ABSTRACT
Five Screenhouse experiments and one field experiment were conducted at the Teaching and Research Farm of the Department of Crop Science, Faculty of Agriculture, University of Calabar between 2008 and 2010. Experiment I evaluated the host status of five Mucuna  species to Meloidogyne incognita.  It was laid out in a completely randomized design (CRD) having six treatments represented by five Mucuna  species (M. pruriens utilis, M. ghana, M. cochichinensis, M. jaspaeda  and M. pruriens  1R2) plus a check (susceptible tomato cv. Roma VF) which were inoculated with 5,000 eggs of M. incognita / plant.  In Experiments II , III, V and VI, tomato seedlings (cv. Roma VF) were inoculated with 5,000 eggs of M. incognita  per plant. Experiment II consisted of five rates: 2, 4, 6, 8 and 10 t/ha on dry matter basis of each Mucuna  species with fresh foliage applied as green manure and soil without amendment served as control (0 t/ha). The 26 treatments were laid out in a CRD with three replications. Experiment III was a 6 x 6 factorial laid out in a CRD with three replications. The treatments were combinations of the five species of Mucuna  amendment at 8 t/ha each with five species of arbuscular mycorrhizal fungus (AMF):  Glomus etunicatum, Glomus mosseae, Glomus clarum, Glomus deserticola  and  Gigaspora gigantea plus their respective controls. The tomato seedlings were inoculated with the AMF species at the nursery stage. Experiment IV was done in the field and was laid out as a split-plot in randomized complete block design with three replications. The main- plots were the Mucuna  species planted and ploughed-in as green manures. Naturally fallowed plots served as control. AMF- inoculated tomato seedlings were transplanted to the sub- plots and uninoculated seedlings served as the control. Tomato grown in the field were naturally infected with M. incognita.  In experiment V, top soils were collected from Calabar, Ikom, Obubra and Ogoja (Cross River State), Nsukka (Enugu State), Umudike (Abia state) and Uyo (Akwa Ibom state) and the experimental design was 3 x 6 factorial in CRD with three replications. Three frequencies of bioformualted Paecilomyces lilacinus  application were combined with six levels of AMF species. Experiment VI was a 6 x 6 x 2 factorial laid out in CRD with three replications and the treatments included six levels each of Mucuna species and AMF species and two levels of P. lilacinus  application. The tomato plants were grown to full maturity and data were collected on number of galls and eggmasses/ root system, gall index (0-5 scale), nematode larvae/200 g of soil, mycorrhizal root colonization (%), weight (g) of fresh root, dry shoot and total fresh fruit/ plant. Mineral contents of the Mucuna species were determined. Data collected were subjected to analysis of variance and means separated with Fisher’s least significant difference and Duncan’s new multiple range test at 5% probability level. Tomato responses to rates of Mucuna were tested with linear or curvilinear regression model at 1% probability level. The results obtained showed that the roots of the Mucuna  spp in both Screenhouse and field trials were neither galled nor had egg masses and were rated immune to infection with a gall index (GI) = 0.00. The tomato plant (check) was highly susceptible, with GI rating of 5.00. The number of nematode larvae on tomato rhizosphere was significantly (p≤ 0.05) higher than that of Mucuna species. In all the Mucuna  species, successive increase in the rate of amendment resulted in a significant (P0.05) decrease in the number of galls, eggmases, nematode larvae but with a significant (p  0.05) enhancement in growth, dry matter, and fresh fruit yield. Mucuna jaspaeda  and M. ghana  amendment produced plants with the fewest galls and eggmasses. These two Mucuna species had the lowest C:N ratio. Number of galls and fresh fruit yield responded in a highly significant (p<0.01) negative (r <- 0.80) and positive (r > 0.70) linear relationship, respectively with Mucuna amendment rate. In both Screenhouse and field, AMF inoculation and Mucuna amendment significantly (p ≤ 0.05) suppressed galling and eggmass production but enhanced growth and fruit yield of tomato compared with their respective controls. Mucuna  amendment significantly (p ≤0.05) enhanced root colonization by AMF. Combined application of both control agents was more effective than in sole applications. The highest fresh fruit yield of 409.00 g/plant was obtained in plots inoculated with Gi. gigantea  and amended with M. jaspaeda.  Application of P. lilacinus  or AMF inoculation significantly ( p ≤ 0.05) inhibited root galling and eggmass production by M. incognita   in the soils from all locations with a significant ( p ≤ 0.05) enhancement in growth and fresh fruit yield of tomato. Double application of the bionematicide was significantly ( ≤.0.05) more effective than single. The most effective AMF species in gall suppression across the soil types was G.   etunicatum,  while  G. deserticola  was the most effective in fresh fruit yield enhancement. Combined application of the three control agents significantly (p  0.05) inhibited galling with a significant (p 0.05) increase in growth and fruit yield of tomato relative to sole applications. The highest fresh fruit yield of 139.46 and 136. 06 g/plant were obtained from G. mosseae  and Gi. gigantea  inoculated plants, respectively grown in M. jaspaeda  amended soils with P. lilacinus  applied. The trials have shown that Mucuna  could be used as a short- term rotation/green manure crop in combination with early inoculation of tomato seedlings with effective AMF species and application of the bioformualted P. lilacinus  to manage root- knot disease on tomato in a more sustainable and eco- friendly way.
