Biological control potential of plant growth‐promoting rhizobacteria suppression of Meloidogyne incognita on cotton and Heterodera glycines on soybean: A review

Over the past decade, we have seen an increasing market for biopesticides and an increase in number of microbial control studies directed towards plant‐parasitic nematodes. This literature survey provides an overview of research on biological control of two economically important plant‐parasitic nematodes, Meloidogyne incognita (Kofoid & White) Chitwood (southern root‐knot nematode) and Heterodera glycines Ichinohe (soybean cyst nematode) using spore‐forming plant growth‐promoting rhizobacteria (PGPR). In this review, the current biological control strategies for the management of those cotton and soybean nematodes, the mechanism of using BacillusPGPR for biological control of plant‐parasitic nematode including induced systemic resistance and antagonism and the future of biological control agents on management of plant‐parasitic nematodes are covered.     

Extracellular enzymes and the pathogenesis of nematophagous fungi

Nematophagous fungi are an important group of soil microorganisms that can suppress the populations of plant-parasitic nematodes. The pathogenic mechanisms of nematophagous fungi are diverse: They can be parasitical–mechanical through producing specialized capturing devices, or toxin-dependent. During infections, a variety of virulence factors may be involved against nematodes by nematophagous fungi. In this review, we present up-to-date information on the modes of infection by nematophagous fungi. The roles of extracellular hydrolytic enzymes and other virulence factors involved in infection against nematodes were summarized. The biochemical properties and peptide sequences of a special group of enzymes, the serine proteases, were compared, and their implications in infections were discussed. We also discussed the impact of emerging new techniques on our understanding of this unique group of fungi.     

食线虫菌物胞外蛋白酶基因工程研究进展

在病原线虫的生物防治中 ,利用食线虫菌物在侵染过程中分泌的胞外蛋白酶 (重要毒力因子 )固定线虫并降解线虫体壁显示出巨大的潜力。综述了近年来食线虫菌物胞外蛋白酶的研究概况、目前开展蛋白酶基因工程研究存在的问题及解决策略;对真菌胞外蛋白酶应用于线虫生防和生物医药领域中的前景也进行了评述。     

Toward Practical Biological Control of Parasitic Nematodes in Domestic Animals

In a series of laboratory and field experiments where the nematophagous fungus Arthrobotrys oligospora was mixed directly with feces it has been demonstrated that it is possible to use nematophagous fungi for biological control of animal parasitic nematodes. A procedure used for selection of nematophagous fungi that can pass the digestive tract of ruminants, horses, and pigs is described. The selected fungus, Duddingtonia flagrans, has been used in further field experiments, and the results have confirmed that by the addition of D. flagrans to feed supplement it is possible to reduce the parasitic burden significantly.     

Oesophagostomum dentatum: potential as a model for genomic studies of strongylid nematodes, with biotechnological prospects

There are substantial gaps in the knowledge of the molecular processes of development and reproduction in parasitic nematodes, despite the fact that understanding such processes could lead to novel ways of treating and controlling parasitic diseases, through blocking or disrupting key biological pathways. Biotechnological advances through large-scale sequencing projects, approaches for the analysis of differential gene and protein expression and functional genomics (e.g., double-stranded RNA interference) now provide opportunities to investigate the molecular basis of developmental processes in some parasitic nematodes. The porcine nodule worm, Oesophagostomum dentatum (order Strongylida), may provide a platform for testing the function of genes from this and related nematodes, given that this species can be grown and maintained in culture in vitro for periods longer than other nematodes of the same order. In this article, we review relevant biological, biochemical and molecular biological and genomic information about O. dentatum and propose that the O. dentatum - pig system provides an attractive model for exploring molecular developmental and reproductive processes in strongylid nematodes, leading toward new intervention methods and biotechnological outcomes.     

