Nematophagous fungi of Toxocara canis eggs in a public park of La Plata, Argentina

Fungi have showed a great potential for the biological control of nematodes. However, they have not been evaluated for the control of animal and/or human parasites transmitted by egg contaminated soils. Environmental contamination with Toxocara spp. eggs is a public health problem. Accidental swallowing of Toxocara canis eggs (a nematode of dogs) usually results on a zoonotic infection (toxocarosis). The objectives of this research were: 1) To test the presence of antagonistic fungi against T. canis in the soil in public places of La Plata city, Argentina, infected with eggs of this parasite, 2) To determine the possible association between biotic and abiotic factors of the soil with the presence of fungal parasites of egg nematodes. Soil samples were tested for: textural type, organic matter (%), pH, presence of egg-parasite fungi, of larvae and of nematode eggs, in particular of Toxocara spp. The studied area showed the following characteristics: pH: 6.6-8.0, organic matter: 1.2-70%, with a predominantly loam texture. The following antagonistic fungal genera were identified: Acremonium, Aspergillus, Chrysosporium, Fusarium, Humicola, Mortierella, Paecilomyces and Penicillium. A prevalence of 70% was detected for nematode eggs, of 33% for Toxocara spp. eggs and of 90% for larvae. No association between the presence of egg-parasite fungi and the considered factors was found. More studies are necessary to know the natural antagonism factors to T. canis eggs for its in situ biological control.     

Mycophagous mites and their internal associated bacteria cooperate to digest chitin in soil

The greater bulk of soil nitrogen is immobilized in chitinous cell walls of fungi. Mycophagous soil mites participate in chitin decomposition and, hence, in the subsequent mobilization of nitrogen. The source of the chitinolytic enzymes was searched in this study. A multimethodical approach was designed for these studies. Histology, plating and identification of bacteria from mite homogenate and, finally, homogenate and bacterial treatment of the soil fungi were applied. Here the presence and activity of chitinolytic bacteria inside mycophagous mites are reported. These bacteria form an extraintestinal group within the mite’s body and pass their enzymes into the mite’s gut. Our results demonstrate that true mycophagous mites, defined by their ability to digest chitin (i.e. the fungal cell wall), achieve this through internal “cooperation” with chitinolytic bacteria that provide the necessary chitinolytic enzymes. The nitrogen from chitin is thus made available to other soil organisms and plants.     

Chitinases of filamentous fungi: a large group of diverse proteins with multiple physiological functions

Chitin is the second most abundant natural biopolymer and the main structural component of invertebrate exoskeletons and cell walls of filamentous fungi. Fungal chitinases have multiple physiological functions including the degradation of exogenous chitin and cell wall remodelling during hyphal growth, but the regulation of the chitinolytic systems of filamentous fungi is not well understood. Fungi have on average between 10 and 25 different chitinases, but only the increasing number of fungal genome sequencing projects in the last few years has enabled us to assess the whole range and diversity of fungal chitinases. In this review the variety, domain architecture and subgroups of chitinases of filamentous fungi are shown, and how these data integrate with that from molecular biological studies on chitinases are discussed.     

Extracellular Chitinases of Fluorescent Pseudomonads Antifungal to Fusarium oxysporum f. sp. dianthi Causing Carnation Wilt

Vascular wilt of carnation caused by Fusarium oxysporum f. sp. dianthi (Prill. & Delacr.) W. C. Synder & H.N. Hans inflicts substantial yield and quality loss to the crop. Mycolytic enzymes such as chitinases are antifungal and contribute significantly to the antagonistic activity of fluorescent pseudomonads belonging to plant-growth-promoting rhizobacteria. Fluorescent pseudomonads antagonistic to the vascular wilt pathogen were studied for their ability to grow and produce chitinases on different substrates. Bacterial cells grown on chitin-containing media showed enhanced growth and enzyme production with increased anti-fungal activity against the pathogen. Furthermore, the cell-free bacterial culture filtrate from chitin-containing media also significantly inhibited the mycelial growth. Both the strains and their cell-free culture filtrate from chitin-amended media showed the formation of lytic zones on chitin agar, indicating chitinolytic ability. Extracellular proteins of highly antagonistic bacterial strain were isolated from cell-free extracts of media amended with chitin and fungal cell wall. These cell-free conditioned media contained one to seven polypeptides. Western blot analysis revealed two isoforms of chitinase with molecular masses of 43 and 18.5 kDa. Further plate assay for mycelial growth inhibition showed the 43-kDa protein to be antifungal. The foregoing studies clearly established the significance of chitinases in the antagonism of fluorescent pseudomonads, showing avenues for possible exploitation in carnation wilt management.     