TABLE OF CONTENTS
Certification                                                                                                                           iii
Dedication                                                                                                                             iv
Acknowledgement                                                                                                                 v
Abstract                                                                                                                                 vii
Table of Contents                                                                                                                  ix
Introduction                                                                                                                           1
Literature Review                                                                                                                  4
Materials and Methods                                                                                                          13
Experiment site                                                                                                                      13
Source of Experimental Materials                                                                                          13
Building up of Nematode Population                                                                                    14
Nematode Inoculum Preparation                                                                                           14
Multiplication of Arbuscular Mycorrhizal Fungi Inoculum                                                  17
Inoculation of Tomato Seedlings with Arbuscular Mycorrhizal Fungus                              18
Collection of Soil Samples                                                                                                     18
Soil Extraction for Pre-planting population density of Nematodes                                      19
Soil Analysis to Determine Arbuscular Mycorrhizal Fungus (AMF) Spore Density 19
Soil Analysis for physical and chemical properties                                                               20
Experiment 1: Evaluation of the host status of Five Mucuna spp to Meloidogyne
 incognita inoculation                                                                                                             21
Experiment II: Effects of five species of Mucuna used as green manures in the
management of  M. incognita infecting tomato                                                                    22
Experiment III: Greenhouse Evaluation of the Effects of Mucuna spp Green Manure
Amendment and Arbuscular Mycorrhizal Fungi (AMF) on the Pathgogenicity of
M.incognita on tomato                                                                                                            24
Experiment IV: Field Evaluation of the Effects of Mucuna spp Green Manure and AMF
on the pathogenicity of M. incognita on Tomato                                                                   27
Experiment V: Evaluation of the Effects of Paecilomyces lilacinus and AMF
against M. incognita on Tomato                                                                                             29
Experiment VI: Evaluation of the Effects of P. lilacinus (PL GoldTM), Arbuscular
Mycorrhizal Fungi and Mucuna Green Manure on the Pathogenicity of M. incognita on
tomato                                                                                                                                    30
Statistical Analysis                                                                                                                  31
Results and Discussion                                                                                                           32
Physico-chemical properties, Arbuscular Mycorrhizal spore Density and Pre-plant
Nematode Density of the soils used for the Experiments                                                      32
Climate Data                                                                                                                           33
Experiment I: Evaluation of the Host Status of Five Mucuna spp to Meloidogyne
incognita inoculation                                                                                                                36
Experiment II: Effects of five species of Mucuna used as green manures in the
management of  M. incognita infecting tomato                                                                       40
Mineral contents and carbon-to-Nitrogen Ratios of the Different Mucuna Species               48
Experiment III: Greenhouse Evaluation of the Effects of Mucuna spp Green
Manure soil Amendment and Arbuscular Mycorrhizal Fungi on the Pathogenicity
of M. incognita on Tomato                                                                                                     50
Experiment IV: Field Evaluation of Effects of Mucuna spp Green Manure and AMF
on the Pathogenicity of M. incognita on Tomato                                                                   59
Experiment V: Evaluation of the Effects of Paecilomyces lilacinus and AMF
against M. incognita on Tomato                                                                                              70
Experiment VI: Evaluation of the Effects of P.lilacinus (PL GoldTM), Arbuscular
Mycorrhizal Fungi and Mucuna Green Manure on the Pathogenicity of M. incognita
on Tomato                                                                                                                               103
Discussion                                                                                                                               122
Summary, Conclusion and Recommendations                                                                       133
Summary                                                                                                                                 133
Conclusion                                                                                                                              136
Recommendations                                                                                                                  136
References                                                                                                                              138
CHAPTER ONE
INTRODUCTION
The commercial tomato (Solanum lycopersicum L.) belongs to the family Solanaceae and it is one of the most highly cherished fruit vegetables in Nigeria(Yayock et al.,1998). Tomato is ranked 15th among the world’s food crops (Vietmeyer, 1986). The total area under tomato production in tropical Africa is about 300,000ha with an estimated annual production of 2.3 million tonnes (Van der Vossen et al., 2004). Nigeria is the largest producer in Tropical Africa with 126,000ha yielding 879,000 tonnes of fresh fruits annually (FAO, 2004). Tomato fruit is very rich in vitamins A and C, providing between 20% and 40% of an adult’s requirements based on an average consumption of 100-125g of fresh fruits (Janes, 1994). It is also a good source of thiamine, riboflavin, niacin, potassium and sodium (Holland et al., 1991). The fruit can be eaten raw or cooked. Large quantities are used to produce soups, juices, sauces, ketchups, purees and pastes. The seeds extracted from the pulp contain 24% of a semi-drying edible oil (Yayock  et al., 1988).
