ABSTRACT
The root-knot nematode (Meloidogyne incognita) is one of the plant parasitic nematodes that causes serious economic damage on tomato plant. Due to loss of effectiveness and environmental hazard of chemical nematicide, the search for new agents of control has become paramount. Therefore, nematicidal efficacy of soil fungal isolates on second stage juvenile of Meloidogyne incognita was carried out. Soil samples for the isolation of fungi and nematode were collected from botanical garden and infested soil of tomato farms. Fungal species were isolated using serial dilution method while nematode was extracted using pie-pan method. The in vitro antagonistic effects of fungi against M. incognita were assessed in potato dextrose broth. In vivo was carried out in screen house where healthy and sterilized tomato seeds were sown in a 7 litre plastic buckets containing 5 kg of sterilized loamy soil. The buckets were arranged in a completely randomized block design on a bench with three (3) replicates. After germination of the tomato seeds, two weeks from sowing,each bucket was thinned to one plant. One thousand (1000) juvenile second stage (J2) M. incognita were inoculated in four holes round the stem of each plant in a bucket and on the next day 10 ml of fungi culture filtrates was inoculated into the same hole. Four fungal species isolated were; Aspergillus niger, Aspergillus nidulans, Penicillium chrysogenum and Rhizopus  stolonifer while nematode isolated was M. incognita. The results of fungicidal activity against M. incognita after 24 and 48 hrs were A. niger (39.28 and 78.47 %), A. nidulans (29.12 and 57.49 %), P. chrysogenum (18.59 and 47.48 %) and R. stolonifer (8.05 and 26.43 %). In in vivo experiment,  A. niger gave the highest values for average plant height at maturity, number of fruits per plant and number of leaves per plant (46.43 cm, 10.33, and 69.67 respectively) and lowest value for number of galls per plant root (3.67). Lowest values were recorded for R. stolonifer in average plant height, number of fruits per plant and number of leaves per plant (26.13 cm, 5.33 and 46.33 respectively) and were the highest value for number of galls per plant roots (16.67). The values recorded for A. niger and R. stolonifer differed significantly (P< 0.05). Disease incidence was highest in the control at harvest after treatment with the value 39.49 % and A. niger had the lowest value of 7.18 % at harvest after treatment, the values recorded for the control and A. niger differed significantly (P< 0.05). The results of this study conclude that these fungal species could serve as biological agent for effective management of root-knot nematodes and bio-fertilizer for the enhancement of plant growth parameters.
CHAPTER ONE
1.0Â Â Â Â Â Â INTRODUCTION
1.1 Background to the Study
Tomato (Solanum lycopersicum) is an annual crop belonging to the family solanaceae. It is one of the most important vegetable crops which is being cultivated worldwide. The crop is a rich source of lycopene, used in the cancer treatment especially in prostate cancer (Singh et al., 2011). According to the National Cancer Institute, there is enough data to show that people who consume large amount of tomato products have significantly decreased risk of prostate, lung, and stomach cancer (Adam et al., 2015). Nigeria is ranked to be the second largest producer of tomato in Africa with an annual production of 17,500,000 metric tons (FAO, 2012). However, large number of phyto-pathogens (sting caused by Belonolaimus longicaudatus and stubby-root caused by Trichodorus spp.) have been reported to be associated with reduction in tomato productivity. Among them, root-knot nematodes which had been reported to be the major pathogen affecting the crop (Singh et al., 2011; Keshari and Gupta, 2015).
Meloidogyne incognita causes root-knot disease in tomato plant by abnormal expansion of root cell  forming giant  cells (Singh and Patel, 2013). M. incognita is one of the key nematodes which is widely distributed (Siddiqui and Shaukat, 2014) and difficult to control (Chitwood, 2016) because of its high reproduction rate, resulting into massive yield loss of tomato fruits ranging from 32 to 40 % (Anwaar and Mckenry, 2012). M incognita has also been identified as one of the three most economically damaging genera of plant parasitic nematodes on horticultural and field crops. Obligate parasites of roots of thousands of plant species, including monocotyledonous, dicotyledonous, herbaceous and woody plants. The genus includes more than 90 species (Moens et al., 2015), with some species having several races. M. incognita occurs in 23 of 43 crops listed as having plant parasitic nematodes of major importance, ranging from field crop, through pasture and grasses to horticultural, ornamental  and vegetable  crops  (Acquaah,  2012). If  root-knot  nematodes become established in deep-rooted, perennial crops, control is difficult, and options are limited.
