method found antifungal effect of forty four plant extracts and eight plant oils against Rhizoctonia solani. Dawar et al; (2007) obtained best results by paper disc and well diffusion methods against Fusarium spp. and M. phaseolina. On the other, Shrestha and Tiwari (2009) and Uzama et al; (2012) assessed in vitro activity of crude extracts of
7 plants against potato dry rot through poisoned food technique and diffusion method and reported similar results. The oil of Syzygium aromaticum showed strong inhibition in well diffusion method (Uzama et al., 2012). At 25mg/ml the extract of the leaves of
Eucalyptus saligna was very active against Candida albicans. Aslam et al; (2010) reported that the growth of R. solani (damping-off) was suppressed by 44.73% with neem when supplemented in potato dextrose agar medium.
3.2. Dilution method
Dilution methods including agar dilution, micro and macro dilution techniques are useful in determining the MIC - Maximum Inhibitory Concentration of large number of samples (Tenover et al., 1995). MIC is the lowest concentration of antimicrobials that reduce the least noticeable growth of microorganisms. Inhibition values recorded a distinctive deviation between neem and chinaberry when different concentrations of aqueous, ethyl acetate and ethanol extracts of chinaberry and neem were mixed to agar medium before inoculation of Alternaria solani and Fusarium oxysporum (Hassanein et al., 2010). Hadizadeh et al; (2009) conducted agar dilution bioassay for evaluation of antifungal effectiveness of the ethanol extracts prepared from Ziziphus spina, Citrullus colocynthis, Urtica dioica and Nerium oleander floral parts against A. alternata, F. oxysporum, F. solani and R. solani. Similarly Viuda-Martos et al; (2008) used agar dilution
34 method to evaluate the efficiency of essential oils of citrus plants on growth of food spoilage moulds.
4. MAJOR ANTIMICROBIAL PHYTOHEMICALS
Phytoalexins
Phytoalexins are low molecular weight compounds that are produced in response to herbivorous, microbial and environmental stimuli. These include flavonoids, simple phenylpropanoid derivative, terpenoids and isoflavonoids (Bailey and Mansfield 1982;
Grayer and Harborne 1994).
Phytoanticipins
These are present in plants and stored in vacuoles of plant cells and include phenolic glycosides and saponins. These are also present in healthy plants in low concentrations but significantly increase after the challenge or microbial attacks. When the microorganisms interrupt and interfere with the integrity of the cell, glycosides reacts quickly with hydrolyzing enzymes and release toxic aglycones (Osbourn, 1999).
4.1. Phenolics and Polyphenolics
The phenolic compounds present in the plants constitute an important class of plant secondary metabolites. These are the substances that possess an aromatic ring with one or more hydroxyl groups and contain functional derivates. Among the aromatic compounds, phenols or their derivatives have oxygen substitution (Geissman, 1963). Some important phenolic compounds include phenolic antioxidants, alkyl esters of parabens and terpene fractions of essential oils, e.g., eugenol, carvacrol, thymol and vanillin. Sesquiterpenes
35 with monoterpenes are imperative parts of essential oils in plants. Simplest forms of phenolic compounds consist of monophenols as cresol, diphenols as hydroquinone and triphenols as gallic acid. Davidson (1997) stated that gallic acid occurs in plants as hydrolyzable tannins or quinic acid esters or tannic acid. Phenolic compounds possessing antibacterial and antifungal properties have been evaluated against several model organisms and pathogens including Xanthomonas, Penicillium spp, Rhizopus spp. and
Geotrichum candidum. The mechanism of phenolics centre’s cellular membranes. Phenols interrupt the cytoplasmic membrane and cause escape of cells. Phenolic compounds may also restrain cellular proteins directly or indirectly. On the other hand, some researchers found that phenolic compounds may have sound mechanisms of action or targets which directly control inhibition of microbes (Davidson, 1997). Phenolics have long been assumed in disease resistance in many horticultural crops (Barkai-Golan, 2001) and callus deposition in cell walls of immature fruits of tomato. It was demonstrated by Barkai-Golan
(2001) that the presence of phenolic compounds in the inoculated plants captured development of Botrytis cinera, thereby preventing decay of fruits.
Mostly the phenolic compounds are water soluble because they are commonly present in combination with sugars as glycosides (Harborne, 1998), and play very significant role in defense against microbial activities, infections and for growth development. In case of injuries they give oxidative stabilities to the plants (Cetkovic et al., 2007). Simple phenolics such as gallic acid, caffeic acid, cinnamic acid, chlorogenic acid, pyrogallol, and catechol and hydroxyl benzoic acid are recognized antimicrobial agents (Noriaki et al., 2005; Bowles and Miller, 2006). In an experiment the methanol leaves extract of Eucalyptus darlympleana was consecutively partitioned with CH2Cl2, nBuOH, EtOAc, and H2O, for chemical detection of the antifungal substances. Two phenolic compounds (3, 4-dihydroxybenzoic acid and Gallic acid) and three flavonoids
36
(quercetin-3-O-α-L-rhamnoside, quercetin and quercetin-3-O-β-D-glucoside) were identified by spectroscopy. Among them, gallic acid was only found to be effective in suppressing mycelial growth and spore germination of Botrytis cinerea at moderately high concentrations (Oh et al., 2008). According to Doherty et al; (2010) the phytochemical analysis revealed that in both Aframomum melegueta seeds extract and Zingiber officinale extracts terpenes, tannins, phlobatannins, flavonoids, steroids, saponins, and alkaloids are present. Thus phenoles have been widely used in the disinfection.They form the standard with which other fungicides are compared). These compounds also act as electron donors and are voluntarily oxidized to an electron acceptor quinine or phenolate ion. According to Lambert et al; (2001) phenolic as a major component and non-phenolic compounds as minor component in the alcohol extracts are considered to be usually antifungal agents.
The antifungal activity of leaves extract of Syzigium cumini may be caused by tannins and some other phenolic components. Gallic and ellagic acid polyphenol derivatives are rich in S. cumini (Chattopadhyay et al., 1998). Also, kaempferol, acylated flavonol glycosides, myricetin and other polyphenols have been isolated from leaves of S. cumini (Mahmoud et al., 2001; Timbola et al., 2002) which may be accountable for antifungal activities.
4.1.1. Tannins
The tannins are polymeric phenolic substances, are able of tanning leather. Water soluble hydrolysable tannins derived from a flavonoids monomer contain gallic acid usually in the form of ester. Tannins can form complexes with polysaccharides and inactivate microbial adhesions, cell envelop transport proteins and enzymes. A number of studies revealed antimicrobial activity of tannins against phytopathogenic fungi and bacteria (Alstrom, 1992). Five tannins isolated from Terminalia citrine fruit showed inhibitory effect on microbial strains (Burapadaja and Bunchoo, 1995). Polyphenols have also been described as inhibitors of polygalacturonase activity of Sclerotinia fructicola in
37 apples and other pathogen / host combinations (Barkai-Golan, 2001). Tannin-rich plant extracts have usually been used as medicines against infectious diseases of human
(Tegegne et al., 2008).
4.1.2. Flavonoids
Flavonoides constitute the largest group of polyphenolic compounds which are generally distributed throughout the plant kingdom and synthesized by plants in response to microbial infections (Dixon, 1999). Four major groups of flavonoids are: anthocyanins flavones, flavonols and isoflavones, which are hydroxylated phenolic structure with one carbonyl group, and occur as a C3-C6 unit, linked to an aromatic ring. Flavonoids are complexed with cell wall proteins and found to be very useful against microorganisms
(Cowan, 1999). Several forms of flavonoids show broad spectrum antimicrobial activity and often found effectual as antimicrobial material (in vitro) against a wide range of microorganisms (Orner and Jha 1993; Dixon 1999; Gurjar et al., 2012). According to
Duthie and Crozier (2000), flavonoids function as free radical scavengers and direct antioxidants having the ability to alter enzymatic activities and reduce cell propagation. In plants, they appear to play a defensive role against invading pathogens including fungi, bacteria and viruses (Sohn et al., 2004). An important source of polymethoxylated flavonoids is reported in citrus spp. (Afek et al., 1986). It is reported that peels of mandarin fruits, Citrus reticulate, yielded two flavones (Tripoli et al., 2007; Wang et al., 2008).
Earlier study described the isolation and antifungal activity of two new polymethoxylated flavonoids from lime, Citrus aurantifolia (Johann et al., 2007). They are often used to develop phylogenetic associations among plants (Mizuno et al., 1991).
4.1.3. Coumarins
38
Coumarins are phenolic substances comprise of fused benzene and α-pyrone rings.
The pure sap, explosive and crucial oil extracted from entire plant or specific plant parts like seeds, leaves, stem, flowers, fruits and roots are generally used for preparing the antimicrobial substances that are extensively used against different plant pathogens
(Gurjar et al., 2012). 4.1.4. Terpenoids
Secondary metabolites consisting of isoprene units and essential oils are known as terpenoids. Terpenoid compounds are supplementary with oxygen and show microbial activity against bacteria and fungi (Rao et al., 1993; Kubo et al., 1995). Brahmachari
(2004) reported that the active ingredients of neem also include triterpenoides, e.g,
Nimbin, Nimbidine, Azadirachtin etc.
4.2. Alkaloids
Alkaloids have good antimicrobial activity and heterocyclic nitrogen compounds.
Effective antibacterial activity from Samanea saman was shown by different solvent extracts, aqueous extract and isolated fractions of alkaloids against human pathogenic bacterial strains (Raghavendra et al., 2008). These innate compounds have great significance due to their interesting biological and pharmacological actions.
4.3. Saponins/Steroids
Saponins are plant steroids, often glycosylated and triterpene glycosides. Because of their soap like properties they are called saponins. It was first isolated in 1820 from potato and other hosts of family Solanaceae and documented with fungicidal and pesticidal properties. The saponin, tomatine, is a secondary metabolite formed in tomato leaves and unripe fruits as mentioned by van Baarlen et al; (2007). The rind of green tomatoes
39 contains high concentrations of saponins that interact with sterols in membranes and act as antifungal and insecticidal compound (van Baarlen et al., 2007). Saponins inhibit mycelial growth of grey mould (B. cinerea) but do not affect germination of conidia.
During infection of plants fungi may often be exposed to many antifungal compounds. The pathogens of tomato may encounter not only preformed steroidal glycoalkaloida-tomatine, but also phytoalexins such as rishitin, and develop resistance to more than one of these compounds which may be essential for pathogen entrance and virulence (Suleman et al.,
1996). Saponins from some vascular plants parts as green berries of Phytolacca dodecandra, alfalfa roots (Medicago sativa) and fruits of Swartzia medagascariens have fungicidal properties against several pathogenic fungi (Oleszek
1999; Waller 1999).
5. SECONDARY METABOLITES
Plants also produce a wide variety of secondary metabolites which regulate growth, protect from infections and tolerate predations and environmental stresses (Ebel, 1986).
The important secondary metabolites are alkaloids, steroids, polyphenols, and saponins some of which are already described above (Cragg et al., 1997; Banso and Olutimayin
2001). These natural compounds have great importance due to their interesting biological and pharmacological actions. Their activity depends upon the method and solvent used for extraction, its concentration and structure (Kishore et al., 2001). The secondary metabolites in vitro studies have revealed adequate antimicrobial properties specifically or widely with broad spectrum (Dahanukar et al., 2000). These metabolites may be preformed compounds which are present in healthy plants and which may build chemical barriers to infectivity by the potential pathogens, otherwise, phytoanticipins may be manufactured in response to phytoalexins (Osbourn, 1999). Extracts of various plants also contain low-
40 molecular weight as well as complex compounds as isoflavonoids, vanogenic glycosides, sesquiterpenes, cyclic hydroxamic acids, sulfur-containing indole derivatives and many other compounds which inhibit fungal growth in vitro (Van Etten et al., 1994; Kuc’ 1995).
Sasidharen and Menon (2010) analyzed fresh and dried rhizomes of two ginger cultivars which were found to contain volatile oil as a major component. In recent years many proteins or peptides have been isolated from green plants (Huynh et al., 2001) which are involved in defense mechanisms against fungi and bacteria. Attention has been focused on the exploitation and development of novel plant products with low mammalian toxicity but increased antimicrobial peptides (Subramanyam and Roesli 2000; Jamil et al., 2007).
Data is available on the antifungal effects of essential oils against insect pests (Lajide et al., 1998; Sahayaraj 2008), they are regarded as safe, non toxic and highly systemic than chemical pesticides (Elgayyar et al., 2001; Canillic and Mourey 2001; Suhr and Nielson
2003; Thangavelu 2004)). Siva et al; (2008), Amadi et al; (2010) and Gurjar et al; (2012) have identified several antifungal compounds but Duke et al; (2000) added that natural products can also exhibit structural complexes and may contain halogenated atoms.
6. MODE OF ACTION OF ANTIMICROBIAL PHYTOCHEMICALS
The phytochemicals (Mizutani, 1991) which are intimately associated with plants and have allelopathic effects are generally known as Allelochemicals (Whittaker and
Feeny, 1971). Their production in the plants varies in quality and quantity throughout the year (Devi et al., 1997) and also depends upon the age and the specie of the donor plant
(Burgos et al., 1999). The chemicals are synthesized in plants as secondary metabolites and have no direct role in the growth and development but serve in defensive mechanism as antimicrobials (Harborne 1989; Seigler 1996), and in few cases boost reproductive ability of the plants (Taiz and Zeiger, 1998). As discussed by Nascimento et al; (2000),
41 the interacting compounds have different structures and modes of action when compared with conventionally used chemicals. These not only directly dismiss harmful insects, pests and pathogens, but release certain compounds which reduce the microbial growth (Naqui et al., 1994). Essential oils and their derivatives have different mechanisms. Juven et al;
(1994) stated that the phenolic compounds sensitize the phospholipid bilayer of the microbial cytoplasmic membrane causing increased permeability and unavailability of essential intracellular components. The chemicals are released from the plant through leaching, volatilization, root exudation and decay or kill the targets through biotic or abiotic means (Anaya, 1999). This process also involves a number of physicochemical and metabolic steps (Mizutani 1999; Waller et al., 1999). In general, inhibitory action on molds and microorganisms is due to disintegration precipitation of protoplasm, cytoplasm granulation, rupturing of cytoplasmic membrane and inactivation of intercellular and extracellular enzymes. These biological procedures may take place independently or simultaneously and cause inhibition of germination and growth of pathogen (Campo et al.,
2003).
Abyaneh et al; (2005) observed morphological changes in a toxicogenic isolate of
Aspergillus parasiticus when exposed to various concentrations of aqueous leaf and seed extracts of neem. The mycelium in culture produced 90 and 75% less aflatoxins at 50% concentration respectively. Under these conditions, semi-thin longitudinal and cross sections of the mycelium and vesicles showed attenuation of the cell wall at different intervals causing deformation and uneven shape and vacoulation of the mycelium cytoplasm and vesicles, some mycelia indicated a cleft between the cell wall and cytoplasm.
7. SOME ANTIFUNGAL PROPERTIES OF TARGET BOTANICALS
42
Shinwari and Malik (1989) recorded about 6000 plants having medicinal properties in
Pakistan. Majority of the plants are rich storehouse of natural biochemical compounds and drugs (Hashim and Devi 2003; Okigbo and Nmeka 2005; Okigbo and Omodamiro 2006).
Under field conditions, methanolic extracts of 133 out of 183 medicinal plants were reported to be effective in controlling plant diseases (Oleszek 1999; Waller 1999; Kim et al., 2004). Higher plants and their parts are used for the treatment of diseases in man, animals and plants in the form of extracts, powder, decoction and infusions (Nostro et al.,
2000). In view of unavailability of effective or cheap fungicides, soil-borne diseases caused by Phaseolina, Rhizoctonia and Fusarum spp; can be economically managed by the plant extracts. Some antifungal properties of the target botanicals are described briefly.
7.1. Garlic (Allium sativum). Cross-section of garlic revealed the presence of
allinase enzyme and allicin substrate (Koch and Lawsone 1996, Milner 2005).
According to Ameh et al; (2013), methanolic extracts of bulbs of A. sativum
contain maximum amount of chemical compounds such as carbohydrates,
glycosides and proteins while reducing sugars, oils, steroids and flavonoids in
medium to lesser quantities, but resins, tannins and terpenoids are absent. The
strong odor of fresh and crushed garlic is attributed to oxygenated sulpher
compound, allicin, which possesses antimicrobial properties (Stephen, 2005).
Allicin has strong antifungal activity and successfully controlled various plant
diseases including late blight of potato and tomato, seed-borne fungi in carrot,
downy mildew of Arabidopsis thaliana (Slusarenko et al., 2008). Garlic extract
also inhibited the formation of mycotoxins such as aflatoxin produced by
Aspergillus parasiticus (Lawson, 1996). Garlic extracts at different concentrations
indicated antibacterial activity against bacterial pathogens of tomato; Clavibacter
43
michiganensis sub sp. michiganensis, Pseudomonas syringae pv, tomato,
Xanthomonas vesicatoria, causing bacterial specks, spots and canker. Under
greenhouse conditions, garlic extracts showed best results at 106cfu/ml and
reduced disease incidence by 58% as against 30% with other plant extracts
(Balestra et al., 2009). Jagtap et al; (2012) tested crude extracts of six plants
against root rot, fruit rot and gummosis of citrus caused by Phytophthora spp. and
achieved best results with garlic which restricted mycelial growth at 5, 10 and 15%
concentration. Fungi like
Botrytis cinerea, F. oxysporum f. sp. lycopersici and R. solani were also controlled
(Alkhail 2005; Agbenin and Marley 2006). Similarly, Shrestha and Tiwari (2009) recorded complete inhibition of mycelial growth of F.solani at 40% concentration of garlic but extracts of other botanicals were not effective.
7.2. Neem (Azadirachta indica). It contains at least 35 biologically active
compounds like azadirachtin and nimbin which are present in seeds, bark and
leaves and have strong ability to kill pathogens and pests (Pennington and
Styles1975; Mulla et al., 1999). According to Siddiqui et al; (2000), secondary
metabolites such as terpenoids, quercetin, desactylimbin and sitosterol present in
extracts of neem are responsible for antimicrobial activity. Extracts in the form of
leaf diffusates and powder formulations from leaf, bark and fruit were highly
effective against soil borne fungi as P. infestans, R. solani, R. stolonifera and F.
oxysporum (Paul and Sharma 2002, Naz et al., 2006, Yeni et al., 2010). Rathode
et al., (2010) using poisoned food technique found it very useful against charcoal
rot of soyabean. Mishra et al; (2009) reported high efficacy of Neemazol against
phytopathogenic fungi, maximum growth inhibition occurred at 2000 ppm. Neem
44
oil reduced the mycelial growth and sclerotial survival of Macrophomina
phaseolina (Dubey et al., 2009). Hassanein et al; (2008) found it effective against
Alternaria solani and F. oxysporum causing early blight and wilt of tomato,
respectively, and controlled powdery mildew of cucumber (Aboellil, 2007). Its
natural product “Trilogy” retarded the growth of Podosphaera xanthii. Neem
extracts along with those of other plants controlled seed borne fungi in different
crops (Mondali et al., 2009) including African yam bean against Aspergillus
flavus, A. niger, Botryodiplodia theobromae and Fusarium moniliformae. Plant
extracts can be prepared in different solvents but Javed and Rahman (2011)
recommended chloroform and ethyl acetate for the management of Macrophomina
phaseolina, the cause of charcoal rot diseases. Alcoholic and aqueous extracts of
neem were more effective in the inhibition of mycelial growth of Aspergillus spp.
but not against R. stolonifer. Rahman et al; (2011) compared 24 plant extracts with
strains of
Trichoderma against chili anthracnose and observed 100% inhibition of conidial germination with neem leaf extract. It was concluded that the commercial neem products are safer and can be used against human, insect and plant diseases.
7.3. Ginger (Zingiber officinale). Ginger contains several phenols, and the
gingerol and its pleasant aromatic odor is because of essential oil (Anukwuorji et
al., 2013), and presence of chemical compounds like caprylic acid in ginger make
it a source with potent antifungal properties (Ernst and Pittler 2000; Okigbo and
Nmeka 2005). Rodrigues et al; (2007) evaluated aqueous extract of ginger at
different concentrations on radial growth and sclerotial production of Sclerotinia
sclerotiorum Accumulation of phytoalexins and glyceollin, mycelial growth and
sclerotial production inhibition all this indicated its antimicrobial activity and
45 presence of elicitor compounds in the ginger extract. In vitro study carried by
Abdel Kader et al; (2012) and Fawzi et al; (2009) confirmed antifungal activity of various plant extracts including ginger against F. solani, A. solani, A. alternata,,
F. oxysporum, R. solani, Pythium sp and M. phaseolina. The results revealed that ginger extract had maximum inhibition on the growth of fungal pathogens and attributed to presence of gingerol (Fricker et al., 2003).
7.4. Mhendi, Henna (Lawsonia inermis). Various parts of Lawsonia are traditionally used in cosmetics and medicines and indicate antifungal properties
(Sharma and Sharma, 2011). Ambikapathy et al; (2011) tested aqueous and organic solvent extracts of five medicinal plants along with Lawsonia against
Pythium debaryanum. The results indicated maximum antifungal activity of methanolic extract of lawsonia against pythium damping off disease. On the other hand, Sharma and Sharma (2011) found that the acetone extract of lawsonia were more effective in controlling plant pathogenic and human fungal species such as
Drechslera halodes, A. solani, R. solani, F. solani, F. moniliformae, Curvularia lunata, A. flavus, A. fumigatus, A. parasiticus var. globosum, Trichophyton rubrum and C. albicans.
7.5. Tulsi (Ocimum sanctum, O. basilicum). All parts of ocimum (leaves, frowers, roots, seeds) are used in medicine. Ocimum extracts contains biologically active compounds having antimicrobial activities (Moreles and Simon 1996;
Dubey et al., 2000, Amadi et al., 2010) and are used in ayruvedic treatments.
Chuku et al; (2010) recorded antifungal activity of ethanol and crude aqueous extract of ocimum against seed-borne mycoflora of melon followed by A. indica, and Ogbo and Oyibo (2008) found fungistatic effect on postharvest pathogens of avocado. Eugenol is the main constituent of essential oil obtained from Ocimum
46 gratissimum and its antifugal activity was determined by Faria et al; (2006) against
A. solani and Penicillium digitatum.
7.6. Turmeric (Curcuma longa). Turmeric is closely and historically associated with folklore medicines and spice in relation to human life since earlier civilization
(Nair et al., 2005, Agarwal et al., 2007, Lakshmi, 2012)). Antimicrobial properties of herbal extracts of turmeric have been recognized throughout the world
(Chattopadhyay et al., 2004). It is a true medicinal plant and contains analgesic natural compounds (Lee et al., 2003). The rhizomes contain several compounds which include monoterpenoids, sesquiterpenoids and curcuminoids (Tang and
Eisenbrand, 1992). The phytochemical analysis of turmeric also revealed presence of ar-Turmerone and sesquiterpene. According to Hatcher et al; (2008) and Singh and Jain (2011) yellow pigmentation of turmeric is due to curcuminoid also known as curcumin which is the main bioactive compound with wide range of antifungal, insecticidal and antibacterial properties (Chander et al., 1992; Rai et al., 2008).
Aqueous extracts of turmeric rhizomes and four curcumin solutions at different concentrations inhibited mycelial growth and sporulation of A. solani, higher concentration being more effective (Balbi-pena et al., 2006). Lee et al; (2003) assessed the fungicidal property of turmeric against six fungal species and achieved best results with methanol fraction. Aqueous extracts of turmeric inhibited conidial germination of Colletotrichum gleosporioides, the cause of mango anthracnose (Imtiaj et al., 2005), food associated molds (Pundir and Jain,
2010) and ethanolic extract restricted growth of R. stolonifer and Mucor sp. indicating antifungal potential of turmeric. Earlier researches documented that the turmeric oil, termenol, an alkaloid; curcumins, curcumenoids and veleric acid present in turmeric contributed antimicrobial properties (Cikrikci et al., 2008).
47
7.7. Marigold (Tagetes minuta, T. erecta and T. patula). A substance called
alphaterthienyl with pungent odor is produced by marigolds which have
antifungal, antibacterial, antiviral, nematicidal and insecticidal properties (Khan
et al., 1971; Soule 1993; Franzener et al., 2007; Shafique et al., 2011). It was
demonstrated by Shafique et al., (2011) that the aqueous and methanol extracts of
flowers and shoots T. erectus reduced mycelial growth of Ascochyta rabiei.
Antifungal activity of extracts T. erectus at different concentrations was also
checked against A. alternata, and maximum inhibition was recorded with
methanol extract at 4 % concentration followed by aqueous and n-hexane extract
(Shafique and Shafique, 2012).
7.8. Eucalyptus (Eucalyptus globules). A number of biologically active
compounds have been found in Eucalyptus tree possessing antibacterial (Ghalem
and Mohammad, 2008), antifungal (Bluma et al., 2008) and antagonistic activities
against phytopathogens (Delaquis et al., 2002; Schelz et al., 2006; Oh et al., 2008).
Dawar et al; (2007) examined efficacy of leaves, stem, bark and fruit extracts using
paper disk and well diffusion methods against root fungi of mungbean and
chickpea. Maximum growth inhibition of
Fusarium sp. and M. phaseolina was obtained at higher concentration than R. solani. Earlier work of Khalil (2001) had depicted antifungal activity of E. rostrata on spore germination and mycelial growth of A. solani. Similarly effect of E. camaldulensis was studied on Botriodiplodia theobromae (Sahi et al., 2012). Moreover, Oh et al; (2008) evaluated different species of Eucalyptus viz. E. globules, E. darlympleana, E. gunnii and
E. unigera against soft rot fungi of kiwi fruit (Diaporthe actinidiae, B. cineria and
Botrosphaeria dothidea). Among four species of eucalyptus, methanolic extracts of E. darlympleana and E. unigera effectively reduced growth of test fungi.
48
7.9. Chinaberry (Melia azedarach. Several workers have reported that Melia
spp; possess medicinal and insecticidal properties (Ali et al., 2001; Khan et al.,
2001). Carpinella et al; (2005) stated that the antifungal compounds present in M.
azedarach are like vanillin, hydroxycoumarin scopoletin, pinoresinol and 4-
hydroxy-3-methoxycinnamaldehyde which are responsible for antifungal activity.
Aqueous leaf extracts of M. azedarach and three similar species were tested
against M. phaseolina. The results showed that aqueous extracts of M. azedarach
and A. indica at 5-20% concentrations considerably reduced growth of the test
fungus (Ashraf and Javaid, 2007). Carpinella et al; (2003) evaluated extracts from
seed kernels, senescent leaves and fruit of M. azedarach which manifested
antifungal activity against F. solani, A. flavus, Sclerotinia sclerotiorum, F
.oxysporum, F. verticillioides and Diaporthe phaseolorum var. meridionales.
Recently Jabeen et al; (2008) reported leaf extract of M. azedarach inhibited in
vitro growth of chick pea blight.
7.10. Ashoka tree (Polyalthia longifolia var. Pendula). Dandge et al; (2012)
tested seed extract of P. longifolia and P. longifolia var. Pendula against 6
Deuteromyceteous fungi like Curvularia lunata, A. alternata, Mucor racemosus,
Phoma nebulosa, Fusarium equiseti and Botryodiplodia theobromae.
Antimicrobial metabolites were detected with variable effects, maximum with P.
nebulosa and minimum with B. theobrome, C. lunata,
A. alternata and R. solani. Leaf extracts were used as antimicrobial agent against some fungi and bacteria (Murthy et al., 2005; Nair and Chanda 2006).
7.11. Periwinkle (Catharanthus roseus). Ethanolic extract of periwinkle and several other medicinal plants checked mycelial growth and sporulation of A. niger, A. flavus and A.
49 fumigates isolated from poultry feed. The radial growth of A. flavus was partially restricted than A. niger and A. fumigates by datura extract (Islam et al., 2003).
7.12. Mango (Mangifera indica). A phenolic compound named C-glucoside xanthone mangiferin is widely distributed in bark, leaves, fruits and roots of family Anacardiaceae
(Desai et al., 1966; El Ansari et al., 1971; Yoshimi et al., 2001). Kanwal et al; (2010) quantified five types of flavonoids from mango leaves. Based on ethnopharmacological knowledge mango tree is rich in antioxidant, antibacterial, antifungal, anti-inflammatory and immunomodulatory properties (Aqil and Ahmad 2003; Nuñez-Selles 2005; Stoilova et al., 2005).
7.13. Lemon (Citrus limon). Lemon is a vital medicinal plant and has the ability to produce alkaloids-flavonoids which are biological active having antifungal, antibacterial, antiviral, antidiabetic and anticancer properties (Ortuno et al., 2006; Dhanavade et al., 2011). Pure extracts from leaves, stem, flowers and roots have been clinically evaluated against important bacterial strains (Kawaii et al., 2000). Viuda-Martose et al; (2008) used agar dilution method to find effect of the essential oils of lemon and some other citrus species against of molds like A. niger, A. flavus, P. chrysogenum and P. verrucosum associated with fruit spoilage. Lemon oil was considered suitable alternatives to chemical additives.
Dried peel contained antimicrobial metabolites in soxhlet extracts with cold and hot water, methanol, ethanol, ethyl acetate and acetone which appeared effective against bacterial pathogens except hexane extracts. However, methanol extract was effective against fungal pathogens and growth inhibition (Khushwaha et al., 2012).
7.14. Jambolan, Jaman (Syzgium cumini). It has received much attention and recognition in folk medicines and pharmaceutical formulations as antimicrobial, astringent, stomachic, diuretic, antiscorbutic and antidiabetic in chronic diarrhea and swelling of the spleen
50
(Shafi et al., 2002; Migliato 2005; Jabeen and Javaid 2010). Its seeds are used by the villagers for the cure of illnesses due to fungal, bacterial and viral pathogens
(Chandrasekaran and Venkatesalu, 2004). Leaf extracts with organic solvents
(methanol, chloroform and ethyl acetate) appreciably reduced biomass of M. phaseolina
(Javaid and Rehman, 2011), chloroform extract being most effective. Ethanol, n-hexane and aqueous extracts from leaves, stem-bark, root-bark and fruits of S. cumini had fungistatic activity against Ascochyta rabiei causing chick pea blight (Jabeen and Javaid,
2011).
7.15. Moringa oleifera. It is locally known as Swanjna and is the only cultivated species as a crop (Veeraragava 1998; Olson 2002). Its leaves are used in different biomasses, livestock fodder, green manure, biogas, plant growth enhancer, medicines and biopesticides (Nouman et al; 2012). The plant was reported to possess various fatty acids, amino acids, vitamins and nutrients (Nesamani, 1999). Its extracts stimulated plant and seedling growth, increased plant vigor, prolonged storage life and also suppressed fungal growth. Along with 8 other medicinal plants, extract of moringa isolated through disc diffusion method was highly effective against plant pathogens, and antifungal activity of extracts was attributed to protein or peptides contents Das et al; (1997). The fungicidal effects of leaf extracts have been documented on some soil-borne fungi such as
Rhizoctonia, Pythium and Fusarium spp. (Stoll et al., 1988). Pre-treatment of okra seeds
(Abelmoschus esculentus) with three concentrations of leaf extract and bleach reduced fungal activities (Nawakburuka et al., 2012). The antifungal activity of extracts of
Tinospora cordifolia (leaves), M. oleifera (bark) and Trachyspermum ammi (seeds) at high concentration was expressed and established by poisoned food technique against F. oxysporum f. sp. lycopersici and F. solani (wilt of tomato and eggplant) as inhibition of mycelial growth (Dwivedi and Enesba, 2012). The aqueous and ethanolic extracts of A.
51 cordifolia, Cassia alata and M. oleifera were capable of controlling fruit rot pathogens of tomato as F. verticillioides and M. phaseolina, ethanolic extracts were superior over aqueous phase (Enikuomehin and Oyedeji, 2010). Maji et al; (2005) reported that moringa extracts inhibited conidial germination of Phyllactinia corlea. It was concluded by Howell et al; (1997), McLean et al; (2005) and Dwivedi and Enesba (2012) that the extracts of M. oleifera were feasible for the management of plant diseases. Seeds and leaves of moringa also possess medicinal and antifungal properties against skin diseases as dermatophytes caused by Epidermophyton xoccosum, Trichophyton rubrum, T. mentagrophytes and
Microsporum canis.
8. GENERAL BENEFICIAL IMPACTS OF ANTIMICROBIAL AGENTS
The investigations on medicinal plants carried so far have shown that the botanicals have antifungal and antibacterial activity, mainly as inhibition of germination, suppression of multiplication or inactivation of the pathogens (Sehajpal et al., 2009). Earlier workers tested efficacy of plant extracts against plant pathogens both under laboratory and field conditions (Singh et al., 1995; Pirthiviraj et al., 1996; Sarma et al., 1999), although their application under field conditions seem to be limited. However, antifungal or antimicrobial activities of plant extracts of Zingiber officinales, Azadirachta indica, Allium cepa, A. sativum, Pisum sativum. Arachis hypogeae, Aleo vera and Lippea javanicum have been reported against a wide range of pre and postharvest plant pathogens (Lee et al., 1990;
Barkai-Golan 2001; Makeredza et al., 2005; Otanga 2005). Kanwal et al;
(2010), Muto et al; (2005), Jabeen and Javaid (2010) and Riaz et al; (2010) reported that both the crude extracts and purified compounds from medicinal plants can be used for the effective management of plant diseases as compared to synthetic agrochemicals.
Methanolic extracts of 183 medicinal plants were screened against several fungal
52 pathogens under field conditions and 90% of them manifested highly satisfactory, encouraging and desired results (Oleszek 1999; Waller 1999; Kim et al., 2004).
Extracts of lemon grass leaves and chili fruits (Khair and Wafaa, 2007) and water or acetone extracts of Inula viscose, effectively reduced incidence of late blight disease of potatoes and tomatoes (Wang et al., 2004). Nwachukwu and Umechuruba (2001) reported that the plant extracts have played important role in inhibiting seed-borne fungi (F. oxysporum) and improving seed quality of the treated plants. Aqueous extracts of
Withania somnifera, Datura stramonium and Ranunculus arvensis inhibited mycelial growth of Fusarium oxysporum f. sp. lycopersici and Rhizoctonia solani (Qasem 1999;
Yadav et al., 2005). According to Anukwaurji et al; (2013), Okigbo and Nmeka (2005),
Okigbo et al; (2009a; 2009b), the extracts of Chromolena odorata, Ocimum gratissimum,
Cymbopogon citrates and Zingiber officinales, were highly effective against potato rotting fungi (Botryodiplodia theobromae, Ceratocystis fimbriata, Rhizopus stolonifer and
Fusarium solani). Maximum inhibition was obtained at 10 and 7.5% extracts of C. odorata and Z. officinale against the fungus C. fimbriata. Farrag et al; (2013) found that damping off of cucumber was controlled through cucumber seeds treated with 2% extract of peppermint. Seedlings raised from treated seeds were vigorous than the untreated seeds.
Alcoholic extract of Capsicum frutescence inflicted 100% inhibition of colony growth, and extracts of Zingiber officinale applied at 3000ppm controlled Penicillium digitatum,
Fusarium spp. and Aspergillus niger (Singh et al., 2011).
