Mycoparasitism studies of Trichoderma harzianum strains against Rhizoctonia solani: evaluation of coiling and hydrolytic enzyme production

The genus Trichoderma is a potential biocontrol agent against several phytopathogenic fungi. One parameter for its successful use is an efficient coiling process followed by a substantial production of hydrolytic enzymes. The interaction between fifteen isolates of Trichoderma harzianum and the soil-borne plant pathogen, Rhizoctonia solani, was studied by light microscopy and transmission electron microscopy (TEM). Macroscopic observations of fungal growth in dual cultures revealed that growth inhibition of the pathogen occurred soon after contact with the antagonist. All T. harzianum isolates tested exhibited coiling around the hyphae of R. solani. The strains ALL23, ALL40, ALL41, ALL43 and ALL49 did not differ in coiling frequency and gave equal coiling performances. No correlation between coiling frequency and the production of cell wall-degrading chitinases, N-acetyl-β-d-glucosaminidase and β-1,3-glucanases, was found.     

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.     

Synthesis of enzymes connected with mycoparasitism by ectomycorrhizal fungi

The production of enzymes involved in mycoparasitism by several strains of ectomycorrhizal fungi: Amanita muscaria (16-3), Laccaria laccata (9-12), L. laccata (9-1), Suillus bovinus (15-4), S. bovinus (15-3), S. luteus (14-7) on different substrates such as colloidal chitin, mycelia of Trichoderma harzianum, T. virens and Mucor hiemalis was examined. Chitinases and β-1,3-glucanases were assayed spectrophotometrically by measuring the amount of reducing sugars releasing from suitable substrate by means of Miller’s method. β-glucosidases were determined by measuring the amount of p-nitrophenol released from p-nitrophenyl-β-D-glucopyranoside. It was observed that A. muscaria (16-3) and L. laccata (9-12) biosynthesized the highest activity of enzymes in contrast to the strains of S. bovinus and S. luteus. The mycelium of T. harzianum turned out to be the best substrate for the induction of β-1,3-glucanases and β-glucosidases for both strains of L. laccata, although the difference in the induction of chitinases in the presence of mycelia of different species of Trichoderma was not indicated.     

Antifungal effect of Trichoderma spp. β-1,3-glucanase on Phytophthora parasitica: Hyphal morphological distortions

Phytopathogenic fungi devastate agricultural crops worldwide. The biological agents, such as Trichoderma spp., antagonize phytopathogenic fungi by secreting various cell wall-degrading enzymes, for example, endochitinase and β-1,3-glucanase that target glycosidic linkages in β-glucan and chitin polymers of fungal cell walls, thus inhibiting pathogen growth. In this study, two antifungal genes endochitinase and β-1,3-glucanase cloned from local Trichoderma spp. were ligated in pET28a+ expression vector individually to generate two recombinant vectors. The vectors were mobilized into Escherichia coli host strain Rosetta-gami 2 for protein expression, and the 6xHis-tagged recombinant proteins were purified through Ni-NTA affinity chromatography. The purified proteins were individually confronted in vitro with pure cultures of Phytophthora parasitica (destructive pathogen affecting several hundred plant species worldwide) for analyzing their effect on pathogen growth. In vitro confrontation assay revealed P. parasitica growth inhibition by purified β-1,3-glucanase. The pathogen growth inhibition was due to hyphal morphological distortions, such as breakages, swelling, and holes evinced through electron micrography confirming direct role of β-1,3-glucanase in pathogen structural degradation.     

Control of fusarium wilt of <Emphasis Type="Italic">Solanum melongena</Emphasis> by <Emphasis Type="Italic">Trichoderma</Emphasis> spp.

Biological control of wilt of egg plant (Solanum melongena L.) caused by Fusarium solani was made with the application of five Trichoderma species, T. harzianum, T. viride, T. lignorum, T. hamatum and T. reesei. The effect of volatile and non-volatile antibiotics of Trichoderma origin on growth inhibition of the wilt pathogen was studied. T. harzianum showed maximum growth inhibition (86.44 %) of the pathogen through mycoparasitism. The non-volatiles produced by the Trichoderma species exhibited 100 % growth inhibition of the pathogen under in vitro condition. Production of siderophores and fungal cell wall degrading enzymes, chitinase and β-1,3-glucanase were found. Treatments with two most efficient Trichoderma species, T. harzianum and T. viride resulted in the decreasing population of Fusarium solani in soil thereby deterring disease incidence in field condition.     

