Trichoderma species are free-living fungi that are common in soil and root ecosystems. Some strains establish root colonization and enhance growth and development, crop productivity, resistance to abiotic stresses and uptake and use of nutrients. Trichoderma species can antagonize and control a wide range of economically important plant pathogenic fungi, viruses, bacteria and nematodes. Root-knot nematodes, Meloidogyne spp., are sedentary, obligatory root endoparasites of great economic importance, and polyphagous species, such as M. javanica and M. incognita are among the major limiting factors of crops production worldwide. Therefore, these nematodes have been the main target for nematode biocontrol by Trichoderma. Several Trichoderma species and isolates have been evaluated as biocontrol agents against the nematodes with various crops and experimental conditions. Significant results of nematode control and plants growth were achieved. Aiming to improve the biocontrol process, modes of action of the fungus against the root-knot nematodes have been investigated and are described in this chapter. Mechanisms such as parasitism, enzymatic lysis, antibiosis and induced resistance were studied. Understanding the fungus-nematode-plant interactions and the mechanisms of the biocontrol process might contribute to improve the implementation of this biocontrol agent.

The outer surface of nematodes act as an external skeleton and is covered by a tough, but flexible, multi-layered, extracellular cuticle which protects them from the external environment, maintains body shape and is involved in locomotion and defence against their host or microorganism attack. This chapter highlights the role of the nematode surface cuticle, during the various life-stages, with their environment, including their host and other microorganism. A comprehensive appraisal is presented of the complex interactions between nematodes and microbial antagonists, as the surface cuticle is believed to be involved in the host-recognition events determining the specificity of such interactions.

Monoclonal and polyclonal antibodies that bind to eggs and/or second-stage juveniles of the nematode Meloidogyne javanica were tested for their effects on the parasitic interactions between this nematode and the fungus Trichoderma. Parasitism of Trichoderma asperellum-203 and Trichoderma atroviride on nematode egg masses, eggs and juveniles was enhanced when antibodies were incorporated into in vitro parasitism bioassays. Parasitism on separated eggs (without gelatinous matrix) and their hatched juveniles was also improved, compared to controls without antibodies that did not attach fungal conidia. Improved parasitism could be due to bilateral binding of the antibodies to the nematodes and conidia, enabling better conidial attachment to the nematodes. Enhanced germination of antibody-bound conidia further improved parasitism. Differences were observed among antibodies in their effects on fungal parasitism and their interaction with Trichoderma species. We focused mainly on the egg- and juvenile-binding monoclonal antibody MISC that exhibited a stronger reaction with T. asperellum-203 than with T. atroviride. Pretreatment of this antibody with fucose inhibited its binding to nematodes and conidial attachment to nematodes, as well as conidial agglutination in the presence of the antibody. Antibody binding to juveniles affected their movement and viability, especially gelatinous matrix-originated juveniles. The fucose-specific lectin Ulex europaeus-I enhanced conidial attachment to nematode life-stages, and conidial agglutination occurred in its presence. These phenomena were inhibited by preincubating lectin with fucose. Our results suggest that carbohydrate residues, such as fucose, on the surface of the nematode and fungal conidia are involved in the antibody- and lectin-mediated improved parasitism.

The predatory nematode Koerneria sudhausi reproduced on bacteria that developed spontaneously on a nutrient medium in Petri dishes. The nematode population had a female/male ratio of 2.9:1, but female nematodes could reproduce asexually on the bacteria. Each female produced about 60 progeny at 25 degrees C in 9 days without mating. The generation time (egg to egg) at the same temperature was 8 days. Koerneria sudhausi reproduced at 17 degrees C at the same rate as at 25 degrees C, if the bacteria were available as a food source. When the nematode was introduced to a monoxenic culture of Meloidogyne javanica on tomato roots on agar, the nematode fed on second-stage juveniles and eggs. The nematodes introduced to M. javanica-inoculated soil before transplanting tomato seedlings reduced the root galling index in pot experiments. The results suggested that this predatory nematode, K. sudhausi, may serve as a candidate for a biological control agent against root-knot nematodes, because the nematode can be easily cultured on bacteria and achieve a high reproduction rate.