Essential oils extracted from higher plants have antimicrobial activities. Eugenol and Thyme extracted from clove and Thymus vulgaris, respectively, are important oils with antimicrobial activity (Imelouane et al., 2009). Similarly, there are plants species rich in essential oils viz. garlic, clove, ginger, onion and pepper (Arora and Kaur 1999; Indu et al., 2006).
53
Anand and Bhaskaran (2009) reported antifungal activity of 44 plant species against fruit rotting fungi of chilli. Leaf extract of Aegle marmelos and Abrus precatorius were highly effective against Alternaria alternata, spore germination was inhibited by about 75%, except extracts from A. marmalos and A. precatorius.
Aqueous extracts of twenty plants were tested by Khalil (2001) on spore germination of Alternaria solani and zoosporic fungal species, best results were obtained with extracts of Eugenia aromatica which showed strong degree of inhibition on spore germination and mycelia growth of all fungi. Islam (2003) checked antifungal activity of five indigenous plants of Potohar region of Pakistan against three pathogenic fungi of commercial crops. Studies revealed that all extracts exhibited considerable inhibition on mycelial growth of tested fungi.
Agbenin et al; (2005) and Hadian et al; (2011) recorded nematicidal and antifungal activity of neem seed powder in green house tomatoes. It also controlled Fusarium and
Root-knot nematode disease. Results indicated that all the treatments significantly improved the plant growth, increase plant length and weight and reduced disease severity as compared to control (untreated). Zia-Ul-Haq et al; (2011, 2012) checked methanolic and ethanolic extracts of seeds of Ipomoea hederacea, Ferula assafoetida, Grewia asiatica,
Lepidium sativum and Terminalia chebula against various species of fungi, bacteria and nematode.
Extract of Abrus precatorius sprayed twice on chili controlled Alternaria alternata and Colletotrichum capsici. First spray was done at the time of fruit setting and the second after twenty days of fruit set. It lowered disease incidence (Anand and Bhaskaran, 2009).
Al-Samarrai et al; (2012) observed effectiveness of two concentrations (4000 and
5000ppm) of neem, chili and pong-pong on stored citrus fruits. Chourasiya (2013) evaluated the efficacy of chemical fungicides (Mancozeb, Carbendazim and Zineb) and
54 botanicals (neem, garlic and eucalyptus) against early blight of tomato in the field. Among chemical sprays Mancozeb was superior and produced the highest yield of 246.40 q/ha followed by Carbendazim (221.50 q/ha). Neem leaf extract effectively lowered disease severity (30.66 PDI) and was followed by garlic (32.44 PDI) and eucalyptus (34.07 PDI).
3. MATERIALS AND METHODS
These investigations were carried out in the Plant Pathology Department, Faculty of
Agricultural Science and Technology, Bahauddin Zakariya University (BZU) Multan
55
Pakistan, during three consecutive years, 2011- 2014. Analytical work was done in the
Laboratory of Food Science and Technology and field trials were conducted at the
Experimental Farm and greenhouse of the University.
3.1. General methods
Plants, growth conditions and inoculations
All plants were raised in an insect-free and protected glasshouse maintained at 2030oC.
Tomato cultivar ‘Money Maker’ was used throughout the study. Potting mixture consisted of field soil, farm yard manure and sand (1:1:1), which was treated with 5% commercial formalin (1L/sq.m), under plastic cover for 2-3 days with frequent turnings. Pots (earthen
35 x 50 cm, accommodating 3 kg soil or plastic pot 10 cm in diameter with 1 kg capacity) were filled with the soil mixture. Tomato seeds were surface disinfected by 12% NaOCl2 for two minutes followed by three washings with sterile distilled water for one minute.
Tomato seedlings thus raised were transplanted in the pots, irrigated with distilled water and covered with plastic bags after each treatment or as and when required.
Glassware and chemicals
Pyrex Petri plates, 90 mm in diameter with 9-12 ml volume and glass tubes of 25 ml volume were used in this study. PDA medium was used for the isolation and multiplication of fungi. The medium was autoclaved at 121oC for 20 minutes, cooled to 55oC, and amended before solidification with antibiotic streptomycin (50mg/1000ml) to restrict bacterial growth. In order to stimulate fungal sporulation, specific culture media as carrot agar (CA), carnation leaf agar (CLA), V8 juice agar (Appendix-1) were also used at different times (Dhingra and Sinclair, 1985). All the chemical, biochemical, solvents, and stains were of analytical reagent grade and obtained from Merck or Sigma. Topsin-M
(Life Sciences Company) a broad spectrum systemic fungicide with active ingredient of
56
Thiophenate methyl (250g/100L) was included and dispensed at I ml to 9 ml substrate.
3.2. Sample Collection
Leaves, roots and fruit samples of tomato manifesting characteristic symptoms of fungal diseases were collected in plastic bags, labeled properly and stored in a refrigerator at 4oC until processed (Olivain et al., 2006). The collection was made from the irrigated tomato fields and open vegetable areas at the University campus. Tomato seeds were obtained from local seed market and growers and tested for the association of mycoflora. Selected plant extracts were dissolved in (20 g/L) sterile distilled water, allowed to settle for one hour, filtered through muslin cloth and the filtrate was used for foliar spray.
3.3. Isolation of fungi
3.3.1. Isolation from plant tissues
Fungi associated with different parts of tomato plant were isolated using standard methods of isolation (Pathak 1987; Agrios 2005). Symptomatic leaf, fruit and root samples, and healthy tissue as control, were cut into small segments (1cm x 1cm), treated with sodium hypochlorite, washed with tap water; surface sterilized with ethyl alcohol (70%) for one minute followed by three washings with sterile distilled water (Baudoni 1988; Waudo et al., 1995). Excessive moisture was blot dried between double layered sterilized filter paper. Isolation was carried out by taking 400 pieces from each plant sample; ten segments were aseptically placed on PDA in ten Petri dishes and replicated four times. Inoculated plates were incubated at 25oC and observed at times for fungal growth, and incidence of colonies was calculated as percentage.
3.3.2. Isolation from seed
57
Standard Blotter (SBM) and Agar Plate Methods as described by the International Seed
Testing Association (ISTA, 1996) were used for the isolation of pathogen associated with tomato seeds.
Blotter test: Four hundred seeds of tomato were randomly selected, surface sterilized with sodium hypochlorite (1%) for 2 minutes and rinsed twice with sterile distilled water.
Twenty seeds were plated in each 9cm petri plate which was lined with three layered moistened filter paper. Experiment was run in quadruplicate having five petri plates per replication. The plates were incubated under near ultraviolet light (NUV) altered with 12 hours darkness for seven days at 25±2oC. Infection was expressed in percentage.
Agar plate method: Fungal infestation of tomato seeds were determined by agar plating method (Lacey and Mercadier, 1998). Similarly, four hundred seeds were surface sterilized with 2% NaOCl for 3 minutes and rinsed thrice in sterile distill water. Twenty seeds, replicated four times, were placed on each of PDA medium under aseptic conditions and the plates incubated under 12 hour light and 12 hour darkness period at 25±2oC for seven days. The charged plates were observed under binocular stereo-microscope for fungal growth and percentage extent of infected seed was calculated.
3.4. Identification, maintenance and multiplication of fungi
Identification of fungal isolates were carried out morphologically on the basis of spore shapes, types, color, colony texture and other growth characteristics according to criteria/keys described by Ellis (1971), Barnett and Hunter (1972) and Dhingra and
Sinclair (1985). Among the isolated fungi, 5 fungal species were selected for further work.
These were: Alternaria solani, Colletotrichum coccodes, Fusarium oxysporum f.sp. lycopersici, F. solani and Phytophthora infestans.
58
Fungi isolated from different plant parts and seeds of tomato were transferred aseptically to PDA plates by hyphal tip method (Brown, 1924). Hyphal tips of fungal species were marked under the dissecting microscope and transferred individually along with bits of agar to separate PDA slants using sterile inoculating needle. The purified cultures were then maintained at 4oC and revived monthly. Mycoflora isolated in this study was preserved for further studies by cryopreservation and filter paper discs (Chandler 1994;
Madigan et al., 2000; Morales 2008). Hyphal fragments from pure cultures were also transferred into the cryovials having glass micro beads, filled with 500µl 20% glycerol and stored at ˗80oC. Sterilized filter paper discs (1cm2) were cut, dried under aseptic conditions, placed on agar surface, inoculated with isolated fungi and incubated at 28oC for 10-15 days. When the filter discs were completely covered by fungal growth, the pieces were dried and stored in plastic bags at -20oC for further studies.
3.5. Pathogenicity tests of fungi
Fungi isolated from plant parts
Healthy tomato seedlings were raised and transplanted in earthen pots and inoculated with test pathogens by cutting a flap on the basal portion of the stem with the help of a sterilized blade. A 6mm inoculum plug from one week old cultures of test fungus was placed in the cut portion of stem and then inoculated portion was wrapped with Para film.
Plants without fungal plug served as control. Alternatively, spore suspensions
(5x104spores/ml) were sprayed with a hand atomizer on the leaves to point of runoff. In order to maintain humidity all the inoculated and control plants were covered with polyethylene bags. After 48 hours the bags were removed and pots were kept in greenhouse. The plants were monitored for the development of disease symptoms as observed on the original infected plants, thus fulfilling the Koch’s postulates (Dhingra and
59
Sinclair, 1985).
Seed-borne fungi
Spore suspension and inoculation
The test fungi were multiplied on PDA medium for 10-15 days in an incubator at
25±2°C. Conidia were harvested by flooding the petriplates with sterile distilled water
(10ml) and by gently agitating the cultures with inoculating loop. Conidia were then filtered through double layered cheese cloth in each case, adjusted to desired concentration
(1x106spores/ml) by Neauber haemocytometer. The fungal suspension was further diluted with 200ml sterile distilled water for seed inoculation. Plastic pots were filled with formalin treated soil and disinfected tomato seeds were dipped in spore suspension of
15day old culture of respective fungi and then sown in pots (five seeds per pot). Surface sterilized seeds without fungal infestation served as control. The pots were placed in the green house at 26±2oC, irrigated with distilled water during the course of experiment and frequently monitored for disease development (Dhingra and Sinclair, 1985). The experiment was conducted in completely randomized design with three replications.
3.6. Assessment of antifungal properties of plant extracts
Collection of plant material:
Fifteen species of medicinal plants, locally cultivated and easily available, were marked at the university campus and identified by the Plant Taxonomist, the Department of
Botany, Bahauddin Zakariya University Multan (Table 3.1). These plants have also been described in the Flora of Pakistan (Ali, 1980) and thus comply with those accepted under the International Code of Botanical Nomenclature. Fresh and apparently healthy leaves, flowers and rhizomes from these plants were collected in paper bags and stored in a refrigerator.
60
Table 3.1: List of Medicinal Plants used as sources of extracts Sr. No Plant species and code Family Common Local name Part used name 1 Citrus limon (L.) Burm.f. (Cl) Rutaceae Lemon Lemon Leaves
61
2 Allium sativum L (As) Alliaceae Garlic Lahsan Bulbs
3 Melia azedarach L.(Ma) Myrtaceae Chinaberry Drek Leaves
4 Ocimum sanctum L. (Os) Labiatae Holy basil Tulsi Leaves
5 Lawsonia inermis L. (Li) Lythraceae Henna Mehndi Leaves
6 Zingiber officinale Rosc. Zingiberaceae Ginger Adrak Rhizome (Zo) 7 Azadirachta indica A. Juss. Meliaceae Neem tree Nim Leaves (Ai) 8 Eucalyptus globules Labill. Myrtaceae Gum tree Sufaida Leaves (Eg) 9 Curcuma longa/domestica Zingiberaceae Turmeric Haldi Rhizome Val. (Cd) 10 Tagetes minuta L. (Tm) Compositae Marigold Gainda Flowers
11 Moringa oleifera Lamk. (Mo) Moringaceae Drumstick Sohanjana Leaves tree 12 Catharanthus roseus (L.) G. Apocynaceae Periwinkle Sada bahar Leaves Don (Cr) 13 Mangifera indica L. (Mi) Anacardiaceae Mango Am Leaves
14 Polyalthia longifolia (Sonner) Annonaceae Ashok tree Ashok Leaves Thw. (Pl) 15 Syzgium cumini (L.) Skeels Myrtaceae Jambolana Jaman Leaves (Sc)
3.7. Processing of tissue and Extraction The leaves, bulbs, flowers and rhizomes of selected medicinal plants were thoroughly washed with running water and then with sterile distill water. The samples were soaked in
1% NaOCl for 2-3 minutes, rinsed with sterile distilled water and blot dried. Modified methods of Okigbo et al; (2009b) and Seema et al; (2011) were followed for aqueous extraction while organic solvent extraction was done by the method of
Ambikapathy et al; (2011).
62
Preparation of aqueous and dry powder extracts The fresh samples (100g) of each plant was chopped and macerated in an electric grinder with 100ml of sterile distilled water (1:1w/v). The extract was first passed through sterilized double layered muslin cloth and centrifuged at 6000rpm for 15 minutes at 4oC.
The supernatant was filtered through Whatman No.1 filter paper and used as 100% stock solution and refrigerated. The crude extracts were further diluted with sterile distilled water to concentrations of 20%, 40%, 60% and 80%. For dry powder preparation, 100 gm plant tissue was shade dried for 15 days and ground to fine powder using tissue grinder.
Dry powder of each plant part was homogenized, suspended in a medium, centrifuged and supernatant as mother extract was stored at 4oC in airtight bottles for further studies.
Preparation of organic solvent extract and dry powder
For the aqueous phase, fresh leaves, rhizomes, bulbs and flowers (100g) of plants were sterilized with sodium hypochlorite (1%) solution and rinsed in two changes with sterile distilled water. The tissue was separately chopped in small pieces and macerated with
100ml methanol (1:1w/v). Solvent extract of each plant was centrifuged at 6000rpm for
10-15 min. It was evaporated at 45oC in a water bath to get 100% crude extract which was then diluted with sterile distilled water to make further concentrations at 20%, 40%, 60% and 80%, and stored for antifungal properties. In order to prepare powder, 100g material of selected plants was shade-dried, homogenized with 100ml methanol and subjected to centrifugation, filtration and evaporation as above. The supernatant thus obtained was regarded as Mother Extract, further diluted and preserved aseptically in sterile bottles at
4oC and used within four days.
3.8. Phytochemical testing of plant materials
63
Preparation of Extracts
Dried plant material (25g) of each sample was refluxed for 2 hours with (150ml) water and methanol separately. Both fractions of extracts were then filtered using Whatman filter paper No.42. Subsequently, the solvents were completely evaporated using rotary evaporator under reduced pressure at 40oC. The extracts were stored at 4oC in crude form to be used for chemical analysis. Qualitative and quantitative chemical analysis were carried out for screening of bioactive compounds present in test medicinal plants using the standard procedures adapted by Harborne (1998), Sofowora (1993), Trease and Evans
(2009). The following biochemical constituents were identified following the procedure of Edeoga et al; (2005).
Flavonoids test: Few drops of NaOH were added to the extracts of test plants. Appearance of initially deep yellow color which became colorless with addition of few drops of HCl indicated the occurence of Flavonoids.
Terpenoids test (Salkowski test): Five ml (1mg/ml) of each plant extract was mixed to 2ml of chloroform, and 3ml of concentrated H2SO4 was added carefully to form a layer. A reddish brown layer at the boundary / interface indicated the presence of terpenoids.
Saponins test: Two g of plant material was boiled in 20 ml of solvent in a water bath and filtered. After cooling filtrate extract (10 ml) was mixed with 5 ml of distilled water and vigorously shaken to the form a froth which remained persistent and stable for few minutes. An emulsion layer was formed when 3 drops of olive oil were added.
Steroids test: Half a gm of methanolic aqueous extracts of each sample was added to acetic anhydride (2 ml) and then with H2SO4 (2 ml). The color changed from violet to blue or green indicating the presence of steroids.
64
Tannins test: 0.5 g of each fraction was boiled in 20 ml of distilled water, filtered and few drops of 0.1% FeCl2 were added. Formation of brown green or blue black coloration indicated the presence of tannins.
Phlobatannins test: Fractions of each plant sample were boiled with 1% of aqueous HCl which resulted in deposition of red precipitates indicating the presence of phlobatannins.
Cardiac Glycosides test (Keller-Killani test): About 5 ml of each extracts was dissolved in
2 ml of glacial acetic acid (CH3COOH) containing one drop of FeCl3. The above mixture was taken with 1 ml of concentrated H2SO4. Appearance of brown ring at the interface indicated the presence of cardiac glycosides.
Determination of Total Phenolic Compounds / (TPC) contents
These were determined using protocol of Singleton and Rossi (1965) with slight modifications. Aliquots of one ml of plant extracts (0.5g/20ml) were taken in test tubes, mixed with 4ml of Folin-Ciocalteau reagent and then after 7 minutes 5ml sodium carbonate (20%) was added, shaken vigorously and replicated tree times. The mixture was incubated at room temperature in darkness for 2 hours. The absorbance was measured at
740nm, using 5ml-cuvettes in a spectrophotometer. Gallic acid (5, 10, 25, 50, 75 and
100ppm) was used as a standard chemical for calibration curve. Quantification of TPC was expressed as Gallic acid equivalent (GAE) mg/g of dried fraction.
3.9. In vitro evaluation of Antifungal activity of plant extracts
Two methods viz, Poisoned Food Technique (Dhingra and Sinclair, 1985) and Agar Well
Diffusion (Perez et al., 1990) as laboratory protocol were adopted for the evaluation of efficacy of aqueous and organic solvent plant extracts.
65
3.9.1. Poisoned Food Technique
Antifungal activity of aqueous and solvent extracts was studied against test fungi following the procedure of Okigbo et al; (2009b) with slight amendment. Aqueous stock extracts and powdered samples of fresh plants were serially diluted to four concentrations
(20%, 40%, 60% and 80%). One ml of each concentration of extract and Topsin-M was dispensed in 3-replicated Petri dish each containing 9ml medium, which provided a
PDAextract mixture with resultant 2, 4, 6 and 8% extract and 2.5 mg/ml fungicide concentrations, respectively. PDA plates without extract served as negative controls while positive control was set up using fungicide Topsin-M. Four concentrations of fresh and dry powder samples in methanol were prepared in the same manner as aqueous extracts.
The Petri plates were gently swirled for thorough mixing of extract and a medium. The negative controls were maintained using blank agar plates and the fungicide (Topsin-M) as positive control. Agar disk was removed by a sterilized cork borer of 6 mm diameter from the periphery of one week old culture of each test fungi at the center of each plate containing extract-medium mixture. The plates were placed in an incubator at 25±2oC for
7 days and the diameter of fungal colony was measured. The average radial growth and the antifungal activity of extracts in term of percent growth reduction of test organism were computed by the formula of Vincent (1947);
I = (C − T)/C ×100, where ‘I’ is percentage growth inhibition (mm), ‘C’ is mean diameter of fungal colony in control and ‘T’ is mean diameter of fungal colony with plant extract.
3.9.2. Agar Well Diffusion Method
Agar Well Diffusion method was employed to test antifungal activity of aqueous and methanol extracts against predominantly occurring tomato fungi. The cultures of test fungus were evenly spread over PDA medium with a sterile glass rod. Wells were cut with
66 a sterile cork borer of 6 mm diameter and charged with 100ul of aqueous and methanol extracts at each concentration (20%, 40%, 60% and 80%). The plates were left at room temperature for ten minutes to allow diffusion of extract into the agar (Rios et al., 1988).
Antifungal activity of dry powder and aqueous solvent was tested in a similar way. Wells filled with distilled water served as negative control and those filled with Topsin-M plus extract as positive control. All the plates were incubated at 27oC for 72 hours and then observed for the formation of growth inhibition zone which indicated the effect or presence of antifungal component. The experiment was conducted in completely randomized design
(CRD) with three replications.
3.10. Spore germination inhibition test
The antifungal effectivness of ethnobotanical extracts on spore germination of test fungi
was determined using the cavity slide method suggested by Nair and Ellingboe (1962).
One drop (0.1ml) of each extract at 80% concentration was mixed with a drop of spore
suspension of test fungus (105spores/ml) to a sterile cavity slide. Positive and negative
control treatments were prepared as a film of recommended fungicide and sterile distilled
water instead of plant extract. Three slides were used as replicates for each treatment.
The slides were kept in moist chamber lined with filter paper and incubated for 48 hours
at 25±2oC. A drop of lactophenol-cotton blue was dispensed to wells immediately after
incubation to stop spore germination. The slides were directly observed under a high
power microscope at 400X, at least 300 spores were marked in four random fields of each
replication and percent inhibition of spore germination was calculated by using the
formula:
67
Inhibition (%) = Conidia germinated in control – Conidia germinated in Treatment x 100 Conidia germinated in control
3.11. In vivo tests of plant extracts and Topsin-M against early blight in greenhouse The protective (1day prior to inoculation) and curative (1 day after inoculation) activities of extracts of three plant species viz. Azadirachta indica (neem), Allium sativum (garlic) and
Ocimum sanctum (Tulsi), showing antifungal properties, were investigated in vivo against early blight in greenhouse studies. Topsin-M treated plants at 250gm/100L served as positive control. Tomato seedlings raised in clay pots were sprayed with plant extracts (20 g/L). The extract were prepared as above, allowed to settle for 1 hour, filtered through muslin cloth and the filtrate was used as foliar spray. Seedlings were maintained in the green house at 25 ± 5oC, 85% relative humidity for 95 days. After harvest, average fruit yield (g/plant) for each treatment was recorded.
Protective activity of treatments
The experiment was carried out twice in the greenhouse in randomized complete block design (RCBD) using three replicates and 5 plants per treatment. 21- day old tomato seedlings were transferred into pots, allowed to establish for about 72 hours and sprayed with 30 ml plant extracts at 80% concentration, until run-off, at 15 day-intervals. These plants were then inoculated with 20 ml suspension of A. solani (5×106 spores/ml) and covered with polyethylene bags for 24 hours to maintain humid conditions, plants inoculated with spore suspension only served as negative control and plants treated with
Topsin-M (250gm/100L) as positive control. All the plants were kept in a climate chamber at a daily temperature of 28±2°C and 85% relative humidity. Disease development was recorded at 15 day intervals from randomly selected leaves of each treatment. The disease
68 severity was recorded on 0–9 Scale as 0= healthy, 1=1-5%, 2=5-10%, 3= 11-25%,
5=2650%, 7= 51-75%, and 9=<76% leaf area infected (Latha et al., 2009). The percent disease index (PDI) was recorded according to formula of Wheeler (1969).
Disease Incidence (%) = Sum of individual disease rating x 100 Number of leaves observed x Maximum disease rating
Curative activity of treatments
Three week-old tomato plants were inoculated with 20ml spore suspension of A. solani
(5x106 spores/ml), and the plants were covered with polyethylene bags for 24 hours to maintain humid conditions. Curative activity of treatments was investigated by application of 30ml of plant extract one day after. Topsin-M treated plants served as positive control and plant simply inoculated as negative control. The experiment was repeated twice in a
RCB design with three replications, comprising five pots per replication. Ten leaves were randomly selected and tagged in each treatment and assessed for disease development at
15-day intervals. The disease severity and PDI were estimated as above.
3.12. Field evaluation of plant extracts and Topsin-M against early blight
A total of 120 plants (60 each for protective and curative treatment) were grown in a plot measuring 25 × 24 meter which was separated by one meter wide drain, plant to plant and row to row distance was maintained at 45 and 60cm, respectively. Four sequential applications of protective and curative plant extracts were made, and untreated plots served as control. The experiment was conducted in randomized complete block design under 3 replication keeping 4 plants per treatment. The trial was repeated twice under similar conditions. Disease progression was recorded at 15-day intervals, four leaves were
69 selected at random in each treatment and severity of early blight disease was recorded on
0-9 scale as mentioned above (Latha et al., 2009; Wheeler 1969). Fruit yield (t/ha) was assessed by selecting three plants from each treatment and the results were averaged.
3.13. Statistical Analysis
The data of different parameters were analyzed by following Analysis of Variance
(ANOVA) to differentiate statistically different means. Fisher’s Least Significant
Difference (LSD) and Tukey’s-Honest Significant Difference (HSD) tests were used for comparison and separation of means at 0.05% level of significance. Standard deviation on different parameters was also applied (Steel et al., 1997). Data were analyzed using general
Statistical Analysis System 8.0 (SAS Institute Cary NC).
4. RESULTS
ISOLATION AND IDENTIFICATION OF TOMATO FUNGI 70
Twenty five fungal species belonging to 16 genera were isolated from various parts of tomato and identified on the basis of standard manuals on fungi. The microscopic and macroscopic characteristics of the fungi involved are presented in Appendix 2. The fungi isolated from leaves, seeds and roots of tomato were cultured, maintained, purified, preserved and used for next inoculations. After the development and establishment of disease, the pathogen was reisolated from the infected host and its identity confirmed, thus
Koch Postulates were fulfilled.
4.1. Association of fungi with tomato seeds
Tomato seeds harbored 17 species of fungi which are distributed in 10 genera (Table 4.1).
Seventeen fungi were isolated by the blotter paper method and 13 fungi in the agar plate method. The two methods of isolation showed significant differences in the frequency and identification of fungi. Fusarium solani was recorded with the highest frequency of 15.0% in blotter test as against 10.6% in the agar plate. It was followed by Alternaria alternata and Aspergillus flavus with frequency of 11.2 and 10.3% in blotter paper method, but only
9.6 and 7.4% in the agar plate method, respectively. The occurrence of Chaetomium globosum, Curvularia lunata and Drecshlera australiensis were 9.85, 7.10 and 6.90% in blotter test but 5.70, 5.20 and 4.50%, respectively, in the agar plale method. A. sulphureus in blotter test and Rhizopus stolonifer in the agar plate were least found (1.35-1.4 %). F. moniliformae, A. fumigatus, Mucor sp and A. sulphureus were not recovered in the agar plate method. In order to overcome contamination problems and excessive culturing, the fungal isolates in question were stored in microbeads and filter paper pieces which enhanced longevity, stability, storability, germination capacity and sporulation.
71
Table 4.1: Frequency % of fungi isolated from tomato seeds using Blotter and Agar plate methods
Blotter Paper Method Agar Plate method Fungal species *Mean Range * Mean Range Alternaria alternate 11.20 b 7-20 7.40 b 7-20 Alternaria tenuissima 5.50 cde 2-14 3.20 efg 2-8 Aspergillus flavus 10.25 b 8-17 9.65 a 7-17 Aspergillus fumigates 2.35 ghi 1-8 0.00 h - Aspergillus niger 6.90 c 3-16 3.70 def 2-16 Aspergillus sulphureus 1.40 i 1-5 0.00 h - Aspergillus terreus 4.10 efg 2-12 2.20 fg 1-9 Chaetomium globosum 9.85 b 3-18 5.70 bc 3-14 Cladosporium sp 4.10 efg 2-12 3.00 efg 1-9 Curvularia lunata 7.10 c 3-18 4.50 cde 4-12 Drecshlera australiansis 6.00 cd 2-13 5.20 cd 3-14 F.moniliformae 3.65 fgh 3-12 0.00 h - F.oxysporumf.sp. lycopersici 4.90 def 2-14 3.25 efg 1-9 Fusarium solani 15.00 a 10-20 10.60 a 5-18 Mucor sp 2.20 hi 1-8 0.00 h - Penicillium digitatum 1.80 i 1-8 4.70 cde 3-12 Rhizopus stolonifer 1.65 i 1-6 1.35 gh 1-4
LSD at P=0.05 1.7707 1.9358 *Values are means of four replications. Mean in each column not sharing a common letter are significantly different at P≤ 0.05 according to Tukey’s HSD test.
4.2. Fungi associated with foliage, fruits and roots All the tomato samples yielded 12 species of filamentous fungi belonging to 13
taxonomic genera (Table 4.2), and non-sporulating were considered as sterile fungi.
Alternaria solani (19%) was found to be predominantly associated with foliar and fruits. It
was followed by F. oxysporum f.sp. lycopersici (17.50%) and A. alternata (15.75%),
whereas occurrence of Phytophthora infestans and Colletotrichum coccodes each was
72
15.25% with non-significant differences. F. solani, Botrytis cineria, Rhizoctonia solani,
Septoria lycopersici, Verticillium albo-atrum, R. stolonifer and Aspergillus spp. were recovered in low frequencies ranging from 2.5 to 12.5%. Tomato leaves yielded high frequency of A. solani (19%) followed by A. alternata, P. infestans and Septoria lycopersici (15.75, 11.5 and 4.50%), respectively. The fungal species belonging to genus
Fusarium, Aspergillus, Rhizoctonia, Botrytis, Rhizopus and Verticillium were not isolated from leaves. The fungi commonly isolated from tomato fruits were: A. solani, C. coccodes and P. infestans but with non-significant differences. Two Fusarium spp. (F. oxysporum f.sp. lycopersici and F. solani) and A. alternata showed moderate appearance of 13.75,
10.25 and 13.0%, respectively, but Rhizopus stolonifer and Aspergillus sp., probably occurring as saprophytic fungi, were observed in low frequency of 2.5-3%. From the tomato roots, only four fungal species were isolated among which F. oxysporum f.sp. lycopersici contributed major share of occurrence (17%) followed by F. solani, R. solani and V. albo-atrum with low share of 12.25, 6 and 3.25%, respectively.
Table 4.2: *Mean Frequency % of fungi isolated from various parts of tomato plant and level of significance
73
Isolated Fungi Parts of tomato plant
Leaf Fruit Root Alternaria alternate 15.75 b 13.00 c 0.00 e Alternaria solani 19.00 a 15.75 b 0.00 e Aspergillus sp. 0.00 e 3.00 ef 0.00 e Botrytis cineria 0.00 e 10.50 d 0.00 e Colletotrichum coccodes 0.00 e 15.25 a 0.00 e Fusarium oxysporum f. sp. lycopersici 0.00 e 13.75 c 17.50 a F. solani 0.00 e 10.25 d 12.25 b Phytophthora infestans 11.50 c 15.25 b 0.00 e Rhizoctonia solani 0.00 e 0.00 g 6.00 c Rhizopus stolonifer 0.00 e 2.50 f 0.00 e Septoria lycopersici 4.25 d 0.00 g 0.00 e Verticillium albo-atrum 0.00 e 0.00 g 3.25 d Non sporulating fungi 0.50 e 0.75 g 0.25 e
LSD at P=0.05 0.9589 1.3707 1.6793 *Values are means of four replications. Mean in each column not sharing a common letter are significantly different at P≤ 0.05 according to Tukey’s HSD test.
74
A B C
1 2 3
D E
4 5
Plate 4.1: Colony growth on PDA medium, of fungi isolated from tomato: (A) Alternaria solani, (B) Phytophthora infestans, (C) Colletotrichum coccodes, (D) Fusarium oxysporum f. sp. lycopersici, (E) Fusarium solani. Reprouctive structures of fungi isolated from tomato: (1) A. solani, (2) P. infestans, (3) C. coccodes, (4) Fusarium oxysporum f.sp. lycopersici, (5) F. solani.
75
4.3. PATHOGENICITY TEST
The results of pathogenicity tests revealed that the isolated fungi were pathogenic on tomato and the reisolated fungi were the same as used for pathogenicity tests, thus full filling the Koch’s postulates (Appendix 3).
4.4. PHYTOCHEMICAL ANALYSIS OF MEDICINAL PLANTS
In order to assess antifungal properties of the medicinal plants it was desirable to better conduct phytochemical analysis of plant extracts and test them biologically for the presence of antifungal properties and main components.
4.4.1. Preliminary phytochemical analysis
Among fifteen plants crude extracts of Azadirachta indica and Allium sativum indicated the presence of flavonoids, saponins, terpenoids, cardiac glycosides, tannins and coumarins while phlobatannins were found absent in both garlic and neem extracts.
Ocimum sanctum when subjected to phytochemical tests revealed the presence of terpenoids, flavonoids, saponins, coumarins, tannins and steroids. Tannins, cardiac glycosides, steroids, flavonoids and terpenoids were found in extracts of Melia azedarach whereas saponins, phlobatanins and coumarins were absent. The extract of Lawsonia inermis revealed the presence of flavonoids, terpenoids, tannins, cardiac glycosides and saponins. Phytochemical tests of Moringa oleifera and Zingiber officinales revealed the presence of flavonoids, terpenoids, tannins and saponins but other constituents were absent. Only three chemical compounds were found in Tagetes minuta, Eucalyptus globules, Citrus limon and Curcuma longa and only two compounds in Syzgium cumini,
Mangifera indica and Polyalthia longifolia. All the chemical constituents were absent in
Catharenthus roseus except cardiac glycosides. Presence (+) or the absence (-) of the 76 components in various test plants is indicated in Table 4.3.
77
Table 4.3: Qualitative analysis of medicinal plants
Plant species Flavonoids Terpenoids Saponins Steroids Cardiac Phlobatannins Tannins Caumarins Glycosides
Azadirachta indica L. + + + + + - + + Allium sativum L. + + + + + - + + Curcuma longa L. + + + - - - - - Ocimum sanctum L. + + + + - - + + Zingiber officinale Rosc + + + - - - + - Eucalyptus globules Labill. - + - - - + + - Syzgium cumini L. + - - - - - + - Lawsonia inermis L. + + - + - + - Melia azedarach L + + - + + - + - Moringa oleifera Lam. + + + - - - + - Tagetes minuta L. - + - + + - - - Catharanthus roseus L. - - - - + - - - Citrus limon (L.)Burm.f. + + - - + - - - Mangifera indica L. + - - - - - + - Polyalthia longifolia Sonn + - - - + - - -
Presence = (+), Absence = (-). Each datum is the average of two independent determinations.
63
4.4.2. Estimation of total phenol contents (TPC) All plants showed appreciable amount of total phenol contents extracted from different plant materials. The results presented in Table 4.4 showed a significant difference
(P<0.05) in TPC amount extracted with different solvents. Intercept (0.0936) and slope
(0.0209) values derived from gallic acid standard curve (Fig 4.1) were used to quantify total phenolic acid in GAE mg/g. The highest yield was obtained from aqueous extract of
Azadirachta indica leaves (56.43 GAE mg/g) followed by bulbs of Allium sativum and
Ocimum sanctum leaves (54.25 GAE mg/g, 53.38 GAE mg/g), respectively, whereas,
Mangifera indica yielded lowest contents of phenols in methanol extract (7.82 GAE mg/g).
The maximum amount of polyphenols was detected in water extract of Azadirachta indica
(56.43 GAE mg/g) while the methanolic extract of A. indica also showed good amount
(51.25 GAE mg/g). Bulbs of Garlic and leaves of Ocimum sanctum also showed considerable high amounts of TPC in water (54.25GAE mg/g) and (53.38GAE mg/g) which are comparable to the TPC of their methanolic extract (50.30 GAE mg/g) respectively. Total phenol contents in terms of GAE mg/g in aqueous extracts from Melia azedarach and Lawsonia inermis were found to be 42.05 and 40.52, respectively. On the other hand the extracts with methanol showed polyphenol contents of Melia and Lawsonia to be 38.83 and 35.78 GAE mg/g.
The rhizomes of Zingiber officinale and leaves of Moringa oleifera yield good amount of
TPC in water extract (29.80 and 29.37GAE mg/g) as compared to methanol extract which showed lower amount of TPC from Zingiber officinale (26.98 GAE mg/g) and Moringa oleifera (25.10 GAE mg/g).