Biocontrol: Fungi as Nematode Control Agents

The fungal antagonists of nematodes consist of a great variety of organisms belonging to widely divergent orders and families of fungi. They include the nematode-trapping fungi, endoparasitic fungi, parasites of nematode eggs and cysts, and fungi which produce metabolites toxic to nematodes. The diversity, adaptations, and distribution of nematode-destroying fungi and taxonomic problems encountered in their study are reviewed. The importance of nemato-phagous fungi in soil biology, with special emphasis on their relationship to populations of plant-parasitic nematodes, is considered. While predacious fungi have long been investigated as possible biocontrol agents and have often exhibited spectacular results in vitro, their performance in field studies has generated little enthusiasm among nematologists. To date no species has demonstrated control of any plant pest to a degree achieved with nematicides, but recent studies have provided a much clearer concept of possibilities and problems in the applied use of fungal antagonists. The discovery of new species, which appear to control certain pests effectively under specific conditions, holds out some promise that fungi may be utilized as alternatives to chemical control after a more thorough and expanded study of their biology and ecology.     

Mutual relationships between soils and biological carrier systems

Improved viability and antagonistic activity of biocontrol agents during soil inoculation is of crucial importance to their effective application. The chitinolytic bacterium Serratia marcescens was used as a model organism to study the efficacy of freeze-dried alginate beads (in comparison to their non-dried counterparts) as possible carriers for immobilized biocontrol agents. The release of bacteria and chitinolytic enzyme from alginate beads, before and during their application in soil, was examined, and the beads' physical properties characterized. Dispersal of the alginate bead-entrapped S. marcescens in the soil resulted in high soil cell densities throughout the 35 days of the experiment. Chitin inclusion in the beads resulted in significantly higher chitinolytic activity of S. marcescens, increased dry-bead porosity and decreased stiffness. Rehydration of the dried beads (after immersion in soil) resulted in a sixfold increase in weight due to water absorption. No significant differences were found in bacterial count inside the non-dried (gel) versus dried beads. However, higher cell densities and chitinase activity were detected in soil containing dried beads with chitin than in that containing their non-dried counterparts. The biological performance of S. marcescens was examined in the greenhouse: a free cell suspension reduced bean (Phaseolus vulgaris L.) disease by 10%, while immobilized bacteria found in the dried, chitin-containing beads reduced disease by 60%.     

Potential for Nematode Control by Mycofloras Endemic in the Tropics

Results of mycological surveys of root-knot and cyst nematodes from tropical regions indicate that most fungal species associated with females or cysts of species of Globodera, Heterodera, and Meloidogyne are those found with nematodes from temperate areas. Some fungal species, however, were found in higher frequency in tropical regions than in temperate countries; e.g., Cylindrocarpon destructans and Ulocladium atrum were the most common species associated with G. pallida and G. rostochiensis cysts in Peru. These fungi are not so frequent in nematodes from temperate areas. Fungi associated with diseased nematodes in the tropics vary greatly in nutritional requirements and include thermophilic species as well as cold-tolerant fungi. Multi-cropping systems possible in most tropical regions may be designed to increase the frequency of occurrence of microbial species antagonistic to phytonematodes.     