The predatory nematode Koerneria sudhausi reproduced on bacteria that developed spontaneously on a nutrient medium in Petri dishes. The nematode population had a female/male ratio of 2.9 : 1, but female nematodes could reproduce asexually on the bacteria. Each female produced about 60 progeny at 25°C in 9 days without mating. The generation time (egg to egg) at the same temperature was 8 days. Koerneria sudhausi reproduced at 17°C at the same rate as at 25°C, if the bacteria were available as a food source. When the nematode was introduced to a monoxenic culture of Meloidogyne javanica on tomato roots on agar, the nematode fed on second-stage juveniles and eggs. The nematodes introduced to M. javanica -inoculated soil before transplanting tomato seedlings reduced the root galling index in pot experiments. The results suggested that this predatory nematode, K. sudhausi , may serve as a candidate for a biological control agent against root-knot nematodes, because the nematode can be easily cultured on bacteria and achieve a high reproduction rate.

Trichoderma (T. asperellum-203, 44 and GH11; T. atroviride-IMI 206040 and T. harzianum-248) parasitism on Meloidogyne javanica life stages was examined in vitro. Conidium attachment and parasitism differed beween the fungi. Egg masses, their derived eggs and second-stage juveniles (J2) were parasitized by Trichoderma asperellum-203, 44, and T. atroviride following conidium attachment. Trichoderma asperellum-GH11 attached to the nematodes but exhibited reduced penetration, whereas growth of T. harzianum-248 attached to egg masses was inhibited. Only a few conidia of the different fungi were attached to eggs and J2s without gelatinous matrix; the eggs were penetrated and parasitized by few hyphae, while J2s were rarely parasitized by the fungi. The gelatinous matrix specifically induced J2 immobilization by T. asperellum-203, 44 and T. atroviride metabolites that immobilized the J2s. A constitutive-GFP-expressing T. asperellum-203 construct was used to visualize fungal penetration of the nematodes. Scanning electron microscopy revealed the formation of coiling and appressorium-like structures upon attachment and parasitism by T. asperellum-203 and T. atroviride. Gelatinous matrix agglutinated T. asperellum-203 and T. atroviride conidia, a process that was Ca2+-dependent. Conidium agglutination was inhibited by carbohydrates, including fucose, as was conidium attachment to the nematodes. All but T. harzianum could grow on the gelatinous matrix, which enhanced conidium germination. A biomimetic system based on gelatinous-matrix-coated nylon fibers demonstrated the role of the matrix in parasitism: T. asperellum-203 and T. atroviride conidia attached specifically to the gelatinous-matrix-coated fibers and parasitic growth patterns, such as coiling, branching and appressoria-like structures, were induced in both fungi, similarly to those observed during nematode parasitism. All Trichoderma isolates exhibited nematode biocontrol activity in pot experiments with tomato plants. Parasitic interactions were demonstrated in planta: females and egg masses dissected from tomato roots grown in T. asperellum-203-treated soil were examined and found to be parasitized by the fungus. This study demonstrates biocontrol activities of Trichoderma isolates and their parasitic capabilities on M. javanica, elucidating the importance of the gelatinous matrix in the fungal parasitism.