The results indicated that moderate amount of total phenol contents were observed from
Citrus limon, Tagetes minuta and Curcuma longa (27.60, 26.55 and 25.45 GAE mg/g), respectively. While less polyphenols were detected in methanol extracts of Citrus limon,
80
Tagetes minuta and Curcuma longa with TPCs 24.43, 22.98 and 21.75 GAE mg/g, respectively.The water extract yield low TPCs from Catharanthus roseus, Eucalyptus globules, Syzgium cumini, Polyalthia longifolia and Mangifera indica (14.10, 13.02,
11.10, 10.70 and 10.50 GAE mg/g). On the other hand the extracts with methanol showed less total polyphenols (11.50, 10.93, 9.80, 8.60 and 7.82GAE mg/g) as compared to water extract of these plants (Table 4.4).
81
Table 4.4: Total Phenol Contents in aqueous and methanol extracts of plants
Phenol Contents (GAE mg/g)* Sr. No. Plant samples Aqueous Methanol 1 Citrus limon 27.60 ± 0.90c 24.43 ± 0.42c 2 Allium sativum 54.25 ± 0.15a 50.30 ± 0.56a 3 Melia azedarach 42.05 ± 0.50b 38.83 ± 0.33b 4 Ocimum sanctum 53.38 ± 0.13a 48.03 ± 0.85a 5 Lawsonia inermis 40.52 ± 0.43b 35.78 ± 0.32b 6 Zingiber officinale 29.80 ± 0.45c 26.98 ± 0.31c 7 Azadirachta indica 56.43 ± 0.10a 51.25 ± 0.13a 8 Eucalyptus globules 13.02 ± 0.34d 10.93 ± 0.47d 9 Curcuma longa 25.45 ± 0.48c 21.75 ± 0.48c 10 Tagetes minuta 26.55 ± 0.38c 22.98 ± 0.28c 11 Moringa oleifera 29.37 ± 0.28c 25.10 ± 0.39c 12 Catharanthus roseus 14.10 ± 0.58d 11.50 ± 0.36d 13 Mangifera indica 10.50 ± 0.33d 7.82 ± 0.82d 14 Polyalthia longifolia 10.70 ± 0.23d 8.60 ± 0.50d 15 Syzgium cumini 11.10 ± 0.23d 9.80 ± 0.39d LSD at P=0.05 9.664 5.369
*Data are means (n=3) ± Standard Deviation. Mean in each column not sharing a common letter are significantly different at P≤ 0.05 according to Tukey’s HSD test.
82
2.5 y=0.0209x+0.0936 R2 =0.9986 2
1.5
1
0.5
0
0 20 40 60 80 100 120
Gallic acid concentration (ppm)
Fig 4.1: Gallic acid calibration curve 4.5. DETERMINATION OF ANTIFUNGAL ACTIVITIES OF PLANT
EXTRACTS USING POISONED FOOD TECHNIQUE (IN VITRO)
Laboratory assay of aqueous and solvent extracts of fifteen botanicals, prepared in fresh and dry formulations at different concentrations, were carried out against five target fungal pathogens of tomato. Positive and negative controls were included in each case. Results are expressed as Means (average of 3-4 replications), significance as L.S.D. was found at
P= 0.05 (Fisher’s LSD test). The effects of plant extracts were categorized as ineffective
(0-10%), least (10-30%), moderate (30-50%), effective (50-90%) and highly effective (90-
100%). The results are presented in Tables 4.5- 4.8. The interactive effects of plant extracts and concentrations showed highly significant differences.
4.5.1. EFFICACY OF AQUEOUS EXTRACTS
4.5.1.1. Aqueous fresh extracts
4.5.1.1.1. Neem, Azadirachta indica extract
Neem leaf extract inhibited and suppressed mycelial growth of test fungi ranging between 90 to 100% at 20, 40, 60 and 80% concentrations. Results presented in Table 4.5
83 showed highly significant difference in antifungal activity against target fungi with respect to formulation and extract concentrations. Freshly prepared leaf extract completely inhibited mycelial growth of test fungi except P. infestans. The effectiveness increased with increase in the concentration. The leaf extract at 60% and 80% concentration completely inhibited growth of A. solani. Similarly 100% inhibition was also achieved at
80% concentration against F. oxysporum f. sp. lycopersici and C. coccodes followed by
F.solani (98.61%) but least inhibition of 0.65-9.08% was observed in case of P. infestans at 20, 40, 60 and 80% concentrations.
4.5.1.1.2. Garlic, Allium sativum
The aqueous extract of A. sativum bulbs showed complete inhibition of the mycelial growth of A. solani, F. oxysporum f. sp. lycopersici and F. solani at higher concentration.
Extract found highly effective against C. coccodes and P. infestans inhibiting the mycelial growth up to 96.81%. Inhibitory activity increased with the increase of concentrations.
The results obtained were highly significant (Table 4.5).
4.5.1.1.3. Tulsi, Ocimum sanctum
The data (Table 4.5) indicated that aqueous extract of Ocimum leaves was highly effective against all target fungi. Extract at higher concentrations offered complete inhibition of mycelial growth of A. solani, F. solani and C. coccodes. Aqueous extract showed maximum inhibition ranging from 90.60% to 99.27% against P. infestans and F. oxysporum f. sp. lycopersici respectively.
4.5.1.1.4. Chinaberry, Melia azedarach
Results summarized in Table 4.5 suggest that leaf extract of Melia azedarach at various concentrations were effective in inhibiting the mycelial growth of test fungi. Maximum
84 inhibition of 88.61% was observed against F. solani followed by A. solani (81.96%) and the lowest of 61.10% against P. infestans at 80% concentration.
4.5.1.1.5. Henna, Lawsonia inermis
The results (Table 4.5) reveal that maximum inhibition range (65.27 to 86.52%) of growth was observed against F. solani, followed by F. oxysporum f. sp. lycopersici (63.14-
80.19%) at 20, 40, 60 and 80 percent concentrations, while lowest inhibition of
50.40% was observed against P. infestans at 80% concentration.
4.5.1.1.6. Ginger, Zingiber officinale
Results presented in Table 4.5 depicted that aqueous extract of Zingiber rhizome at various concentrations was significantly but moderately effective (30.33-50%) against A. solani, C. coccodes, F. solani and F. oxysporum f. sp. lycopersici but ineffective against
P. infestans.
4.5.1.1.7. Moringa oleifera
Aqueous extract of Moringa leaves at different concentrations was moderately effective
(47.96-29.50%) against F. oxysporum f. sp. lycopersici, F. solani, A. solani, P. infestans and C. coccodes (Table 4.5).
4.5.1.1.8. Lemon, Antifungal activity of Citrus limon
Leaf extract of Citrus limon was moderately antifungal at various concentrations against
A. solani,, C. coccodes and P. infestans, its activity was low against F. oxysporum f. sp. lycopersici and was ineffective to F. solani at all concentrations (Table 4.5).
4.5.1.1.9. Tagetes minuta
Leaf extract of Tagetes minuta was moderately effective against all fungi (Table
85
4.5). Maximum growth inhibition occurred in A. solani and F. oxysporum f. sp. lycopersici
(48.72, and 46.80%) respectively followed by C. coccodes (43.95 percent), minimum against F. solani and none against P. infestans.
4.5.1.1.10. Curcuma longa (domestica)
Aqueous extract of Curcuma rhizomes at various concentrations was moderately effective against A. solani, P. infestans, F. oxysporum f. sp. lycopersici and C. coccodes, but ineffective against F. solani (Table 4.5).
4.5.1.1.11. Periwinkle. Catharanthus roseus
Results given in Table 4.5 indicate that the aqueous extract of Catharenthus leaves was least effective in inhibiting the growth of all fungi. Mycelial growth of fungi in general was inhibited by 29.93% at 80% concentration; only 28.01% reduction was noted for F. oxysporum f sp. lycopersici.
4.5.1.1.12. Eucalyptus globules
Aqueous extract of Eucalyptus leaves was least effective against all fungi, as the mycelial growth of A. solani was inhibited by 29.26%, but inhibitoty activity was very slight against rest of the fungi (Table 4.5).
4.5.1.1.13. Polyalthia longifolia, Syzgium cumini and Mangifera indica
Though the results illustrated in table (4.5) were statistically significant at all concentrations but aqueous leaf extract of Polyalthia longifolia, Syzgium cumini and
Mangifera indica were found to be ineffective against all the test fungi.
4.5.1.2. Aqueous dry powder extracts
Table 4.6 revealed that strong inhibition of test fungi was observed with aqueous dry powder extract of neem as compared to negative control. Maximum reduction
86
(98.74%) in mycelial growth was observed against F. solani at highest concentration followed by C. coccodes, P. infestans, F. oxysporum f. sp. lycopersici and A. solani.
Similarly, dry powder of garlic bulbs at all concentrations also resulted in maximum inhibition of mycelial growth of fungi, 80% concentration of powder extract was highly effective against all fungi particularly, C. coccodes (100%), F. solani (98.60%),
A. solani (98.46%) and F. oxysporum f.sp. lycopersici (98.45%), and ineffective against
P. infestans (Table 4.6).
Dry formulation of Ocimum sanctum at all concentrations was also highly inhibitory against all fungi but moderately effective against A. solani and P. infestans
(Table 4.6). F. solani was significantly inhibited by Tulsi extract upto 99.02% followed by F. oxysporum f.sp. lycopersici (98.59%) and C. coccodes (98.57%).
Results presented in table 4.6 depicted that dry formulations of Melia leaves are also effective against all fungi resulting into inhibition of mycelia growth of test fungi ranging between 50.43-74.04%. Similar results are also observed with dry formulation of
Lawsonia inermis at significant level (Table 4.6). Maximum inhibition (78.06%) was observed against F. oxysporum f. sp. lycopersici followed by A. solani, F. solani, C. coccodes and P. infestans.
Dry powder extract of Zingiber officinale was found moderately effective in reducing mycelia growth of A. solani, C. coccodes, F. solani and F. oxysporum f.sp. lycopersici (31.22-50.63%) but was ineffective against P. infestans. Dry powder extract of Moringa oleifera leaves also showed moderate activity against all fungi. Similarly,
Citrus limon was found moderately effective in inhibiting the growth of test fungi except
F. solani. The Tagetes minuta showed moderate inhibition (31.00 to 45.99%) against A. solani, F. oxysporum f. sp. lycopersici and C. coccodes. Least inhibition was found against
87
F. solani and no inhibition observed against P. infestans (Table 4.6).Whereas, dry powder extract of Curcuma longa moderately suppressed mycelial growth of F. oxysporum f. sp. lycopersici (46.83%), F. solani (46.16%) and C. coccodes (40.82%) and slightly effective against A. solani. But the extract found ineffective against P. infestans (Table 4.6).
Results presented in table 4.6 showed that aqueous dry powder extract of
Catharanthus roseus was least effective against all the test fungi.Conversely, dry powder extract of Eucalyptus globules showed effective inhibition only against A. solani (71.39%) at 80% concentration. Polyalthia longifolia, Syzgium cumini and Mangifera indica
Similarly, dry powder extract also showed no inhibition against mycelial growth of fungi at various concentrations.
88
Table 4.5: Antifungal effect of aqueous fresh plant extracts at different concentrations against important tomato fungi (Percent growth reduction)
*Conc. F.oxysporum f. sp. Extract A. solani F. solani C. coccodes P. infestans (%) lycopersici 20 91.05c (72.59) 95.37d (77.58) 91.94d (73.51) 90.13d (71.68) 0.65d (4.64) 40 94.03b (75.86) 97.69c (81.25) 94.44c (76.36) 91.88c (73.44) 2.59c (9.27) Azadirachta indica 60 100.00a (90.0) 98.69b (83.45) 96.80b (79.70) 97.13b (90.0) 6.65b (14.94) 80 100.00a (90.0) 98.61a (83.23) 100.00a (80.25) 9.00a (17.53) **LSD 0.67 1.35 1.32 1.25 20 92.04d (73.61) 90.31d (71.86) 91.11d (72.65) 90.60c (72.15) 53.48c (46.99) 40 94.17c (76.03) 92.91c (74.56) 93.05c (74.72) 93.31b (75.01) 90.92b (72.46) Allium sativum 60 98.58b (83.16) 96.38b (79.04) 95.41b (77.63) 96.17a (78.72) 91.73b (73.29) 80 100.00a (90.0) 100.00a (90.0) 96.81a (79.72) 94.8a (76.83) LSD 0.94 0.76 1.42 1.62 20 93.03d (74.70) 90.60c (72.15) 91.94c (73.51) 93.31c (75.01) 90.60c (72.14) 40 95.31c (77.49) 92.77b (74.40) 95.69b (78.02) 96.02b (78.49) 93.03b (74.69) Ocimum sanctum 60 98.58b (83.15) 98.98a (85.02) 100.00a (90.0) 100.00a (90.0) 95.78a (78.150 80 99.27a (86.56) 100.00a (90.0) 100.00a (90.0) 96.27a (78.86) LSD 1.07 0.54 0.76 1.12 20 54.68d (47.68) 52.30d (46.31) 60.83d (51.25) 52.54d (46.46) 50.40d (45.23) 40 63.49c (52.82) 56.06c (48.48) 69.99c (56.78) 60.03c (50.78) 52.03c (46.16) Melia azedarach 60 78.69b (62.51) 66.61b (54.70) 82.91b (65.58) 71.65b (57.83) 59.32b (50.37) 80 72.25a (58.21) 88.61a (70.27) 79.61a (63.16) 61.10a (51.41) LSD 0.94 1.00 1.39 1.26
Continued ….
89
20 51.13d (45.65) 63.14d (52.61) 65.27d (53.89) 56.68d (48.84) 42.79c (40.85) 40 57.67c (49.41) 67.04c (54.96) 77.77c (61.87) 60.03c (50.78) 47.00b (43.28) Lawsonia inermis 60 63.49b (52.82) 76.73b (61.15) 79.72b (63.23) 62.73b (52.38) 50.57a (45.32) 80 80.19a (63.57) 86.52a (68.46) 68.15a (55.64) 50.40a (45.23) LSD 1.12 1.19 1.26 1.51 20 31.81d (34.33) 30.33d (33.41) 37.77d (37.92) 38.53d (38.37) 1.30cd (6.55) 40 38.35c (38.26) 33.94c (35.63) 43.05c (41.00) 42.19c (40.51) 2.43c (8.98) Zingiber officinale 60 44.60b (45.0) 39.87b (39.15) 45.55b (42.45) 46.49b (42.99) 4.05b (11.62) 80 44.49a (41.83) 48.47a (44.12) 49.68a (44.81) 7.78a (16.20) LSD 1.17 1.58 1.96 1.61 20 30.82d (33.72) 38.42d (38.30) 33.05d (35.09) 33.75d (35.52) 29.50d (32.89) 40 36.65c (37.25) 39.87c (39.15) 42.22c (40.52) 35.98c (36.86) 33.87c (39.25) Moringa oleifera 60 39.34b (38.84) 45.79b (42.58) 44.02b (41.56) 38.21b (38.18) 40.03b (35.59) 80 47.96a (43.83) 47.77a (43.72) 43.63a (41.34) 46.51a (43.0) LSD 1.26 1.34 1.68 1.52 20 31.81d (34.33) 14.43d (22.32) 0.00b (0.0) 35.98c (36.86) 29.99c (33.20) 40 37.64c (37.84) 18.76c (25.67) 0.00b (0.0) 38.85b (38.55) 33.55b (35.39) Citrus limon 60 45.45b (42.39) 24.69b (29.79) 0.00b (0.0) 39.81b (39.11) 30.79c (33.70) 80 50.00a (45.0) 26.86a (31.21) 2.22a (8.57) 44.74a (41.98) 35.01a (36.27) LSD 1.21 1.12 1.75 1.01 20 36.40c (37.10) 12.22d (20.46) 32.16d (34.55) 0.17a (2.35) 40 38.49c (43.04) 43.05b (41.08) 15.28c (22.99) 35.82c (36.76) 0.17a (2.35) Tagetes minuta 60 46.59b (38.34) 43.19b (41.00) 21.80b (27.83) 38.53b (38.37) 0.17a (2.35) 80 46.80a (43.17) 26.39a (30.91) 43.95a (41.52) 0.17a (2.35) LSD 1.70 1.47 1.45 0.00
90
Continued ….
20 30.96d (33.81) 32.93d (35.02) 2.91c (9.83) 30.89d (33.76) 32.41d (34.70) 40 37.35c (37.67) 38.57c (38.39) 5.27b (13.28) 33.27c (35.23) 36.14c (36.95) Curcuma longa 60 43.04b (40.99) 41.02b (39.83) 7.77a (16.19) 38.53b (38.37) 43.76b (41.41) 80 50.00a (45.0) 47.67a (43.66) 8.61a (17.06) 41.24a (39.95) 47.81a (43.74) LSD 1.70 1.06 1.26 1.41 1.75
20 12.36d (20.58) 15.44d (23.13) 16.39c (23.88) 20.86c (27.17) 12.97d (21.11) Catharenthus roseus 40 18.18c (25.23) 17.61c (24.81) 16.66c (24.09) 21.97c (27.95) 15.24c (22.97)
60 22.01b (27.98) 23.52b (28.33) 18.61b (25.55) 25.00b (30.0) 17.02b (24.36)
80 29.26a (32.74) 28.01a (31.95) 25.55a (30.36) 29.93a (33.17) 19.45a (26.17)
LSD 1.33 1.37 1.13 1.62 20 14.14d (22.08) 11.94d (20.21) 15.60d (23.26) 17.99a (25.10)
Eucalyptus globules 40 22.38b (28.23) 15.14c (22.89) 17.83c (24.98) 19.45a (26.17) 18.89c (25.76) 60 24.55a (29.70) 17.50b (24.72) 24.36b (29.57) 15.24b (22.97) 23.86b (29.24) 80 16.74c (24.15) 19.99a (26.56) 28.34a (32.16) 18.15a (25.22) LSD 1.36 1.41 0.95 1.46 20 2.55d (8.94) 1.85c (7.82) 0.83c (4.43) 1.90b (7.94) 1.62c (6.80) 40 4.83c (12.69) 4.02b (11.57) 3.33b (10.51) 2.86b (9.74) 4.06b (11.62) Syzgium cumini 60 6.67b (14.97) 4.89b (12.77) 6.39a (14.64) 7.00a (15.34) 5.67b (13.78) 80 9.09a (17.54) 9.80a (18.24) 7.36a (15.74) 8.43a (16.88) 8.92a (17.37) LSD 1.82 1.85 1.20 1.59 2.35
91
20 1.70bc (6.46) 1.56bc (6.91) 0.69cd (3.99) 0.63cd (4.57) 0.17c (2.35) 40 3.69b (10.85) 3.01b (9.99) 1.94c (7.69) 1.59c (7.24) 3.73b (11.14) Polyalthia longifolia 60 5.82a (13.96) 5.61a (13.70) 4.44b (12.17) 5.57b (13.65) 4.05b (11.62) 80 7.95a (16.38) 7.63a (15.95) 7.50a (15.89) 7.96a (16.38) 7.62a (15.87) LSD 2.13 2.33 1.57 1.56 2.51
Continued ….
20 4.54c (12.30) 6.33c (14.57) 1.80d (7.72) 0.47d (2.76) 2.43cd (8.98) 40 6.81b (15.13) 8.36b (16.80) 4.44c (12.17) 4.62c (12.40) 4.86c (12.74) Mangifera indica 60 6.96b (15.29) 8.50ab (16.95) 6.39b (14.64) 6.53b (14.80) 7.94b (16.37) 80 9.94a (18.38) 9.95a (18.38) 8.88a (17.34) 9.23a (17.69) 10.54a (18.94) LSD 1.54 1.48 1.27 1.15 2.53 Topsin - M 100.00a (90.0) Control negative 0.00e (0.0) to Fisher’s LSD test. Data in parenthesis are arcsine transformed values. *Conc. = Concentration. **LSD= Least significance difference.
92
Table 4.6: Antifungal effect of aqueous dry plant extracts at different concentrations against important tomato fungi (Percent growth reduction) *Conc. F.oxysporum f. sp. Extract A. solani F. solani C. coccodes P. infestans (%) lycopersici 20 90.04d (71.64) 90.99c (72.56) 90.09d (71.68) 90.82d (72.37) 89.98d (71.56) 40 91.86c (73.44) 92.96b (74.63) 92.89c (74.53) 92.24c (73.86) 93.09c (74.79) Azadirachta indica 60 94.39b (76.30) 96.62a (79.53) 95.54b (77.81) 95.72b (78.13) 95.33b (77.60) 80 96.35a (79.03) 97.18a (80.46) 98.74a (83.59) 98.41a (82.80) 97.23a (80.67) **LSD 1.36 1.4 1.06 1.33 1.75 20 91.86c (73.45) 90.71c (72.27) 90.10d (71.70) 90.66d (72.22) 2.25c (8.21) 40 95.51b (77.84) 90.98c (73.58) 93.02c (74.70) 93.35c (75.11) 4.49b (12.16) Allium sativum 60 97.61a (81.26) 95.49b (77.82) 96.79b (79.76) 94.93b (77.05) 6.74a (14.75) 80 98.46a (82.94) 98.45a (82.93) 98.60a (83.25) 100.00a (90.0) 8.46a (16.90) LSD 1.33 1.31 1.48 1.23 2.15 20 61.71d (51.77) 90.85c (72.42) 90.93d (72.50) 90.82d (72.37) 68.04c (55.58) 40 66.06c (54.37) 91.56c (73.13) 92.88c (74.55) 93.51c (75.28) 71.85b (57.95) Ocimum sanctum 60 77.14b (61.44) 95.49b (77.81) 96.65b (79.47) 95.88b (78.34) 63.39d (52.76) 80 84.01a (66.44) 98.59a (83.22) 99.02a (85.11) 98.57a (83.17) 77.89a (61.96)
93
LSD 1.15 1.45 1.31 1.04 1.95 20 54.00d (47.29) 51.20d (45.68) 52.86d (46.64) 54.11d (47.36) 50.43d (45.24) 40 59.47c (50.45) 55.84c (48.35) 58.71c (50.02) 60.12c (50.84) 55.44c (48.12) Melia azedarach 60 67.18b (55.05) 60.48b (51.05) 64.85b (53.64) 68.51b (55.86) 59.93b (50.73) 80 72.09a (58.11) 65.68a (54.14) 73.22a (58.83) 74.04a (59.37) 62.52a (52.25) LSD 1.58 1.26 1.27 1.53 1.72
Continued ….
20 52.60d (46.48) 55.84d (48.35) 50.63d (45.36) 51.58d (45.90) 31.95d (34.42) 40 58.49c (49.88) 64.70c (53.54) 57.46c (49.29) 56.32c (48.63) 38.00c (38.05) Lawsonia inermis 60 65.08b (53.77) 72.85b (58.60) 64.43b (53.39) 63.13b (52.61) 44.73b (41.97) 80 72.79a (58.56) 78.06a (62.07) 69.73a (56.62) 66.45a (54.61) 49.91a (44.95) LSD 0.86 1.33 1.45 1.84 1.72 20 32.40d (34.69) 31.22d (33.97) 35.29d (36.44) 34.01d (35.67) 1.60c (6.76) 40 37.03c (37.48) 33.75c (35.52) 41.70c (40.22) 38.76c (38.50) 3.46b (10.68) Zingiber officinale 60 43.48b (41.25) 37.55b (37.79) 45.88b (42.64) 50.31a (45.18) 4.67ab (12.41) 80 50.63a (45.36) 46.13a (42.78) 47.28a (43.44) 46.83b (43.18) 5.87a (13.94) LSD 1.29 1.02 1.17 1.36 1.82 20 31.42d (34.09) 34.88d (36.20) 30.82d (33.72) 33.86c (35.58) 30.92d (33.78) 40 35.77c (36.72) 37.83c (37.96) 36.40c (37.10) 38.13b (38.13) 39.55b (38.96) Moringa oleifera 60 41.10b (39.87) 44.30b (41.73) 46.16a (42.80) 37.81b (37.94) 33.50c (35.36) 80 45.86a (42.62) 45.99a (42.70) 44.21b (41.67) 41.14a (39.89) 43.00a (40.98) LSD 1.40 1.33 1.59 1.70 2.12
94
20 32.82d (34.95) 33.33d (35.26) 0.84abc (4.64) 36.55b (37.19) 30.92b (33.78) 40 35.91c (36.81) 38.68c (38.45) 0.70c (4.23) 37.02b (37.46) 34.89a (36.20) Citrus limon 60 44.88b (42.06) 43.46b (41.24) 1.54ab (6.96) 40.66ab (39.60) 31.95b (34.42)
80 47.97a (43.83) 46.97a (43.26) 1.81a (7.62) 44.62a (41.90) 36.27a (37.03) LSD 1.35 1.58 1.09 4.34 1.94 20 31.00d (33.82) 34.46d (35.94) 11.58d (19.87) 32.75c (34.90) 0.53ab (4.15) 40 35.77c (36.72) 40.08c (39.28) 14.65c (22.49) 34.96bc (36.24) 0.61a (4.24) Tagetes minuta 60 38.15b (38.14) 42.47b (40.67) 21.90b (27.89) 36.55b (37.19) 0.70a (4.69) 80 43.48a (41.25) 45.99a (42.70) 25.94a (30.62) 42.24a (40.53) 0.87a (5.10) LSD 1.28 1.04 1.37 2.49 0.59
Continued ….
20 14.45d (22.33) 33.33d (35.26) 30.54d (33.55) 30.38c (33.43) 0.00b (0.0) 40 17.96c (25.06) 38.68c (38.45) 35.43c (36.52) 34.17b (35.76) 0.00b (0.0) Curcuma longa 60 24.41b (29.60) 43.60b (41.32) 43.10b (41.03) 36.23b (37.0) 0.00b (0.0) 80 28.75a (32.42) 46.83a (43.18) 46.16a (42.80) 40.82a (39.70) 7.25a (15.59) LSD 0.86 1.37 1.28 2.76 0.81 20 11.36d (19.68) 13.08d (21.19) 13.95d (21.92) 20.56c (26.95) 13.30c (21.34) 40 15.29c (23.01) 17.86c (24.99) 17.43c (24.67) 21.51bc (27.61) 15.03bc (22.79) Catharenthus roseus 60 18.23b (25.26) 21.38b (27.54) 19.11b (25.91) 24.68ab (29.76) 17.10b (24.41) 80 25.67a (30.43) 25.32a (30.21) 23.85a (29.23) 27.37a (31.53) 19.52a (26.21) LSD 1.35 1.52 1.29 3.23 2.23 20 52.60d (46.48) 14.35d (22.25) 11.72d (20.0) 15.50b (23.11) 12.61d (20.78) 40 55.82c (48.34) 20.68b (27.04) 15.20c (22.94) 18.35b (25.32) 17.79b (24.94) Eucalyptus globules 60 66.48b (54.62) 24.33a (29.55) 17.57b (24.78) 24.05a (29.34) 15.20c (22.93) 80 71.39a (57.66) 16.32c (23.82) 20.64a (27.02) 26.58a (31.02) 20.90a (27.19)
95
LSD 1.25 0.86 0.75 4.08 2.03 20 1.13d (5.59) 0.99c (5.39) 0.98d (5.29) 3.48bc (10.69) 0.53c (4.15) 40 3.23c (10.27) 3.80b (11.14) 2.93c (9.78) 3.79b (10.97) 3.29b (10.19) Syzgium cumini 60 5.19b (13.14) 5.21b (13.13) 5.02b (12.91) 5.85ab (13.04) 4.67b (12.21) 80 8.14a (16.55) 8.02a (16.40) 6.83a (15.12) 8.86a (17.21) 7.43a (15.76) LSD 1.28 1.64 1.31 3.68 1.86 20 1.27d (5.91) 1.83d (7.66) 1.15d (5.09) 0.00c (0.0) 0.53b (4.15) 40 3.23c (10.30) 3.94c (11.34) 2.51c (8.98) 2.05bc (6.94) 1.91b (7.32) Polyalthia longifolia 60 6.87b (15.14) 6.33b (14.53) 3.77b (11.15) 4.58ab (12.0) 6.39a (14.13) 80 7.86a (16.25) 8.86a (17.30) 6.84a (15.15) 6.64a (14.65) 8.46a (16.76) LSD 0.97 1.58 1.16 3.09 3.46
Continued ….
20 2.25d (8.48) 3.52c (10.64) 2.23c (7.75) 0.00c (0.0) 1.56c (6.92) 40 4.35c (11.99) 5.63b (13.70) 4.47b (12.04) 3.64b (10.66) 4.67b (12.39) 60 7.44b (15.80) 8.30a (16.74) 6.14b (14.31) 7.12a (15.30) 8.12a (16.47) 80 8.98a (17.42) 8.86a (17.31) 8.51a (16.94) 8.70a (17.05) 10.02a (18.33) LSD 1.43 1.36 1.84 2.85 2.72 Topsin - M 100.00a (90.0) Control negative 0.00e (0.0) Mangifera indica
to Fisher’s LSD test. Data in parenthesis are arcsine transformed values. *Conc. = Concentration. **LSD= Least significance difference.
96
Plate 4.2: Bioefficacy of aqueous extracts of neem (left) and garlic (right) on mycelial growth of A. solani. .
4.5.2. EFFECTIVENESS OF METHANOL EXTRACTS
In vitro Antifungal activity of methanol extracts of the medicinal plants in aqueous and dry powder formulations was assessed with positive (Topsin-M) and negaive controls
Rsults are presented in Tables 4.7 and 4.8. Methanol extracts in general exhibited less inhibitory activity against the target fungi than aqueous extracts. However, freshly
97 prepared methanol extracts were comparatively more effective than dry formulations in methanol.
4.5.2.1. Azadirachta indica
Data presented in Table 4.7 revealed that methanol extracts of fresh leaves of neem were less effective but concentration effect was significant over controls. Maximum reduction of radial growth (91.61-100%) was found in F. oxysporum f. sp. lycopersici followed by
A. solani (90.91-97.73%) and F. solani (90-98.61%) at 20-80% concentration. Neem leaf extract effectively suppressed mycelial growth of C. coccodes but was ineffective against
P. infestans over negative control, but positive control also indicated effectiveness. Dry formulation exhibited significant inhibitory effect on fungal growth of test fungi. It effectively suppressed the growth of A. solani (98.74%) followed by F. oxysporum f. sp. lycopersici (96.29%), C. coccodes (97.46%) and F. solani (95.26%) at
80% concentration (Table 4.8).
4.5.2.2. Allium sativum
Methanol extracts of fresh bulbs of A. sativum showed significantly strong inhibition (90.46 -98.58%) against A. solani, F. solani, F. oxysporum f. sp. lycopersici, P. infestans and C. coccodes. Likewise dry formulation of extracts significantly and greatly reduced the growth of all fungi except P. infestans (Table 4.7, 4.8).
4.5.2.3. Ocimum sanctum
The results presented in Table 4.7 revealed that methanol solvent extract of O. sanctum leaves significantly reduced the mycelial growth of all test fungi. The highest
98 concentration of 80% greatly or completely suppressed the growth of F. solani (100%) followed by F. oxysporum f.sp. lycopersici (98.69%) over control. Likewise, dry powder extract of Tulsi leaves also showed strong inhibition against all fungi.
4.5.2.4. Melia azedarach
Leaves of M. azedarach freshly prepared in methanol solvent significantly reduced the colony growth of all test fungi in a range of 50.08 - 85.11% at different concentrations.
Among the fungi, F. oxysporum f. sp. lycopersici was effectively suppressed by (65.02-
69.93, 71.81 and 85.11%) by the solvent extract at 20%, 40%, 60% and 80%, respectively
(Table 4.7). Similarly, dry powder extract of Melia also showed adverse effects on mycelial growth of all fungi (Table 4.8).
4.5.2.5. Lawsonia inermis
Both the formulations of methanol extract of L. inermis significantly reduced the growth of all fungi except P. infestans which was less inhibited as compared to control. Maximum reduction of 74.16% (fresh) and 74.96% (dry) was noticed in radial growth of F. solani and F. oxysporum f.sp. lycopersici respectively (Table 4.7 and 4.8).
4.5.2.6. Zingiber officinales
Methanol extract of fresh rhizomes of Z. officinale showed statistically significant effects on all test fungi at various concentrations. It moderately reduced the mycelial growth of test fungi but ineffective against A. solani and P. infestans. Similarly, dry powder extract of Z. officinale significantly declined mycelial growth of C. coccodes by 49.52%, but
99 ineffective against P. infestans (Table 4.8). There was a gradual reduction in fungal growth with increase of concentration from 20-80%.
4.5.2.7. Moringa oleifera
The fresh methanol extract of M. oleifera leaves significantly reduced the mycelial growth of test fungi in a range of 30.39 to 47.82% over control (Table 4.7). But it was ineffective against P. infestans at different concentrations. Dry formulation of Moringa leaves extract inhibited the growth of different fungi at moderate level but the differences were statistically significant.
4.5.2.8. Citrus limon
Methanolic solvent of C.limon leaves were significantly but moderately effective against
A.solani and C. coccodes (31-50%) at 20, 40, 60 and 80% concentrations, least effective against F. oxysporum f. sp. lycopersici and P. infestans but no activity was observed against F. solani. Dry powder extract moderately reduced the radial growth of F. oxysporum f. sp. lycopersici, C. coccodes, and P. infestans. Extract showed least inhibition against A. solani but was ineffective against F. solani (Table 4.7, 4.8).
4.5.2.9. Tagetes minuta
Methanol extract of fresh and dry Tagetes flowers were effective at all concentrations against different fungi. They moderately reduced the mycelial growth of A. solani, F.
100 oxysporum f. sp. lycopersici, C.coccodes, but least inhibition against F. solani and none against P. infestans were noticed (Table 4.7, 4.8).
4.5.2.10. Curcuma longa (domestica)
The solvent extracts from fresh and dry rhizomes of C. longa were moderately effective against some fungi at different concentrations and inactive against F. solani and
P. infestans. Inhibition capacity increased with increase in concentration. Dry formulation gave maximum growth inhibition of 49.93% against A. solani but fresh extract was effective (47.82%) against F. oxysporum f. sp. lycopersici at 80% concentration (Table
4.7 and 4.8).
4.5.2.11. Catharanthus roseus
Fresh and dry leaf methanol extracts of periwinkle exhibited least inhibitory effect against all test fungi at various concentrations. Fresh leaves extract was ineffective against P. infestans (Table 4.7, 4.8).
4.5.2.12. Eucalyptus globules
Fresh and dry formulations of E. globules at different concentrations showed least inhibition against all fungi (Table 4.7, 4.8).
4.5.2.13. Syzgium cumini, Polyalthia longifolia and Mangifera indica
101
Methanol extracts of Syzgium cumini, Polyalthia longifolia and Mangifera indica were found almost ineffective against all test fungi with both fresh and dry formulations
(Table 4.7 and 4.8).