Wide-range antifungal antagonism of Paenibacillus ehimensis IB-X-b and its dependence on chitinase and beta-1,3-glucanase production

Previously, we isolated a strain of Bacillus that had antifungal activity and produced lytic enzymes with fungicidal potential. In the present study, we identified the bacterium as Paenibacillus ehimensis and further explored its antifungal properties. In liquid co-cultivation assays, P. ehimensis IB-X-b decreased biomass production of several pathogenic fungi by 45%-75%. The inhibition was accompanied by degradation of fungal cell walls and alterations in hyphal morphology. Residual medium from cultures of P. ehimensis IB-X-b inhibited fungal growth, indicating the inhibitors were secreted into the medium. Of the 2 major lytic enzymes, chitinases were only induced by chitin-containing substrates, whereas beta-1,3-glucanase showed steady levels in all carbon sources. Both purified chitinase and beta-1,3-glucanase degraded cell walls of macerated fungal mycelia, whereas only the latter also degraded cell walls of intact mycelia. The results indicate synergism between the antifungal action mechanisms of these enzymes in which beta-1,3-glucanase is the initiator of the cell wall hydrolysis, whereas the degradation process is reinforced by chitinases. Paenibacillus ehimensis IB-X-b has pronounced antifungal activity with a wide range of fungi and has potential as a biological control agent against plant pathogenic fungi.     

Pharyngeal polysaccharide deacetylases affect development in the nematode C. elegans and deacetylate chitin in vitro

Chitin (β-1,4-linked-N-acetylglucosamine) provides structural integrity to the nematode eggshell and pharyngeal lining. Chitin is synthesized in nematodes, but not in plants and vertebrates, which are often hosts to parasitic roundworms; hence, the chitin metabolism pathway is considered a potential target for selective interventions. Polysaccharide deacetylases (PDAs), including those that convert chitin to chitosan, have been previously demonstrated in protists, fungi and insects. We show that genes encoding PDAs are distributed throughout the phylum Nematoda, with the two paralogs F48E3.8 and C54G7.3 found in C. elegans. We confirm that the genes are somatically expressed and show that RNAi knockdown of these genes retards C. elegans development. Additionally, we show that proteins from the nematode deacetylate chitin in vitro, we quantify the substrate available in vivo as targets of these enzymes, and we show that Eosin Y (which specifically stains chitosan in fungal cells walls) stains the C. elegans pharynx. Our results suggest that one function of PDAs in nematodes may be deacetylation of the chitinous pharyngeal lining.     

Extracellular proteases of Trichoderma species. A review

Cellulolytic, xylanolytic, chitinolytic and beta-1,3-glucanolytic enzyme systems of species belonging to the filamentous fungal genus Trichoderma have been investigated in details and are well characterised. The ability of Trichoderma strains to produce extracellular proteases has also been known for a long time, however, the proteolytic enzyme system is relatively unknown in this genus. Fortunately, in the recent years more and more attention is focused on the research in this field. The role of Trichoderma proteases in the biological control of plant pathogenic fungi and nematodes has been demonstrated, and it is also suspected that they may be important for the competitive saprophytic ability of green mould isolates and may represent potential virulence factors of Trichoderma strains as emerging fungal pathogens of clinical importance. The aim of this review is to summarize the information available about the extracellular proteases of Trichoderma. Numerous studies are available about the extracellular proteolytic enzyme profiles of Trichoderma strains and about the effect of abiotic environmental factors on protease activities. A number of protease enzymes have been purified to homogeneity and some protease encoding genes have been cloned and characterized. These results will be reviewed and the role of Trichoderma proteases in biological control as well as their advantages and disadvantages in biotechnology will be discussed.     

Chitinolytic enzymes: An appraisal as a product of commercial potential

Chitin, its deacetylated form, chitosan and chitinolytic enzymes viz. endo‐chitinase, N‐acetylglucosaminidase, chitosanase, chitin deacetylase (CDA) are gaining importance for their biotechnological applications. Presently, chitin degrading enzymes constitute high‐cost low‐volume products in human health care and associated research. Indeed chitinases and CDA‐chitosanase complex possesss tremendous potential in agriculture to control plant pathogenic fungi and insects. The success in exploring chitinases especially for agriculture, i.e. as a high‐volume low‐cost product, depends on the availability of highly active preparations with a reasonable cost. Therefore, a reconsideration in terms of understanding the roles of chitinolytic enzymes in applications, e.g. host–pathogen interaction for biocontrol, different mechanisms of chitin degradation, and identification of new enzymes with varying specificities, may make them more useful in a variety of commercial processes in the near future. The possible issues and challenges encountered in the translation of proof of concept into a commercial product will be appraised in this review. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:833–846, 2013     

