In vitro association of nematophagous fungi Duddingtonia flagrans (AC001), Monacrosporium thaumasium (NF34) and Pochonia chlamydosporia (VC1) to control horse cyathostomin (Nematoda: Strongylidae)

In vitro effects of nematophagous fungi Duddingtonia flagrans (AC001), Monacrosporium thaumasium (NF34) and Pochonia chlamydosporia (VC1) were evaluated against eggs and third-stage infective larvae (L₃) of horse cyathostomin (Nematoda: Strongylidae). The following percentage reductions compared with the control group were observed after a 20-day exposure period: AC001, 61.6%; NF34, 66.1%; VC1, 73.2%; group AC001 + VC1, 86.8%; NF34 + VC1, 77.3%; AC001 + NF34, 92.4%. The results showed that the fungal isolates (VC1, AC001 and NF34), acting alone or in conjunction, were efficient in controlling horse cyathostomin under in vitro conditions.     

Influence of different storage methods on the predatory capacity of the fungi Arthrobotrys robusta and Monacrosporium thaumasium after passage through the bovine gastrointestinal tract

The biological control of helminth parasites of bovines by nematophagous fungi is an alternative to the use of drugs with the principal objective of reducing the source of infection available on pastureland. The maintenance of predatory activity of the fungal isolates is one of the basic prerequisites to ensure the success of this form of control. In this study behaviour of the isolates I31 of Arthrobotrys robusta and NF34a of Monacrosporium thaumasium was investigated following three storage methods: stored at 4 °C, cryopreserved with or without cryoprotectants or preserved in silica gel. All samples were subsequently passed through the gastrointestinal tract of calves. The latter involved the administration of 20 g of mycelia to the animals. This quantity was sufficient to recover fungal material from the faeces. The peak reduction in the number of infective larvae in the faeces occurred 24 h after administration of the samples (P < 0.05). The storage at 4 °C was the treatment that produced the greatest reduction in larvae for NF34a (81.3%) and I31 (65.1%) isolates. Nf34a isolate was responsible for the highest percentage reduction of larval helminth populations (P < 0.05). Cryopreservation appears to be an efficient method of preserving isolates, although diminished predatory capacity compared to storage at 4 °C was seen only for isolate NF34a (73.2%). Cryopreservation did not interfere in predatory activity of I31 isolate (P < 0.05). Maintenance of isolates in silica gel showed the lowest reduction throughout the experiment (P < 0.05).     

Effect of refrigeration storage of nemathophagous fungi embedded in sodium alginate pellets on predatory activity against asinine gastrointestinal nematodes

ABSTRACT

The objective of this study was to evaluate the effectiveness of pelleted formulations of Duddingtonia flagrans and Monacrosporium thaumasium sodium alginate matrix stored for two and five years, by refrigeration of 2–8°C, on the predation of nematode infective larvae after passage of the gastrointestinal tract of asinines. Asinines were divided into seven groups, each group containing eight animals, in which each animal received a single dose of 100?g of pellets (containing 20?g of fungal mycelia) along with commercial feed to facilitate ingestion: GI – received D. flagrans pellets stored for five years; GII- received pellets of D. flagrans stored for two years; GIII – received newly produced D. flagrans pellets; GIV – received pellets of M. thaumasium stored for five years; GV – received pellets of M. thaumasium stored for two years; GVI – received pellets of newly-stocked M. thaumasium; and Control – received pellets without nematophagous fungi. It was observed that after passage of the pellets containing D. flangras (AC001) and M. thaumasium (NF34) by the gastrointestinal tract of the asinines, regardless of pellet storage time in assays A (Petri dishes) and B (coprocultures), there was a significant larval reduction (p?<?0.01) up to 72?h. It was concluded that the use of sodium alginate matrix pellets containing D. flagrans and M. thaumasium stored for two and five years were effective on the predation of infective nematode larvae after passage of the gastrointestinal tract from asinines.     