102
Table 4.7: Antifungal effect of methanol fresh plant extracts at different concentrations against important tomato fungi (Percent growth reduction) *Conc. F.oxysporum f. sp. Extract A. solani F. solani C. coccodes P. infestans (%) lycopersici 20 90.91d (72.47) 91.61d (73.18) 90.00d (71.56) 66.56d (54.67) 0.17d (2.35) 40 93.04c (74.72) 93.78c (75.59) 92.08c (73.69) 72.13c (58.14) 3.25c (9.99) Azadirachta indica 60 95.02b (77.15) 96.38b (79.16) 95.14b (77.30) 79.61b (63.16) 6.81b (15.07) 80 97.73a (81.36) 100.00a (90.0) 98.61a (83.26) 87.10a (68.97) 9.08a (17.51) **LSD 1.10 1.11 1.03 1.51 1.49 20 91.76c (73.33) 90.46c (72.01) 90.83c (72.38) 91.24d (72.79) 90.60c (72.15) 40 92.04c (73.65) 92.91b (74.59) 93.33b (75.07) 92.35c (73.98) 93.03b (74.77) Allium sativum 60 96.16b (78.77) 95.37a (77.59) 95.14a (77.30) 94.58b (76.57) 96.11a (78.64) 80 98.58a (83.18) 96.24a (78.86) 96.39a (79.11) 95.70a (78.10) 96.11a (78.64) LSD 1.34 1.12 1.41 1.03 1.38 20 90.62c (72.20) 90.31d (71.88) 90.00d (71.58) 91.72c (73.28) 52.84c (46.62) 40 93.18b (74.89) 91.61c (73.18) 94.86c (76.94) 94.58b (76.57) 90.60b (72.17) Ocimum sanctum 60 96.16a (78.77) 96.53b (83.47) 96.66b (79.49) 98.41a (82.78) 92.05b (73.66) 80 96.44a (79.19) 98.69a (79.35) 100.00a (90.0) 98.57a (83.15) 94.48a (76.47) LSD 1.53 0.87 0.77 0.68 1.71 20 52.27d (46.30) 65.02d (53.74) 58.75d (50.0) 52.70d (46.55) 50.57b (45.32) 40 57.67c (49.41) 69.93c (56.75) 65.27c (53.89) 57.48c (49.30) 52.03b (46.16) Melia azedarach 60 63.63b (52.91) 71.81b (57.93) 73.33b (58.91) 61.46b (51.62) 59.48a (50.46) 80 68.74a (56.01) 85.11a (67.31) 78.47a (62.35) 79.45a (63.05) 61.26a (51.51) LSD 1.75 1.39 1.11 1.44 1.78
Continued ….
103
20 50.00d (44.99) 52.44d (46.40) 55.27d (48.02) 51.75d (46.0) 32.25d (34.60) 40 56.81c (48.91) 56.92c (48.98) 67.91c (55.50) 53.82c (47.19) 39.22c (38.77) Lawsonia inermis 60 58.80b (50.07) 62.42b (52.19) 69.99b (56.79) 57.32b (49.21) 42.79b (40.85) 80 63.77a (52.99) 70.65a (57.20) 74.16a (59.45) 63.05a (52.57) 47.81a (43.74) LSD 1.76 1.26 1.30 1.39 1.78 20 0.00c (0.0) 30.33d (33.41) 34.16d (35.76) 35.66d (36.67) 0.17c (2.35) 40 0.00c (0.0) 32.78c (34.93) 38.33c (38.25) 42.51c (40.69) 1.62c (6.80) Zingiber officinale 60 3.12b (10.03) 39.87b (39.15) 44.58b (41.89) 50.00a (45.0) 3.73b (11.0) 80 6.24a (14.42) 44.06a (41.58) 48.88a (44.36) 46.02b (42.71) 6.65a (14.88) LSD 1.07 1.47 1.04 1.62 1.63 20 30.39d (33.45) 38.42d (38.30) 33.33d (35.26) 33.43d (35.32) 0.17c (2.35) 40 35.93c (36.83) 39.87c (39.15) 41.94c (40.36) 36.30c (37.05) 2.43b (8.68) Moringa oleifera 60 40.91b (39.76) 45.36b (42.33) 44.30b (41.72) 38.53b (38.37) 0.17c (2.35) 80 46.30a (42.88) 47.82a (43.75) 47.77a (43.72) 43.79a (41.43) 3.89a (11.30) LSD 1.63 1.25 1.15 1.92 1.33 20 31.67d (34.24) 14.14d (22.08) 0.00b (0.0) 35.98c (36.85) 17.67c (24.84) 40 37.21c (37.59) 17.46c (24.69) 0.00b (0.0) 38.37b (38.27) 22.04b (27.99) Citrus limon 60 45.02b (42.14) 24.83b (29.88) 0.00b (0.0) 39.96b (39.21) 28.69a (32.38) 80 50.00a (45.0) 26.86a (31.21) 2.22a (0.0) 44.90a (42.07) 29.17a (32.68) LSD 1.74 1.12 0.82 1.82 1.65 20 32.67d (34.85) 36.25c (37.02) 11.94d (20.20) 32.64d (34.84) 0.17a (2.35) 40 46.59b (43.04) 43.05b (41.0) 15.69c (23.33) 36.14c (36.95) 0.17a (2.35) Tagetes minuta 60 38.35c (38.26) 43.48b (41.25) 22.22b (28.12) 38.53b (38.37) 0.17a (2.35) 80 49.00a (44.43) 46.52a (43.0) 26.53a (30.99) 44.11a (41.61) 0.17a (2.35) LSD 1.54 0.97 1.38 1.72 2.17
Continued ….
104
20 30.39d (33.45) 32.78d (34.93) 3.19c (10.21) 32.00b (34.44) 30.79d (33.70) 40 37.35c (37.67) 38.42c (38.30) 5.55b (13.57) 33.91b (35.61) 35.05c (36.27) Curcuma longa 60 39.77b (39.09) 42.18b (43.75) 7.50a (15.86) 38.85a (38.55) 42.63b (40.76) 80 44.46a (41.81) 47.82a (40.50) 7.22a (15.55) 40.13a (39.30) 46.68a (43.09) LSD 0.98 1.39 1.25 2.49 1.27 20 18.32a (25.34) 14.28d (22.19) 11.11d (19.45) 21.97d (27.94) 0.17a (2.35) 40 19.03a (25.85) 19.20c (25.98) 14.16c (22.09) 24.84c (29.89) 0.17a (2.35) Catharenthus roseus 60 15.48b (23.16) 23.39b (28.92) 25.41a (25.75) 27.54b (31.65) 0.17a (2.35) 80 18.32a (25.33) 26.71a (31.12) 18.88b (30.27) 29.77a (33.06) 0.17a (2.35) LSD 1.57 1.27 1.42 1.71 2.17 20 17.04d (24.37) 16.74d (24.14) 10.83d (19.20) 15.28d (23.0) 10.21d (18.62) 40 22.44c (28.27) 23.39c (28.91) 14.44c (22.33) 18.31c (25.32) 13.78c (21.77) Eucalyptus globules 60 25.99b (30.65) 25.99b (30.65) 17.63b (24.82) 24.83b (29.88) 19.77b (26.39) 80 28.97a (32.56) 28.88a (32.50) 21.80a (27.83) 28.34a (32.16) 24.47a (29.65) LSD 1.42 1.72 1.25 1.97 1.60 20 1.98c (7.78) 0.99d (5.54) 1.94d (7.69) 2.23b (8.10) 1.46cd (6.50) 40 3.41c (10.45) 3.01c (9.96) 4.02c (11.54) 3.02b (9.90) 3.89c (10.66) Syzgium cumini 60 5.25b (13.21) 5.03b (12.92) 8.61a (16.99) 6.53a (14.76) 6.97b (15.26) 80 7.95a (16.34) 7.20a (15.51) 6.11b (14.26) 7.96a (16.37) 10.54a (18.85) LSD 1.56 1.26 1.54 1.62 2.51 20 0.00d (0.0) 0.56c (4.27) 1.11b (5.0) 0.16c (1.14) 1.63c (6.52) 40 3.12c (10.03) 4.31b (11.87) 3.89a (11.03) 2.23b (7.33) 3.73b (11.05) Polyalthia longifolia 60 6.81b (15.10) 6.62a (14.83) 4.72a (12.48) 5.57a (13.60) 5.35b (13.22) 80 8.09a (16.51) 7.78a (16.16) 6.38a (14.38) 7.00a (15.29) 9.08a (17.44) LSD 1.23 1.64 2.70 1.88 1.83
105
Continued ….
20 3.12b (10.12) 3.73c (11.05) 2.08c (8.14) 0.63d (3.13) 2.76cd (8.71) 40 7.95a (16.33) 5.75b (13.84) 4.16b (11.74) 3.18c (10.20) 5.51bc (13.43) 60 8.09a (16.51) 8.36a (16.76) 7.36a (15.71) 7.32b (15.59) 8.59ab (16.88) 80 8.80a (17.25) 9.37a (17.81) 9.16a (17.53) 9.07a (17.49) 11.67a (19.85) LSD 1.46 1.19 1.81 1.72 3.63 Topsin -M 100.00a (90.0) Control negative 0.00e (0.0) Mangifera indica
to Fisher’s LSD test. Data in parenthesis are arcsine transformed values. *Conc. = Concentration. **LSD= Least significance difference.
106
Table 4.8: Antifungal effect of methanol dry plant extracts at different concentrations against important tomato fungi (Percent growth reduction) *Conc. F.oxysporum f. sp. Extract A. solani F. solani C. coccodes P. infestans (%) lycopersici 20 92.00c (73.59) 91.13c (72.70) 90.09c (71.67) 90.82c (72.37) 55.61d (48.22) 40 92.14c (73.75) 92.96b (74.63) 92.19b (73.80) 92.40b (74.03) 62.87c (52.45) Azadirachta indica 60 97.19b (80.42) 95.63a (78.0) 92.74b (74.46) 93.51b (75.28) 70.98b (57.41) 80 98.74a (83.57) 96.20a (78.80) 95.26a (77.44) 97.46a (81.03) 84.45a (66.79) **LSD 1.25 1.08 1.14 1.49 1.51 20 91.58d (73.15) 90.57c (72.13) 90.09d (71.68) 90.98d (72.55) 0.53a (4.15) 40 93.97c (75.80) 90.85c (72.41) 93.44c (75.19) 93.35c (75.08) 0.53a (4.15) Allium sativum 60 95.23b (77.41) 92.96b (74.63) 95.53b (77.90) 95.09b (77.25) 0.53a (4.15) 80 97.76a (81.41) 95.49a (77.82) 98.60a (83.25) 98.25a (82.50) 0.53a (4.15) LSD 0.87 0.91 1.39 1.28 0.00 20 89.62d (71.21) 90.29d (71.86) 91.21d (72.77) 91.14c (72.70) 67.70c (52.97) 40 93.41c (75.15) 92.26c (73.87) 92.74c (74.41) 93.35b (75.13) 72.19a (55.37) Ocimum sanctum 60 95.23b (77.41) 95.07b (77.22) 95.26b (77.44) 94.93ab (77.05) 63.73d (56.97) 80 97.61a (81.27) 96.06a (78.60) 98.74a (83.59) 96.36a (79.08) 70.29b (58.18) LSD 1.06 0.87 1.19 1.85 1.34 20 51.19d (45.68) 52.88d (46.65) 50.76d (45.44) 51.74d (45.99) 50.08d (45.05) 40 58.20c (49.72) 54.99c (47.86) 57.32c (49.21) 59.49c (50.47) 52.68c (46.53) Melia azedarach 60 65.92b (54.28) 60.48b (51.05) 64.15b (53.22) 65.98b (54.32) 57.86b (49.52) 80 70.83a (57.31) 64.55a (53.46) 70.01a (56.79) 73.25a (58.86) 62.35a (52.15)
107
LSD 0.96 0.93 0.85 1.39 1.93
Continued ….
20 51.61d (45.92) 54.011d (47.30) 50.49d (45.28) 50.31d (45.18) 30.57d (33.56) 40 55.68c (48.26) 63.43c (52.79) 56.62c (48.80) 55.22c (47.99) 36.27c (37.03) Lawsonia inermis 60 62.55b (52.27) 71.87b (57.97) 63.04b (52.56) 61.86b (51.86) 41.62b (40.17) 80 70.27a (56.95) 74.96a (59.98) 70.43a (57.06) 65.82a (54.22) 48.88a (44.35) LSD 0.76 1.03 1.49 1.63 1.56 20 31.00d (33.83) 29.96d (33.18) 32.08d (34.49) 31.48d (34.13) 0.53cd (4.15) 40 37.03c (37.48) 34.88c (36.20) 40.59c (39.57) 39.24c (38.78) 2.51bc (8.12) Zingiber officinale 60 42.92b (40.92) 37.55b (37.79) 43.10b (41.03) 49.52a (44.72) 3.29ab (10.29) 80 45.86a (42.62) 44.86a (42.05) 47.00a (43.28) 46.83b (43.18) 5.36a (13.21) LSD 1.07 1.28 1.51 1.65 2.31 20 31.70d (34.26) 32.49d (34.74) 31.52d (34.15) 34.17c (35.77) 30.74d (33.67) 40 33.52c (35.37) 37.55c (37.79) 36.12c (36.94) 37.81b (37.94) 38.86b (38.56) Moringa oleifera 60 41.52b (40.11) 39.94b (39.19) 46.44a (42.96) 38.13b (38.13) 33.85c (35.57) 80 46.14a (42.78) 44.58a (41.89) 43.93b (41.51) 41.77a (40.26) 42.83a (40.87) LSD 0.78 1.21 0.89 1.78 1.64 20 13.75d (21.76) 33.75d (35.52) 0.70ab (4.23) 36.86c (37.38) 30.74c (33.67) 40 18.09c (25.16) 38.96c (38.62) 0.70ab (4.56) 37.97c (38.04) 33.33b (35.26) Citrus limon 60 24.69b (29.79) 43.32b (41.16) 0.42ab (3.49) 40.19b (39.34) 32.30b (34.63) 80 29.31a (32.77) 46.55a (43.02) 1.08a (5.26) 44.30a (41.72) 36.10a (36.92) LSD 1.43 1.42 0.79 1.42 1.48
108
20 31.42d (34.09) 34.74d (36.11) 0.00c (0.0) 32.59c (34.81) 0.00b (0.43) 40 35.49c (36.56) 38.96c (38.62) 0.00c (0.0) 34.17c (35.77) 0.00b (0.43) Tagetes minuta 60 38.43b (38.31) 42.19b (40.50) 21.90b (27.89) 37.66b (37.85) 0.70ab (4.69) 80 43.34a (41.17) 45.43a (42.37) 26.08a (30.71) 41.61a (40.17) 1.39a (6.27) LSD 0.71 1.21 0.75 1.70 0.95
Continued ….
20 30.58d (33.56) 33.61d (35.43) 31.38d (34.06) 30.38d (33.44) 0.53a (4.15) 40 34.36c (35.88) 38.68c (38.45) 38.35c (38.26) 34.01c (35.67) 0.53a (4.15) Curcuma longa 60 39.97b (39.21) 43.74b (41.40) 45.33b (42.32) 38.13b (38.13) 0.53a (4.15) 80 49.93a (44.96) 47.25a (43.42) 49.37a (44.64) 40.50a (39.52) 0.53a (4.15) LSD 0.79 1.08 0.83 1.44 0.00 20 10.24d (18.65) 12.94d (21.07) 12.41d (20.61) 13.60d (21.64) 11.23d (19.55) 40 15.71c (23.34) 15.61c (23.27) 14.92c (22.72) 21.36b (27.52) 15.54c (23.21) Catharenthus roseus 60 18.94b (25.78) 20.25b (26.74) 19.81b (26.42) 24.05a (29.36) 21.25b (27.44) 80 24.97a (29.97) 22.93a (28.60) 21.62a (27.70) 15.98c (23.54) 24.70a (29.79) LSD 0.87 0.84 0.72 1.70 1.77 20 16.55d (23.99) 12.24d (20.46) 13.11d (21.22) 15.03d (22.80) 13.13c (21.23) 40 20.20c (26.70) 17.58c (24.79) 14.64c (22.49) 18.51c (25.47) 15.37b (23.07) Eucalyptus globules 60 25.53b (30.34) 21.10b (27.33) 17.43b (24.67) 23.73b (29.14) 21.07a (27.32) 80 29.31a (32.77) 27.00a (31.30) 20.36a (26.82) 26.58a (31.03) 16.41b (23.88) LSD 1.39 1.38 0.70 1.79 1.67 20 8.42c (16.78) 0.99d (5.39) 0.56c (3.90) 2.69c (9.21) 0.53d (4.15) 40 9.68bc (18.08) 2.96c (9.75) 0.84c (4.88) 4.11c (11.55) 3.46c (10.41) Syzgium cumini 60 11.08ab (19.40) 4.93b (12.81) 3.07b (10.02) 6.01b (14.13) 6.39b (14.37) 80 12.48a (20.66) 7.74a (16.11) 5.72a (13.80) 8.86a (17.29) 10.37a (18.73)
109
LSD 2.58 1.19 1.09 1.75 2.45 20 9.68c (18.06) 2.25c (8.46) 1.95c (7.82) 0.00c (0.0) 1.73cd (6.93) 40 10.24ab (18.61) 4.08b (11.62) 2.65c (9.31) 2.21b (8.21) 4.84bc (12.44) Polyalthia longifolia 60 10.80ab (19.13) 6.47a (14.71) 4.05b (11.50) 3.63b (10.91) 6.39ab (14.06) 80 12.34a (20.53) 7.46a (15.83) 7.11a (15.45) 6.01a (14.13) 8.98a (17.22) LSD 2.47 1.07 1.15 1.45 3.75
Continued ….
20 7.86b (16.19) 0.99c (5.39) 2.93c (9.67) 1.58d (6.15) 3.46c (10.59) 40 9.26ab (17.64) 3.10b (10.11) 3.91c (11.29) 4.11c (11.43) 6.05c (13.74) 60 11.36a (19.66) 6.75a (15.03) 6.42b (14.64) 7.43b (15.74) 9.85b (18.10) 80 11.50a (19.77) 7.46a (15.85) 8.09a (16.50) 10.12a (18.44) 12.96a (21.04) LSD 2.59 0.73 1.29 2.46 2.92 Topsin - M 100.00a (90.0) Control negative 0.00e (0.0) Mangifera indica
to Fisher’s LSD test. Data in parenthesis are arcsine transformed values.*Conc. = Concentration. **LSD= Least significance difference.
110
Plate 4.3: Bioefficacy of methanol extract of neem on mycelial growth of A. solani. 4.6. CONFIRMATORY TEST FOR THE ANTIFUNGAL ACTIVITY OF PLANT EXTRACTS THROUGH AGAR WELL METHOD
Aqueous and solvent extracts of fifteen plants in fresh and dry formulations were tested against test fungi through agar well method in order to confirm the efficiency of poisoned food technique. The wells in agar plate were charged with the reactants and the results are based on the formation and measurement of growth inhibition zones which developed around the wells after 72 hours of incubation. There was a significant difference among effectiveness of various plant extracts against different fungi. Comparitively aqueous
111 extracts manifested strong antifungal activity as compared to methanol extracts. Fresh formulation was more effective than dry powder of aqueous and methanol solvents at all concentrations. However, inhibition zone diameter increased with increase in concentration of plant extracts. Maximum zone (18.0mm) was observed in positive control
(Topsin-M) while no zone was developed in negative control (DW).
4.6.1. Fresh formulation of aqueous extracts
The fresh aqueous extract of 7 plants exhibited antifungal property more or less against
A. solani. The growth inhibition zone varied greatly in size (Table 4.9). Among the seven plants leaf extracts of Azadirachta indica were highly effective against all fungi.
Maximum inhibition zone ranging between 12.67 to 17.33mm diameters developed against F. oxysporum f.sp. lycopersici followed by C. coccodes, A. solani, F. solani and
P. infestans. Zone size increases as the concentration increased. Allium sativum was also found effective for antifugal activity against all test fungi. Maximum zone of inhibition
(16.67mm) was observed at 80% concentration against F. oxysporum f.sp. lycopersici followed by A. solani, C. coccodes, F. solani and P. infestans. Leaf extracts of Ocimum sanctum showed greater zone against A. solani followed by F. oxysporum f. sp lycopersici but no zone was developed against C. coccodes and P. infestans at all concentrations.
Moderate activity of aqueous extract was observed in Melia azedarach against all test fungi at various concentrations. Leaf extract of Lawsonia inermis developed moderate antifungal activity against A. solani, C. coccodes and F. solani, no zone was produced by extract against F. oxysporum f.sp. lycopersici and P. infestans.Whereas, Z. officinale and
Moringa oleifera produce antifungal inhibitory zones against A. solani and F. solani.
4.6.2. Dry powder formulation of aqueous extracts
112
Few plant extracts in dry formulation produced moderate antifungal inhibitory zone against some of the fungi as compared to positive control. Best zone diameter was, however, observed in aqueous leaf extract of Azadirachta indica against F. oxysporum f.sp. lycopersici (16.0mm) and C. coccodes (15.67mm) followed by A. sativum, M. azedarach and M. oleifera (Table 4.10 ).
4.6.3. Fresh formulation of methanol extracts
Out of 15 plants only 6 plants exhibited antifungal property against test fungi with varying degree of inhibition (Table 4.11). Leaf extract of A. indica and bulb extracts of A. sativum were found highly effective against all test fungi producing inhibition zone range of
10.67mm to 16.67mm diameter against F. solani, C. coccodes, P. infestans, A solani and
F. oxysporum f.sp. lycopersici. Methanol extract of M. azedarach showed intermediate activity against all fungi but no activity was observed against P. infestans. However, leaf extracts of Z. officinale, O. sanctum and M. oleifera possessed more or less variable or mild inhibitory effect against A. solani, F. solani and F. oxysporum f.sp. lycopersici.
4.6.4. Dry formulation of methanol extracts
Methanol extracts of garlic and neem powders were equally effective against all test fungi
(Table 4.12). Maximum zone of inhibition was developed against F. oxysporum f.sp. lycopersici measuring 15.33mm and 15.0mm diameters respectively, at 80% concentration. A moderate antifungal activity of melia leaf powder extract was observed against test fungi. Whereas, tulsi powder extract showed low activity only against A. solani, F. solani and F. oxysporum f.sp. lycopersici. No zone was developed agaist rest of the fungi.
113
114
Table 4.9: Antifungal activity of aqueous (fresh) plant extracts at different concentrations against selective tomato fungi using agar well method Mean zone diameter(mm)
Extracts *Conc As Pi Cc Fs Fo (%) 20 10.67 ± 1.15 NZ 11.67 ± 0.58 11.00 ± 1.00 11.33 ± 0.58 40 12.67 ± 0.58 10.33 ± 0.58 11.67 ± 0.58 11.33 ± 1.15 12.67 ± 0.58 Melia azedarach 60 13.33 ± 0.58 11.33 ± 0.58 12.33 ± 0.58 12.33 ± 0.58 13.33 ± 1.00 80 13.67 ± 0.58 12.00 ± 1.00 12.67 ± 0.58 12.67 ± 0.58 13.67 ± 0.58 20 12.00 ± 1.00 NZ NZ NZ NZ 40 12.67 ± 0.58 NZ NZ 10.67 ± 1.15 NZ Zingiber officinale 60 14.00 ± 1.00 NZ NZ 11.33 ± 1.15 NZ 80 14.67 ± 0.58 NZ NZ 11.67 ± 0.58 NZ 20 13.67 ± 0.58 12.67 ± 0.58 14.67 ± 0.58 13.33 ± 0.58 14.33 ± 0.58 40 14.00 ± 1.00 12.67 ± 0.58 15.67 ± 0.58 13.67 ± 0.58 15.33± 0.58 Azadirachta indica 60 14.33 ± 1.15 13.33 ±1.15 16.33 ± 1.15 14.33 ± 1.53 16.33 ± 0.58 80 14.67 ± 1.53 13.67 ± 1.53 16.67 ± 0.58 14.67 ± 1.15 17.33 ± 1.15 20 10.33 ± 0.58 NZ NZ NZ NZ 40 11.00 ± 1.15 NZ NZ 10.67 ± 1.15 NZ Moringa oleifera 60 11.67 ± 0.58 NZ NZ 11.33 ± 1.15 NZ 80 12.00 ± 1.00 NZ NZ 11.67 ± 0.58 NZ 20 11.33 ± 0.58 NZ 11.33 ± 1.15 10.33 ± 0.58 NZ 40 12.67 ± 0.58 NZ 12.33 ± 0.58 11.00 ± 1.00 NZ Lawsonia inermis 60 13.33 ± 0.58 NZ 12.67 ± 0.58 11.33± 0.58 NZ 80 13.67 ± 0.58 NZ 13.33 ± 1.00 12.00 ± 1.00 NZ 20 12.00 ± 1.00 NZ NZ NZ 11.33 ± 0.58 40 12.67 ± 1.53 NZ NZ 11.33 ± 0.58 12.00 ± 1.00 Ocimum sanctum 60 13.33 ± 1.15 NZ NZ 11.67 ± 0.58 12.33 ± 1.15 80 14.00 ± 1.00 NZ NZ 12.67 ± 0.58 12.67 ± 1.00 20 13.67 ± 0.58 NZ 12.67 ± 0.58 11.67 ± 1.15 14.67 ± 0.58 40 14.00 ± 1.00 11.67 ± 0.58 13.33 ± 1.15 12.00 ± 1.00 15.33 ± 0.58 Allium sativum 60 15.67 ± 0.58 12.33 ± 0.58 14.33 ± 0.58 13.33 ± 0.58 16.33 ± 0.58 80 16.33 ± 0.58 13.33 ± 0.58 14.33 ± 0.58 16.67 ±0.58 Topsin-M
Control negative NZ Values are means of three replicates (mean ±SD). NZ= No Zone. *Conc= Concentration. Alternaria solani (As), Phytophthora infestans (Pi), Colletotrichum coccodes (Cc), Fusarium solani (Fs), Fusarium oxysporum f. sp.lycopersici (Fo).
115
Table4.10: Antifungal activityof aqueous (dry) plant extracts at different concentrations against selective tomato fungi using agar well method
Extracts Mean zone diameter(mm)
*Conc As Pi Cc Fs Fo (%) Melia azedarach 20 10.00 ± 1.00 NZ NZ NZ NZ 40 11.00 ± 1.15 10.33 ± 1.15 11.67 ± 0.58 11.33 ± 1.15 11.33 ± 0.58 60 12.33 ± 0.58 10.00 ± 1.00 11.00 ± 0.58 11.33 ± 0.58 12.33 ± 0.58 80 12.67 ± 0.58 11.33 ± 0.58 11.67 ± 0.58 11.33 ± 0.58 12.67 ± 0.58 Azadirachta indica 20 11.33 ± 1.53 NZ 12.33 ± 0.58 12.67 ± 0.58 NZ 40 12.67± 0.58 12.67 ± 0.58 15.67 ± 1.15 13.67 ± 0.58 13.33 ± 1.53 60 13.33 ± 1.00 12.33 ± 0.58 15.00 ± 1.15 13.33 ± 0.58 15.33 ± 0.58 80 13.67 ± 1.15 13.00 ± 1.00 15.33 ± 0.58 13.67 ± 1.00 16.00 ± 1.00 Moringa oleifera 20 NZ NZ NZ NZ NZ 40 10.33 ± 0.58 NZ NZ 10.67 ± 1.15 NZ 60 11.00 ± 1.15 NZ NZ 10.67 ± 1.15 NZ 80 11.00 ± 1.00 NZ NZ 11.00 ± 0.58 NZ Allium sativum 20 12.00 ± 0.58 NZ NZ 11.33 ± 1.52 12.33 ± 1.00 40 12.00 ± 1.00 11.67 ± 0.58 13.33 ± 0.58 12.00 ± 1.00 14.67 ± 0.58 60 14.67 ± 1.53 11.67 ± 0.58 13.00 ± 1.00 12.33 ± 1.00 15.33 ± 1.15 80 15.33 ± 1.15 12.33 ± 1.15 14.33 ± 1.00 13.33 ± 0.58 15.67 ± 0.58 Topsin-M 18.00 ± 1.00
Control negative NZ Values are means of three replicates (mean ±SD). NZ= No Zone. *Conc= Concentration. Alternaria solani (As), Phytophthora infestans (Pi), Colletotrichum coccodes (Cc), Fusarium solani (Fs), Fusarium oxysporum f. sp. lycopersici (Fo).
116
Table 4.11: Antifungal activity of methanol (fresh) plant extracts at different concentrations against selective tomato fungi using agar well method
Extracts Mean zone diameter(mm)
*Conc As Pi Cc Fs Fo (%) Melia azedarach 20 11.33 ± 0.58 NZ NZ 10.33 ± 0.58 NZ 40 12.00 ± 1.00 NZ 11.00 ± 1.00 11.00 ± 1.00 12.00 ± 1.00 60 12.67 ± 0.58 NZ 11.33 ± 0.58 11.67 ± 0.58 12.67 ± 0.58 80 13.33 ± 0.58 NZ 12.33 ± 0.58 12.33 ± 0.58 13.00 ± 1.00 Zingiber officinale 20 NZ NZ NZ 10.33 ± 0.58 NZ 40 NZ NZ NZ 10.33 ± 0.58 12.67 ± 1.53 60 13.67 ± 0.58 NZ NZ 10.67 ± 1.15 NZ 80 14.33 ± 0.58 NZ NZ 10.33 ± 1.15 NZ Azadirachta indica 20 12.67 ± 1.53 11.67 ± 1.53 13.67 ± 1.53 12.33 ± 0.58 12.67 ± 1.15 40 13.33 ± 0.58 12.00 ± 1.00 15.00 ± 0.58 13.33 ± 0.58 14.67 ± 0.58 60 14.00 ± 1.00 12.67 ± 0.58 15.67 ± 0.58 14.00 ± 1.00 15.67 ± 1.00 80 14.00 ± 1.73 13.33 ± 1.15 16.00 ± 1.00 14.33 ± 0.58 16.67 ± 0.58 Moringa oleifera 20 10.00 ± 1.00 NZ NZ NZ 10.33 ± 1.53 40 10.67 ± 1.53 NZ NZ 10.67 ± 1.15 NZ 60 11.33 ± 0.58 NZ NZ 11.00 ± 1.15 NZ 80 11.33 ± 0.58 NZ NZ 11.33 ± 0.58 NZ Ocimum sanctum 20 12.00 ± 1.00 NZ 11.33 ± 1.15 9.33 ± 1.00 NZ 40 12.33 ± 1.53 NZ NZ 11.00 ± 0.58 11.67 ± 1.15 60 13.00 ± 1.00 NZ NZ 11.00 ± 1.00 12.00 ± 1.15 80 13.33 ± 0.58 NZ NZ 12.00 ± 1.00 12.33 ± 0.58 Allium sativum 20 12.67 ± 1.53 NZ 12.00 ± 1.00 10.67 ± 0.58 13.33 ± 1.15 40 13.33 ± 1.15 11.33 ± 0.58 12.67 ± 0.58 11.67 ± 1.15 15.00 ± 1.00 60 15.00 ± 0.58 12.00 ± 0.58 13.67 ± 0.58 12.67 ±0.58 16.00 ± 1.00 80 15.67± 0.58 12.67 ± 0.58 14.67 ± 0.58 13.67 ± 0.58 16.00 ± 1.00 Topsin-M 18.00 ± 1.00
117
Control negative NZ Values are means of three replicates (mean ±SD). NZ= No Zone. *Conc= Concentration. Alternaria solani (As), Phytophthora infestans (Pi), Colletotrichum coccodes (Cc), Fusarium solani (Fs), Fusarium oxysporum f. sp. lycopersici (Fo).
Table 4.12: Antifungal activity of methanol (dry) plant extracts at different concentrations against selective tomato fungi using agar well method
Extracts Mean zone diameter(mm)
*Conc As Pi Cc Fs Fo (%) Melia azedarach 20 NZ NZ NZ 10.00 ±1.00 NZ 40 10.67 ± 1.00 10.33 ± 1.53 11.67 ± 0.58 11.33 ± 1.15 11.00 ± 1.00 60 11.33 ± 0.58 10.00 ± 1.00 10.67 ± 0.58 11.00 ± 1.00 12.00 ± 1.00 80 12.33 ± 0.58 11.00 ± 1.00 11.33 ± 0.58 11.00 ± 1.00 12.00 ± 1.15 Azadirachta indica 20 12.00 ± 1.00 NZ NZ NZ NZ 40 12.00 ± 0.58 12.67 ± 0.58 15.67 ± 1.15 13.67 ± 0.58 13.00 ± 1.53 60 12.67 ± 0.58 12.00 ± 0.58 14.67 ± 0.58 12.67 ± 0.58 14.67 ± 1.15 80 13.33 ± 1.00 12.67 ± 1.15 14.67 ± 1.15 13.00 ± 1.15 15.00 ± 0.58 Ocimum sanctum 20 NZ NZ NZ NZ NZ 40 NZ NZ NZ NZ NZ 60 10.67 ± 1.15 NZ NZ 11.00 ± 1.15 11.67 ± 1.15 80 13.00 ± 1.15 NZ NZ 12.00 ± 0.58 11.67 ± 1.15 Allium sativum 20 12.00 ± 1.00 NZ NZ 9.00 ± 1.00 NZ 40 11.67 ± 1.15 11.67 ± 0.58 13.33 ± 0.58 12.00 ± 1.00 13.67 ± 1.15 60 14.33 ± 0.58 11.33 ± 0.58 12.67 ± 0.58 11.67 ± 0.58 14.33 ± 1.53 80 15.00 ± 0.58 12.00 ± 1.00 14.00 ± 1.15 13.00 ± 1.00 15.33 ± 1.15
Topsin-M 18.00 ± 1.00
Control negative NZ Values are means of three replicates (mean ±SD). NZ= No Zone. *Conc= Concentration. Alternaria solani (As), Phytophthora infestans (Pi), Colletotrichum coccodes (Cc), Fusarium solani (Fs), Fusarium oxysporum f. sp. lycopersici (Fo).
4.7. INHIBITION OF FUNGAL SPORULATION BY PLANT EXTRACTS
118
Ten plant extracts which showed inhibitory effect on mycelial growth of fungal pathogens were assessed against fungal sporulation.
4.7.1 Effect of aqueous extracts on spore germination
Extracts of 10 plants showed more or less inhibitory effect against spore germination of target
fungi. The leaf extract of neem (Azadirachta indica) significantly and highly inhibited spore
germination of target fungi, but of Curcuma longa had minimum inhibition (Fig. 4.2). The highest
concentration of plant extracts (80%) markedly inhibited spore germination of tested fungi as
compared to control. The aqueous leaf extracts of Azadirachta indica, Allium sativum and Ocimum
sanctum exhibited maximum (upto 100%) inhibition against spore germination of Alternaria
solani, Fusarium oxysporum f.sp. lycopersici, F. solani and C. coccodes. A. indica and O. sanctum
effectively retard sporulation of P. infestans but A. sativum showed intermediate effects.
Maximum spore inhibition (91%, 85.66%) was observed by leaf extract of Lawsonia inermis
against F. solani and F. oxysporum f.sp. lycopersici respectively, but rest of the fungi showed
intermediate effects. Leaf extract of Melia azedarach retarded the germination of F. oxysporum
f.sp. lycopersici by 90% while germination of F. solani, A. solani and C. coccodes ranged between
78-88%. However, it has more or less intermediary effect on P. infestans. Additionally, Moringa
oleifera, Zingiber officinales, Citrus limon and Tagetes minuta showed intermediate to least
inhibition against all these fungi (Fig. 4.2).
4.7.2. Inhibitory effect of methanol extracts
The results presented in Fig. 4.3 revealed that methanolic extracts of A. sativum and A. indica
were equally effective against spore germination. They completely inhibited sporulation of A.
solani, F. solani, F. oxysporum f.sp. lycopersici and C. coccodes. Similarly, significant inhibition
by leaf extract of Azadirachta indica was observed against spore germination of P. infestans.