刀孢轮枝菌胞外几丁质酶的基因克隆及系统发育分析

食线虫真菌是植物寄生线虫的重要天敌,它们所产生的胞外水解酶(蛋白酶、几丁质酶和胶原蛋白酶等)能够降解线虫体壁和卵壳中的蛋白质及几丁质等结构成分并在侵染过程中发挥着重要的作用。本文中,我们发现刀孢轮枝菌Lecanicillium psalliotae对南方根结线虫Meloidogyne incognita卵具有较强的侵染能力。为了进一步研究刀孢轮枝菌胞外几丁质酶的性质,我们通过简并引物设计和DNA walking方法从刀孢轮枝菌的基因组中成功地克隆得到一个内切几丁质酶基因Lpchi1,该几丁质酶编码基因含有3个内含子,编码423个氨基酸。同源性和系统发育分析表明,不同生防真菌来源的几丁质酶具有较高的同源性并根据分子量的大小形成三个不同的进化分枝。     

Genomic comparison of chitinolytic enzyme systems from terrestrial and aquatic bacteria

Chitin degradation ability is known for many aquatic and terrestrial bacterial species. However, differences in the composition of chitin resources between aquatic (mainly exoskeletons of crustaceans) and terrestrial (mainly fungal cell walls) habitats may have resulted in adaptation of chitinolytic enzyme systems to the prevalent resources. We screened publicly available terrestrial and aquatic chitinase‐containing bacterial genomes for possible differences in the composition of their chitinolytic enzyme systems. The results show significant differences between terrestrial and aquatic bacterial genomes in the modular composition of chitinases (i.e. presence of different types of carbohydrate binding modules). Terrestrial Actinobacteria appear to be best adapted to use a wide variety of chitin resources as they have the highest number of chitinase genes, the highest diversity of associated carbohydrate‐binding modules and the highest number of CBM33‐type lytic polysaccharide monooxygenases. A ctinobacteria do also have the highest fraction of genomes containing β‐1, 3‐glucanases, enzymes that may reinforce the potential for degrading fungal cell walls. The fraction of bacterial chitinase‐containing genomes encoding polyketide synthases was much higher for terrestrial bacteria than for aquatic ones supporting the idea that the combined production of antibiotics and cell‐wall degrading chitinases can be an important strategy in antagonistic interactions with fungi.     

刀孢轮枝菌胞外几丁质酶的基因克隆及系统发育分析

Nematophagous fungi, one of the natural enemies of nematodes, have been employed in biological control. Extracellular enzymes secreted from nematophagous fungi, including protease, chitinase and collagenase serve as virulence factors of infection. In this study, we found Lecanicillium psalliotae can penetrate the eggs of the root-knot nematode Meloidogyne incognita and influence development of the eggs. A chitinase gene Lpchi1 was isolated from L. psalliotae using degenerate primers and DNA-walking technique. Comparison of the chitinase amino acid sequences from different pathogenic fungi revealed that the enzymes were highly similar. The phylogenetic analysis demonstrated that the chitinases derived from different fungi were clustered into three main clades corresponding to different molecular weight.     

The nematode-trapping efficacy of two chlamydospore-forming fungi against Haemonchus contortus in sheep

An in vitro study was carried out to determine efficacy of Indian isolates of the nematode-trapping fungi Arthrobotrys musiformis and Duddingtonia flagrans to capture infective larvae of Haemonchus contortus. These fungi have previously been screened and selected for their survival in the gastrointestinal tract of sheep without losing growth and nematode capturing potential. Following the feeding of chlamydospores of these two fungi alone or in combination in sheep experimentally infected with Haemonchus contortus, coprocultures were set up to enumerate the infective third stage larvae. The number of larvae captured from faeces of fungus-fed sheep was significantly higher compared with fungus-unfed controls irrespective of the fungus used. The fungal combination produced no antagonistic effect and thus can be used as efficiently as the fungi alone in the biological control of animal parasitic nematodes.     