Insect-parasitic nematodes: from lab curiosities to model organisms

Interest in studying insect-parasitic nematodes was originally focused on their potential as biological control agents of insects and other arthropod pests. Now, after 30 years of intense basic and applied research, realization of the practical use of insect-parasitic nematodes, particularly of entomopathogenic nematodes and their symbiotic bacteria, has spurred developments across a far broader scientific front. We are now entering a new era of discovery in which tools of molecular genetics are being increasingly used to address a range of biological questions. The knowledge gained from these efforts will directly benefit the practical application of insect-parasitic nematodes as more effective biopesticides. Moreover, these studies will advance these nematodes as unique and intrinsically interesting biological model systems not only for basic research but also in applied fields such as plant health, human medicine, pharmaceutical bioprospecting, and genetic engineering. In this review, the past and current state of insect-parasitic nematode research is summarized. Future research priorities and goals are identified and discussed.     

The living strategy of nematophagous fungi

The infection structures, trophism, and ecological character of nematophagous fungi are reviewed in this article on the basis of data extracted from the literature and the most recent experiments conducted in this area. Traditionally, nematophagous fungi are classified into four groups according to their modes of attacking nematodes: nematode-trapping fungi using adhesive or mechanical hyphal traps, endoparasitic fungi using their spores, eggparasitic fungi invading nematode eggs or females with their hyphal tips, and toxin-producing fungi immobilizing nematodes before invasion. In the present review, we focus on the first two groups. The living strategies of these nematophagous fungi depend on the diversity of their infection structures, such as different traps and spore types, which determine the modes of infecting nematodes. The diversity of trophic modes of nematophagous fungi is an important prerequisite for fungal survival and activity in soil. The abundance and activity of Hirsutella rhossiliensis and H. minnesotensis, representatives of endoparasites and potential biocontrol agents against nematodes, are highly dependent on environmental factors. Comprehensive understanding of the survival and activity of nematophagous fungi in soil is fundamental for the exploitation of these fungi as successful biocontrol agents.     

Genetic diversity and recombination in natural populations of the nematode‐trapping fungus Arthrobotrys oligospora from China

Nematophagous fungi can trap and capture nematodes and other small invertebrates. This unique ability has made them ideal organisms from which to develop biological control agents against plant‐ and animal‐parasitic nematodes. However, effective application of biocontrol agents in the field requires a comprehensive understanding about the ecology and population genetics of the nematophagous fungi in natural environments. Here, we genotyped 228 strains of the nematode‐trapping fungus Arthrobotrys oligospora using 12 single nucleotide polymorphic markers located on eight random DNA fragments. The strains were from different ecological niches and geographical regions from China. Our analyses identified that ecological niche separations contributed significantly, whereas geographic separation contributed relatively little to the overall genetic variation in our samples of A. oligospora. Interestingly, populations from stressful environments seemed to be more variable and showed more evidence for recombination than those from benign environments at the same geographic areas. We discussed the implications of our results to the conservation and biocontrol application of A. oligospora in agriculture and forestry.     

The rhizosphere: a playground and battlefield for soilborne pathogens and beneficial microorganisms

The rhizosphere is a hot spot of microbial interactions as exudates released by plant roots are a main food source for microorganisms and a driving force of their population density and activities. The rhizosphere harbors many organisms that have a neutral effect on the plant, but also attracts organisms that exert deleterious or beneficial effects on the plant. Microorganisms that adversely affect plant growth and health are the pathogenic fungi, oomycetes, bacteria and nematodes. Most of the soilborne pathogens are adapted to grow and survive in the bulk soil, but the rhizosphere is the playground and infection court where the pathogen establishes a parasitic relationship with the plant. The rhizosphere is also a battlefield where the complex rhizosphere community, both microflora and microfauna, interact with pathogens and influence the outcome of pathogen infection. A wide range of microorganisms are beneficial to the plant and include nitrogen-fixing bacteria, endo- and ectomycorrhizal fungi, and plant growth-promoting bacteria and fungi. This review focuses on the population dynamics and activity of soilborne pathogens and beneficial microorganisms. Specific attention is given to mechanisms involved in the tripartite interactions between beneficial microorganisms, pathogens and the plant. We also discuss how agricultural practices affect pathogen and antagonist populations and how these practices can be adopted to promote plant growth and health.     