119
Conidia of F. oxysporum f.sp. lycopersici, F. solani, A. solani and C. coccodes were highly inhibited (92%, 90%, 89%, 87%, respectively) with methanolic leaf extract of Ocimum. Leaf extract of Melia azedarach ranked next showing moderate inhibition against F. oxysporum f.sp. lycopersici, A. solani, F. solani, and C. coccodes ranging from 84 to 71 percent (Fig. 4.3).
Similarly, Lawsonia inermis, Moringa oleifera and Zingiber officinale extract have intermediate effect whereas Citrus limon, Curcuma longa and Tagetes minuta exhibited least inhibition against spore germination of tested fungi.
120
Fig 4.2: Inhibition percentage (means±SD) of aqueous extracts on spore germination of different fungi.
121
Fig4.3: Inhibitionpercentage (means±SD) of methanol extracts on spore germination of different fungi.
4.8. EFFECTIVENESS OF PLANT EXTRACTS AGAINST EARLY BLIGHT OF TOMATO (IN VIVO)
122
Field observations and laboratory investigations revealed that Alternaria solani, the causal agent
of early blight, is an important pathogen. It was isolated from leaves, fruit and seed of tomato in
the highest frequency. The pathogen potentially infects potato, tomato and other solanaceous hosts,
develops epiphytotic conditions under favourable environments and inflicts colossal losses in
production. The fungus reacted positively with the aqueous and methanol extracts of medicinal
plants in all formulations which suppressed mycelial growth and prevented sporulation, thus
manifested properties of antifungal substances. It was, therefore, imperative to test the efficacy of
antifugal material or components against early blight of tomato under greenhouse and field
conditions where the disease incidence was recorded periodically at15 days intervals.
4.8.1. Greenhouse experiments
Plant extracts of Azadirachta indica, Allium sativum and Ocimum sanctum were applied at 80%
concentration and compared with Topsin-M fungicide and negative control in two consecutive
years 2012-2014. After treatment, PDI (%) of early blight severity was recorded at 15-day
intervals. Results summarized in Table 4.13 and 4.14 showed promising results against Alternaria
solani in in vivo conditions. The protective and curative application of plant extracts and Topsin-
M fungicide significantly reduced the PDI by 22-64% and 25-64%, respectively over untreated
control. In one day protective application maximum reduction (64.38%) in disease intensity was
shown by Topsin-M with 14.44% PDI while the least reduction of disease intensity was achieved
by Ocimum sanctum (22.39%) with 57.78 PDI. Similarly curative application of Topsin-M
resulted in 64% reduction in disease with 12.78% PDI. While lowest disease intensity (25.37%)
was recorded in O. sanctum.
It was also observed that rate of increase in PDI was slow in case of botanicals and fungicide
treatments compared to untreated control. Leaf extract of Azadirachta indica has a potent protective
123
activity in reducing the early blight of tomato resulting in 28.80 to 56.92 percent disease severity
with 15.56-49.44% PDI from 50 to 95 DAP and in the control the maximum disease index was
74.44%. Similarly bulb extract of A. sativum reduced early blight disease up to 53.42% with lowest
PDI of 18.89%. The curative activities depicted that neem leaf extract and garlic extract had
displayed relatively high curative activity against the disease. At 50 days after planting, greatest
reduction of disease severity was achieved by A. indica followed by A. sativum (55 and 53% with
16.11 and 16.67PDI, respectively), table 4.13. In the second year, Topsin-M appeared to be most
effective in reducing the disease severity in both protective (71%) and curative (72%) with 9.44
and 10.56 PDI. It was followed by leaf exracts of Azadirachta indica and Allium sativum, but that
of Ocimum sanctum was ineffective (Table 4.14). Significant difference was noticed in the
protective and curative applications. Neem leaf extract significantly reduced the severity of early
blight (41.32-61.02%) over untreated control. Whereas, no significant (P < 0.05) difference in
decreasing the early blight severity was noticed among curative activity of neem and garlic
(62.32%) with 14.44PDI at 50 DAP. Extract of O. sanctum showed least protective and curative
activity, 27.27 and 34.96%, respectively.
4.8.2. Field Evaluations
The disease severity and yield parameters varied in the two seasons due to change in
environmental conditions in the field. Results presented in Table 4.15 and 4.16 demonstrated that
the teatments significantly reduced severity and incidence of early blight and there was increase in
disease index (PDI) from 50 to 95 DAP in two seasons. The protective effect of plant extracts and
Topsin-M (positive control) was variable with respect to disease reduction which ranged between
32.86 to 79.10% in the first and 37.97 to 84.78% in the second year. A similar trend was observed
in the curative application, 50.75-83.87% and 51.33-100%, respectively, in both years
124
(Table 4.15, 4.16). In the second cultivation season (2012-2013) protective application of Topsin-
M reduced the disease intensity up to 79.10% (7.78PDI). Minimum PDI (12.22) was recorded in
Neem leaf extract which was statistically at par with garlic bulb extract (12.78) at 80%
concentration. Least reduction in the disease was observed with application of Ocimum sanctum
32.86% with highest PDI (52.22). A similar trend in curative application of fungicide and plant
extracts was also noticed.
In the second season of 2013-14, maximum protective activity was shown by Topsin-M followed
by Azadirachta indica and Allium sativum which significantly reduced severity of early blight
disease (84.78, 78.25 and 73.90%) with 3.89, 5.56 and 6.67PDI, respectively. Among botanicals,
A. sativum had a potent curative activity against the disease and reduced disease intensity up to
80.95% (4.44PDI) at 50 DAP over untreated control plots. Neem ranked second to garlic in
reducing disease severity (66.67%) with 7.78PDI after 50 days of planting. Least reduction of
disease severity was observed when tomato plants were treated with protective and curative
treatment with Ocimum sanctum leaf extract, 37.97 and 51.33%, respectively, at 95DAP with
37.22 and 30.56PDI (Table 4.16).
4.8.3. Effect on yield
Results indicated that application of botanicles against early blight disease enhanced tomato yield
under greenhouse conditions (Table 4.13, 4.14). In the first season plants sprayed with fungicide
(Topsin-M), Azadirachta indica and Allium sativum increased the fruit yield by
125
57.62, 55.76% and 51.30%, respectively, over untreated control, and similar order of higher yield
(47%, 46.29 and 45.94%) was observed in the second season. Application of Ocimum sanctum
produced the lowest fruit yield in the two seasons (46.47 and 42.40%).
Picking of fruits (95 DAP) indicated that fruit yield was significantly higher in all treatments;
(3.17 to 3.70 t/ha and 3.13 to 3.70t/ha)) as against (2.13 and 2.43t/ha) in untreated controls in the
two cultivation seasons, respectively. In the year of 2012-13 plots sprayed with A. sativum
produced increased fruit yield by 73.28%, followed by Topsin-M (64.22%) and Azadirachta indica
(63.91%) but low yield increase was found with O. sanctum (48.44%). In the second season
maximum increase in fruit yield was obtained in plots sprayed with Topsin-M (52.33%) followed
by A. sativum (44.11%) and A. indica (31.78%) over untreated control (Table
4.15, 4.16).
Table 4.13: Efficacy of plant extracts on early blight disease under Green House conditions (2012-13)
Protective activity Curative activity Yield Treatments Reduction (%) Reduction Increase DAP PDI PDI g/plant (%) (%) D1 15.56 kl 56.92 50.00 16.11 kl 55.38 D2 24.44 hi 40.19 26.67 h 45.45 D3 35.56 ef 28.80 34.44 f 42.06 D4 49.44 c 43.33 d 37.60 Azadirachta indica 139.67 55.76
D1 18.89 ijk 16.67 jk 53.42 43.62 53.84 D2 29.44 fg 25.00 gh 32.74 52.13 D3 42.22 e 40.56 e 29.85 35.40 D4 52.22 d 48.89 c Allium sativum 29.60 135.67 51.30
126
50.77 D1 21.11 hij 17.78 jk 47.95 36.17 40.91 D2 33.33 f 28.89 gh 30.09 33.64 D3 43.89 e 39.44 de 22.39 25.37 D4 57.78 bc 55.56 cd Ocimum sanctum 131.33 46.47 64.62 D1 14.44 k 12.78 l 64.38 54.26 55.68 D2 23.89 hi 21.67 ij 47.79 44.86 D3 32.78 f 32.78 fg 38.81 39.20 D4 45.56 e 42.22 d Topsin- M 141.33 57.62 D1 40.56 e 0.00 36.11 ef 0.00 0.00 D2 52.22 d 0.00 48.89 c 0.00 D3 62.78 b 0.00 59.44 b 0.00 89.67 - D4 74.44 a 0.00 69.44 a Control negative
LSD at P=0.05 5.1616 4.8857
Values are means of three replications. Means within columns not sharing common letters are significantly different at P≤ 0.05 according to Tukey’s HSD test. PDI= Percent Disease Index. DAP= days after planting. D1= 50DAP, D2=65DAP, D3=80DAP, D4=95DAP.
Table 4.14: Efficacy of plant extracts on early blight disease under Green House conditions (2013-14).
Treatments Protective activity Curative activity Yield Reduction Reduction Increase DAP PDI PDI g/plant (%) (%) (%) D1 12.78 kl 61.02 14.44 lm 62.32 D2 20.00 ij 56.10 21.67 jk 57.14 D3 28.89 gh 48.00 32.78 gh 45.87 D4 39.44 de 41.32 40.56 de 40.65 Azadirachta indica 138.00 46.29
127
D1 13.89 jkl 57.63 14.44 lm 62.32 D2 22.78 hi 50.00 23.33 j 53.85 D3 30.00 g 46.00 34.44 fgh 43.12 D4 39.44 de 41.32 42.78 de 37.40 Allium sativum 137.67 45.94
D1 18.33 ijk 44.07 16.67 kl 56.52 D2 28.89 gh 36.59 23.89 ij 52.75 D3 38.33 ef 31.00 33.89 gh 44.04 34.96 D4 48.89 c 27.27 44.44 d Ocimum sanctum 134.33 42.40
D1 9.44 l 71.19 10.56 m 72.46 D2 18.33 ijk 59.76 19.44 jkl 61.54 51.38 D3 28.33 gh 49.00 29.44 hi 41.46 D4 38.33 ef 42.98 40.00 def Topsin- M 138.67 47.00
D1 32.78 fg 38.33 efg D2 45.56 cd 0.00 0.00 50.56 c 0.00 0.00 D3 55.56 b 0.00 60.56 b 0.00 - Control negative D4 67.22 a 0.00 68.33 a 0.00 94.33 LSD at P=0.05 5.7542 4.2629
Values are means of three replications. Means within columns not sharing common letters are significantly different at P≤ 0.05 according to Tukey’s HSD test. PDI= Percent Disease Index. DAP=days after planting. D1=50DAP, D2=65DAP, D3=80DAP, D4=95DAP.
128
Control Neem
Plate 4.4: Untreated control plant (Left) and Neem extract treated plant (Right).
Table 4.15: Efficacy of plant extracts on early blight disease under Field conditions (2012- 13).
Treatments Protective activity Curative activity Yield Reduction (%) Reduction Increase DAP PDI PDI g/plant (%) (%) D1 12.22 kl 67.17 55.06 9.44 jk 72.58 D2 22.22 j 49.11 15.56 hi 67.44 D3 31.67 i 41.43 25.56 g 58.93 D4 45.56 de 32.22 e 56.72 Azadirachta indica 3.50 63.91
129
D1 12.78 k 7.78 jk 77.42 65.66 56.18 D2 21.67 j 17.22 h 63.95 52.68 60.71 D3 29.44 i 24.44 g 43.57 58.21 D4 43.89 ef 31.11 ef Allium sativum 3.70 73.28
D1 14.44 k 11.67 ij 66.13 61.20 40.45 D2 29.44 i 19.44 h 59.30 34.82 D3 40.56 fg 27.78fg 55.36 32.86 50.75 D4 52.22 c 36.67 d Ocimum sanctum 3.17 48.44
D1 7.78 l 5.56 k 83.87 79.10 68.54 D2 15.56 k 11.67 ij 75.58 68.54 68.75 D3 15.56 k 19.44 h 57.86 64.18 D4 32.78 hi 26.67 g Topsin- M 3.50 64.22
D1 37.22 gh 34.44 de D2 49.44 cd 0.00 0.00 47.78 c 0.00 0.00 D3 62.22 b 0.00 62.22 b 0.00 - Control negative D4 77.78 a 0.00 74.44 a 0.00 2.13 LSD at P=0.05 4.4572 3.5319 Values are means of three replications. Means within columns not sharing common letters are significantly different at P≤ 0.05 according to Tukey’s HSD test. PDI= Percent Disease Index. DAP=days after planting. D1=50DAP, D2=65DAP, D3=80DAP, D4=95DAP.
Table 4.16: Efficacy of plant extracts on early blight disease under Field conditions (2013- 14)
Treatments Protective activity Curative activity Yield Reduction Reduction Increase DAP PDI PDI g/plant (%) (%) (%) D1 5.56 k 78.25 7.78 lmn 66.67 D2 13.89 ghi 60.31 13.89 ijk 61.54 D3 23.33 ef 52.81 20.56 gh 57.95 D4 32.78 cd 45.37 30.00 de Azadirachta indica 52.21 3.21 31.78
130
D1 6.67 jk 73.90 4.44 no 80.95 D2 15.56 gh 55.54 10.56 klm 70.77 60.23 D3 25.56 e 48.30 19.44 ghi 54.87 D4 35.00 c 41.67 28.33 def Allium sativum 3.51 44.11
D1 9.44 ijk 63.07 5.56 mno 76.19 D2 18.33 fg 47.63 12.78jkl 64.62 54.55 D3 27.78 de 43.81 22.22 gh 51.33 D4 37.22 c 37.97 30.56 cd Ocimum sanctum 3.13 28.77
100.00 D1 3.89 k 84.78 0.00 o D2 11.67 hij 66.66 9.44 klmn 73.85 63.64 D3 18.89 fg 61.79 17.78 hij 61.06 D4 26.11 e 54.82 24.44 efg Topsin- M 3.70 52.19
D1 25.56 e 23.33 fgh D2 35.00 c 0.00 0.00 36.11 c 0.00 0.00 D3 49.44 b 0.00 48.89 b 0.00 - Control negative D4 60.00 a 0.00 62.78 a 0.00 2.43 LSD at P=0.05 5.5637 5.1765 Values are means of three replications. Means within columns not sharing common letters are significantly different at P≤ 0.05 according to Tukey’s HSD test. PDI= Percent Disease Index. DAP=days after planting. D1=50DAP, D2=65DAP, D3=80DAP, D4=95DAP.
131
A
B C
Plate 4.5: Untreated control (A), Neem (B) and Garlic (C) extracts treated tomato plants in experimental field.
5. DISCUSSION
Tomato is the most common, important, widely consumed and extensively grown
132 vegetable crop all over the world including Pakistan. It is highly nutritive and constitutes essential
component of our diet. However, tomato crop in the country at present is confronted with
challenges as low production, vulnerability to diseases, increased consumption by the exploding
population and crisis of marketing, high pricing and imports. With a production level of about
9.8kg per unit area and per capita consumption of 3.4kg/annum, tomato production has to be
doubled by 2025 (GOP, 2013). Increase in production is possible through adoption of improved
production technology, reducing post harvest losses and control of minor and major diseases at
cheaper cost. Survey of production ecology at Multan revealed that the fungal diseases are
predominantly occurring in tomato and inflicting heavy losses which are roughly estimated at 15-
25%. Important, existing, emerging and re-emerging fungal diseases are damping off affecting
crop stand in the field, early blight, late blight and anthracnose on foliage, wilts and root rot in the
field.
Investigations on diseases of tomato on systematic, scientific and organized basis revealed the presence of a number of diseases. Tomato seeds harbored 17 fungal species distributed in 10 taxonomic genera (Table 4.1), best isolated by the blotter paper method. Majority of seed borne fungi cause seed rot and damping off of seedlings which result in poor stand of crop in the field.
The fungal flora associated with tomato seeds has been studied at large by many workers including
Jones et al; (1991), Perveen and Ghaffar (1995), Bankole (1996), Neergard (1977), Agarwal (1981) and Afroz et al; (2008) and the results obtained are in full agreement with these workers. Twelve fungal species were isolated from foliage, fruits and roots of tomato (Table 4.2) and some associated with post harvest problems. Main pathogenic species were Alternaria solani,
Phytophthora infestans, Colletotrichum coccodes, Fusarium oxysporum f.sp. lycopersici and F. solani. These pathogens and diseases are of common distribution in the world and our records are
133 in conformity with those of Ali et al; (2005), Sudhamoy et al; (2009), Amini and Sidovik (2010),
Derbalah et al; (2011), Chavan and Twade (2012) and Haggag and El-Gamal (2012).
Plant diseases, including tomato diseases are generally managed through indiscriminate and
extensive chemicals and fungicidal applications which are expensive and reported to create many
problems relating to environment, pollution and human and animal health (Osman and
AlRehiayani 2003; Kumar et al., 2007). Moreover, chemicals induce development of resistance
against diseases and production of resistant strains. Therefore, the scientists are finding alternative
approach as use of plant extracts against pest and diseases (Riaz et al., 2010) that are ecofriendly,
sustainable and fit in integrated disease management practices. Approximately 6000 medicinal
plants possessing antifungal properties have been reported from Pakistan (Shinwari and Malik,
1989) but only few have been tested against plant diseases. This study represents for the first time
the involvement and use of extracts of 15 medicinal plants against target fungi of tomato through
different in vitro and in vivo techniques. The formulations consisted of fresh and dry extracts in
water and methanol at different concentrations (20-80%). Before the application of the extracts
their phytochemical analysis was conducted which revealed the presence of eight compounds i.e
Flavonoids, terpenoids, saponins, steroids, cardiac glycosides, phlobatannins, tannins and
coumarins. It was found that antifungal activities of the extracts of medicinal plants varied greatly
and some fungi were highly sensitive to extracts than others. The order of effectiveness of
medicinal plants was Azadirachta indica > Allium sativum > Ocimum sanctum > Melia azedarach
> Lawsonia inermis > Zingiber officinale > Moringa oleifera > Citrus lemon > Tagetes minuta >
Curcuma longa > Catharenthus roseus > Eucalyptus globules > Syzigium cumini > Polyalthia
longifolia > Mangifera indica.
Secondary metabolites of plants are the factual source of the medicinal value of plants producing
interesting effects on plant’s physiology (Harborne, 1973a). Most likely the mechanism of disease
134
suppression by plant products might be due to the secondary metabolites present in plant extracts
that may either act directly on the target pathogen (Amadioha, 2000) or induce systemic resistance
in plants, hence reducing disease development (Kagale et al., 2004). Plants are naturally gifted
with defense genes which are dormant in healthy plants. Certain factors stimulated these genes to
induce systemic resistance against disease. A number of scientists mentioned that effect of plant
extracts on disease incidence may be due to presence of secondary metabolites (Alkaloids,
flavonoids, phenols, tannins, terpenoids, steroids and saponins) that could adversely influence
fungal growth and disease development but also serve as a defensive mechanism (Harborne 1989;
Halama and Haluwin 2004; Mohamed and El-Hadidy 2008; Katalinic et al., 2006; Balestra et al.,
2009).
Therefore, preliminary phytochemical analysis was performed on the basis of these activities and
their qualitative analysis revealed the presence of more or less various compounds in all the tested
botanicals. Results indicated that Azadirachta indica and Allium sativum are rich in flavonoids,
saponins, terpenoids, cardiac glycosides, steroids, tannins and coumarins whereas phlobatannins
were absent and cardiac glycosides was not present in O. sanctum (Table 4.3). Present results are
in analogy with previous work (Chhetri et al., 2008; Ayeni and Yahaya 2010; Chaudhary et al.,
2010; Koche et al., 2010; Joshi et al., 2011). Tannins, cardiac glycosides, steroids, flavonoids and
terpenoids were found in extracts of Melia azedarach whereas saponins, phlobatanins and
coumarins were absent. Only flavonoids, terpenoids, saponins, cardiac glycosides and tannins were
present in Lawsonia inermis while tannins were found absent in
Curcuma longa. These results are in consistency with Chhetri et al; (2008) and Philip et al;
(2011). Flavonoids and tannins are phenolic compounds that act as antioxidants (Ayoola et al.,
2008). It was noticed that flavonoids were appeared in all selected plants except eucalyptus
135
globules, tagetes minuta and catharenthus roseus. Similarly, terpenoids were present in all the
selected plants but found absent in syzgium cumini, catharenthus roseus, mangifera indica and
polyalthia longifolia. Similar results were documented by Kavishanker et al; (2011) and Wang et
al; (2011). It was observed that phlobatannins were absent in all crude extracts except Eucalyptus
globules. There is a correlation between concentration of secondary compounds and their
bioactivities. Therefore it has been reported that amount of phenolic compounds vary in different
parts of same plant, family and species (Kim et al., 2003; Cetkovic et al., 2007; Adedapo et al.,
2009).
The antimicrobial activities of plant extracts greatly depend on quantity and type of phenolics
present in plant tissues and intrinsic resistance of the pathogen (Martini et al., 2004). Phenolic
compounds that possess an aromatic ring having one or more hydroxyl elements are most
ubiquitous group of secondary metabolites present in medicinal as well as edible plants. Phenolics
tend to be water soluble, since they normally occur as glycosides when combined with sugar
(Harborne, 1973b, 1998). In fact variation in antioxidant activity exhibited by plants is due to
phenols either by preventing decomposition of hydroperoxides into free radicals or by inactivating
lipid free radicals (Cai et al., 2004; Pitchaon et al., 2007). In this study, quantitative data obtained
from Folin-Ciocalteu method gives better information about the amount of soluble phenolic
compounds in plant extracts. A. indica followed by A. sativum and O. sanctum yielded highest
contents of equivalent mg Gallic acid/g dry weight both in aqueous and methanol solvents (Table
4.4). This pointed out that high solubility of phenolic compound extracted from plant parts may
fractioned with solvents of increasing polarity (Bautista et al., 2003; Naczk and Shahidi 2004;
Turkmen et al., 2006; Mohsen and Ammar 2008; Ghasemzadeh et al., 2011).
Present results are in analogy with Maruska et al; (2010), Hismath et al; (2011) and Arzoo et al;
(2012). Although there was a significant difference in the phenolic contents of Mangifera indica
136
extract, the TPC was very low unlike A. indica extract. But these finding are in disparity to
Vighasiya et al; (2011) who yielded highest phenol contents in methanol extract of Mangifera
indica. This might be due to the production of phytochemicals by different plant species,
correlation among the presence or absence of different contents of a secondary compound in one
or the more vegetal parts of plants. The quantity of TPC certainly depends on climatic conditions,
experimental conditions, solvent type and extraction method.
Based on results of phytochemical analysis, selected medicinal plants were further investigated for their antifungal activities under laboratory conditions against five target fungi. In
vitro screening of fifteen plants revealed that twelve allelopathic plants showed antifungal
activities against test fungal species of tomato. Among them fresh and dry aqueous extracts of
Azadirachta indica, Allium sativum and Ocimum sanctum exhibited maximum antifungal activity
against all test fungi. These findings are in consistent with earlier scientists (Lee et al., 2003;
Makeredza et al., 2005; Otanga 2005; Tomar and Chandel 2006). Leaf extracts of Azadirachta
indica showed significant effects on mycelial growth particularly of Fusarium oxysporum f. sp
lycopersici, Alternaia solani, Fusarium solani (Table 4.5). Similar results were obtained previously
by number of researchers (Nwachukwu and Umechuruba 2001; Aboellil 2007; Hassanein et al.,
2008; Phalesteen et al., 2008; Siva et al., 2008; Rathode et al., 2010;
Ravikumar and Garampalli 2013). This might be due to presence of Azadirachtin in neem leaves
(Dubey and Kumar 2003; Bajwa and Ahmad 2012). Interestingly, growth of Phytophthora
infestans was reduced with freshly ground leaf extract but no inhibition was recorded with
powdered leaf extract of A. indica. These findings are in partial agreement with Mirza et al; (2000),
Rashid et al; (2004), Agbenin and Marley (2006), Naz et al; (2006) and Yeni et al; (2010).
Bulbs of Allium sativum exhibited substantial antifungal potential against all of the tested fungi (Table 4.5). There are previous reports on antifungal activity of Allium sativum against wide
137 array of plant fungal pathogens (Aba-Alkhail 2005; Sealy et al., 2007; Daniela et al., 2008; Yi et al., 2008; Shrestha and Tiwari 2009; Taskeen-Un-nisa et al., 2011; Ting-Ting et al., 2011).
It is assumed that the antimicrobial activity of garlic may be ascribed to the presence of allicin
(Singh and Singh, 2005; Ameh et al., 2013) having strong antifungal potential effectively
controlled various plant diseases including Late blight of tomato and potato (Slusarenko et al.,
2008).
The present study showed that aqueous leaf extracts of Ocimum sanctum reduced the mycelial
growth of A. solani, Colletotrichum coccodes, F. oxysporum f. sp lycopersici, F. solani (Table
4.5). Shinde and Dhale (2011) reported maximum inhibition of Fusarium oxysporum with
alcoholic extract of O. sanctum rather than aqueous extract. Recently, several studies have been
reported to use different kinds of plants in controlling the same or other pathogen with similar
effects (Abo-Elyousr and Asran 2009; Baraka et al., 2006; Haikal 2007; Nagaraja et al., 2008,
Bhardwaj 2012).
However, Melia azedarach and Lawsonia inermis were effective in reducing the fungal growth of
more or less all test fungi (Table 4.5). These findings are in consistence with that of Yasmeen et
al; (2008), Jabeen et al; (2008), Ambikapathy et al., (2011), Sharma and Sharma (2011) and Javaid
and Samad (2012) who also observed good antimicrobial activity of Lawsonia inermis and Melia
azedarach. Whereas, other plant extracts such as Zingiber officinale, Moringa oleifera, Curcuma
longa, Citrus limon and Tagetes minuta exhibited moderate antifungal activity. But these results
are in contrary to Balbi-pena et al; (2006), Fawzi et al; (2009), Singh and Jain (2011) and Dwivedi
and Enesba (2012) who reported excellent antifungal activity of ginger and moringa against
Fusarium oxysporum f.sp. lycopersici and Alternaria solani.
The remaining plant extracts showed least or no fungitoxicity. These findings are in conformity
with Uzama et al; (2012) who also observed low activity of Polyalthia longifolia. Several factors
138
may be responsible for the differences in antifungal activities of plants, for example, kind of
solvent, extraction method, part and age of plants used. All these factors can affect quantity and
kind of secondary compounds obtained from the plant. Joseph et al; (2008) and Dawar et al; (2007)
noticed that Eucalyptus globules effectively reduced the radial growth of Fusarium solani f. sp.
melongenae. These results are not analogous with present findings. Consequently all the
suppressive / inhibitory activity of the extracts might be attributed to the inborn natural bioactive
materials (Khalil, 2001).
Inhibitory magnitude of plant extracts to tested fungal isolates was directly proportional to applied
concentrations and crude extracts of selected plant extracts exhibited dose dependent activity.
Higher concentrations of plant extracts exerted maximum inhibitory effects on growth of different
fungi. It was noticed in present investigations that efficacy of all extracts increased with the
increase in concentration. Similar results were in accordance with those reported by previous
researchers (Hassanein et al., 2008; Aslam et al., 2010; Nashwa and Abo-Elyousr 2012; Farrag et
al., 2013).
In contrast to aqueous extracts, methanol extracts of test plant species proved less inhibitory to all fungal isolates as compared to aqueous extracts. Our results are similar to earlier workers
(Hassanein et al; (2008) and Mondali et al; (2009) who also quoted less activity of solvent extract of neem compared to aqueous extract against early blight and wilt diseases of tomato and seedborne fungi of different crops. The variation in antifungal activity of same plant parts in aqueous and solvent extracts could be due to differential polarity natures of the solvents. In addition the variable activity might be due to the fact that different chemicals dissolve in different solvents. But presently antifungal activity of methanol extracts of fresh and dry leaves of Azadirachta indica,
Ocimum sanctum, Melia azedarach, and bulbs of Allium sativum were found effective particularly
139 against A. solani, F. oxysporum f. sp lycopersici (Table 4.7). These findings are in consistence with those of Upsana et al; (2002), Tegegne et al; (2008) and Shafique and Shazia Shafique (2012).
There are various methods to evaluate antimicrobial potential of medicinal plants. These bioassays
are the simplest tools that can be used to determine the activity of plant extracts. Most general of
them are poisoned food technique and agar well diffusion method (Kanwal et al., 2010; Bhagwat
and Datar 2013; Birhanu et al., 2013). In our study poisoned food technique exhibited satisfactory
results as compared to agar well method. Maximum numbers of fungal isolates were inhibited
through this technique while agar well assay showed lesser efficacy. These results are in
accordance with Jagtap et al; (2012) who evaluated efficacy of garlic and neem against mycelial
growth of three species of Phytophthora using poison food technique. However, agar well
diffusion assay also depicted that neem, garlic, melia and tulsi in fresh and dry formulations
performed superior than other test plant extracts, with varying degree of inhibition zone size at
different concentrations. Aqueous and solvent extracts of neem leaves followed by garlic and melia
extracts produced maximum zone of inhibition against Colletotrichum coccodes, Fusarium
oxysporum f. sp. lycopersici, Alternaria solani and F. solani
(Table 4.9). These results are in consistency with Charimbu et al; (2009) and Ambikapathy et al;
(2011).
The systematic search of medicinal plants has depicted that botanicals have the antifungal activity to inhibit spore germination of fungal species (Babu et al., 2003; Anwar et al., 2007;
Sehajpal et al., 2009). Aqueous and solvent extracts of Neem, garlic, tulsi, melia and hena completely inhibited the sporulation of Alternaria solani, Fusarium oxysporum f. sp. lycopersici,
F. solani and Colletotrichum coccodes (Fig 4.2, 4.3). Similar kind of results were reported by earlier investigators (Dwivedi and Shukla 2000; Khalil 2001; Anand and Bhaskaran 2009; Mishra et al., 2009; Rahman et al., 2011).
140
Disease problems are changing due to change in climatic conditions and minor diseases can assume the status of major diseases. Therefore, line of control has to be changed with particular emphasis on use of botanicals. Laboratory analysis revealed that A. solani, the causal agent of early blight of tomato is isolated in highest frequency, suppressed by many plant extracts, is an important pathogen. Based on in vitro results three plant extracts showing maximum antifungal activity were subjected to greenhouse and field experimentations against early blight disease. Plant extracts of
Azadirachta indica and Allium sativum greatly reduced the disease intensity when applied to plants both in greenhouse and field, thus increasing the yield of tomato (Table 4.13, 4.14, 4.15, 4.16).
These findings are in close agreement with Patil et al; (2001) and Hassanein et al; (2008) who depicted that tomato plants when sprayed with aqueous neem leaf extract greatly lowered the disease incidence at higher concentration, thus increasing its yield. Use of plant extracts along with sanitation has some positive influence on greenhouse and field plants which corroborate with the findings of previous researchers (Amadioha 2000; Kim et al., 2004; Agbenin et al., 2005; Batish et al., 2007; Afroz et al., 2008; Itako et al., 2008; Hadian et al., 2011; Pattnaik et al., 2012; Al-
Samarrai et al., 2012). It was interestingly observed that garlic extract proved more effective in reducing disease intensity in field as compared to greenhouse. Parallel findings were achieved by
Wszelaki and Miller (2005), Latha et al; (2009), Nashwa and Abo-Elyousr (2012) and Arzoo et al;
(2012). Recently, Chourasiya et al; (2013) evaluated the efficacy of chemical fungicides and botanicals against early blight of tomato in a field experiment. They observed that neem leaf extract effectively reduced disease incidence followed by garlic with increased yield. These findings support our results. Presumably plant extracts affected sporulation of fungal pathogens, which may resulted in inhibiting disease producing activity of pathogen and increased induced resistance in plant. This results into overall better growth of plant eventually increasing the yield.
141
It is concluded that most of the plants studied and their extracts possess good antifungal activity
under laboratory and field conditions with varying efficacy. Aqueous extracts of Azadirachta
indica, Allium sativum and Ocimum sanctum showed significant antifungal potential as compared
to extracts of other plants. A correlation is present among phenol contents and antifungal activity,
plants with higher phenolics showed superior capability. Extracts of these plants may be used as a
structural escort for the preparation of biofungicides against destructive diseases of plants. Further
studies may be carried out by using various mixtures of these compounds to exploit their
synergistic effects in the integrated disease management and easy availability to the farmers.
6. SUMMERY
Investigations were carried out on the prevalence of fungi on tomato crop in typical specific production ecology near Bahauddin Zakariya University, Multan, with particular emphasis on their identity and reactivity to the extracts of selected medicinal plants.
• Twenty five fungal species distributed in 16 taxonomic genera were isolated from different
parts of tomato; seeds (1.35-15%), leaves (0.5-19.0%), fruits (0.75-15.75%) and roots
(0.25-17.5%).
• Tomato seeds harbored 17 fungal species, best and significantly isolated by the Blotter
Paper than Agar Plate method.
• All the fungi isolated were subjected to pathogenicity test and fulfillment of Koch’s
postulates. Predominantly occurring fungi included species of Alternaria, Fusarium,
Colletotrichum, Phytophthora, Chaetomium, Curvularia and Aspergillus. Two diseases;
142
blights and wilts are re-emerging as serious problem. Five fungal species were selected for
subsequent studies on antifungal properties of indigenous plant extracts.
• Fifteen medicinal plants of tropical and subtropical type were selected at the campus. Three
formulations of plant extracts at various concentrations were evaluated for the antifungal
activity against the target fungi. The assessment was based on the relative reduction of
mycelial growth, sporulation and formation of inhibitory zone in the medium and their
efficacy in controlling early blight disease.
• Phytochemical analysis of the extracts of medicinal plants revealed the presence of
flavonoids, terpenoids, saponins, steroids, cardiac glycosides, phlobatannins, tannins and
caumarins and phenolic compounds, to which antifungal activity is attributed.
• A positive correlation was found between the biological activity of test fungi and antifungal
activity of extracts at various concentrations.
• Extracts of twelve out of fifteen medicinal plants proved effective against the target fungi.
Fresh aqueous extracts showed better efficacy than the dry powder and methanol extracts.
• Botanicals in general exhibited pronounced antifungal effects; (80-100%), moderate or
intermediate (50-70%) and negligible or variable (20-40%). Aqueous and methanol
extracts were highly effective at higher concentration (80%).
• Relative antifungal activity of extracts from various plants followed the order of
Azadirachta indica > Allium sativum > Ocimum sanctum > Melia azedarach > Lawsonia
inermis > Zingiber officinale > Moringa oleifera > Citrus lemon > Tagetes minuta >
Curcuma longa > Catharenthus roseus > Eucalyptus globules > Syzigium cumini >
Polyalthia longifolia > Mangifera indica.
143
• The best antifungal results were obtained with extracts of Azadirachta indica, Allium
sativum, Ocimum sanctum, Melia azedarach and Lawsonia inermis against foliar
pathogens and wilt with the exception of Phytophthora. In general the target pathogens
were sensitive or vulnerable to antifungal property of the extracts.