A Novel Antifungal <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> Isolated from Potato Soils in Greenland

A rhizobacterium with high antifungal activity was isolated from a potato field at Inneruulalik, South Greenland. Phylogenetic analysis based on multi locus sequence typing showed that the bacterium was affiliated with strains of Pseudomonas fluorescens. The bacterium, denoted as Pseudomonas fluorescens In5, inhibited in vitro a broad range of phytopathogenic fungi, and the antifungal activity increased with decreasing temperature. Microcosm experiments demonstrated that P. fluorescens In5 protected tomato seedlings from Rhizoctonia solani. Transposon mutagenesis showed that the major cause for the antifungal activity of P. fluorescens In5 was a novel non-ribosomal peptide synthase (NRPS) gene. In addition, transposon mutagenesis showed that P. fluorescens In5 also contained a putative quinoprotein glucose dehydrogenase gene, which was involved in growth inhibition of phytopathogenic fungi. Although P. fluorescens In5 contained the capacity to synthesize hydrogen cyanide, β-1,3-glucanase, protease, and chitinase, these did not seem to play a role in the in vitro and microcosm antifungal assays.     

Efficacy ofTrichoderma chitinases againstRhizoctonia solani, the rice sheath blight pathogen

Thirty-five strains ofTrichoderma viride andT. harzianum were screened for their antagonistic ability against the rice sheath blight pathogen,Rhizoctonia solani. The strains that inhibited/overgrew the phytopathogenic fungus were considered effective. Light microscopic studies showed the antagonism of the hyphae of effectiveTrichoderma strains towards their host hyphae. Chitinase activity ofTrichoderma culture filtrates was enhanced, when colloidal chitin was used as the sole carbon source, instead of glucose. Chitinase pattern differed among the four select strains. The chitinase isoforms are induced differentially by carbon sources. The chitin affinity column fraction ofTrichoderma culture filtrate inhibited,in vitro, the growth ofR. solani.     

Effect of<Emphasis Type="Italic">Trichoderma</Emphasis> species on growth of Fusarium<Emphasis Type="Italic">proliferatiom</Emphasis> and production of fumonisins,fusaproliferin and beauvericin

Trichoderma species has been suggested as potential biocontrol agent forFusarium verticillioides on maize. In this cereal,F. verticillioides and F. proliferatum contributed to fumonisin accumulation. In addition,F. proliferatum could produce beauvericin and fusaproliferin. The aim of this work was to evaluate the effect ofTrichoderma spp. on growth and fumonisin B¹ fusaproliferin and beauvericin production byF. proliferatum. Dual cultures of F.proliferatum andT. harzianum ITEM 3636 andT. longibrachiatum ITEM 3635 on maize meal agar at 0.995 aw were done. The effect ofTrichoderma spp. on the lineal growth ofF. proliferatum was determined. The effect ofTrichoderma species on fumonisin B¹, fusaproliferin and beauvericin production byF. proliferatum was determined on co-inoculated maize kernels by HPLC.T. harzianum suppressedF. proliferatum growth once contact between the colonies occurred.T. longibrachiatum showed a less antagonistic effect againstF. proliferatum. A reduction on fumonisin B¹ production of 98% and 88% was observed in the co-incubation ofF. proliferatum withT. harzianum andT. longibrachiatum, respectively. The decrease of FB¹ production was significant even in maize kernels on whichF. proliferatum had been growing 7 days prior to the addition ofTrichoderma spp. The concentration of beauvericin and fusaproliferin produced during 30 days coincubation ofF. proliferatum with bothTrichoderma spp. did not differ to those produced byF. proliferatum alone. These mycotoxins might enter the food chain causing so far unknown consequences to the health of domestic animals and humans. For this reason it is important, when a potential biocontrol agent is under study, to test the effect on the fungal growth and on the putative mycotoxin produced. Part of the information was presented at the Mycotoxin Prevention Cluster Dissemination Day and Mycoglobe Launch Conference, Brussels, Belgium, Oct 20–21, 2004 Financial support: Agenda Córdoba Ciencia, grant N^o 0279–000431/00     

Comparative Characterization of Laminarinases from the Filamentous Marine Fungi Chaetomium Indicum Corda and Trichoderma Aureviride Rifai

Marine filamentous fungi (103 strains) isolated from various marine habitats were studied for their ability to produce extracellular O-glycosylhydrolases. Cultural filtrates of these strains were shown to contain a series of glycanases (laminarinases, amylases, cellulases, pustulanases) and glycosidases (β-glucosidases, N-acetyl-β-glucosaminidases, β-galactosidases, α-mannosidases).Two species of marine fungi from different habitats were chosen for isolation of laminarinases and detailed study on enzyme properties. The fungus Chaetomium indicum associated with the alga Fucus evanescens C. Agardh was collected near the Kuril Islands, and T. aureviride was sampled from bottom deposits of South China Sea. Properties of extracellular laminarinases were similar: temperature optimums (40–45 ^∘C), molecular masses (54–56 kDa), K _m (0.1–0.3 mg ml⁻¹). Temperature stability of laminarinase of C. indicum was significantly higher than those from T. aureveride. It is shown that these enzymes are specific to β-1,3-bonds in glucans, release predominantly glucose from laminaran and do not catalyze reaction of transglycosylation. Accoding to these data enzymes are exo-1,3-β-D-glucan-glucanohydrolases (EC 3.2.1.58). Inhibitor analysis demonstrated the significant role of tryptophan and tyrosine residues in the catalytic activity of enzymes. Molecules of T. aureviride laminarinase contained the functionally important thiol group.     