Organic amendments and green manure are potential alternatives to the harmful chemical control means currently used against plant-parasitic nematodes. In this work, Chrysanthemum coronarium was applied to the soil as a green manure to control the root-knot nematodes Meloidogyne incognita and M. javanica. Chrysanthemum coronarium significantly reduced nematode infection of tomato roots and improved plant-top fresh weight, both in the greenhouse and in microplots. Other green manures, derived from Anthemis pseudocotula, wild chickpea (Cicer pinnatifidum), Geranium spp. and wheat, were not as effective as C. coronarium. Chrysanthemum coronarium, retained its nematicidal activity even when applied as a dried material. Only mature C. coronarium plants, in their flowering stage, exhibited nematode control activity, but the green plant parts were more effective than the flowers. An aqueous extract of C. coronarium exhibited in vitro, nematostatic activity towards M. incognita and M. javanica second-stage juveniles and inhibited their hatching from eggs and egg-masses; its nematostatic activity was expressed also against other phytonematode species such as Heterodera avenae and Pratylenchus mediterraneus, but did not affect the beneficial entomopathogenic nematode Steinernema feltiae.

The surface coat of the 2nd-stage juveniles (J2) of plant-parasitic nematodes is considered to be involved in interactions with microorganisms in the soil and rhizosphere, as well as with the host plant. Characterization of surface antigens might be important in the development of new nematode control strategies. In this study, polyclonal and monoclonal antibodies raised against Meloidogyne javanica, M. incognita and other plant-parasitic nematodes were tested for their binding to the surface coat and secreted-excreted products of M. javanica. Some of the monoclonal and polyclonal antibodies raised against M. incognita showed cross-reactivity with the surface coat of M. javanica. Western blot analysis of M. javanica surface coat extracts revealed labelling of several polypeptides with a 48 kDa main band for the polyclonal antibody IACR-PC Mi 373, and a 55 kDa main band for PC Mj E2. Further characterization of the antigens recognized by the polyclonal antibody PC Mj E2, in planta, showed that they were present in the parasitic stages J2 and J3 and that the surface coat was shed during root penetration. The hypodermis of the infective juveniles was labelled by PC Mj E2 and the monoclonal antibody IACR-Misec 3F.4, suggesting that these surface antigens are produced in the hypodermis. Nematode behaviour was affected by all the antibodies that bound to the surface coat of the pre-parasitic J2, and we demonstrated that the movement pattern of the M. javanica J2 was affected by these antibodies. Continuous binding of the antibodies to the M. javanica surface inhibited the infection of Arabidopsis thaliana roots on agar plates.

ABSTRACT The fungal biocontrol agent, Trichoderma harzianum, was evaluated for its potential to control the root-knot nematode Meloidogyne javanica. In greenhouse experiments, root galling was reduced and top fresh weight increased in nematode-infected tomatoes following soil pretreatment with Trichoderma peat-bran preparations. The use of a proteinase Prb1-transformed line (P-2) that contains multiple copies of this gene improved biocontrol activity in the greenhouse experiments compared with the nontransformed wild-type strain (WT). All the Trichoderma strains showed the ability to colonize M. javanica-separated eggs and second-stage juveniles (J2) in sterile in vitro assays, whereas P-2 also penetrated the egg masses. This protease-transformed line presented the same nematicidal and overall proteolytic activity as the WT in in vitro tests in which concentrated soil extracts from Trichoderma-treated soils immobilized the infective J2. However, the J2 immobilization and proteolytic activities of both P-2 and the WT were higher than those obtained with strain T-203. Characterization of the activity of all Trichoderma strains soil extracts on J2 showed that it was heat resistant and restricted to the low-molecular-weight fraction (less than 3 kDa). It is suggested that improved proteolytic activity of the antagonist may be important for the biological control of the nematodes.

New nematode control measures are required due to the likely removal of effective nematicides from the market because of increasing concerns of possible effects on human health and the environment. Biological control, organic and inorganic soil amendments, naturally occurring nematicides, induced resistance, interruption of host recognition and transgenic plants will be a part of integrated management of plant-parasitic nematodes in the near future. The paper reports work carried out in Israel on these topics. (C) 2000 Society of Chemical Industry.

Nematode control methods without using nematicides have been studied. Biological control by nematode antagonists, use of soil amendments to build up antagonistic microorganisms and release toxic compounds to nematodes, detection of naturally occurring nematicides in plants, interruption of nematode's host-recognition process and study of nematode collagen genes are described here.