• Growth behavior of the target organisms against extracts was investigated through
Poisoned Food and Agar Well Diffusion techniques. Fresh formulations were highly
effective because of their better diffussibility in the medium and produced maximum
inhibition zone (more than 17mm), methanol extracts (11-16mm) than dry powder
(1015mm). The results obtained were generally uniform, consistent, stable and visible with
the extracts and test pathogens.
• The study revealed that Alternaria blight is continued as a serious disease and emerging as
a serious production problem of tomato. In order to test the antifungal activity, three
medicinal plant extracts at 80% concentration along with commonly used fungicide
Topsin-M were tested as foliar spray against early blight in the green house and field. Under
controlled green house conditions, curative activity of A. indica, A. sativum and O.
sanctum extracts reduced disease incidence by 37.60, 29.60 and 25.37% respectively, as
against 39.20% by Topsin-M, promoted growth and development of the plant and increased
the yield by 55.76, 51.30 and 46.47% over untreated control in 2012-13. In the field where
the micro and macroclimate change, the disease parameters and yield spectrum may also
alter. Results indicated that treatments (Topsin-M, A. sativum, A. indica and O. sanctum)
when applied curatively, reduced severity and incidence of blight by 64.18, 58.21, 56.72
and 50.75% and produced yield by 3.50, 3.70, 3.50 and 3.17 t/ha, respectively, in 2012-13.
Similar trend was noticed during second year of cultivation both in protective and curative
144
applications. It was concluded that the botanicals were as effective as the chemical control
against early blight and can easily replace fungicide at a cheaper cost.
• The investigations carried out so far may be considered of preliminary nature but constitute
a strong base in future for selection and identification of more promising plants with
enhanced antifungal properties, the development and formulation of stable products (as
powders, tablets and liquids) for marketing, awareness and easy availability to the farmers
and their application in the integrated disease management. So far, extracts from medicinal
plants have proved quite effective against the foliar diseases but these can hardly be used
against soil-borne diseases as Fusarium and Verticillium wilts for which more work is
required. Moreover antifungal plants and their extracts need to be identified for the control
of Phytophthora blight which seems to have developed many races
resistant to fungicides.
145
7. REFERENCES
Aba Alkhail, A. A. 2005. Antifungal Activity of Some Extracts Against Some Plant Pathogenic
Fungi. Pak. J. Biol. Sci. 8, 413-417.
Abdel-Kader, M. M., N. S. El-Mougy, N. G. El-Gammal, F. Abd-El-Kareem M. A. Abd-Alla.
2012. Laboratory evaluation of some chemicals affecting pathogenic fungal growth. J.
Appl. Sci. Res. 8, 523-530.
Abdullah, M. T., N. Y. Ali and P. Suleman. 2008. Biological control of Sclerotinia sclerotiorum
(Lib.) de Bary with Trichoderma harzianum and Bacillus amyloliquefaciens. Crop. Prot.
27, 354-359.
Aboellil, A. H. 2007. Trilogy, a Product of Neem (Azadirachta indica) Induces Resistance in
Cucumber Against Podoshaera xanthii. Res. J. Microbiol. 2, 402-414.
Abo-Elyousr, K.A. and M. R. Asran. 2009. Antibacterial activity of certain plant extracts against
bacterial wilt of tomato. Arch. Phytopathol. Plant Prot. 42, 573-578.
146
Abyaneh, M. R., A. Allameh,T. Al-Tiraihi and M. Shams. 2005. Studies on the Mode of Action of
Neem (Azadirachta indica) Leaf and Seed Extracts on Morphology and Aflatoxin
production Ability of Aspergillus parasiticus. Acta Hort. 675, 123-127.
Adedapo, A. A., F. O. Jimoh, A. J. Afolayan and P. J. Masika. 2009. Antioxidant potential of
methanol extracts of the leaves and stems of Celtis Africana. Rec. Nat. Prod. 3(1), 23-
31.
Afek, U., A. Sztejnberg and S. Carmel. 1986. 6,7-dimethoxycoumarin, a Citrus phytoalexin
conferring resistance against Phytophthora gummosis. Phytochemistry. 25, 1855-1856.
Afroz, M., M. Ashrafuzzaman, M. Ahmed, M. Ali and M. Azim. 2008. Integrated management
of major fungal diseases of tomato. Int. J. Sustain Crop Prod. 3, 54-59.
Agarwal, V. K. 1981. Seed-borne fungi and viruses of some important crops. Research Bulletin,
Govind Ballabh Pant University of Agriculture and Technology. pp. 147.
Agarwal, B. B., C. Sundaram, N. Malani and H. Ichikawa. 2007. Curcumin: The Indian solid
gold. Adv. Exp. Med. Biol. 595, 1-75.
Agbenin, N. O., A. M. Emechebe, P. S. Marley and A. D. Akpa. 2005. Evaluation of nematicidal
action of some botanicals on Meloidogyne incognita in vivo and in vitro. J. Agric. and Rur.
Dev. Tropic. Subtropics (JARTS). 106, 29-39.
Agbenin, O. N and P. S. Marley. 2006. In vitro assay of some plant extracts against Fusarium
oxysporum f. sp. lycopersici, causal agent of Tomato Wilt. J. Pl. Prot. Res. 46, 215-220.
Agrios, G. N. 2005. Plant Pathology. 5th ed. Acadamic Press, NY, US. pp. 922.
Alexopoulos, C. J., C.W. Mims and M. Blackwell. 1996. Phylum Oomycota. Introductory
Mycology, 4th Edi. 683-737.
Ali, S. I. 1980. Flora of Pakistan. Pakistan Agricultural Research Council.
147
Ali, N. A. A., W. D. Jülich, C. Kusnick and U. Lindequist. 2001. Screening of Yemeni medicinal
plants for antibacterial and cytotoxic activities. J. Ethnopharmacol. 74(2), 173-179.
Ali, S., V. Rivera and G. Secor. 2005. First report of Fusarium graminearum causing dry rot of
potato in North Dakota. Plant Dis. 89, 105.
Alkhail, A. A. 2005. Antifungal activity of some extracts against some plant Pathogenic
Fungi. Pak. J. Biol. Sci. 8(3), 413-417.
Alström, S. 1992. Antibacterial activity of tea and coffee wastes against some plant pathogenic
Pseudomonas syringae strains. J. Phytopathol. 136(4), 329-334.
Al-Samarrai, G., H. Singh and M. Syarhabil. 2012. Evaluating eco-friendly botanicals (natural
plant extracts) as alternatives to synthetic fungicides. Ann. Agric. Environ. Med. 19,
673676.
Amadi, J. E., S. O. Salami and C. S. Eze. 2010. Antifungal properties and phytochemical screening
of extracts of African Basil (Ocimum gratissimum L.). Agric. Biol. J. North Am. ISSN
Print: 2151-7517, Online 2151-7525.
Am adioha, A. C. 2000. Controlling rice blast in vitro and in vivo with extracts of Azadirachta
indica. Crop Prot. 19, 287-290.
Ambikapathy, V., S. Gomathi and A. Panneerselvam. 2011. Effect of antifungal activity of some
medicinal plants against Pythium debaryanum (Hesse). Asian J. Plant Sci. 1, 131-134.
Ameh, G.I., S. C. Eze and F.U. Omeje. 2013. Phytochemical screening and antimicrobial studies
on the methanolic bulb extract of Allium sativum L. Afr. J. Biotechnol.12,1665-1668.
Amini, J. and D. Sidovich. 2010. The effects of fungicides on Fusarium oxysporum f. sp.
lycopersici associated with Fusarium wilt of tomato. J. Pl. Prot. Res. 50(2), 172-178.
Anand, T. and R. Bhaskaran. 2009. Exploitation of plant products and bioagents for ecofriendly
management of chili fruit rot disease. J. Pl. Prot. Res. 49(2), 195-203.
148
Anaya, A. L. 1999. Allelopathy as a tool in the management of biotic resources. Crit. Rev. Pl. Sci.
18, 697-739.
Anju, R. M., N. Bhat and B. P. Singh. 2007. Effect of fungicides on growth and germination
of zoosporangia and zoospores of Phytophthora infestans. J. Mycol. Pl. Pathol. 37, 527529.
Anukwuorji, C. A., C. L. Anuagasi and R. N. Okigbo. 2013. Occurrence and control of fungal pathogens of sweet potato (Ipomoea batatas L. Lam) with plant extracts. Ph. Tech. Med.
2(3), 278-289.
Anwar, M. N., J. Begum, M. Yusuf, J. U. Chowdhury, S. Khan and M. Nural. 2007. Antifungal
activity of forty higher plants against phytopathogenic fungi. Bangladesh J. Microbiol.
24(1),76-78.
Aqil, F. and I. Ahmad. 2003. Broad-spectrum antibacterial and antifungal properties of certain
traditionally used Indian medicinal plants. World J. Microbiol. Biotech. 19(6), 653-657.
Arora, D. S. and J. Kaur. 1999. Antimicrobial activity of spices. Int. J. Antimicrobiol Agents. 12,
257-262.
Arzoo, K., S. K. Biswas and M. Rajik. 2012. Fusarium Wilt induced by plant extracts. Pl. Pathol.
J. 11(2), 42-50.
Ashraf, H., and A. Javaid. 2007. Evaluation of antifungal activity of Meliaceae family against
Macrophomina phaseolina. Mycopathologia. 5, 81-84
Aslam, A., F. Naz, M. Arshad, R. Qureshi and C. A. Rauf. 2010. In vitro fungal activity of
selected medicinal plant diffusates against Alternaria solani, Rhizoctonia solani and
Macrophomina phaseolina. Pak. J. Bot. 42, 2911-9.
Ayeni, K. E. and S. A. Yahya. 2010. Phytochemical screening of three medicinal plants- Neem
leaf (Azadirachta indica), Hibiscus leaf (Hibiscus rosasinensis) and Spear Grass leaf
(Imperata cylindrical) Continental J. Pharma. Sci. 4, 47 – 50.
149
Ayoola, G. A., H. A. B. Coker, S. A. Adesegun, A. A. Adepoju-Bello, K. Obaweya, E. C.
Ezennia and T. O. Atangbayila. 2008. Phytochemical Screening and Antioxidant
Activities of Some Selected Medicinal Plants Used for Malaria Therapy in Southwestern
Nigeria. Trop. J. Pharma. Res. 7(3), 1019-1024.
Azam, F and S. J. Shah. 2003. Exploring the role of farmer led management practices on various
tomato and cucumber diseases in Peshawar and Dragai areas of NWFP. In: Final Reports
on PHP funded research projects. pp. 1-75.
Babu, K. S., P. V. Srinivas, B. Praveen, K. H. Kishore, U. S. Murty and J. M. Rao. 2003.
Antimicrobial constituents from the rhizomes of Rheum emodi. Phytochemistry. 62(2),
203-207.
Bailey, J. A., and J. W. Mansfield. 1982. Phytoalexins. Blackie Glasgcow, UK. pp 334.
Bajwa, A. A. and A. Ahmad. 2012. Potential applications of Neem based products as
biopesticides. The Health. 3(4), 116-120.
Balbi-Peña M. I. B., A. Becker, J. R. Stangarlin, G. Franzener, M. C. Lopes, and K. R. F. Schwan-
Estrada. 2006. Control of Alternaria solani in tomato by Curcuma longa and curcumin-I.
In vitro evaluation. Fitopat. Brasil. 31, 310-314.
Baldauf, S. L., A. J. Roger, I. Wenk-Siefert and W. F. Doolittle. 2000. A kingdom-level phylogeny
of eukaryotes based on combined protein data. Science. 290(5493), 972-977. DOI:
10.1126/science.290.5493.972.
Balestra, G. M., A. Heydari, D. Ceccarelli, E. Ovidi and A. Quattrucci. 2009. Antibacterial effect
of Allium sativum and Ficus carica extracts on tomato bacterial pathogens. Crop Prot.
28, 807–811.
Bankole, S. A. 1996. The distribution and pathogenicity of the seed mycoflora of two tomato
varieties cultivated in western Nigeria. Afr. Crop Sci. J. 4, 491-496.
150
Banso, A., and T. Olutimayin. 2001. Phytochemical and Antimicrobial Evaluation of Aqueous
extracts of Daniella oliveri and Nauclea latifolia. Nigeria J. Biotechnol. 12, 114118.
Baraka, M. A., S. A. Omar, E. El-Barougy and A. H. Zian. 2006. Controlling seedling dampingoff,
root rot and wilt diseases of lupine (Lupinus albus L.). Agric. Res. J. Suez Canal Univ. 6,
57-68.
Barkai-Golan, R. 2001. Postharvest Diseases of Fruits and Vegetables. Development and
Control. Elsevier, Amsterdam NL. pp. 418.
Barnett, H. L. and B. B. Hunter. 1972. Illustrated Genera of Imperfect Fungi (4th Edi.). Burgess
Publication Ltd. St.Paul, Minnesota, USA, 1-216.
Batish, D. R., K. Arora, H. P. Singh and R. K. Kohli. 2007. Potential utilization of dried powder
of Tagates minuta as a natural herbicide for managing rice weeds. Crop Prot. 26, 566571.
Baudoni, A. B. A. M. 1988. Diagnosis of disease and proof of pathogenicity (Koch’s Postulates)
In: Laboratory exercises in Plant Pathology. An Instructional kit, Baudoni A.B.A.M. (Ed).
APS Press, St Paul M.N. pp. 213.
Bautista, B.S., E. García, L. Barrera, N. Reyes and C. Wilson. 2003. Seasonal Evaluation of the
postharvest fungicidal activity of powders and extracts of huamúchil (Pithecellobium
dulce): action against Botrytis cinerea, Penicillium digitatum and Rhizopus stolonifer of
strawberry fruit. Postharvest Biology & Technology. 29, 81-92. ISSN 0925-5214.
Baysal, Ö., M. Siragusa, H. Ikten, I. Polat, E. Gümrükcü, F. Yigit and J. T. da Silva. 2009. Fusarium
oxysporum f. sp. lycopersici races and their genetic discrimination by molecular markers
in West Mediterranean region of Turkey. Physiol. and Mol. Pl. Pathol. 74, 68-75.
Bhagwat, M. K., and A. G. Datar. 2013. Antifungal activity of herbal extracts against plant
pathogenic fungi. Arch. Phytopath. Plant Prot. 1-7.
151
Bhardwaj, S. K. 2012. Evaluation of plant extracts as antifungal agents against Fusarium solani
(Mart.) Sacc. World. J. Agric. Sci. 8(4), 385-388. ISSN 1817-3047. DOI:
10.5829/idosi.wjas.2012.8.4.1676.
Bianchi, A., A. Zambonelli, A. Z. D'Aulerio and F. Bellesia. 1997. Ultrastructural studies of the
effects of Allium sativum on phytopathogenic fungi in vitro. Pl. Dis. 81(11), 1241-1246.
Birhanu, S., M. S. Akhtar and D. Muleta. 2014. Management of Post-Harvest Spoilage Fungi by
Some Spice Extracts. Arch. Phytopathol. Pl. Prot. 47(17), 2124-2140.
Bluma, R., M. R. Amaiden, and M. Etchverry. 2008. Screening of Argentine plant extracts:
Impact on growth parameters and aflatoxin B1 accumulation by Aspergillus section Flavi.
Int. J. Food Microbiol. 122, 114-125.
Booth, C. 1971. The genus Fusarium. Common wealth Mycol. Inst. Kew, Surrey, UK. pp 237.
Bouwmeester, K., P. M. J. A.Van Poppel and F. Govers. 2009. Genome biology cracks enigmas
of Oomycete plant pathogens. Annu. Plant Rev. 34, 102-133.
Bowles, B. L., and A. J. Miller. 2006. Caffeic acid activity against Clostridium botulinum
Spores. J. Food Sci. 59, 905-908.
Brahmachari, G. 2004. Neem-An Omnipotent Plant: A Retrospection. Biol. Chem.Wiley US,
408-421.
Brent, K. J., and D. W. Hollomon. 2007. Fungicide resistance in crop protection, how can it be
managed. FRAC Monograph. 1, 2nd revised, Crop life International, Brussels. pp 56.
Brown, W. 1924. Two mycelial methods. II. A method of isolation single strains on fungi by
cutting out a hyphal tip. Ann. Bot. 38, 402-404.
Burapadaja, S., and A. Banchoo. 1995. Antimicrobial activity of tannins from Terminalia citrine.
Planta Med. 61, 365-366.
152
Burgos, N. R., R. E. Talbert and J. D. Mattice. 1999. Cultivar and age differences in the production
of allelochemicals by Secale cereale. Weed Sci. 47, 481-485.
Cai, Y., Q. Luo, M. Sun and H. Corke. 2004. Antioxidant activity and phenolic compounds of
112 traditional Chinese medicinal plants associated with anticancer. Life sci. 74,
21572184.
Campo, J., C. Nguyen-The, M. Sergent and M. J. Amito. 2003. Determination of most bioactive
phenolic compounds from rosemary against Listeria monocytogenes- influence of
concentration, pH and NaCl. J. Food Sci. 68, 2066–2071.
Canillac, N. and A. Mourey. 2001. Antibacterial activity of the essential oil of Picea excelsa on
Listeria, Staphylococcus aureus and coliform bacteria. Food Microbiol. 18, 261-268.
Carpinella, M. C., L. M. Giorda and C. G. Ferrayoli. 2003. Antifungal effects of differen organic
extracts from Melia azedarach L. on phytopathogenic fungi and their isolated active
components. J. Agric. Food Chem. 51, 2506-2511.
Carpinella, M. C., C. G. Ferrayoli and C. G. Ferrayoli. 2005. Antifungal synergistic effect of
scopoletin, a hydroxycoumarin isolated from Melia azedarach L., fruits. J. Agric. Food
Chem. 53, 2922-2927.
Cetkovic, G. S, J. M. Brunet, S. M. Bjilas, V. T. Tumbas, S. L. Markov and D. D. Cetkovic.
2007. Antioxidant potential, lipid peroxidation inhibition and antimicrobial activities of
Satureja montana . sub sp. Kitaibelli Extracts. Int. J. Mol. Sci. 8, 1013-1026.
Chander, H., S. G. Kulkarni and S. K. Berry. 1992. Studies on turmeric and mustard oil as
protectants against infestation of red flour beetle, Tribolium castaneum (Herbst) in stored
rice. J. Insect Sci. 5, 220-222.
Chandler, D. 1994. Cryopreservation of fungal spores using porous beads. Mycol. Res. 98, 525-
153
526.
Chandrasekaran, M., and V. Venkatesalu. 2004. Antibacterial and antifungal activity of
Syzygium jambolanum seeds. J. Ethnopharmac. 91, 105-108.
Charimbu, M. K., I. N. Wagara and D. O. Otaye. 2009. Antifungal activity of various crude plant
extracts against Phaeoisariopsis griseola pathogenic on common bean. 9th African Crop
Science,Conference Proceedings, Cape Town, South Africa. 683–685. ISSN
1023070X/2009.
Chattopadhyay, D., B. K. Sinha, L. K. Vaid. 1998. Antibacterial activity of Syzygium species.
Fitoterapia. 69, 356-367.
Chattopadhyay, I., K. Biswas, U. Bandyo Padhyayand R. K. Banerjee. 2004. Turmeric and
curcumin: Biological actions and medicinal applications. Curr. Sci. 87, 44-53.
Chavan, R. and S. Tawade. 2012. Studies on fungi associated with tomato rots. Multilogic in
Science., 2, 85-87. ISSN 2277-7601
Chhetri, H. P., N. S. Yogol, J. Sherchan, K. C. Anupa, S. Mansoor and P. Thapa. 2008.
Phytochemical and antimicrobial evaluation of some medicinal plants of Nepal.
Khatmandu Univ. J. Sci. Eng. Tech. 1, 49-54.
Chourasiya, P. K., A. A. Lal and S. Simon. 2013. Effect of certain fungicides and botanicals against
Early blight of Tomato caused by Alternaria solani (Ellis. and Martin) under Allahabad
Utter Pradesh, India conditions. Int. J. Agric. Sci. Res. 3, 151-156.
Chaudhry,G.,S. Goyal and P. Poonia. 2010. Lawsonia inermis Linnaeus:A Phytopharmacological
Review. Int. J. Pharma. Sci. Drug Res. 2(2), 91-98.
Chuku, E. C., J. A. Osakwe and J. Eke. 2010. Fungi Flora Of Melon Seeds (Citrullus
colocynthis) And Their Response To Seed Treatment. Scientia Africana. 9(2), 77-82.
Cikricki, S., E. Mozioglu and H. Yýlmaz. 2008. Biological activity of curcuminoids isolated
154
from Curcuma longa. Rec. Nat. Prod. 2, 19-24.
Conn, E. E. and P. K. Stumpy. 1970. Out lines of Biochemistry (3rd ed.). John Wiley and
Sons. N.Y, US. 7-9.
Cowan, M. M. 1999. Plant products as antimicrobial agents. Clin. Microbiol. Rev. 12, 564-582.
Cragg, G. M., D. J. Newman and K. M. Sander. 1997. Natural products in drug discovery and
development. J. Natl. Prod. 60, 52-60.
Cuevas ,V. C, A.M. Sinohin and J. I. Orajay. 2005. Performance of selected Philippine species of
Trichoderma as biocontrol agents of damping off pathogens and as growth enhancer of
vegetables in farmers' field. Phillip. Agric. Sci .88, 63-71.
Cuthbertson, A. G. S., and A. K. Murchie. 2005. Economic spray thresholds in need of revision
in Northern Irish Bramley orchards. Biodivers. News. 32, 19.
Dahanukar, S. A., R. A. Kulkarni, N. N. Rege. 2000. Pharmacology of Medicinal Plants and
Natural Products. Ind. J. Pharmacol. 2, S81-S118.
Dandge, V. S., S. P. Rothe, A. S. Pethe and K. V. Kothale. 2012. Antifungal activity of
Polyalthia longifolia (Sonnef) Thw. against some fungal pathogens. Int. J. Sc. Pharma
Educ. Res.1, 73-77.
Daniela, P., K. Eckhard and J.S. Alan. 2008. Effects of garlic (Allium sativum) juice containing
allicin on Phytophthora infestans and downy mildew of cucumber caused by
Pseudoperonospora cubensis. Eur. J. Plant Pathol. 122, 197-206.
Darout, I., A. Cristy, N. Skaug and P. Egeberg. 2000. Identification and quantification of some
potential antimicrobial anionic components in miswak extract. Ind. J. Pharmacol. 32, 11-
14.
Dawar, S., S. M. Younus, M. Tariq and M. J. Zaki. 2007. Use of Eucalyptus sp. in the control of
root infecting fungi on mungbean and chickpea. Pak. J. Bot. 39(3), 975-979.
155
Das, S. R., B. K. Pani and S. Kar. 1997. Evaluation of some plant extracts against Sclerotium
rolfsii causing stem rot in tuberose. Environ. Ecol. 5, 975-976.
Datar, V. V., and C. D. Mayee. 1981. Assessment of loss in tomato yield due to early blight.
Ind. Phytopath. 34, 191-195.
Davidson, P. M. 1997. Chemical preservatives and natural antimicrobial compounds. In: Food
Microbiology-Fundamentals and Frontiers. (Eds. M. P.Doyle, L.R.Beuchat and T.
J.Montville). Washington D.C, US. pp 520-556.
De Ávila, Z. R., S.C.M. de Mello, Z. M. A. Ribeiro and E. M. G. Fontes. 2000. Production of
Alternaria cassiae inoculum. Pesq. Agropec.bras. Brasilia. 35(3), 533-541.
De Curtis, F., G. Lima, D. Vitullo, and V. De Cicco. 2010. Biocontrol of Rhizoctonia solani
on tomato by delivering antagonistic bacteria through a drip irrigation system. Crop Prot.
29, 663-670.
Delaquis, P. J., K. Stanich, B. Girard and G. Mazza. 2002. Antimicrobial activity of individual
and mixed fractions of dill, cilantro, coriander and eucalyptus essential oils. Int. J. Food
Microbiol.74, 101-109.
Dellavalle, P. D., A. Cabrera, D. Alem, P. Larranaga, F. Ferreira and M. D. Rizza. 2011.
Antifungal activity of medicinal plant extracts against Phytopathogenic fungus
Alternaria spp. Chil. J. Agric. Res. 231-239.
Derbalah, A., M. El-Mahrouk and A. El-Sayed. 2011. Efficacy and Safety of Some Plant Extracts
against Tomato Early Blight Disease Caused by Alternaria solani. Pl. Pathol. J.
10, 115-121.
Desai, P. D, A. K. Ganguly, T. R. Govindachari, B. S. Joshi, V. N. Kamat, A. H. Manmade, P. A.
Mohamed, S. K. Nagle R. H. Nayak, A. K. Saksena, S. S. Sathe and N.
Vishwanathan. 1966. Chemical investigation of some Indian plants: Part II. Ind. J.
156
Chem. 4, 457-549.
Devi, S. R., F. Pellissier, and M. N.V. Prasad. 1997. Allelochemicals. In:Plant Ecophysiology,
(ed. M.N.V. Prasad). John Wiley & Sons, N , US. pp. 253-303.
Dhanavade, M. J., C. B. Jalkute, J. S. Ghosh and K. D. Sonawane. 2011. Study Antimicrobial
Activity of Lemon (Citrus limon (L.) Burm.Peel Extract. Br. J. Pharmacol. Toxicol. 2,
119-122.
Dhingra, O. D., and J. B. Sinclair. 1985. Basic Plant Pathology Methods. 2nd, CRC Press. Inc:
Boca Raton. FL.
Dillard, H. R. 1989. Effect of temperature, wetness duration, and inoculums density on infection
and lesion development of Colletotrichum coccodes on tomato fruit. Phytopathology.
79, 1063-1066.
Dixon, R. A. 1999. Isoflavonoids: biochemistry, molecular biology, and biological functions. In:
Comprehensive Natural Products Chemistry. Netherlands, Elsevier Sci., 1, pp. 773-823.
Dorgan, J. F., A. Sowell, C. A. Swanson, N. Potischman, R. Miller, N. Schussler and
H.E.Stephenson Jr. 1998. Relationship of serum carotenoids, retinol, a-tocopherol and
selenium with breast cancer risk: results from a prospective study in Columbia,
Missouri. Cancer Causes Control. 9, 89-97.
Doherty,V. F., O. O. Olaniran and U. C. Kanife. 2010. Antimicrobial activities of Aframomum
melegueta (Alligator pepper). Int. J. Biol. 2, 126-131.
Dubey, N. K., T. N. Tiwari, D. Mandin, H. Andriamboavonjy and J. P. Chaumont. 2000.
Antifungal properties of Ocimum gratissimum essential oil (ethyl cinnamate chemtype).
Fitoterapia, 71, 567-569.
Dubey, R.C. and R. Kumar. 2003. Efficacy of Azadirachtin and fungicides on growth and
157
survival of sclerotia of Macrophomina phaseolina causing charcoal rot in soyabean. Ind.
Phytopath. 56, 216-217.
Dubey, S. C., M. Suresh and B. Singh. 2007. Evaluation of Trichoderma species against Fusarium
oxysporum f. sp. ciceris for integrated management of chickpea. Biol.
Control. 40, 118-127.
Dubey, R. C., H. Kumar and R. R. Pandey. 2009. Fungitoxic Effect of Neem Extracts on Growth
and Sclerotial Survival of Macrophomina phaseolina in vitro. J. Amer. Sci. 5, 17-24.
Duthie, G., and A. Crozier. 2000. Plant-derived phenolic antioxidants. Curr. Opinion. Lipidol.
11, 43-47.
Duke, S. O., F. R. Dayan, J. G. Romaine and A. M. Rimando. 2000. Natural products as sources
of herbicides: Status and Future Trends. Weed Res. 40, 99-111.
Dwivedi, R. S., and S. P. Pathak. 1981. Effect of certain chemicals on the population dynamics
of Fusarium oxysporium f. sp. lycopersici in tomato field soil. Proc. Ind. Natl. Sci. Acad.
47, 751-755.
Dwivedi, S. K., and A. Enespa. 2012. Effectiveness of extract of some medicinal plants against
soil-borne Fusaria causing diseases on Lycopersicon esculentum and Solanum
melongena plants. Intr. J. Pharaco. Biol. Sci. 3, 1171-1180.
Ebel, J. 1986. Phytoalexin synthesis: The biochemical analysis of the induction process. Annu.
Rev. Phytopath. 24, 235-264.
Edeoga, H.O., D. E. Okwu and B.O. Mbaebie. 2005. Phytochemical constituents of some
Nigerian medicinal plants. Afr. J. Biotechnol. 4, 685-688.
Eilenberg, J., A. Hajek and C. Lomer. 2001. Suggestions for unifying the terminology in
biological control. Biocontrol. 46, 387-400.
Elad, Y., and G. Zimand. 1993. Use of Trichoderma harzianum in combination or
158
alternation with fungicides to control cucumber grey mould (Botrytis cinerea) under
commercial greenhouse conditions. Plant Pathol. 2, 324-332.
El Ansari, M. A., K. K. Reddy, K. N. S. Sastry and Y. Nayudamma. 1971. Dicotyledonae,
anacardiaceae polyphenols of Mangifera indica. Phytochemistry. 10, 2239-2241.
Elgayyar, M., F. A. Draughon, D. A. Golden and J. R. Mount. 2001. Antimicrobial activity of
essential oils from plants against selected pathogenic and saprophytic microorganisms. J.
Food Prot. 64(7), 1019-1024.
Elliott, M., S. F. Shamoun, G. Sumampong, D. James, S. Masri, and A. Varga. 2009. Evaluation
of several commercial biocontrol products on European and North American populations
of Phytophthora ramorum. Biocontrol Sci. Technol. 99, 1007-21.
Ellis, M. B. 1971. Dematiaceous hyphomycetes. Commonwealth Mycol. Inst. Kew, England,
464–497.
Eloff, J. N. 1998. Which extractant should be used for the screening and isolation of
antimicrobial components from plants. J. Ethnopharmac. 60, 1-8.
Enikuomehin, O. A., and E. O. Oyedeji. 2010. Fungitoxic effect of some plant extracts against
tomato fruit rot pathogens. Arch. Phytopath. Plant Prot. 43, 233–240.
Erdemgil, F. Z., M. Yilmaz and M. Kivanc. 2004. Antimicrobial activity of Thalictrum
orientale’s extracts. J. Marmara. Pure and Appl. Sci. 19, 23-33.
Ernst, E. and M. H. Pittler. 2000. Efficacy of ginger for nausa and vomiting: a systematic review
of randomized clinical trials. Brit. J. Anesthesia. 84 (3), 367-371.
Falodun, A., L. O. Okunrobo and N. Uzoamaka. 2006. Phytochemical Screening and
AntiInflamatory Evaluation of Methanolic and Aqueous extracts of Euphorbia
heterophylla Lim. Afr. J. Biotech. 5(6), 529-531.
Fakir, G. A. 2001. An annotated list of seed-borne disease in Bangladesh. Seed Pathology
159
Laboratory. Department of Plant Pathology, BAU, Mymensingh. p 41.
Farrag, E. S. H., M. H.A. Moharam and El-Sayed H. Ziedan. 2013. Effect of plant extracts on
morphological and pathological potential of seed-borne fungi on cucumber seeds. J. Agric.
Tech. 9, 141-149.
Faria, T. J., R. S. Ferreira, L. Yassumoto, J. Roberto, P. de Souza, N. K. Ishikawa and A. de Melo
Barbosa. 2006. Antifungal Activity of Essential Oil Isolated from Ocimum
gratissimum L. (eugenol chemotype) against Phytopathogenic Fungi. Braz. Arch. Biol.
Tech. 49 (6), 867-871.
Fawzi, E. M., A. A. Khalil and A. F. Afifi. 2009. Antifungal effect of some plant extracts on
Alternaria alternata and Fusarium oxysporum. Afr. J. Biotechnol. 8(11), 2590-2597.
Fernandez-Ruiz, V., M. C. Sanchez-Mata, M. Camara, M. E. Torija, S. Rosello and F. Nuez.
2002. Lycopene as a bioactive compound in tomato fruits. Symposium on “Dietary
Phytochemicals and Human Health”. Phytoch. Soc. Eur. Salamanca. 193-194.
Franzener, G., A. S. Martinez-Franzener, J. R. Stangarlin, C. Furlanetto and K. R. F. Schwan
Estrada. 2007. Protection of tomato plants by Tagetes patulaaqueous extract against
Meloidogyne incognita. Nematologia Brasileira. 31, 27-36.
Fravel, D. R., D. J. Rodes and P. R. Larkin. 1999. In: Production and commercialization of
biocontrol products. (Eds; R, Albajes, M. L. Gullino, J. C. van Lenteren, Y. Elad),
Kluwer Wageningen NL. pp 365-376.
Fravel, D. R., K. L. Deahl and J. R. Stommel. 2005. Compatibility of the biocontrol fungus
Fusarium oxysporum strain CS-20 with selected fungicides. Biol. Control. 34,165–169.
Fricker, C.E., M. L. Smith and K. Akpagana. 2003. Bioassay-guided isolation and identification
of antifungal compounds from ginger. J. Phytother. Res. 17, 897-903.
Fry, W. E., S. B. Goodwin, A. T. Dyer, J. M. Matuszak, A. Drenth, P. W. Tooley, L. S.
160
Sujkowski, Y. J. Koh, B. A. Cohen, L. J. Spielman, K. L. Deahl, D. A. Inglis, and K. P.
Sandlan. 1993. Historical and recent migrations of Phytophthora infestans: Chronology,
pathways, and implications. Plant Dis.77(7), 653-661.
Gangawane, L. V. and S. S. Kamble. 2001. Use of other agrochemicals in the management of
charcoal rot of potato caused by Macrophomina phaseolina resistant to carbendazim.
Frontiers in Fungal Biotechnology and Plant Pathogen Relations. 58-62.
Geissman, T. A. 1963. Flavonoids compounds, tannins, lignins and related compounds. In:
Pyrrole pigments, Isoprenoids compounds and phenolic plant constituents.(eds; M.
Flarkin and E. H. Stotz). Elsevier NY,US. 9, 265.
Gentilcore, D. 2010. Pomodoro!: A History of the Tomato in Italy. Columbia University Press.
Ghalem, B. R., and B. Mohammad. 2008. Antibacterial activity of leaf essential oils of
Eucalyptus. Afr. J. Pharmacol. 2, 211-215.
Ghasemzadeh, A., H. Z. E. Jaafer and A. Rahmat. 2011. Effects of solvent type on phenolics and
flavonoids content and antioxidant activities in two varieties of young ginger (Zingiber
officinale Roscoe) extracts. J. Med. Pl. Res. 5(7), 1147-1154.
GOP. 2012-13. Agricultural Statistics of Pakistan. Statistics Division, Govt. Pakistan, Islamabad.
Goss, R. W. 1936. Fusarium Wilts of potato, their differentiation and the effect of environment
upon their occurrence. Amer.J. Potato Res.13(7), 171-180.
Govers, F. and M. Latijnhouwers. 2004. Late blight. Encyclopedia of Plant and Crop
Science.(ed; R.M. Goodman). Dekker Encyclopedias, NY US. 1-5.
Govers, F. 2005. Late blight: The perspective from the pathogen. In: Potato in progress: Science
meets practice.(eds. A. J. Havenkort and P.C. Strik). AP.Wageningen, NL. pp. 45-54.
Govindachar, T. R., G. Suresh and S. Masilamani. 1999. Antifungal activity of Azadirachta
indica leaf hexane extract. Fitoterapia. 70, 417–420.