Effects of Paecilomyces lilacinus protease and chitinase on the eggshell structures and hatching of Meloidogyne javanica juveniles

A serine protease and an enzyme preparation consisting of six chitinases, previously semi-purified from a liquid culture of Paecilomyces lilacinus strain 251, were applied to Meloidogyne javanica eggs to study the effect of the enzymes on eggshell structures. Transmission electron microscopic studies revealed that the protease and chitinases drastically altered the eggshell structures when applied individually or in combination. In the protease-treated eggs, the lipid layer disappeared and the chitin layer was thinner than in the control. The eggs treated with chitinases displayed large vacuoles in the chitin layer, and the vitelline layer was split and had lost its integrity. The major changes in the eggshell structures occurred by the combined effect of P. lilacinus protease and chitinases. The lipid layer was destroyed; the chitin layer hydrolyzed and the vitelline layer had lost integrity. The effect of P. lilacinus protease and chitinase enzymes on the hatching of M. javanica juveniles was also compared with a commercially available bacterial chitinase. The P. lilacinus protease and chitinase enzymes, either individually or in combination, reduced hatching of M. javanica juveniles whereas a commercial bacterial chitinase had an enhancing effect. Some juveniles hatched when the eggs were exposed to a fungal protease and chitinase mixture. We also established that P. lilacinus chitinases retained their activity in the presence of endogenous protease activity.     

Induction and purification of a thermophilic chitinase produced byAeromonas sp. DYU-too7 using glucosamine

In the presence of chitin,Aeromonas sp. DYU-Too7 can produce extra-cellular, chitin-degrading enzymes. Chitin analogues and other carbon sources can be used to cultivate this bacterial strain. The chitinases produced by the strain were higher in the GIcN (glucosamine) medium than those in other media. The maximal chitinase activity occurred in the medium containing 0.1% GIcN. Cultivation ofAeromonas sp. DYU-Too7 in the GIcN medium sped up the chitinase production; however the same result did not appear when it was cultivated in the (Chitin+GIcN) medium. This result may indicate that GIcN can be utilized byAeromonas sp. DYU-Too7 as a carbon source and an inducer to produce chitinases. A chitinase with a molecular mass of 36 kDa was further purified and characterized to have an optimal reacting pH of 5.0 and an optimal reacting temperature of 50°C. This chitinase showed high stability in the proximity of 30°C and also high stability in the proximity of pH 7.0. The hydrolysates of colloidal chitin, with the aid of the 36-kDa chitinase, were analyzed by an HPLC and found to be chitobiose.     

Biotechnological aspects of chitinolytic enzymes: a review

Chitin and chitinases (EC 3.2.1.14) have an immense potential. Chitinolytic enzymes have wide-ranging applications such as preparation of pharmaceutically important chitooligosaccharides and N-acetyl d-glucosamine, preparation of single-cell protein, isolation of protoplasts from fungi and yeast, control of pathogenic fungi, treatment of chitinous waste, and control of malaria transmission. In this review, we discuss the occurrence and structure of chitin, the types and sources of chitinases, their mode of action, chitinase production, as well as molecular cloning and protein engineering of chitinases and their biotechnological applications.     

Chitinases: in agriculture and human healthcare

Abstract

Biological control of phytopathogenic fungi and insects continues to inspire the research and development of environmentally friendly bioactive alternatives. Potentially lytic enzymes, chitinases can act as a biocontrol agent against agriculturally important fungi and insects. The cell wall in fungi and protective covers, i.e. cuticle in insects shares a key structural polymer, chitin, a β-1,4-linked N-acetylglucosamine polymer. Therefore, it is advantageous to develop a common biocontrol agent against both of these groups. As chitin is absent in plants and mammals, targeting its metabolism will signify an eco-friendly strategy for the control of agriculturally important fungi and insects but is innocuous to mammals, plants, beneficial insects and other organisms. In addition, development of chitinase transgenic plant varieties probably holds the most promising method for augmenting agricultural crop protection and productivity, when properly integrated into traditional systems. Recently, human proteins with chitinase activity and chitinase-like proteins were identified and established as biomarkers for human diseases. This review covers the recent advances of chitinases as a biocontrol agent and its various applications including preparation of medically important chitooligosaccharides, bioconversion of chitin as well as in implementing chitinases as diagnostic and prognostic markers for numerous diseases and the prospect of their future utilization.     