Fungal chitinases and their biological role in the antagonism onto nematode eggs. A review

Chitin, the most abundant aminopolysaccharide in nature, is a rigid and resistant structural component that contributes to the mechanical strength of chitin-containing organisms. Chemically, it is a linear cationic heteropolysaccharide composed of N-acetyl-D-glucosamine and D-glucosamine units. The enzymatic degradation of chitin is performed by a chitinolytic system with synergistic and consecutive action. Diverse organisms (containing chitin or not) produce a great variety of chitinolytic enzymes with different specificities and catalytic properties. Their physiological roles involve nutrition, parasitism, chitin recycling, morphogenesis, and/or defense. Microorganisms, as the main environmental chitin degraders, constitute a very important natural source of chitinolytic enzymes. Nowadays, the most used method for pest and plant diseases control is the utilization of chemical agents, causative of significant environmental pollution. Social concern has generated the search for alternative control systems (i.e., biological control), which contribute to the generation of sustainable agricultural development. Interactions among the different organisms are the natural bases of biological control. Interest in chitinolytic enzymes in the field of biological control has arisen due to their possible involvement in antagonistic activity against pathogenic chitin-containing organisms. The absence of chitin in plants and vertebrate animals allows the consideration of safe and selective “target” molecules for control of chitin-containing pathogenic organisms. Fungi show appropriate characteristics as potential biological control agents of insects, fungi, and nematodes due to the production of fungal enzymes with antagonistic action. The antagonistic interactions between fungi and plant nematode parasites are among the most studied experimental models because of the high economic relevance. Fungi which target nematodes are known as nematophagous fungi. The nematode egg is the only structural element where the presence of chitin has been demonstrated. In spite of being one of the most resistant biological structures, eggs are susceptible to being attacked by egg-parasitic fungi. A combination of physical and chemical phenomena result in their complete destruction. The contribution of fungal chitinases to the in vitro rupture of the eggshell confirms their role as a pathogenic factor. Chitinases have been produced by traditional fermentation methods, which have been improved by optimizing the culture conditions for industrial processes. Although wild-type microorganisms constitute an alternative source of chitinolytic enzymes, the advances in molecular biology are allowing the genetic transformation of fungi to obtain strains with high capability as biocontrol agents. Simultaneously, a better understanding of rhizosphere interactions, additional to the discovery of new molecular biology tools, will allow the choosing of better alternatives for the biological control of nematodes in order to achieve an integrated management of the soil ecosystem.     

Attachment tests of Pasteuria penetrans to the cuticle of plant and animal parasitic nematodes, free living nematodes and srf mutants of Caenorhabditis elegans.

Populations of Pasteuria penetrans isolated from root-knot nematodes (Meloidogyne spp.) and cyst nematodes (Heterodera spp.) were tested for their ability to adhere to a limited selection of sheathed and ex-sheathed animal parasitic nematodes, free living nematodes, including Caenorhabditis elegans wild type and several srf mutants, and plant parasitic nematodes. The attachment of spores of Pasteuria was restricted and no spores were observed adhering to any of the animal parasitic nematodes either with or without their sheath or to any of the free living nematodes including C. elegans and the srf mutants. All spore attachment was restricted to plant parasitic nematodes; however, spores isolated from cyst nematodes showed the ability to adhere to other genera of plant parasitic nematodes which was not the case with spores isolated from root-knot nematodes. The results are discussed in relationship to cuticular heterogeneity.     

A family of glycosyl hydrolase family 45 cellulases from the pine wood nematode Bursaphelenchus xylophilus

We have characterized a family of GHF45 cellulases from the pine wood nematode Bursaphelenchus xylophilus. The absence of such genes from other nematodes and their similarity to fungal genes suggests that they may have been acquired by horizontal gene transfer (HGT) from fungi. The cell wall degrading enzymes of other plant parasitic nematodes may have been acquired by HGT from bacteria. B. xylophilus is not directly related to other plant parasites and our data therefore suggest that horizontal transfer of cell wall degrading enzymes has played a key role in evolution of plant parasitism by nematodes on more than one occasion.     