161
Grayer, R. J., and J. B. Harborne. 1994. A survey of antifungal compounds from plants, 1982-
1993. Phytochemistry. 37, 19-42.
Green, R. J. 2004. Antioxidant activity of peanut plant tissues. Master Thesis, North Carolina
State University.
Gunnell, P. S., and W. D. Gubler. 1992. Taxonomy and morphology of Colletotrichum species
pathogenic to strawberry. Mycologia. 84, 157-165.
Gurjar, M. S., S. Ali., M. Akhtar and K. S. Singh. 2012. Efficacy of plant extracts in plant
disease management. Agric. Sci. 425-433.
Hadian, S., K. Rahnama, S. Jamali and A. Eskandari. 2011. Comparing Neem extract with
chemical control on Fusarium oxysporum and Meloidogyne incognita complex of tomato.
Adv. Environ. Biol. 5, 2052-2057.
Hadizadeh, I., B. Peivastegan and M. Kolahi. 2009. Antifungal activity of nettle (Urtica dioica
L.), colocynth (Citrullus colocynthis (L.) Kuntze, Oleander (Nerium oleander L.) and
konar (Ziziphus spina-christi L.) extracts on plants pathogenic fungi. Pak. J. Biol. Sci.
12, 58-63.
Hafiz, A. 1986. Plant disease. Pakistan Agriculture Research Council, Islamabad. pp 552.
Haggag, K. H. and N. G. El-Gamal. 2012. In vitro Study on Fusarium solani and Rhizoctonia solani
Isolates Causing the Damping off and Root Rot Diseases in Tomatoes. Nat. & Sci. 10, 16-
25.
Haikal, N. Z. 2007. Improving biological control of Fusarium root-rot in cucumber (Cucumis
sativus L.) by allelopathic plant extracts. Int. J. Agric. Biol. 9, 459-561.
Halama, P. and C.Van Haluwin. 2004. Antifungal activity of lichen extracts and lichenic acids.
Biocontrol. 49, 95-107.
162
Han, D. Y., D. L. Coplin, W. D. Bauer and H. A. J. Hoitink. 2000. A rapid bioassay for
screening rhizosphere microorganisms for their ability to induce systemic resistance.
Phytopathology. 90, 327-332.
Hanafi, A. 2003. Integrated production and protection in greenhouse crops in Morocco p.
192-197 In: Tomato sous Abri. Scientific Publication of CTIFL. 2003. Editions Center
Technique Inter professionnel des Fruits et Legumes. 232.
Harborne, J. B. 1973 a. Phytochemical Methods: A Guide to Modern Techniques of plant
Analysis. Chapman and Hall Ltd, London. p. 279.
Harborne, J. B. 1973b. Phenolic compounds. In: Phytochemical Methods. Springer Netherlands .
pp. 33-88.
Harborne, J. B. 1989. Introduction to Biochemical Ecology. AP. NY, US.
Harborne, J. B. 1998 . Phytochemical Methods. A guide to modern techniques of plant analysis
3rd edition. Chapman and Hall, London. UK.
Hassanein, N. M., M. A. Abouzeid, K. A. Youssef and D. A.Mahmoud. 2008. Efficacy of leaf
Extracts of Neem (Azadirachta indica) and Chinaberry (Melia azedrach) Against Early
Blight and Wilt Diseases of Tomato. Austr. J. Basic and Appl. Sci. 2, 763-772.
Hashim, M. S. and K. S. Devi. 2003. Insecticidal action of the polyphenolic rich fraction from
the stem bark of Sterlus asper on Dysdercus cingulatus. Fitoterapia. 74, 670-676.
Hatcher, H., R. Planalp, J. Cho, F. M. Torti and S. V. Torti. 2008. Curcumin: from ancient
medicine to current clinical trials. Cell Mol. Life Sci. 65, 1631-1652.
Hismath, I., W. M. Wan Aida and C. W. Ho. 2011. Optimization of extraction conditions for
phenolic compounds from neem (Azadirachta indica) leaves. Intr. Food Res. J. 18(3), 931-
939.
163
Hobson, G. E., J. N. Davies. 1971. The Tomato. In: The biochemistry of fruits and their products.
AP, NY. US. 2, 437-82.
Howell, C. R., J. E. DeVay, R. H. Garber, W. E. Batson. 1997. Field control of cotton seedling
diseases with Trichoderma virens in combination with fungicide seed treatments. J.
Cotton. Sci. 1, 15- 20.
Huynh,Q. K., J. R. Borgmeyer, C.E. Smith, L. D. Bell and D. M. Shah. 2001. Isolation and
Characterization of a 30 kDa protein with antifungal activity from leaves of Engelmannia
pinnatifida. J. Biol. Chem. 316, 723-727.
Imelouane, B., H. Amhamd, J. P. Wathelet, M. Ankit, K. Khedid and A. E. Bachiri. 2009. Chemical
composition and antimicrobial activity of essential oil of thyme (Thymus vulgaris) from
eastern Morocco. Int. J. Agric. Biol. 11, 205-208.
Imtiaj, A., S. A. Rehman, S. Alam, R. Parvin, K. M. Farhana, S-B. Kim and T-S. Lee. 2005. Effect
of Fungicides and Plant Extracts on the Conidial Germination of Colletotrichum
gloeosporioides Causing Mango Anthracnose. Mycobiology. 33, 200-205.
Indu, M. N., A. A. M. Hatha, C. Abirosh, U. Harsha and G. Vivekanandan. 2006. Antimicrobial
activity of some of the south Indian spices against serotypes of Escherichia coli,
Salmonella, Listeria monocytogenes and Aeromonas hydrophila. Braz. J. Microbiol. 37,
153-158.
Intana, W., T. Suwanno, C. Chamswarng, K. Chantrapromma and C. Ngamriabsakul. 2007.
Increased efficacy for controlling Anthracnose of chili using antifungal metabolites from
mutant strains of Trichoderma harzianum. Thai J. Agric. Sci. 40, 65- 72.
Islam, M. R., M. S. Alam, M. Z. Rahman, S. P. Chowdhury, M. F. Begum, N. Akhter, M. S.
Alam, K. D. Han and M. W. Lee. 2003. Effects of Plant Extracts on Conidial
164
Germination, Mycelial Growth and Sporulation of Fungi Isolated From Poultry Feed.
Mycobiology. 31(4), 221-225.
ISTA. 1996. International rules for seed testing. Seed Sci. Tech. 24, 1-335.
Itako, A.T., K. R. F. Schwan-Estrada, J. B. T. Júnior, J. R. Stangarlin and M. E. S. Cruz. 2008.
Antifungal activity and protection of tomato plants by extracts of medicinal plants. Trop.
Plant Pathol. 33(3), 241-244.
Ivanovska, N., S. Philipov, R. Istatkova and P. Georgieve. 1996. Antimicrobial and immunological
activity of ethanol extracts and fractions Isopyrum thalictroides. J. Ethnopharmacol. 54,
143-151.
Jabeen, K., A. Javaid and M. Athar. 2008. Fungistatic activityof aqueous and organic solvent
extracts of Melia azedarach against Ascochyta rabiei. Pak. J. Phytopathol. 20, 143-149.
Jabeen, K., and A. Javaid. 2010. Antifungal activity of Syzygium cumini against Ascochyta
rabiei– the cause of chickpea blight. Nat. Product Res. 24(12), 1158–1167.
Jagtap, G. P., M. C. Dhavale and U. Dey. 2012. Evaluation of natural plant extracts,
antagonists and fungicides in controlling root rot, collar rot, fruit (brown) rot and
gummosis of citrus caused by Phytophthora spp. in vitro. Sci. J. Microbiol. 1(2), 27-
47.
Jamil, A. M. S., M. M. Khan and M. Ashraf. 2007. Screening of some medicinal plants for
isolation of antifungal proteins and peptides. Pak. J. Bot. 39, 211-221.
Javaid, A., and H. A. Rehman. 2011. Antifungal activity of leaf extracts of some medicinal trees
against Macrophomina phaseolina. J. Med. Plant. Res. 5(13), 2868-2872.
Javaid, A. and S. Samad. 2012. Screening of allelopathic trees for their antifungal potential
against Alternaria alternata strains isolated from dying-back Eucalyptus spp. Nat. Prod.
Res:FormallyNat.Prod.Letters.26(18),1697-1702, DOI:10.1080/14786419.2011.602638.
165
Johann, S., A. Smania-Jr, M. G. Pizzolatti, I. Schripsema, R. Braz-Filho and A. Branco. 2007.
Complete 1H and 13C NMR assignments and antifungal activity of two 8-hydroxy
flavonoids in mixture. Ann. Acad Bras. Cienc. 79, 215-222.
Jones, J. B. 2008. Tomato plant culture: in the field, greenhouse, and home garden. 2nd. ed. CRC
Florida USA.
Jones, J. B., J. P. Jones, R. E. Stall and T. A. Zitter. 1991. Compendium of tomato diseases.
AP, Minisota.US.
Jones, J. P., and S. S. Woltz. 1981. Fusarium-incited diseases of tomato and potato and their
control. In Fusarium: Diseases, Biology, and Taxonomy. (eds. P.E. Nelson,T.A.
Toussoun and R.J. Cook), Pennsylvania State University Press, University Park, 157168.
Joseph, B., M. A. Dar and V. Kumar. 2008. Bioefficacy of Plant Extracts to Control Fusarium
solani f. Sp. Melongenae Incitant of Brinjal Wilt. Global J. Biotechnol. Biochem. 3 (2),
56-59, ISSN 2078-466X.
Joshi, B., G. P. Sah, B. B. Basnet, M. R. Bhat, D. Sharma, K. Subedi, J. Pandey and R. Malla.
2011. Phytochemical extraction and antimicrobial properties of different medicinal plants:
Ocimum sanctum (Tulsi), Eugenia caryophyllata (Clove), Achyranthes bidentata
(Datiwan) and Azadirachta indica (Neem). J. Microbiol. Antimicrobials. 3(1), 1-7.
Juven, B. J., J. Kanner, F. Sched and H. Weisslowicz. 1994. Factors that interact with the
antibacterial of thyme essential oil and its active constituents. J. Appl. Microbiol.76, 626–
631.
Kagale, S., T. Marimuthu., B. Thayumanavan, R. Nandakumar and R. Samiyappan. 2004.
Antimicrobial activity and induction of systemic resistance in rice by leaf extract of Datura
metel against Rhizoctonia solani and Xanthomonas oryzae pv. oryzae. Physio.
Mol. Pl. Pathol. 65, 91–100.
166
Kanwal, Q., I. Hussain, H. L. Siddiqui and A. Javaid. 2010. Antifungal potential of flavonoids
isolated from mango (Mangifera indica L.) leaves. Nat. Prod. Res. 24, 1907-1914.
Katalinic, V., M. Milos, T. Kulisic and M. Jukic. 2006. Screening of 70 medicinal plant extract for
antioxidant capacity and total polyphenols. Food Chem. 94, 550-557.
Kavishankar, G. B., N. Lakshmidevi and S. M. Murthy. 2011. Phytochemical analysis and
antimicrobial properties of selected medicinal plants against bacteria associated with
diabetic patients. Intr. J. Pharma and Bio Sci. 2(4), 509-518.
Kawaii, S., T. Yasuhiko, K. Eriko, O. Kazunori,Y. Masamichi, K. Meisaku, ChihiroIto and F.
Hiroshi. 2000. Quantitative study of flavonoids in leaves of Citrus plants. J. Agric. Food
Chem. 48, 3865-3871.
Ketelaar, J. W. and P. Kumar. 2002. Vegetable Integrated Production and Pest Management: The
Case for Farmers as IPM Experts. International Conference on Vegetables; ITC Hotel
Windsor Sheraton and Towers, Bangalore, India, 1-14.
Khair, A. H., and M. H. Wafaa. 2007. Application of some Egyptian medicinal plant extracts
against potato late blight and early blights. Res. J. Agric. Biol. Sci. 3, 166-175.
Khalil, A. R. M. 2001. Phytofungitoxic properties in the aqueous extracts of some plants. Assiut J.
Agric. Sci. 32, 135-143.
Khan, A. M., S. K. Saxena and Z.A. Siddiqui. 1971. Efficacy of Tagetes erecta in reducing root
infesting nematodes of tomato and okra. Ind. Phytopath. 24, 166-169.
Khan, R., B. Islam, M. Akram, S. Shakil, A. Ahmad, S.M. Ali, M. Siddiqui and A.U. Khan.
2009. Antimicrobial activity of five herbal extracts against multi drug resistance, strains
(MDR) of bacteria and fungus of clinical origin. Molecules. 14, 586-597.
Khushwaha, A., R. P. Singh, V. Gupta and M. Singh. 2012. Antimicrobial properties of peels of
167
citrus fruits. Int. J. Univ. Pharmacy and Life Sci. 2, 24-38.
Kim, D. O., O. K. Chun, Y. J. Kim, H. Y. Moon and C. Y. Lee. 2003. Quantification of
Polyphenolics and their antioxidant, capacity in fresh plums. J. Agri. and Food Chem. 51,
6509-6515.
Kim, J-C., G. J. Choi, S-W Lee, J-S Kim, K. Y. Chung and K. Y. Cho. 2004. Screening extracts of
Achyranthes japonica and Rumex crispus for activity against various plant pathogenic
fungi and control of powdery mildew. Pest Manage Sci. 803–808.
Kishore, G. K., S. Pande, and J. N . Rao. 2001. Control of late leaf spot of groundnut (Arachis
hypogaea) by extracts from non-host plant species. Pl. J. Pathol. 17(5), 264-270.
Koch, H. P., and L. D. Lawsone.1996. Garlic, the science and therapeutic application of Allium
sativum L.and related species. (eds. D.C. Retford, Williams and Wilkins), Baltimore. 1–
233.
Koche, D., R. Shirsat, S. Imran and D. G. Bhadange. 2010. Phytochemical screening of eight
traditionally used ethnobotanical plants from Akola District (MS) India. Int. J. Pharma.
Bio Sci. 1(4), 253-256.
Kubo, A., C. S. Lunde and L. Kubo. 1995. Antimicrobial activity of the olive oil flavour
compounds. J. Agric. Food Chem. 43, 1629-33.
Kuc´, J. 1995. Phytoalexins, stress metabolism and disease resistance in plants. Annu. Rev.
Phytopath. 33, 275–97.
Kumar, A. S., N. P. E. Reddy, K. H. Reddy and M. C. Devi. 2007. Evaluation of fungicidal
resistance among Colletotrichum gloeosporioides isolates causing Mango Anthracnose in
Agri. Export Zone of Andhra Pradesh, India. Plant Path. Bull. 16, 157-160.
168
Lacey, L. A. and G. Mercadier. 1998. The effect of selected allelochemicals on germination of
conidia and blastospores and mycelial growth of the entomopathogenic fungus,
Paecilomyces fumosoroseus (Deuteromycotina: Hyphomycetes). Mycopathologia.142,
17–25.
Lajide, L., C. O. Adedire, W. A. Muse, S. O. Agele, N. E. S. Lale, N. B. Molta, P. O. Donli, M. C.
Dike, and M. Aminu-Kano. 1998. Insecticidal activity of powders of some Nigerian plants
against the maize weevil (Sitophilus zeamais Motsch).Entomology in the Nigerian
economy. Research focus in the 21st century, 227-235.
Latha, T. K. S., E. Rajeswari and V. Narasimhan. 2000. Management of root-rot disease complex
through antagonists and chemicals. Ind. Phytopath. 53(2), 216-218.
Latha, P., T. Anand, N. Ragupathi, V. Prakasam and R. Samiyappan. 2009. Antimicrobial activity
of plant extracts and induction of systemic resistance in tomato plants by mixtures of
PGPR strains and Zimmu leaf extract against Alternaria solani. Biological Control. 50,
85–93.
Lakshmi, B. S. 2012. Bio Control of Cereal Crop Pathogens by Curcuma longa L. Rhizomes. IJPI’s
J. Biotechnology. Biotherapeutics. 2(8), 7-12. ISSN 2229 – 6824.
Lambert, R. J. W., P. N. Skandamis and P. J. Coote. 2001. A study of the minimum inhibitory
concentration and mode of action of oregano essential oil, thymol and carvacrol. J.
Appl. Microbiol. 91, 453- 462.
Lawson, L. D. 1996. The composition and chemistry of garlic cloves and processed garlic. In:
Garlic: the science and therapeutic application of Allium sativum L (eds; H.P. Koch,
L.D.Lawson). Williams and Wilkins, Baltimore US, 37–108.
LeBoeuf, J., R. Pitblado and P. May. 2008. Identifying the Pathogens Causing Anthracnose of
169
Tomato and Pepper in Southern Ontario, Canada. In: XI International Symposium on the
Processing Tomato. 823, 147-150.
Lee, H. U., C.H. Kim and E. J. Lee. 1990. Effects of pre and mixed cropping with non-host
plants on incidence of Phytophthora blight of red pepper. Kor. J. Pl. Path. 6, 440-446.
Lee, H. S., Kyung-Ja Choi, Kwang-Yun Cho and Young-Joon Ahn. 2003. Fungicidal Activity of
Turmerone Identified in Curcuma longa Rhizome against Six Phytopathogenic Fungi.
Agric.Chem. Biotech. 46, 25-28.
Leslie, J. F., K. A. Zeller and B. A. Summerell. 2001. Icebergs and speciation in species of
Fusarium. Physio. Mol. Plant Pathol. 59, 107-117.
Madigan. M. T., J. M. Martinko and J. Parker. 2000. Biology of microorganism. 9th edi. New
Jersey, USA: prentice-Hall, Inc.
Mahmoud, I. I., M. S. A. Marzouk, F. A. Moharram, M. R. El-Gindi and A. M. K. Hassan. 2001.
Acylated flavonol glycosides from Eugenia jambolana leaves. Phytochemistry. 58, 1239-
1244.
Maji, M. D., S. Chattopadhyay, P. Kumar and B. Saratchandra. 2005. In vitro screening of some
plant extracts against fungal pathogens of Mulberry (Morus sp.). Arch. Phytopathol. Plant.
Prot. 38(3), 157–164.
Majid, K., M. Aslam, M. Shahid and A. Saleem. 1992. Late blight of tomato caused by
Phytophthora infestans(Mont.) de Bary. A new record for Pakistan. Pak. J. Phytopathol.
4, 70.
Makeredza, B., J. Sibiya and R. M. Madakadze. 2005. The effect of garlic, ginger and turmeric
extracts on bean seed borne Xanthomonas axonopodis pv. phaseoli. Afr. Crop Sci.7, 325-
329.
Malkhan, S. G., A. Shahid, A. Masood and S. S. Kangabam. 2012. Efficacy of plant extracts
170
in plant disease management. J. Earth and Environ. Sci. 3, 425-433.
Mancini, F., A. J. Termorshuizen, J. L. S. Jiggins and A. H. C. van Bruggen. 2008. Increasing the
environmental and social sustainability of cotton farming through farmer education in
Andhra Pradesh, India. Ind. Agric. Systems. 96, 16–25.
Margulis, L., and K.V. Schwartz. 1988. Five Kingdoms: An Illustrated Guide to the Phyla of Life
on Earth. New York, W.H. Freeman and Co. pp 376.
Maruška, A., J. Proscevičius, K. Bimbiraitė-Survilienė, O. Kornyšova, O. Ragažinskienė V.
Ratautaitė. 2010.Comparison of phytochemical composition of medicinal plants by means
of chromatographic and related techniques. Procedia Chem. 2(1), 83-91.
Martini, N. D., D. R. P. Katerere and J. F. Eloff. 2004. Biological activity of five antibacterial
flavonoids from Combretum erythrophyllum (Combretaceae). J. Ethnopharmacol. 93,
207-212.
McCue, G. A. 1952. The history of the use of the tomato: An annotated bibliography. Ann.
Missouri Bot. Gard. 39, 289-348.
McLean, K. L., J. Swaminathan, C. M. Frampton, J. S. Hunt, H. J. Ridgway and A. Stewart. 2005.
Effect of formulation on the rhizosphere competence and biocontrol ability of
Trichoderma atroviride C52. Plant Pathol. 54, 212- 218.
Messiaen, C. M. 1992. The Tropical Vegetable Garden. The Macmillan Press Ltd. London, 88-
89.
Migliato, K. F. 2005. Standardization of the extract of Syzygium cumini(L.) skeels fruits through
the antimicrobial activity. Caderno de Farmacia. 21, 55–56.
Milner, J. A. 2005. Garlic (Allium sativum). In: Encyclopedia of Dietary Supplements. Black, M.
and Cragg, G. (Eds.). Marcel Dekker, New York., 229-240.
171
Mirza, J. I., S. Hameed, I. Ahmad, N. Ayub and R.H.C. Strang. 2000. In vitro antifungal activity
of neem products against Phytophthora infestans. Pak. J. Biol. Sci. 3, 824-828.
Mishra, P. K., S. Saha, A. B. Rai and M. Rai. 2009. Antifungal effect of neemazal. A commercial
product of neem (Azadirachta indica).Veg. Sci. 36(1), 60-63.
Mizuno, M., M. Linumo, M. Ohara, T. Tanoka and M. Iwasa. 1991. Chemotaxonomy of the genus
Citrus based on polymethoxy-flavones. Chem Pharm Bull. 39, 945-949.
Mizutani, J. 1999. Selected allelochemicals. Critical Reviews in Plant Sciences. 18, 653-671.
Mogle, U. P. 2011. Efficacy of biofertilizer and plant extracts against anthracnose diseases of
tomato. Biosci Discov. 2, 104–108.
Mohamed, N. H., A. M. El-Hadidy. 2008. Studies of biologically active constituents of Verbascum
eremobium Murb. and its inducing resistance against some diseases of cucumber. Egypt.
J. Phytopathol. 36, 133-150.
Mohsen, M. S. and S. M. A. Ammar. 2008. Total phenolic contents and antioxidant activity of
corn tassel extracts. Food Chem. 112, 595-598.
Mondali, N. K., A. Mojumdar, S. K. Chatterje, A. Banerjee, J. k. Datta and S. Gupta. 2009.
Antifungal activities and chemical characterization of Neem leaf extracts on the growth of
some selected fungal species in vitro culture medium. J. Appl. Sci. Environ. Manage.
13(1), 49 - 53.
Morales, M. R. and J. E. Simon. 1996. New basil selection with compact inflorescence for the
ornamental market. In: J. Janick (ed.), progress in new crops. ASHS press. pp 543-546.
Morales, A. 2008. A simple way to preserve fungal cultures. Factsheet, Cornell mushroom blog.
Cornell University. CIAT, Colombia.
Mulla, M. S., and T. Su. 1999. Activity and biological effects of neem products against
172
arthropods of medical and veterinary importance. J. Am. Mosq. Control. Assoc. 15, 133-
152.
Murthy, M. M., M. Subramaniyam, M. Hima Bindu and J. Annapurna. 2005. Antimicrobial
activity of Clerodone diterpenoids from Polyalthea longifolia seeds. Fitoterapia. 76,
336-339.
Muto, M., H. Takahashi, K. Ishihara, H. Yuassa and J. W. Huang. 2005. Antimicrobial activity
of medicinal plants used by indigenous People in Taiwan. Plant. Pathol. Bull. 14(1), 13–
24.
Naczk, M.and F. Shahidi. 2004. Extraction and analysis of phenolics in food. J. Chromatography
A. 1054, 95-111.
Nagaraja, T.G., S. V. Sarang and D. C. Jambhale. 2008. Evaluation of anti-mycotic activity of
Acacia catechuWilld. (Mimosaceae). J. Biopesticides 1, 197-198.
Naika, S., J. V. L. de Jeude, M. de Goffau, M. Hilmi and B.van Dam. 2005. Cultivation of
tomato.4thed.(Digigrafi publishing,Wageningen,Netherlands:Agromisa).ISBN Agromisa:
90-8573-039-2.
Nair, K. R. S. and A. H. Ellingboe. 1962. Method of controlled inoculations with conidiospores
of Erysiphe graminis var. tritici. Phytopathology. 52, 714.
Nair, R., T. Kalaria and S. Chanda. 2005. Antibacterial activity of some selected Indian
medicinal flora. Turk. J. Bio. 29, 41-47.
Nair, R. R., and S. Chanda. 2006. Evaluation of Polyathia longifolia (Sonn.) Thw. Leaf extracts
for antifungal activity. J. cell. Tissue Reserch. 6, 581-584.
Naqui, S.H.A., M. S. Y. Khan and S. B. Vohora. 1994. Antibacterial, antifungal and anthelmintic
investigation on Indian medicinal plants. Fitoterapia. 62, 221–228.
Nascimento, G. G., J. Locatelli and P. C. Freitas. 2000. Antibacterial activity of plants extracts
173
and phytochemical on antibiotic resistant bacteria. Braz. J. Microbiol. 31, 247–256.
Nash, S. M. and W. C. Snyder. 1962. Quantitative estimations by plate counts of propagules of
the bean root rot Fusarium in field soil. Phytopathology, 52,567–572.
Nashwa, S. M. A., and K. A. M Abo-Elyousr. 2012. Evaluation of various plant extracts against
the early blight disease of tomato plants under greenhouse and field conditions. Plant
Protect. Sci., 48, 74–79.
Naz, F., C.A. Rauf, I.U. Haque and I. Ahmad. 2006. Management of Rhizoctonia solani with
plant diffusates and chemicals. Pak. J. Phytopathol.18, 36-43.
Ncube, N., S. A. J. Afolayan and A. I. Okoh. 2008. Assessment techniques of antimicrobial
properties of natural compounds of plant origin: Current methods and future trends. Afr.
J. Biotech. 7, 1797-1806.
Neergaard, P. 1945. Danish species of Alternaria and Stemphylium: taxonomy, parasitism,
economic significance. Copenhagan Munksgaard, Oxford University Press, London, pp
260-287.
Neergaard, P. 1977. Seed pathology. 1(2), MacMillan, London. 1187.
Nelson, P. E., T. A. Toussoun and W.F.O. Marasas. 1983. Fusarium Species. An Illustrated
Manual of Identification.University Park, Pennsylvania: The State University Press. pp.
193.
Nene, Y. and L. Thapilyal. 2000. Poisoned food technique of fungicides in plant disease control
(3rd eds). Oxford and IBH Publishing Company, New Delhi.
Nesamani, S. 1999. Medicinal Plants (vol. I). State Institute of Languages,Thiruvananthapuram,
Kerala, India, 425.
174
Nishikawa, N., T. Kobayashi, K. Shirata, T. Chibana, K. S. T. Chibana and K. T. Natsuaki. 2006.
Seedborne fungi detected on stored solanaceous berry seeds and their biological activities.
J. Gen. Plant. Path. 72, 305-313.
Nonnecke, I. L.1989. Vegetable production. Springer Science & Business Media. ISBN: 0-442-
26721-5.
Noriaki, K., K.Yukari, I. Kazuya, M. Kyo and F. Tokio. 2005. In Vitro Antibacterial,
antimutagenic and anti-Influenza virus activity of caffeic acid phenyl ethyl esters.
Biocontrol Sci.10, 155-161.
Nostro, A., M. P. Germano, V. Angelo, A. Marino and M. A. Cannatelli. 2000. Extraction methods
and bioautography for evaluation of medicinal plant antimicrobial activity. Letters in
Appl. Microbiol. 30, 379-348.
Nouman, W., M.T. Siddiqui, S. M. A. Basra, H. Farooq, M. Zubair and T. Gull. 2013. Biomass
production and nutritional quality of Moringa oleifera as a field crop. Turk. J Agric For.
37, 410-19.
Nuñez-Selles, A. J. 2005. Antioxidant therapy: Myth or reality? J. Braz. Chem. Soci. 16, 699-
710.
Nwachukwu, E. O., and C. I. Umechuruba. 2001. Antifungal activities of some leaf extracts on
seed -borne fungi of African yam bean seeds, seed germination and seedling emergence.
J. Appl. Sci. and Environ. Manag. 5, 29-32.
Nwangburuka, C. C., K. Oyekale, C. N. Ezekiel, P. C. Anokwuru, and O. Badaru. 2012. Effects of
Moringa oleifera leaf extract and sodium hypochlorite seed pretreatment on seed
germination, seedling growth rateand fungal abundance in two accessions of Abelmoschus
esculentus (L) Moench. Arch. Appl. Sci. Res. 4, 875-881.
175
Ogbo, E.M., and A. E. Oyibo. 2008. Effects of three plant extracts (Ocimum gratissimum,
Acalypha wilkesiana and Acalypha macrostachya) on post harvest pathogen of Persea
americana. J. Med. Plant. Res. 2(11), 311-314.
Oh, S-O, J. A. Kim, H-S. Jeon, J. C. Park, Y. J. Koh, H. Hur and J-S. Hur. 2008. Antifungal
Activity of Eucalyptus-Derived Phenolics Against Postharvest Pathogens of Kiwi fruits.
Plant Pathol. J. 24, 322-327.
Okigbo, R. N., and I. A. Nmeka. 2005. Control of yam tuber rot with leaf extracts of Xylopia
aethiopia and Zingiber officinale. Afri. J. Biotech. 4, 804-807.
Okigbo, R. N., and O. D. Omodemiro. 2006. Antimicrobial effect of leaf extract of pigeon pea
(Cajanus cajan (L.) Mill sp) on some human pathogens. J. Herbs, spices and Med plants.
12, 117-127.
Okigbo, R. N., C. L. Anuagasi, J. E. Amadi and U. Ukpabi. 2009a. Potential inhibitory effects of
some African tuberous plant extracts on Escherichia coli, Staphylococcus aurteus and
Candida albican. Intr. J. Integrative Biology. (IJIB India). 6(2), 91-98.
Okigbo, R. N., R. E. Okorie and R. R. Putheti. 2009b. In vitro effects of garlic (A. sativum) and
African Basil (Ocimum gratissimum) on pathogens isolated from rotten cassava roots.
Inerciencia J. 34, 742-747.
Okusa, P. N., O. Penge, M. Deuleeschouwer and P. Dvez. 2007. Direct and indirect antimicrobial
effect and antioxidant activity of Cardia gilldii. J. Ethno. pharmacol. 112(3), 476-481.
Oleszek, W., R. E. Hoagland and R. M. Zablotowicz. 1999. Ecological significance of plant
saponins. In: Principles and Practices in Plant Ecology: Allelochemical Interactions, (eds.
Inderjit, K.M,. M. Dakshini, and C.L. Foy). Boca Raton, USA: CRC Press., 451-
65.
176
Olivain, C., C. Humbert, J. Nahalkova, J. Fatehi, F. L'Haridon and C. Alabouvette. 2006.
Colonization of tomato root by pathogenic and nonpathogenic Fusarium oxysporum
strains inoculated together and separately into the soil. Appl. environ. microbiol. 72(2),
1523-1531.
Olson, M. E. 2002. Combining Data from DNA Sequences and Morphology for a Phylogeny 696
of Moringaceae (Brassicales). Syst. Bot. 27, 55-73.
Örner, M. W. and H. C. Jha. 1993. Antifungal activity of the flavonoids and their mixtures
against different fungi occurring on grain. Pesticide Sci. 38(4), 347-351.
Ortuno, A. A., P. Baidez, M.C. Gomez, I. Arcas, A.G. Porras and J. A. Del Rio. 2006. Citrus
paradisi and Citrus sinensis flavonoids: Their influence in the defence mechanism against
Penicillium digitatum. Food Chem. 98, 351-358.
Osbourn, A. E. 1999. Antimicrobial Phytoprotectants and Fungal Pathogens, A Review. A
Commentary. Fungal Genetics and Biology. 26, 163–168.
Osman, K.A., and S. Al-Rehiayani. 2003. Risk assessment of pesticide to human and the
environment. Saudi J. Biol. Sci. 10, 81-106.
Otanga, R. N. 2005. Etiology, symptomatology and control of Cercospora leaf spot of maize
(Zea mays L.) in western Kenya. MSc. Thesis, Egerton University, Kenya, 50.
Parekh, J., D. Jadeja and S. Chanda. 2005. Efficacy of aqueous and methanol extracts of some
medicinal plants for potential antibacterial activity. Turk. J. Biol. 29, 203-210.
Pasche, J. S., C. M. Wharam and N. Gudmestad. 2004. Shift in sensitivity of Alternaria solani
in response to QoI fungicides. Plant Dis. 88, 181-187.
Patil, M. J., S. P. Ukey and B. T. Raut. 2001. Evaluation of fungicides and botanicals for the
management of early blight (Alternaria solani) of tomato. PKV Res. J. 25, 49-51.
177
Pathak, V.N. 1987. Laboratory Manual of Plant Pathology. 2 nd Ed. Oxford IBH Publ. Co.
New Dehli. P 23-25
Pattnaik, M. M., M. Kar and R. K. Sahu. 2012. Bioefficacy of some plant extracts on growth
parameters and control of diseases in Lycopersicum esculentum. Asian J. Plant Sci. Res.
2, 129-142.
Paul, P. K. and P. D. Sharma. 2002. Azadirachta indica leaf extract induces resistance in barley
against leaf stripe disease. Physiol. Mol. Plant Pathol. 61, 3–13.
Pegg, G. F. and B. L. Brady. 2002. Verticillium wilts. CABI publishing, NewYork. NY.
Pelttari, E., J. Matikainen and H. Elo. 2002. Antimicrobial activity of the marine alkaloids haminol
and pulo’upone and related compounds. Nat. Res. 57, 548-552.
Pennington, T. D. and B.T. Styles. 1975. Ageneric monograph of the Meliaceae. Blumea. 22,
419-540.
Peralta, I. E. and D. M. Spooner. 2001. Granule-bound starch synthase (GBSSI) gene phylogeny
of wild tomatoes (Solanum L. section Lycopersicon [Mill.] Wettst. subsection
Lycopersicon). Amer. J. Bot. 88(10), 1888-1902.
Peralta, I. E., S. Knapp and D. M. Spooner. 2005. New species of wild tomatoes (Solanum
lycopersicon: Solanaceae) from Northern Peru. Systemic Bot. 30, 424-434.
Perez, C., M. Pauli and P. Bazevque. 1990. An antibiotic assay by the agar well diffusion method.
Acta Biol. Med. Exp. 15, 113-115.
Perveen, S., and A. Ghaffar. 1995. Seedborne mycoflora of tomato. Pak. J. Bot. 27, 201-208.
Persley, D. 1993. Diseases of Fruit Crops. Department of Primary Industries Queensland,
Brisbane, Australia.
Phalisteen, S., S. Ishaq, K. Amardeep, J. Arif and S. Sami. 2008. Evaluation studies of some
medicinal plants and Fungicides against Alternaria solani. Afr. J. Clinl. Exp. Microbiol.
178
9(1), 19-32.
Philip, J. P., G. Madhumitha and A. M. Saral. 2011. Free radical scavanging and reducing power
of Lawsonia inermis L. seeds. Asian Pacific J. Tro.Medicine. 4(6), 457-461.
Pitchaon, M., M. Suttajit and R. Pongsawatmani. 2007. Assessment of phenolic content and free
radical scavenging capacity of some Thai indigenous plants. Food Chem. 100, 14091418.
Prithiviraj, B., S. Khistae, D. Ram and U. P. Singh.1996. Effect of methanol extract of Aegle
`marmalos leaves on mycelial growth and sclerotium formation of Sclerotium rolfsii.
Int. J. Pharmacogn. 36, 148-150.