Three Extracellular Chitinases in Suspension-cultured Rice Cells Elicited by N-Acetylchitooligosaccharides

In rice suspension culture, a large part (about 90% of total activity in the culture) of the chitinase activity was found in the medium. Two extracellular chitinases (which we named RCH-A and -B) were separated from the cell suspension by DEAE-cellulofìne column chromatography. When cells were treated with N-acetylchitooligosaccharides (chitin oligosaccharides) for 3 days, extracellular chitinase activity increased about 3-fold over the control culture. After the treatment, another extracellular chitinase (named RCH-C) appeared in addition to increases in the levels of RCH-A and -B. Partial amino acid sequences of these enzymes indicated that RCH-A (33.5 kDa) and -B (34kDa) were class Ib chitinases but RCH-C (27kDa) was a class III chitinase. RCH-A and -B were capable of actively degrading water-insoluble chitin with high affinities, while RCH-C had less affinity for the substrate. However, when a water-soluble chitin derivative, 6–O-hydroxyethylchitin (glycolchitin) was used, RCH-C as well as RCH-A and -B degraded actively with a high affinity. A synergistic effect was observed when these three chitinases acted simultaneously in the hydrolysis of chitin.     

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.     

Distribution of Chitinases in the Entomopathogen <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> and Effect of <Emphasis Type="Italic">N</Emphasis>-Acetylglucosamine in Protein Secretion

For a long time, fungi have been characterized by their ability to secrete enzymes, mostly hydrolytic in function, and thus are defined as extracellular degraders. Chitin and chitinolytic enzymes are gaining importance for their biotechnological applications. Particularly, chitinases are used in agriculture to control plant pathogens. Metarhizium anisopliae produces an extracellular chitinase when grown on a medium containing chitin, indicating that synthesis is subject to induction by the substrate. Various sugar combinations were investigated for induction and repression of chitinase. N-acetylglucosamine (GlcNAc) shows a special dual regulation on chitinase production. M. anisopliae has at least two distinct, cell-bound, chitinolytic enzymes when cultured with GlcNAc as one of the carbon sources, and we suggest that this carbohydrate has an important role in protein secretion.     

A novel strain of Brevibacillus laterosporus produces chitinases that contribute to its biocontrol potential

A novel strain exhibiting entomopathogenic and chitinolytic activity was isolated from mangrove marsh soil in India. The isolate was identified as Brevibacillus laterosporus by phenotypic characterization and 16S rRNA sequencing and designated Lak1210. When grown in the presence of colloidal chitin as the sole carbon source, the isolate produced extracellular chitinases. Chitinase activity was inhibited by allosamidin indicating that the enzymes belong to the family 18 chitinases. The chitinases were purified by ammonium sulfate precipitation followed by chitin affinity chromatography yielding chitinases and chitinase fragments with 90, 75, 70, 55, 45, and 25 kDa masses. Mass spectrometric analyses of tryptic fragments showed that these fragments belong to two distinct chitinases that are almost identical to two putative chitinases, a 89.6-kDa four-domain chitodextrinase and a 69.4-kDa two-domain enzyme called ChiA1, that are encoded on the recently sequenced genome of B. laterosporus LMG15441. The chitinase mixture showed two pH optima, at 6.0 and 8.0, and an optimum temperature of 70 °C. The enzymes exhibited antifungal activity against the phytopathogenic fungus Fusarium equiseti. Insect toxicity bioassays with larvae of diamondback moths (Plutella xylostella), showed that addition of chitinases reduced the time to reach 50 % mortality upon infection with non-induced B. laterosporus from 3.3 to 2.1 days. This study provides evidence for the presence of inducible, extracellular chitinolytic enzymes in B. laterosporus that contribute to the strain’s antifungal activity and insecticidal activity.     

Protein Engineering of Chit42 Towards Improvement of Chitinase and Antifungal Activities

The antagonism of Trichoderma strains usually correlates with the secretion of fungal cell wall degrading enzymes such as chitinases. Chitinase Chit42 is believed to play an important role in the biocontrol activity of Trichoderma strains as a biocontrol agent against phytopathogenic fungi. Chit42 lacks a chitin-binding domain (ChBD) which is involved in its binding activity to insoluble chitin. In this study, a chimeric chitinase with improved enzyme activity was produced by fusing a ChBD from T. atroviride chitinase 18–10 to Chit42. The improved chitinase containing a ChBD displayed a 1.7-fold higher specific activity than chit42. This increase suggests that the ChBD provides a strong binding capacity to insoluble chitin. Moreover, Chit42-ChBD transformants showed higher antifungal activity towards seven phytopathogenic fungal species.     