However, fumigant and non-fumigant nematicides have been developed for controlling this nematode over years, but there are increasing concerns over risks of these chemicals on the environment and human health which led to the withdrawal or restriction of some chemicals, for instance methyl bromide, the most effective and widely used soil fumigant has already been banned in developed countries (Nyezepir and Thomas, 2016). The use of some non-fumigant nematicides organophosphates and carbamates based has been restricted depending on the region, crop, and production system, such as integrated production and organic farming. Consequently, the search for naturally occurring compounds with nematicidal activity has been stimulated as an alternative to using existing compounds (Chitwood, 2016). Soil fungi that inhabit the rhizosphere may serve as a source for such compounds since they share the same environment as the nematodes and produce metabolites as a strategy for protecting their habitat from plant-parasitic nematodes or other organisms (Moosavi and Zare, 2012).
These fungi may release compounds that directly kill nematodes, suppress nematode motility, reduce egg hatching and interfere with metabolic processes, through such mechanisms, they could regulate nematode populations (Moosavi and Zare, 2012). According to Kumar et al. (2010) who study the culture filtrates of Aspergillus niger and were most effective against second stage juvenile followed by Fusarium moniliforme, F. solani, F. oxysporum and P. debarianum. Satyandra and Nita. (2010) carried out in vitro study to determine  the  efficacy  of indigenous  fungi  Acremonium  strictum, Aspergillus terreus, A. nidulans, A.niger, Chetomium aubense, Chladosporium oxysporum, Fusarium chlamydosporium, F. dimarum, F. oxysporum, F. solani, Paecilomyces lilacinus, Pochonia chlamydosporia, Trichoderma viride and T. harzianumon egg parasitism, egg hatching, mobility and  mortality of  root-knot  nematode,  M.  incognita  in which  all  tested  fungi showed varied effects against the nematode. Kalele et al. (2010) studied the bio-control potential of Paecilomyces lilacinus on M. incognita on tomato under glasshouse conditions.
1.2 Statement of the Research Problem
Despite the production and market value of tomato, several reports indicated that Meloidogyne incognita is the major problem in tomato growing areas which results in poor growth, decline in quality and yield of the crop, whereby, a high level of damage can lead to total crop loss (Esheteu et al., 2014). Chemical nematicides have been used for nematode control for the last 50 years (Nyezepir and Thomas, 2016). However, the most limiting factors in using synthetic nematicides in developing countries is that they are either too expensive, sub-standard or due to lack of technical knowledge. Excessive use and poor handling of these chemicals can lead to health hazard, nematode resistance and destabilization of the ecosystem of the crop and reduced resistant to other stresses (e.g drought, other diseases) (Huang, 2016). Therefore, there is an increasing requirement for developing non-chemical alternative methods for the management of M. incognita.
1.3 Justification for the Study
Tomato is one of the most economically important crops majorly consumed worldwide and worth a tremendous amount of money due to its massive yield (Abdissa et al., 2010).
Bio-management of root-knot nematode by Trichoderma spp. has been found safe for soil biota, it is a well-known alternative to chemical nematicide specially for the purpose of protecting crops against nematodes (Adam et al., 2014).
Paecilomyces lilacinus isolates were comparatively effective in controlling the cyst nematode and their use resulted in a significant increase in tomato yield (Ashoub et al., 2018). Soil fungi that were commercially produced such as Pochonia clamydosporia, Trichoderma longibrachiatum, Paecilomyces lilacinus, they revealed promising results as effective biological control agents on root-knot nematode in tomato as reported by Dallemole-giaretta (2014).
Fungi release compounds such as oxalic acid and kojic acid that directly kill nematodes, suppress the nematode motility, reduce egg hatching and interfere with metabolic processes and through such mechanism, they could regulate nematode populations (Moosavi and Zare, 2012).
It is therefore important to evaluate a secure alternative like the soil fungi which is harmless to tomato and the environment.
1.4 Aim and Objectives of the Study
The aim of this study was to determine the nematicidal efficacy of soil fungal isolates against the root-knot nematode (Meloidogyne incognita) of tomato (Solanum lycopersicum).
Objectives of this research are to:
i. isolate and identify the fungi from soil samples collected from the study site.
ii. extract and identify Meloidogyne incognita from soil samples collected from tomato vendor in Bosso market Niger State.
iii.      to  evaluate  efficacy  of  fungal  culture  against  second  stage  juvenile  (J2s)  of Meloidogyne incognita in vitro.
iv. to find out the impact of these fungal culture on plant growth parameters of tomato plants, root-gall formation and disease incidence in vivo.
This material content is developed to serve as a GUIDE for students to conduct academic research
NEMATICIDAL EFFICACY OF SOIL FUNGAL ISOLATES AGAINST THE ROOT-KNOT NEMATODE (MELOIDOGYNE INCOGNITA) OF TOMATO
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