Impact of the nematophagous fungus Pochonia chlamydosporia on nematode and microbial populations

The microbial and nematode populations associated with two plants (tomato and cabbage) inoculated with the nematophagous fungus, Pochonia chlamydosporia var. chlamydosporia or root knot nematode (Meloidogyne incognita), or both, were compared with those in unplanted controls. The dominant factor affecting culturable microbial populations was found to be the presence or absence of tomato plants. Generally microbial colony counts were lowest in unplanted soil, small increases were associated with cabbage and significantly greater numbers with tomato plants. Differences in microbial diversity (estimated from community profiles of carbon substrate utlisation, using Biolog) were observed between planted and unplanted soils, however, there were few differences between soils with either of the two plants. The presence of P. chlamydosporia was associated with a reduction in the numbers of plant parasitic nematodes (51%-78%) including the migratory ectoparasites, whereas free-living nematodes, culturable bacteria and bacterial populations assessed by Biolog were unaffected by the application of fungus.     

Rhizobacteria with nematicide aptitude: enzymes and compounds associated

The use of rhizobacteria to control plant parasitic nematodes has been widely studied. Currently, the research focuses on bacteria-nematode interactions that can mitigate this complex microbiome in agriculture. Various enzymes, toxins and metabolic by-products from rhizobacteria antagonize plant parasitic nematodes, and many different modes of action have been proposed. Hydrolytic enzymes, primarily proteases, collagenases and chitinases, have been related to the nematicide effect in rhizobacteria, proving to be an important factor involved in the degradation of different chemical constituents of nematodes at distinct developmental stages. Exuded metabolites may also alter the nematode-plant recognition process or create a hostile environment for nematodes in the rhizosphere. Specific bacteria strains responsible for the production of toxins, such as Cry proteins, are one of the strategies used by rhizobacteria. Characterization of the rhizobacteria mode of action could strengthen the development of commercial products to control populations of plant parasitic nematodes. This review aims to provide an overview of different enzymes and compounds produced by rhizobacteria related to the process of antagonism to plant-parasitic nematodes.     

Effect of chitosan on hyphal growth and spore germination of plant pathogenic and biocontrol fungi

Aims: To investigate the toxic effect of chitosan on important root pathogenic and biocontrol fungi (nematophagous, entomopathogenic and mycoparasitic). Methods and Results: We have used standard bioassays to investigate the effect of chitosan on colony growth and developed bioassays to test spore germination. The results showed that the root pathogenic and mycoparasitic fungi tested were more sensitive to chitosan than nematophagous and entomopathogenic fungi. Chitosanases (and perhaps related enzymes) are involved in the resistance to chitosan. Two fungi, one sensitive to chitosan, Fusarium oxysporum f. sp. radicis‐lycopersici, and one less sensitive, Pochonia chlamydosporia, were selected for ultrastructural investigations. Transmission electron microscopy revealed differences in the ultrastructural alterations caused by chitosan in the spores of the plant pathogenic fungus and in those of the nematophagous fungus. Confocal laser microscopy showed that Rhodamine‐labelled chitosan enters rapidly into conidia of both fungi, in an energy‐dependent process. Conclusions: Nematophagous and entomopathogenic fungi are rather resistant to the toxic effect of chitosan. Resistance of nematophagous and entomopathogenic fungi to chitosan could be associated with their high extracellular chitosanolytic activity. Furthermore, ultrastructural damage is much more severe in the chitosan sensitive fungus. Significance and impact of the study: The results of this paper suggest that biocontrol fungi tested could be combined with chitosan for biological control of plant pathogens and pests.     

Insect immune responses to nematode parasites

Host innate immunity plays a central role in detecting and eliminating microbial pathogenic infections in both vertebrate and invertebrate animals. Entomopathogenic or insect pathogenic nematodes are of particular importance for the control of insect pests and vectors of pathogens, while insect-borne nematodes cause serious diseases in humans. Recent work has begun to use the power of insect models to investigate host-nematode interactions and uncover host antiparasitic immune reactions. This review describes recent findings on innate immune evasion strategies of parasitic nematodes and host cellular and humoral responses to the infection. Such information can be used to model diseases caused by human parasitic nematodes and provide clues indicating directions for research into the interplay between vector insects and their invading tropical parasites.     