Pundir, R. K., and P. Jain. 2010. Comparative studies on the antimicrobial activity of black pepper
(Piper nigrum) and turmeric (Curcuma longa). Int. J. Appl. Biol. Pharma.Tech. 1, 491-
501.
Qasem, J. R. 1999. Biological activity of corn buttercup (Ranunculus arvensis L.). In: Recent
Advances in Allelopathy. Vol. 1. A Science for the Future,eds. F. A. Macías, J.C.B.
Galindo, J.M.G. Molinillo, and H.G. Cutler. Universidad de Cádiz, Spain: Servicio de
Publicaciones, 287-300.
Raghavendra, M. P., S. Satish and K. A. Raveesha. 2008. In vitro antibacterial potential of
alkaloids of Samanea saman(Jacq.) Merr. Against Xanthomonas and Human Pathogenic
bacteria. World J.Agri. Sci. 4(1), 100-105.
Rahman, M. A., M. Mostafizur Rahman, A. K. Azad and M. Firoz Alam. 2011. Inhibitory effect
of different plant extracts and antifungal metabolites of Trichoderma strains on the
conidial germination and germ tube growth of Colletotrichum capsici causing chili
anthracnose. Int. J. Agric. Res. 1, 20-28.
179
Raja, M. B. and K. M. Khokhar. 1993. Postharvest horticulture technology and its future prospects.
In: Proceeding of First International Horticulture Seminar, 09-11 January 1992. Pakistan
Agricultural Research Council, Islamabad, p 265-277.
Rai, D., J. K. Singh, N. Roy and D. Panda. 2008. Curcumin inhibits FtsZ assembly: an attractive
mechanism for its antibacterial activity. Biochem. J. 410, 147-155.
Rao, K. V., K. Sreermulu, D. Gunasekar and D. Ramesh. 1993. Two new sesquiterpene lactones
from Ceiba pentandra. J. Nat. Prod. 56(12), 2041-2045.
Rathod, L. R., M. D. Jadhav, M. K. Awate, A. M. Surywanshi and P.S. Deshmukh. 2010.
Utilization of medicinal plants to control seed borne pathogens of selected legumes seeds.
Int. J. Adv. Biotech. Res. 11(2), 57-59.
Rashid, A., I. Ahmad, S. Iram, J. I. Mirza and C. A. Rauf. 2004. Efficiency of different neem
(Azadirachta indica A.Juss) products against various life stages of Phytophthora infestans
(Mont.) De Bary. Pak. J. Bot . 36, 881-886.
Ravikumar, M. C. and R. H. Garampalli. 2013. Antifungal activity of plants extracts against
Alternaria solani, the causal agent of early blight of tomato. Arch. Phytopath. Pl. Prot.
46 (16), 1897-1903, DOI: 10.1080/03235408.2013.780350.
Reddy, B. R. C. 1986. Studies on resistance of fungal pathogens to certain fungicides II. Ph.D.
Thesis, Marathwada University, Aurangabad.
Reni, C., and V. E. Reoland. 2006. Tomato early blight (Alternaria solani): the pathogen,
genetics, and breeding for resistance. J. Phytopath. 72, 335-347.
Riaz, T., S. N. Khan and A. Javaid. 2010. Management of corm-rot disease of gladiolus by plant
extracts. Nat. Prod. Res. 24,1131-1138.
Rice, E. L. 1995. Biological Control of Weeds and Plant Diseases--Advances in Applied
Allelopathy. Norman, USA: University of Oklahoma Press, 439.
180
Rick, C. M. 1979. Biosystematic studies in Lycopersicon and closely related species of Solanum.
In: The Biology and Taxonomy of Solanaceae (eds. J.G. Hawkes, R.N. Lester, and A.D.
Skelding), AP, New York, pp. 667-677.
Rios, J. L., M. C. Recio and A. Villar. 1988. Screening methods for natural products with
antimicrobial activity: a review of the literature. J. Ethnopharmacol. 23, 127-149.
Ristaino, J. B. 2002. Tracking historic migrations of the Irish potato famine pathogen,
Phytophthora infestans. Microbes and Infection. 4, 1369-1377.
Rizvi, S. J. H., M. Tahir, V. Rizvi, R.K. Kohli, and A. Ansari. 1999. Allelopathic interaction
Agroforestry systems. Cri. Rev. Plant Sci. 18, 773-796.
Robin, E., and Y. Choen. 2004. Oospores associated with tomato seed may lead to seed borne
transmission of Phytophthora infestans. Phytoparasitica. 32, 237-245.
Rodrigues, E., K. R. F. Schwan-Estrada, A. C. G. Fiori-Tutida, J. R. Stangarlin and M. E. S.
Cruz. 2007. Fungitoxicity, phytoalexins elicitor activity and protection of grown against
Sclerotinia sclerotiorum by ginger extract. Summa Phytopathologica, 33(2), 124-128.
Rodrigues, T. T. M. S., L. A. Maffia, O. D. Dhingra and E. S. G. Mizubuti. 2010. In vitro
production of conidia of Alternaria solani. Trop. Plant Path. 35, 203-212.
Rotem, J. 1998. The Genus Alternaria; Biology, Epidemiology, and Pathogenicity. APS Press,
St. Paul, Minnesota.
Sahayaraj, K. 2008. Common plant oils in agriculture and storage pests management. Green
Farming. 1(2), 48-49.
Sahi, S. T., A. Habib., M. U. Ghazanfar and A. Badar. 2012. In vitro evaluation of different
fungicides and plant extracts against Botryodiplodia theobromae, the causal agent of
Quick Decline of mango. Pak. J. Phytopathol. 24(2), 137-142.
Saltueit, M. E. 2003. Mature Fruit Vegetables. In: Postharvest Physiology and Pathology of
181
Vegetables (eds. J. A bartc and J. K. Brecht) Marcel Dekker, New York. 332.
Sangalang, A. E., L. W. Burgess, D. Backhouse, J. Duff and M. Wurst. 1995. Mycogeography of
Fusarium species in soils from tropical, arid and mediterranean regions of Australia.
Mycol. Res. 99(5), 523-528.
Sangoyomi, T. E., E. J. A. Ekpo and R. Asiedu. 2009. Fungitoxic effects and attributes of Allium
sativum and Ocimum gratissimum extracts on Rhizoctonia solani, the casual agent of yam
(Dioascorea otundata Poir) rot disease. Nigerian J. Mycology. 2(1), 166-177.
Sarma, B. K., V. B. Pandey, G. D. Mishra and U. P. Singh. 1999. Antifungal activity of berberin
iodide, a constituent of Fumaria indica. Folia Microbiol. 44, 164-166.
SAS Institute. 2002. Version 8.00 SAS Institute Inc; Cary, North Carolina, USA.
Sasidharan, I., and A. N. Menon. 2010. Comparative chemical composition and antimicrobial
activity and dry ginger oils (Zingiber officinale roscoe). Int. J. Curr. Pharm Res. 2, 40-
43.
Sasode, R. S., S. Prakash, A. Gupta, R.K. Pandya and A. Yadav. 2012. In vitro study of some
plant extracts against Alternaria brassicae and Alternaria brassicicola. J. Phytology. 4, 44-
46.
Saywell, L. G., and E. W. Lane. 1933. Comparative effect of tomato and orange juice on urinary
acidity. J. Nutrition. 6, 263-270.
Schelz, Z., J. Molnar and J. Hohmann. 2006. Antimicrobial and antiplasmid activities of essential
oils. Fitoterapia. 77, 279-285.
Sealy, R., M. R. Evans C. Rothrock. 2007. The effect of garlic extract and root substrate on
soilborne fungal pathogens. Hort. Technol. 17(2), 169-173.
Segarra, G., M. Reis, E. Casanova, and M. I. Trillas. 2009. Control of powdery mildew (Erysiphe
polygoni) in tomato by foliar applications of compost tea. J. Plant Pathol. 91, 683-689.
182
Sehajpal, A., S. Arora and P. Kaur. 2009. Evaluation of plant extracts against Rhizoctonia solani
causing sheath blight of rice. J. Plant Prot. Sci. 1, 25-30.
Seigler, D. S. 1996. Chemistry and mechanism of allelopathic interactions. Agron. J. 88, 876-
885.
Seema, M., S. S. Sreenivas, N. D. Rekha and N. S. Devaki. 2011. In vitro studies of some plant
extracts against Rhizoctonia solani Kuhn infecting FCV tobacco in Karnataka Light
Soil, Karnataka, India. J. Agri. Tech. 7, 1321-1329.
Shafi, P. M., M. K. Rosamma, K. Jamil and P. S. Reddy. 2002. Antibacterial activity of Syzygium
cumini and Syzygium travancoricum leaf essential oils. Fitoterapia. 73, 414416.
Shafique, S., S. Shafique, R. Bajwa, N. Akhtar and S. Hanif. 2011. Fungitoxic activity of aqueous
and organic solvent extracts of Tagetes erectus on phytopathogenic fungusAscochyta
rabiei. Pak. J. Bot. 43, 59-64.
Shafique, S., and Shazia Shafique. 2012. Tagetes erectus – A Tool for the Management of
Alternaria alternata Strains of Tomato. Int. Conf. Appl. Life Sci. Turkey, 309-314.
Sharma, A., and K. Sharma. 2011. Assay of Antifungal Activity of Lawsonia inermis L. and
Eucalyptus citriodora Hook. J. Pharmacy Res. 4(5), 1313-1314.
Shidfar, F., N. Froghifar, M. Vafa, A. Rajab, S. Hosseini, S. Shidfar and M. Gohari. 2011. The
Effects of Tomato Consumption on Serum Glucose, Apolipoprotein B, Apolipoprotein A-
I, Homocysteine and Blood Pressure in Type 2 Diabetic Patients". Int. J. Food Sci. and
Nutr. 62, 289-294.
Shinde, V. and D. A. Dhale. 2011. Antifungal properties of extracts of Ocimum tenuiflorum and
Datura stromonium against some vegetable pathogenic fungi. J. Phytol. 3(12), 41-44.
Shinwari, Z. K. and S. Malik. 1989. Plant Wealth of Dera Bughti area, Progressive Farming. 9,
39-42
183
Shrestha, A. K., and R. D. Tiwari. 2009. Antifungal activity of crude extracts of some medicinal
plants against Fusarium solani (Mart.) Sacc. Ecoprint. 16, 75-78.
Shtienberg, D., D. Blachinsky,Y. Kremer, G. Ben-Hador and A. Dinoor. 1995. Integration of
genotype and age-related resistance to reduce fungicide use in management of Alternaria
diseases of cotton and potato. Phytopathology. 85, 995-1002.
Siddiqui, B. S., F. Afshan, T. Gulzar and M. Hanif. 2004. Tetracyclic triterpenoids from the leaves
of Azadirachta indica. Phytochemistry, 65(16), 2363-2367.
Sims, W.L. 1980. History of tomato production for industry around the world. Acta Hort. 100,
25-26.
Simmons, E. 2007. Alternaria: An Identification Manual. CBS Fungal Biodiversity Centre,
Utrecht, Netherlands.
Singh, U. P., V. N. Pandey, K. G. Wagner and K. P. Singh. 1995. Antifungal activity of ajoene, a
constituent of garlic (Allium sativum), on powdery, mildew (Erysiphe pisi) of pea
(Pisum sativum) Z. Pflanzekrankh Pflanzensch. 102, 399-406.
Singh, N., R. P. Saxena, S. P. Pathak, and S. K. S. Chauhan. 2001. Management of Alternaria
leaf disease of tomato. Indian Phytopath. 54, 508.
Singh, M. and R. P Singh. 2005. Management of mushroom pathogens through botanicals. Ind.
Phytopath. 57, 189-193.
Singh, R. P., and D. A. Jain. 2011. Evaluation of Antimicrobial activity of Volatile Oil and total
Curcuminoids extracted from Turmeric. Int. J. Chem. Tech Res. 3, 1172-1178.
Singh, H., G. Fairs and M. Syarhabil. 2011. Anti-fungal activity of Capsicum frutescence and
Zingiber officinale against key post-harvest pathogens in citrus. International Conference
on Biomedical Engineering and Technology IPCBEE. IACSIT Press, Singapore. 11, 1-6.
Singleton, V. L. and J. A. Rossi. 1965. Colorimetry of Total Phenolics with Phosphomolybdic-
184
Phosphotungstic Acid Reagents. Amer. J. Enol. Vitic. 16, 144-158.
Sinha, P. P. and R. K. Prasad. 1991. Evaluation of fungicides for control of early blight of tomato.
Madras Agric. J. 78, 141-143.
Siva, N., S. Ganesan, N. Banumathy and N. Muthuchelian. 2008. Antifungal Effect of leaf extract
of some medicinal plants against Fusarium oxysporum causing Wilt disease of Solanum
melogena L. Ethnobotanical Leaflets. 12, 156-163.
Slusarenko, A. J., A. Patel and D. Portz. 2008. Control of plant diseases by natural products: Allicin
from garlic as a case study. Eur. J. Plant Path.121, 313-322.
Smart, C. D., K. L. Myers, S. Restrepo, G. B. Martin and W. E. Fry. 2003. Partial resistance of
tomato to Phytophthora infestans is not dependent upon ethylene, jasmonic acid, or
salicylic acid signaling pathways. Mol. Plant-Microbe Interactions. 16, 141-148.
Smith, H. C. 1965. The morphology of Verticillium albo-atrum, V. dahliae, and V. tricorpus. New
Zealand J. Agric. Res. 8(3), 450-478.
Smith, B. J., and L. L. Black. 1990. Morphological, cultural, and pathogenic variation among
Colletotrichum species isolated from strawberry. Plant Dis. Rep.74, 69-76.
Smith, A. F. 1994. The tomato in America: early history, culture, and cookery. University of
Illinois Press.
Sohn, H. Y., K. H. Son, C. S. Know and S. S. Kang. 2004. Antimicrobial and cytotoxic activity of
18 prenylated flavonoids isolated from medicinal plants: Morus alba L., Morus mongolica
Schneider, Broussnetia papyrifera (L.) Vent, Sophora flavescens Ait and Echinosophora
koreensis Nakai. Phytomedicine. 11, 666-672.
Sofowora, A. 1993. Medicinal plants and Traditional medicine in Africa. Spectrum Books Ltd,
Ibadan, Nigeria. 289.
185
Song, W., L. Zhou, C. Yang, X. Cao, L. Zhang and X. Liu. 2004. Tomato Fusarium wilt and its
chemical control strategies in a hydroponic system. Crop Prot. 23, 243-247.
Soule, J. 1993. Tagetes minuta: A potential new herb from South America. In: New Crops, (eds. J.
Janick and J. E. Simon). Wiley, NY. pp. 649-654.
Srinivasan, K., G. Gilardi, A. Garibaldi, and M. L. Gullino. 2009. Bacterial antagonists from
used rockwool soilless substrates suppress Fusarium wilt of tomato. J. Plant Pathol. 91,
147-154.
Steel, R.G. D., J. H. Torrie and D. A. Dickey, 1997. Principles and Procedures of Statistics. A
Biometrical approach. 3rd Ed., McGraw Hill Book Co., New York, USA.
Stephen, R. D. 2005. An overview of the antifungal properties of allicin and its breakdown
products- the possibility of safe and effective antifungal prophylactic. Mycoses. 48(2), 95-
100.
Stevenson, R. 1997. Late blight: In: Compendium of tomato diseases. (eds. J.B. Jones, J.P. Jones,
R.E. Stall and T.A. Zitter). Amer. Phytopath. Socy. 3340. Pilot Knob Road, Minnesota
USA, pp 17-18.
Stangarlin, J. R., O. J. Kuhn, L. Assi and K. R. F. Schwan-Estrada. 2011. Control of plant diseases
using extracts from medicinal plants and fungi. Science Against Microbial Pathogens:
Communicating Current Research and Technological Advances, Formatex, Badajoz. 2,
1033-1042.
Stoilova, I., S. Gargova, A. Stoyanova and L. Ho. 2005. Antimicrobial and antioxidant activity
of the polyphenol mangiferin. Herbal Polonica. 51, 37-44.
Stoll, G. 1988. Protection Naturelle des Ve´ge´taux en Zone Tropicale (Natural Protection of
Plants in Tropical Zone). Eds Margraf Verlag., CTA, AGRECOL.
186
Subramanyam, B. and R. Roesli. 2000. Inert dusts. In: Bh. Subramanyam and D.W. Hagstrum,
Editors, Alternatives to Pesticides in Stored-Product IPM, Kluwer Academic Publishers,
Dordrecht. 321-380.
Sudhamoy M., M. Nitupama and M. Adinpunya. 2009. Salicylic acidinduced resistance to
Fusarium oxysporum f. sp. Lycopersici in tomao. Plant Physiol. Biochem. 47, 642–649.
Suhr, K. I. and P. V. Nielson. 2003. Antifungal activity of essential oils evaluated by two
different application techniques against rye bread spoilage fungi. J. Appl. Microbiol. 94,
665-674.
Suleman, P., A. M. Tohamy, A. A. Saleh, M. A. Madkour and D. C. Straney. 1996. Variation in
sensitivity to tomatine and rishitin among isolates of Fusarium oxysporum f.sp.
lycopersici, and strains not pathogenic to tomato. Physiol. Mol. Plant Pathol. 48, 131-
44.
Summerell, B. A., B. Salleh and J. F. A. Leslie. 2003. Utilitarian approach to fusarium
identification. Plant Dis. 87, 117–128.
Sutton, B. C. 1980. The Coelomycetes: Fungi Imperfecti with Pycnidia, Acervuli and Stromata.
C M. I. Kew, England.
Sutton, B. C. 1992. The genus Glomerella and its anamorph Colletotrichum. In: Colletotrichum:
Biology, Pathology and Control (eds. J.A.Bailey M. Jeger), CAB Int.Wallingford, UK, 1-
26.
Snyder, W. C. and H. N. Hansen. 1940. The species concept in Fusarium. Amer. J. Bot. 27, 64-
67.
Taiz, L. and E. Zeiger. 1998. Plant Physiology. 2nd edition. Sunderland, Massachusetts: Sinauer
Associates, Inc.
187
Taylor, I. B. 1986. Biosystematics of the tomato. In: The Tomato Crop: A Scientific Basis for
Improvement (eds. J. G. Atherton and J. Rudich), Chapman and Hall Ltd, London. pp.
134.
Tang, W. and G. Eisenbrand. 1992. Curcuma spp. In: Chinese Drugs of Plant Origin. pp. 401-
415, Springer, NewYork, USA.
Taskeen-Un-Nisa, A. H. Wani, M. Y. Bhat, S. A. Pala and R. A. Mir. 2011. In vitro inhibitory
effect of fungicides and botanicals on mycelial growth and spore germination of
Fusarium oxysporum. J. Biopesticides. 4(1), 53-56.
Tegegne, G., J. C. Pretorius and W. J. Swart. 2008. Antifungal properties of Agapanthus
africanus L. extracts against plant pathogens. Crop Prot. 27(7), 1052-1060.
Tenover, F. C., J. M. Swenson, C. M. O’Hara and S. A. Stocker. 1995. Ability of commercial and
reference antimicrobial susceptibility testing methods to detect vancomycin resistance in
Enterococci. J. Clin. Microbiol. 33(6), 1524-1527.
Than, P. P., H. Prihastuti, S. Phoulivong, P.W. J. Taylor and K. D. Hyde. 2008. Chili anthracnose
disease caused by Collectotrichum species. J. Zhejiang. Uni. Sci. 9, 764778.
Thangavelu, R., P. Sundararaju and S. Sathiamoorthy. 2004. Management of anthracnose disease
of banana caused by Colletotrichum musae using plant extracts. J. Hort. Sci. Biotechnol.
79, 664-668.
Timbola, A. K., B. Szpoganicz, A. Branco, F. D. Monache and M. G. Pizzolatti. 2002. A new
flavonol from leaves of Eugenia jambolana. Fitoterapia. 73, 174-176.
Ting-Ting, W, C. Zhi-Hui, M. A. Khan, M. Qing and H. Ling. 2011. The inhibitive effects of
garlic bulb crude extract on Fulvia Fulva of Tomato. Pak. J. Bot. 43(5), 2575-2580.
Tomar, M. and S. Chandel. 2006. Use of Phytoextracts in the management of gladiolus wilt. J.
Mycol. Plant. Pathol. 36(2), 45-49.
188
Trease, G. E. and W. C. Evans. 2009. Pharmacognosy. 16th Edition, Bailliere Tindall Ltd. London.
Elsevier publishers. pp1-571. ISBN 978-0-7020-2933-2.
Tripoli, E., M. A. Guardia, S. Giammanco, D. D. Majo and M. Giammanco. 2007. Citrus
flavonoids: Molecular structure, biological activity and nutritional properties: A Review.
Food Chem. 104, 466-479.
Tura, D. and K. Robards. 2002. Sample handling strategies for the determination of biophenols
in food and plants. J. Chromatogr. A. 975, 71-93.
Turkmen, N., F. Sari, and Y. S. Velioglu. 2006. Effect of extraction solvents on concentration
and antioxidant activity of black and black mate polyphenols determined by ferrous
tartrate and Folin-Ciocalteu methods. Food Chem. 99, 838-841.
Upasana, S., T . Anurag, A. K.Upadhyay, U. Shukla,and A.Tewari. 2002. In vitro Antimicrobial
study of Neem (A. indica) seed and leaf extracts. J. Ind. Vet. Med. 22(2),109-111.
Uzama, D. R. A., M. B. David and S. A. Thomas. 2012. Comparative study on the antifungal
activities of aqueous extracts of the rhizomes of ginger and the leaves of Eucalyptus
saligna and Polyalthia longifolia. Res. J. Engin. and Appl. Sci.1, 50-53.
Van Baarlen, P., L. Legendre and J. A. van Kan. 2007. Plant defence compounds against Botrytis
infection. In: Botrytis; Biology, pathology and control. pp 143-161. Springer
Netherlands.
Van de vooren, J., G. W. H. Welles and G. Hayman. 1986. Glasshouse crop production. In: The
Tomato Crop: A Scientific Basis for Improvement (Eds. Atherton, J. G. and Rudich, J.),
Chapman and Hall Ltd, London. pp 443-484.
Van Etten, H. D., J. W. Mansfield, J. A. Bailey and E. E. Farmer. 1994. Two classes of plant
antibiotics: Phytoalexins versus Phytoanticipins. Plant Cell. 6, 1191–1192.
Varma, P. K., S. Singh, S. K. Gandhi and K. Chaudhary. 2006. Variability among Alternaria
189
solani isolates associated with early blight of tomato. Commun. Agric. Appl. Biol. Sci.
71, 37-46.
Veeraragava, T. D. 1998. Drumstick (Moringa oleifera Lam.). In: A guide on vegetable culture.
(eds. D.V.Thatham, D. M. Jawaharlal, R. Seemanthini). Suri Associates, Coimbatore,
India., 139–43.
Vega-Sánchez, M. E., L. J. Erselius, A. M. Rodriguez, O. Bastidas, H. R. Hohl, P. S. Ojiambo, J.
Mukalazi, T. Vermeulen, W. E. Fry and G. A. Forbes. 2000. Host adaptation to potato
and tomato within the US-1 clonal lineage of Phytophthora infestans in Uganda and
Kenya. Plant Path. 49, 531-539.
Vighasiya, Y., R. Dave and S. Chanda. 2011. Phytochemical analysis of some medicinal plants
from Western Region of India. Res. J. Med. Pl. 5(5), 567-576.
Vincent J. M. 1947. Distortion of fungal hyphae in the presence of certain inhibitors. Nature.
150, 850.
Viuda-Martos, M., Y. Ruiz-Navajas, J. Fernandez-Lopez and J. Perez-Alvarez. 2008. Antifungal
activity of lemon (Citrus limon (L.)Burm, mandarin (Citrus reticulata Blanco), grapefruit
(Citrus paradisi Macf.) and orange (Citrus sinensis (L) Osbeck essential oils.
Food Control. 19, 1130–1138.
Waller, G. R., M.-C. Feng, and Y. Fujii. 1999. Biochemical analysis of allelopathic compounds:
Plants, microorganisms and soil secondary metabolites. In: Principles and Practices in
Plant Ecology: Allelochemical Interactions (eds. Inderjit, K.M.M. Dakshini and C.L.
Foy). CRC Press: Boca Raton, Florida, 75-98.
Wang, W. Q., B. H. Daniel and Y. Cohen. 2004. Control of plant diseases by extracts of Inula
viscosa. J. Phytopath. 94, 1042-1047.
Wang, Y. C, Y. C. Chuang and H. W. Hsu. 2008. The flavonoid, carotenoid and pectin content in
190
peels of citrus cultivated in Taiwan. Food Chem. 106, 277-284.
Wang, L., Y. Zhang, H-P. He, Q. Zhang, S-F. Li and X-J. Hao. 2011. Three New Terpenoid
Indole Alkaloids from Catharanthus roseus. Planta Med. 77, 754-758.
Waudo, S. W., P. O. Owino and M. Kuria. 1995. Control of Fusarium wilt of tomatoes using soil
amendments. E. Afric Agri. For. J. 60, 207–217.
Weststeijn, G.1973. Soil sterilization and glasshouse disinfection to control Fusarium oxysporum
f. sp lycopersici in tomatoes in the Netherlands. Netherlands J. Plant Pathol. 79(1), 36-
40.
Wheeler, B. E. J. 1969. An Introduction to Plant Diseases. John Wiley and Sons Limited,
London, , 301.
Whittaker, R. H. and P. P. Feeny. 1971. Allelochemics. Chemical interactions between species.
Science. 171, 757-770.
Wszelaki A. L. and S. A. Miller. 2005. Determining the efficacy of disease management products
in organically produced tomatoes. Plant Health Progress. doi:10.1094/PHP2005-0713-01-
RS.
Yadav, V. K., A. V. Zope, P. S. Bainade and V. S. Thrimurthy. 2005. Efficacy of some
medicinal plant extracts. J. Plant Dis. Sci. 1, 123-127.
Yasmin, M., K. S. Hossain and M. A. Bashar. 2008. Effects of some angiospermic plant extracts
on in vitro vegetative growth of Fusarium moniliformae. B'desh J. Bot. 37(1), 85-88.
Yeni, I. J., O. S. Dele, I. J. Ademola and A. J. Adeniran. 2010. Allelopathic effect of leaf extract
of Azardirachta indica and Chromolaena odorata against post harvest and transit rot of
tomato (Lycopersicum esculentum L.). J. Amer. Sci. 6, 1595-1599.
Yi, X.D., S.H. Wei, B. Liu, Z.Y. He, Y.Y. Bai and S. Hu. 2008. Restraining effect of garlic juice
on two tomato fungus diseases. J. Shenyang Agric. Univ. 39(1), 89-91.
191
Yonghao L. 2013. Anthracnose of tomato. New Haven (CT): The Connecticut Agricultural
Experiment Station.
Yoshimi, N., K. Matsunaga, M. Katayama, Y. Yamada, T. Kuno, Z. Qiao, A. Hara, J. Yamahara
and H. Mori. 2001. The inhibitory effects of mangiferin, a naturally occurring
glucosylxanthone, in bowel carcinogenesis of male F344 rats. Cancer Letters. 163,
163170.
Zafar, I., S. Mussarat, H. Farrakh and B. Sheraz. 2002. Antifungal properties if some indigenous
plants from Peshawar valley. Asian. J. Plant Sci. 1(6), 708-709.
Zia-Ul-Haq, M., A. Mansoor, J. Mehjabeen, A. Noor, A. Shakeel, Q. Mughal and M. K.
Inamullah. 2011. Antimicrobial screening of selected flora of Pakistan. Arch. Biol. Sci.
63, 691-695.
Zia-Ul-Haq, M., M. R. Shah, M. Qayum and S. Ercisli. 2012. Biological screening of selected
flora of Pakistan. Biol Res. 45, 375-379.
Živković, S., S. Stojanović, Ž. Ivanović, N. Trkulja, N. Dolovac, G. Aleksić and J. Balaž. 2010.
Morphological and molecular identification of Colletotrichum acutatum from tomato fruit.
Pestic. Phytomed. (Belgrade) 25, 231-239.
192
APPENDIX-1
Culture media: The following media were used for the isolation of fungi and in vitro bioassays. The recipes of culture media were prepared in one liter of distilled water and autoclaved at 121oC at 15psi for 20 minutes. 1.1 Potato Dextrose Agar Medium Potato starch 20gm Dextrose 20gm Agar agar 20gm Distilled water 1000ml 1.2 V 8 Juice Agar Medium V-8 juice 300ml Agar agar 20gm Calcium carbonate 3gm Distilled water 700ml 1.3 Carnation Leaf Agar Medium Carnation leaf pieces 500 pieces (1piece/2ml of medium) Water agar 2% 1.4 Carrot Agar Medium Carrots dices 400gm Agar agar 20gm Distilled water 1000ml Note: Modified tomato juice agar (V8 juice replaced by tomato juice) was used for the growth of Phythophthora infestans (Mont.) de Bary. Equipments
Equipments Manufacturer/Supplier Incubator MIR-153, MLR-351 (Japan) Neauber Haemocytometer MARienfeld- (Germany) Rotary evaporator Buchi, Centrifuge machine Centurion Scientific Ltd (UK) Laminar flow hood ESCO
193
Microscope LABO (Germany) Swift M3200 Autoclave Robus Technologies RTA 85(UK) Electric blender Annex Water bath 4(holes) Mistral 1000, HH-S4 Digital electronic balance WT6002D Freezer -80oC MDF U55V (Japan) Spectrophotometer UV 3000 ORi (Germany)
Programs (soft wares) and statistical packages used Programs Source of the programs Endnote X6 Thomson SAS (version 10) SAS Institute, Inc., NC, USA
194
APPENDIX-2
Macroscopic and Microscopic characteristics of isolated fungi
Sr
. Color Type of Spore Favorable N Fungus name of Spore shape Colony color Phylum/Class Hyphae color Temp o Hyphae Septate, Pale Pale to Obclavate to Flat, downy to cottony Ascomycota/ 25±26 °C 1 Alternaria brown light obpyriform conidia grayish. fast growing dothideomycetes alternata to olive brown short conical beaks. brown 2 Fusarium Septate, Bluish hayline Macroconidia septate White to cream cottony Ascomycota/ 30oC solani branches brown fusiform colonies sordariomycetes Microconidia oval to kidney shape
3 Curvularia Septate Brown Pale Multicelled,slightly Shiny velvety –black fluffy Ascomycota lunata brown curved growth / Euascomycetes 25±30oC
4 Drecshlera Septate Dark Brown Geniculate Initially white becoming Ascomycota/ 25°C australiensis fusiform to ellipsoidal grey to blacklish-brown. Dothidiomycetes
5 Fusarium Septate Hayline Simple Macroconidia "string Initially white Ascomycota/ moniliformae or bean-like", 5-septate Sordariomycetes 25°C branche Microconidia abundant, d septate,oval to clavate,
195
6 F.oxysporum Discrete White to Hayline Macroconidia 3-5 Initially white, salmon to Ascomycota/ f.sp sporodoc purple celled,fusiform,Microc purple Sordariomycetes 25±30° C lycopersici hiaseptat onidia non-septate,2 e celled
7 Aspergillus Septate/ White to Brown Round, obscures Initially white, then Yellow Ascomycota / 30° C niger Biseriate pale to black vesicle to gray Eurotiomycetes yellow conidia
8 Aspergillus Septate Yellow Brown Conidia are round, Granular,velvety,yellow Ascomycota / 37° C flavus brown globose to subglobose, brown Eurotiomycetes or hayaline 9 Alternaria Septate Colorles Brown Club-shaped Blackish Ascomycota 30°C tenuissima s Dothidiomycetes
10 Aspergillus Septate White to Hayline Smooth globose, Velvety, brown, Ascomycota / terreus brown elliptical conidia form cinnamon, orange-brown Eurotiomycetes 25±30°C long chain
11 Aspergillus Septate/ White Dark Club shaped, Granular cottony, velvety Ascomycota / 45 °C fumigatus unseriate to tan green subglobose, conidia a green brown in colour Eurotiomycetes in chains
12 Chaetomium Septate Hyaline Brown Single celled, lemon Wooly whitish grey/olive Ascomycota, 25±30°C globosum to pale shaped on surface Sordariomycetes brown
13 Mucor sp Aseptate Hyaline Purplis Round or ellipsoidal Woolly growth, white in Zygomycota/ 25±30°C h colour but turns a Mucormycotina greyishbrown
196
14 Cladosporiu Septate Brown Brown Single celled and Velvet olive Ascomycota 20±25°C m sp ellipsoidal/round at the brown/brownish black Dothidiomycetes tips
15 Penicillium Septate Hyaline Hyaline Conidia single celled, Green, grayish Ascomycota/ 25°C digitatum round Phialides are green Eurotiomycetes bunched in brush like clusters
16 Rhizopus Unbranc White color Grayish Bit like a very tiny White to cream to Zygomycota/ 25 ±27°C stolonifer hed Broad to a brown walnut shell , an graypigmented Zygomycete Hyphae blackish irregular assymetric Aseptate brown shape.
17 Aspergillus Septate, Hyaline White Round, Uniseriate Greenish Ascomycota/ 37 °C sulphureus Eurotiomycetes
18 Alternaria Septate, Brownis Olive Obclavate, obpyriform, Black to olivaceous-black Ascomycota/ 26±28oC solani hyphae h brown or greyish Dothideomycetes
Septoria Branched Black Dark in Long with several cells Woolly, dark to light grey Ascomycota/ 24°C 19 lycopersici color Dothideomycetes
197
20 Phytophthora Profusely Whitish Hyaline Lemon-shaped/ Cottony, petaloid, Oomycota/ 18 ± 24°C infestans branchin pyriform rosaceous, and stellate Oomycetes g myceliu m
21 Rhizoctonia Septate Buffcolored Scleroti Ellipsoid with one side Whitish becoming dark Basidiomycota/ 20ºC-25ºC solani to dark a flattened brown with sclerotia Agariomycetes brown irregula r shape, light to dark brown
22 Verticillium Septate Hyaline or Hyaline Ovoid or ellipsoid White with a yellow tinge Ascomycota/ 20±25ºC albo-atrum greyish single-celled Deuteromycetes black
23 Coletotrichu Septate Cylindri cal Colorle Ovoid to cylindrical, White to greyish Ascomycota/ 25ºC m coccodes and hayline ss when and single celled Sordariomycetes branched viewed alone, pink or salmon 24 Botrytis Crosswalls Colorles Gray to Hyaline, ellipsoid to Light powdery ash-grey Ascomycota/ 20±25oC cineria branched s to white brown obovoid growth Leotiomycetes hyphae grape-like clusters
198
APPENDIX-3
Table: Pathogenicity of important fungi recovered from tomato under greenhouse conditions
Disease Fungus Symptoms Small brown lesions first appear on older leaves, later lesions enlarged and encircled by concentric rings Early blight Alternaria solani surrounded by yellow haloes, defoliation occurred, lesions also developed on stem and fruits. Small water soaked lesions developed on leaves, later enlarged into pale-green to Late blight Phytophthora infestans brown lesions covering larger areas. Infected foliage become brown, shriveled and dead. Yellowing and wilting of leaves occurred; chlorotic Wilt Fusarium oxysporum f.sp lycopersici symptoms appeared on onehalf of the plant. Small depressed circular lesions developed on fruits, Anthracnose Colletotrichum coccodes lesions were dotted with black microsclerotia formed by the pathogen. Root rot/damping Seed rot, damping off and Fusarium solani off poor crop stand.
185