Comparison of Enzymatic and Antifungal Properties between Family 18 and 19 Chitinases from S. coelicolor A3(2)

Streptomyces coelicolor A3(2) has 13 chitinase genes encoding 11 family 18 and two family 19 chitinases. To compare enzymatic properties of family 19 chitinase and family 18 chitinases produced by the same organism, the four chitinases (Chi18bA, Chi18aC, Chi18aD, and Chi19F), whose genes are expressed at high levels in the presence of chitin, were produced in Eschericha coli and purified. The effect of pH on the hydrolytic activity was very different not only among the four chitinases but also among the substrates. The hydrolytic activity of Chi19F, family 19 chitinase, against soluble substrates was remarkably high as compared with three family 18 chitinases, but was the lowest against crystalline substrates among the four chitinases. On the contrary, Chi18aC, a family 18-subfamily A chitinase, showed highest activity against crystalline substrates. Only Chi19F exhibited significant antifungal activity. Based on these observations, the roles of family 19 chitinases are discussed.     

Identification of two group A chitinase genes in Botrytis cinerea which are differentially induced by exogenous chitin

Chitin-degrading enzymes represent potential targets for pesticides in the control of plant pathogenic fungi. Here we describe the cloning, molecular characterization, and expression analysis of two putative chitinases of Botrytis cinerea, a pathogenic fungus infecting a wide range of plants. On the basis of conserved motifs from family 18 of the glycosyl hydrolases and group A of the fungal chitinases, two fragments (BcchiA and BcchiB) were cloned and sequenced. Expression of BcchiA and BcchiB chitinase genes upon growth under different conditions was analysed using RT-PCR. We observed that BcchiA expression was suppressed by glucose, whereas it was strongly stimulated in the presence of chitin or chitin degradation products. Conversely, BcchiB expression was not suppressed by glucose and was not stimulated by chitin or chitin degradation products. The difference in expression regulation is indicative of a functional divergence between the two chitinase paralogous genes.     

Increased antifungal and chitinase specific activities of<Emphasis Type="Italic"> Trichoderma harzianum</Emphasis> CECT 2413 by addition of a cellulose binding domain

Trichoderma harzianum is a widely distributed soil fungus that antagonizes numerous fungal phytopathogens. The antagonism of T. harzianum usually correlates with the production of antifungal activities including the secretion of fungal cell walls that degrade enzymes such as chitinases. Chitinases Chit42 and Chit33 from T. harzianum CECT 2413, which lack a chitin-binding domain, are considered to play an important role in the biocontrol activity of this strain against plant pathogens. By adding a cellulose-binding domain (CBD) from cellobiohydrolase II of Trichoderma reesei to these enzymes, hybrid chitinases Chit33-CBD and Chit42-CBD with stronger chitin-binding capacity than the native chitinases have been engineered. Transformants that overexpressed the native chitinases displayed higher levels of chitinase specific activity and were more effective at inhibiting the growth of Rhizoctonia solani, Botrytis cinerea and Phytophthora citrophthora than the wild type. Transformants that overexpressed the chimeric chitinases possessed the highest specific chitinase and antifungal activities. The results confirm the importance of these endochitinases in the antagonistic activity of T. harzianum strains, and demonstrate the effectiveness of adding a CBD to increase hydrolytic activity towards insoluble substrates such as chitin-rich fungal cell walls.     

Evidence Supporting a Role for Mammalian Chitinases in Efficacy of Caspofungin against Experimental Aspergillosis in Immunocompromised Rats

Objectives

Caspofungin, currently used as salvage therapy for invasive pulmonary aspergillosis (IPA), strangely only causes morphological changes in fungal growth in vitro but does not inhibit the growth. In vivo it has good efficacy. Therefore the question arises how this in vivo activity is reached. Caspofungin is known to increase the amount of chitin in the fungal cell wall. Mammals produce two chitinases, chitotriosidase and AMCase, which can hydrolyse chitin. We hypothesized that the mammalian chitinases play a role in the in vivo efficacy of caspofungin.

Methods

In order to determine the role of chitotriosidase and AMCase in IPA, both chitinases were measured in rats which did or did not receive caspofungin treatment. In order to understand the role of each chitinase in the breakdown of the caspofungin-exposed cells, we also exposed caspofungin treated fungi to recombinant enzymes in vitro.