Chitinolytic Enterobacter agglomerans Antagonistic to Fungal Plant Pathogens

Three Enterobacter agglomerans strains which produce and excrete proteins with chitinolytic activity were found while screening soil-borne bacteria antagonistic to fungal plant pathogens. The chitinolytic activity was induced when the strains were grown in the presence of colloidal chitin as the sole carbon source. It was quantitated by using assays with chromogenic p-nitrophenyl analogs of disaccharide, trisaccharide, and tetrasaccharide derivatives of N-acetylglucosamine. A set of three fluorescent substrates with a 4-methylumbelliferyl group linked by (beta)-1,4 linkage to N-acetylglucosamine mono- or oligosaccharides were used to identify the chitinolytic activities of proteins which had been renatured following their separation by electrophoresis. This study provides the most complete evidence for the presence of a complex of chitinolytic enzymes in Enterobacter strains. Four enzymes were detected: two N-acetyl-(beta)-d-glucosaminidases of 89 and 67 kDa, an endochitinase with an apparent molecular mass of 59 kDa, and a chitobiosidase of 50 kDa. The biocontrol ability of the chitinolytic strains was demonstrated under greenhouse conditions. The bacteria decreased the incidence of disease caused by Rhizoctonia solani in cotton by 64 to 86%. Two Tn5 mutants of one of the isolates, which were deficient in chitinolytic activity, were unable to protect plants against the disease.     

Purification of a chitinase from Trichoderma sp. and its action on Sclerotium rolfsii and Rhizoctonia solani cell walls

Trichoderma harzianum is an effective biocontrol agent of several important plant pathogenic fungi. This Trichoderma species attacks other fungi by secreting lytic enzymes, including beta-1,3-glucanase and chitinolytic enzymes. Superior biocontrol potential may then be found in strains having a high capacity to produce these enzymes. We have therefore evaluated the capacity of six unidentified Trichoderma spp. isolates to produce chitinolytic enzymes and beta-1,3-glucanases in comparison with T. harzianum 39.1. All six isolates demonstrated substantial enzyme activity. However, while the isolates hereafter called T2, T3, T5, and T7 produced lower amounts of enzymes, the activity of isolates T4 and T6 were 2-3 fold higher than that produced by T. harzianum 39.1. A chitinase produced by the T6 isolate was purified by a single ion-exchange chromatography step and had a molecular mass of 46 kDa. The N-terminal amino-acid sequence showed very high homology with other fungal chitinases. Its true chitinase activity was demonstrated by its action on chitin and the failure to hydrolyze laminarin and p-nitrophenyl-beta-N-acetylglucosaminide. The hydrolytic action of the purified chitinase on the cell wall of Sclerotium rolfsii was convincingly shown by electron microscopy studies. However, the purified enzyme had no effect on the cell wall of Rhizoctonia solani.     

Concepts of nematode-fungus associations in plant disease complexes: a review

Plant parasitic nematodes interact with fungi in a variety of ways to cause plant disease complexes. Even some nonplant parasitic nematodes are able to carry fungal spores internally which not only increases their mobility, but also protects them from fungicides. Plant parasitic nematodes frequently wound plants in the process of penetration and feeding. These wounds become subject to infection by fungal pathogens that require aid in penetrating their host. Other nematodes modify plant tissue in such a way that it becomes a better substrate for the fungus and thus increases their growth and reproduction to the detriment of the host. Quantitative and qualitative changes in root exudate which are induced by certain nematodes stimulate the germination, growth, and reproduction of fungal propagules in the rhizosphere. These exudates may also indirectly inhibit components of the rhizosphere microflora (e.g., actinomycetes) which are antagonistic to some plant pathogens. Depending on the species of nematode and fungus, concomitant infections may stimulate nematode reproduction (Pratylenchus-Verticillium) or inhibit reproduction (Heterodera-Fusarium).     

木霉菌防治植物真菌病害研究进展

木霉菌是一种重要的植物病害生防因子,尤其在防治植物病原真菌病害中一直受到极大的关注。木霉菌依靠其菌株在包括趋向生长、识别、接触、缠绕与穿透等步骤的真菌寄生过程中分泌产生的几丁质酶、葡聚糖酶、纤维素酶、蛋白酶等一系列细胞壁降解酶,进行重寄生作用,拮抗其他植物病原菌,行使其生防功能。我们简要概述了木霉菌的种类、拮抗对象、抑菌机制、诱导抗性、促生作用、基于分子生物学的转基因工程研究,以及木霉菌在植物病原真菌生物防治中的应用。