Manipulation of Auxin Transport in Plant Roots during Rhizobium Symbiosis and Nematode Parasitism

The plant rhizosphere harbors many different microorganisms, ranging from plant growth–promoting bacteria to devastating plant parasites. Some of these microbes are able to induce de novo organ formation in infected roots. Certain soil bacteria, collectively called rhizobia, form a symbiotic interaction with legumes, leading to the formation of nitrogen-fixing root nodules. Sedentary endoparasitic nematodes, on the other hand, induce highly specialized feeding sites in infected plant roots from which they withdraw nutrients. In order to establish these new root structures, it is thought that these organisms use and manipulate the endogenous molecular and physiological pathways of their hosts. Over the years, evidence has accumulated reliably demonstrating the involvement of the plant hormone auxin. Moreover, the auxin responses during microbe-induced de novo organ formation seem to be dynamic, suggesting that plant-associated microbes can actively modify their host''s auxin transport. In this review, we focus on recent findings in auxin transport mechanisms during plant development and on how plant symbionts and parasites have evolved to manipulate these mechanisms for their own purposes.     

Thioredoxin1 regulates conidia formation,hyphal growth,and trap formation in the nematode-trapping fungus Arthrobotrys oligospora

Arthrobotrys oligospora, a model nematophagous fungus that produces specific adhesive networks to capture nematodes, has been proposed as a potentially effective biological agent to control harmful plant-parasitic nematodes. Although thioredoxin has been characterized as playing important roles in many cellular processes in other species, its function in nematophagous fungi has not been studied. Here, the function of a thioredoxin homolog, Aotrx1, was investigated in A. oligospora. The encoding gene of Aotrx1 in the nematophagous fungus A. oligospora was knocked out by homologous recombination; strain growth was assessed. The ΔAotrx1 strain of A. oligospora showed a significant decrease in growth rate on different media (PDA, CMY, and TG), a 70% decrease of conidia production, and a lower germination rate compared with the wild type. The mutant strain was unable to form traps to capture nematodes and was more sensitive to SDS and H2O2. Thioredoxin is involved in conidia development, trap formation, normal mycelial growth, and resistance to environmental stresses in the nematode-trapping fungus A. oligospora.     

Phenotypic and genetic characterization of Paecilomyces lilacinus strains with biocontrol activity against root-knot nematodes

Efficient selection of fungi for biological control of nematodes requires a series of screening assays. Assessment of genetic diversity in the candidate species maximizes the variety of the isolates tested and permits the assignment of a particular genotype with high nematophagous potential using a rapid novel assay. Molecular analyses also facilitate separation between isolates, allowing the identification of proprietary strains and trace biocontrol strains in the environment. The resistance of propagules to UV radiation is an important factor in the survival of a biocontrol agent. We have analyzed 15 strains of the nematophagous fungus Paecilomyces lilacinus using these principles. Arbitrarily primed DNA and allozyme assays were applied to place the isolates into genetic clusters, and demonstrated that some genetically related P. lilacinus strains exhibit widespread geographic distributions. When exposed to UV radiation, some weakly nematophagous strains were generally more susceptible than effective isolates. A microtitre tray-based assay used to screen the pathogenic activity of each isolate to Meloidogyne javanica egg masses revealed that the nematophagous ability varied between 37%-100%. However, there was no clear relationship between nematophagous ability and genetic clusters. Molecular characterizations revealed sufficient diversity to allow tracking of strains released into the environment.     

Effects of Interactions of Auxin-Producing Bacteria and Bacterial-Feeding Nematodes on Regulation of Peanut Growths

The influences of an IAA (indole-3-acetic acid)-producing bacterium (Bacillus megaterium) and two bacterial-feeding nematodes (Cephalobus sp. or Mesorhabditis sp.) on the growth of peanut (Arachis hypogaea L. cv. Haihua 1) after various durations of time were investigated in natural soils. The addition of bacteria and nematodes and incubation time all significantly affected plant growth, plant root growth, plant nutrient concentrations, soil nutrient concentrations, soil microorganisms and soil auxin concentration. The addition of nematodes caused greater increases in these indices than those of bacteria, while the addition of the combination of bacteria and nematodes caused further increases. After 42-day growth, the increases in soil respiration differed between the additions of two kinds of nematodes because of differences in their life strategies. The effects of the bacteria and nematodes on the nutrient and hormone concentrations were responsible for the increases in plant growth. These results indicate the potential for promoting plant growth via the addition of nematodes and bacteria to soil.