            The production of tomato in the tropics is highly constrained by a vast array of pathogenic organisms including the plant parasitic nematodes. Over 60 species representing 19 genera of plant parasitic nematodes attack tomato and the root-knot nematode (Meloidogyne spp) is the most destructive (Valdez, 1979). The most widespread and devastating species are M. incognita, M. javanica  and  M. arenaria (IITA, 1992). M. incognita  and M. arenaria  are more common in southern Nigeria while M. javanica is more prevalent in northern Nigeria (Olowe, 2004). Root-knot nematode is an obligate sedentary endoparasite with visible symptoms of attack as root galls, early senescence, chlorosis, wilting, unthrifty growth, stunted appearance, fruit splitting, reduction in fruit number and size and general susceptibility to rot and wilt-inducing pathogens (Ogbuji, 1978, Sasser, 1980). Galled tomato roots are inefficient in nutrient and water uptake (Meon et al., 1978). Low photosynthetic rate has been attributed to poor carbon (iv) oxide assimilation, poor partitioning and translocation of photo assimilates in tomatoes attacked by M. incognita  and  M. javanica (Meon et al., 1978; Khan and Khan, 1987). Although accurate information on yield losses attributable to root-knot nematode in Nigeria is unavailable, conservative estimates indicated more than 50% losses depending on the cultivar, population density of the nematode species, cultural practices and environmental conditions (Olowe, 2005; Udo  et al., 2008).
Over the years, different control methods have been employed in the management of root-knot disease. Chemical control with synthetic nematicides has proved to be the most effective. However, it is uneconomic, has detrimental effects on beneficial non-target organisms, pollutes ground water, high mammalian toxicity, etc. Of late, emphasis is laid on environmentally sound approaches to pest management. The use of resistant crop cultivars is one of such approaches. In tomato, breeders have identified and incorporated the Mi resistance gene to commercial cultivars with applauded success in root-knot nematode control (Williamson, 1998; Sorribas et al., 2005). However, the emergence of virulent resistance-breaking pathotypes have been reported in some species of Meloidogyne (Tzortzakakis and Gowen, 1996; Castagnone-Sereno, 2002), thus constraining this method of control too. In recent times, there has been a worldwide swing to the use of eco-friendly methods for protecting crops from pests and diseases. The use of potential harmful chemical spray is viewed with contempt in many countries. The removal of some effective chemical nematicides (Methyl bromide, Ethyldibromide, Dibromochloropropane, etc.) from the pesticide market has spurred research on alternative management strategies of root-knot disease. The use of biological control agents, crop rotation with antagonistic crops and green manure/organic amendments of soils are some of the alternatives (Rodriguez-Kabana and Morgan-Jones, 1987; Queneherve  et al., 1998; Hashem and Abo-Elyousr, 2011).
Some fungi are nematophagous. Paecilomyces lilacinus parasitizes the egg of root-knot nematodes and has been reported by many researchers to reduce the damage caused by this pest on several crops (Jatala, 1979, Oclarit and Cumagun, 2009). Arbuscular mycorrhizal fungi (AMF) have been reported to be effective in reducing the malady caused by Meloidogyne spp on several crops too (Diederichs, 1987; Shreenivasa  et al., 2007; Odeyemi et al., 2010). Velvetbean (Mucuna spp) is a tropical leguminous plant used as a rotation, forage and green manure crop in many countries. Most findings have reported it to be effective in reducing root-knot nematode population in the soil and its associated damage on many crops when used as a rotation and/or green manure crop (McSorley and Dickson, 1995; Queneherve et al., 1998).
Despite the promise for economic control of root-knot nematode with biological entities and their combination, little work has been done in Nigeria. Considering the fact that the efficacy of green manure varies with species, rate of application and soil types, there is need to investigate native species of Mucuna and AMF and their combinations with established biocontrol agents under different soils in Nigeria for root-knot disease management. In addition, the variation in the results from combination of factors in root-knot nematode management calls for further investigation. On the bases of these considerations, the present study was initiated with the following objectives.
- To ascertain the host status of Mucuna species on Meloidogyne incognita
- To evaluate the effects of five species of Mucuna used as green manures in the management of incognita infecting tomato.
- To evaluate the efficacy of five species of arbuscular mycorrhizal fungus (AMF) against incognita in selected soil types of southeastern Nigeria.
- To evaluate the efficacy of bioformulated lilacinus against M. incognita in selected soil types of southeastern Nigeria.
- To evaluate the combined effects of bioformulated lilacinus, Mucuna green manure amendment and arbuscular mycorrhizal fungi in the management of root-knot disease on tomato.
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