Results

IPA in immunocompromised rats caused a dramatic increase in chitinase activity. This increase in chitinase activity was still noted when rats were treated with caspofungin. In vitro, it was demonstrated that the action of both chitinases were needed to lyse the fungal cell wall upon caspofungin exposure.

Conclusion

Caspofungin seemed to alter the cell wall in such a way that the two chitinases, when combined, could lyse the fungal cell wall and assisted in clearing the fungal pathogen. We also found that both chitinases combined had a direct effect on the fungus in vitro.     

Lytic Enzymes of Trichoderma and Their Role in Plant Defense from Fungal Diseases: A Review

Lytic enzymes of mycoparasitic fungi of the genus Trichoderma, capable of suppressing a number of fungal phytopathogens that originate in air or soil, are reviewed. The topics analyzed include (1) regulation of production of chitinases, -1,3-glucanases, and proteases; (2) molecular and catalytic properties of purified enzymes; and (3) their in vitro ability to degrade cell walls and inhibit spore germination or germ-tube elongation in various phytopathogenic fungi. Among the results summarized are reports of cloning and expression of genes coding for certain lytic enzymes of Trichoderma spp. These genes are used for obtaining plant transgenes with increased resistance to fungal diseases and Trichoderma transformants that produce higher levels of one lytic enzyme (a chitinase or protease) and thereby exhibit a more pronounced ability to suppress phytopathogenic fungi.     

Cloning and expression analysis of the chitinase gene Ifu-chit2 from Isaria fumosorosea

Entomopathogenic fungi can produce a series of chitinases, some of which function synergistically with proteases and other hydrolytic enzymes to degrade the insect cuticle. In the present study, the chitinase gene Ifu-chit2 from Isaria fumosorosea was investigated. The Ifu-chit2 gene is 1,435-bp long, interrupted by three short introns, and encodes a predicted protein of 423 amino acids with a 22 residue signal peptide. The predicted Ifu-Chit2 protein is highly homologous to Beauveria bassiana chitinase Bbchit2 and belongs to the glycohydrolase family 18. Ifu-Chit2 was expressed in Escherichia coli to verify chitinase activity, and the recombinant enzyme exhibited activity with a colloidal chitin substrate. Furthermore, the expression profiles of Ifu-chit2 were analyzed at different induction times under in vivo conditions. Quantitative real-time PCR analysis revealed that Ifu-chit2 expression peaked at two days post-induction. The expression of chitinase Ifu-chit2 in vivo suggests that the chitinase may play a role in the early stage of pathogenesis.     

Production of chitinase by Fusarium species

The presence of chitinase activity without inducers in the enzymic precipitates from the culture fluid of 25-day-old autolyzed cultures of 17 Fusarium species has been studied. In all cases endochitinase and -N-acetylglucosaminidase activities were found. The chitinase activity as a joint action of these two enzymes with production of N-acetylglucosamine was also determined. A correlation among endochitinase, -N-acetylglucosaminidase, and chitinase was always found. Fusarium oxysporum f.sp. lini, F. subglutinans, and F. moniliforme were the best producers of chitinase activity. Fusarium species could be a good source of chitinases for production by fungi.     

Purification and partial characterization of two chitinases from the mycoparasitic fungus <Emphasis Type="Italic">Talaromyces flavus</Emphasis>

Chitinases were produced by Talaromyces flavus CGMCC 3.4301 when it was grown in the presence of chitin. Two chitinases from the culture filtrate of T. flavus were purified to homogeneity by fractional ammonium sulphate precipitation, ion-exchange chromatography on DEAE–Sepharose and Phenyl–Sepharose hydrophobic interaction chromatography. By SDS–PAGE, the molecular weight of the two enzymes was estimated to be 41 and 32 kDa, respectively. The 41 kDa chitinase (CHIT41) had a 4.0 pH optimum; the 32 kDa chitinase (CHIT32) optimum activity was at pH 5.0. The optimum temperature for the two chitinase activities was 40 °C. The two chitinases had activity against cell wall of Verticillium dahliae, Sclerotinia sclerotiorum and Rhizoctonia solani, and inhibited spore germination and germ tube elongation of Alternaria alternata, Fusarium moniliforme, and Magnaporthe grisea.