Enzymatic studies of the extracellular mucilage of two aquatic hyphomycetes,Lemonniera aquatica andMycocentrospora filiformis

The effect of three carbohydrate-digesting enzymes, β-glucuronidase, lyticase and α-mannosidase and three proteolytic enzymes, α-chymotrypsin, papain and pronase E, on the strength of conidial attachment ofLemonniera aquatica andMycoentrospora filiformis was determined using the LH_Fowler cell Adhesion Measurement Module. Carbohydrate-digesting enzyme treatments showed significant differences in number of attached and detached, conidia versus control samples; little or no effect was observed for the proteolytic enzymes. Scanning and transmission electron microscopy showed different degrees of mucilage digestion by the carbohydrate-digesting enzymes on the germ hyphae, hyphae subtending appressoria, and appressoria of the two species. The loss of mucilage integrity and decrease in mucilage thickness were more pronounced on the hyphal sheaths than on the appressorial sheaths. Lyticase caused the most severe damage to the mucilage and cytoplasm of both fungi, particularlyL. aquatica. β-Glucuronidase and α-mannosidase exhibited more effective mucilage digestion onM. filiformis than onL. aquatica. Results indicate that the mucilage of the two species is mainly polysaccharide, containing more β-1,3-glucans than β-glucuronide and α-mannosyl residues. Variability of mucilage composition exists between these species and also between different structures of the same fungus.     

Enhancement of α- and β-Galactosidase Activity in <Emphasis Type="Italic">Lactobacillus reuteri</Emphasis> by Different Metal Ions

The hydrolysis of oligosaccharides and lactose is of great importance to the food industry. Normally, oligosaccharides like raffinose, stachyose, and verbascose which are rich in different plants like soy bean are considered indigestible by the human gut. Moreover, many humans suffer from lactose intolerance due to the absence of effective enzyme that can digest lactose. α-Galactosidase can digest oligosaccharides like raffinose, while β-galactosidases can hydrolyze lactose. Therefore, selection of microorganisms safe for human use and capable of producing high levels of enzymes becomes an attractive task. The objective of this study was to investigate the enhancement of α- and β-galactosidase activity in Lactobacillus reuteri by different metal ions. Ten millimolar of Na⁺, K⁺, Fe²⁺, and Mg²⁺ and 1 mM of Mn²⁺ were added separately to the growth culture of six strains of L. reuteri (CF2-7F, DSM20016, MF14-C, MM2-3, MM7, and SD2112). Results showed that L. reuteri CF2-7F had the highest α- and β-galactosidase activity when grown in the medium with added Mn²⁺ ions (22.7 and 19.3 Gal U/ml, respectively). 0.0274% of Mn²⁺ ions lead to 27, 18% enhancement of α- and β-galactosidase activity over the control group, and therefore, it could be added to the growth culture of CF2-7F to produce enhanced levels of α- and β-galactosidase activity. The addition of Fe²⁺ led to a significant (P < 0.01) decrease in the activity of both enzymes for most strains. This study shows that modified culture medium with that 0.0274% Mn²⁺ can be used to promote the production for α- and β-galactosidase in L. reuteri CF2-7F, which may lead to enhancement of α- and β-galactosidase activity and have a good potential to be used in the food industry.     

Production and characteristics of the exocellular polysaccharide in mutant strains ofXanthomonas fuscans

Production of the exocellular polysaccharide of the phytopathogenic bacteriumXanthomonas fuscans was investigated with respect to its possible use in utilization of industrial wastes containing lactose. Six stablelac ⁺ mutants were obtained after the treatment withN-methyl-N′-nitroso-N′-nitroguanidine. The mutants were compared with the parent strain. Morphological and cultivation characteristics, as well as production of the exooellular polysaccharide were compared. The production was found to be maximal during the stationary phase of growth in strains cultivated under submerged conditions. Gas chromatography revealed that the polysaccharide of the parent strain is formed by α- and β-D-glucose and α- and β-d-mannose with a small amount ofd-ribose and 6-deoxy-l-mannose. Composition of the polysaccharides produced by the mutant strains (lac ⁺) does not qualitatively differ from that of the parent strain. However, they were found to contain a higher quantity ofd-mannose, which is favourable for their industrial utilization.     

Enzymatic Activities of Trametes versicolor and Pleurotus eryngii Implicated in Biocontrol of Fusarium oxysporum f. sp. lycopersici

The growth of the phytopathogenic fungus Fusarium oxysporum f. sp. lycopersici race 2 (FOL 2) was observed in dual culture with two soil fungi as biocontrol agents, Trametes versicolor and Pleurotus eryngii. In both cases, an interaction zone with the pathogen was found with the Fusarium’s hyphae becoming free of cytoplasmic content. The enzymatic complex of fungi, studied as biocontrol agents, showed β-(1,3)-glucanase activity, and no other important glucanase activities were noted in all of the media studied. As the principal components of F. oxysporum cell walls are glucans, the results of the positive attack on the cell walls of FOL 2 by the T. versicolor and P. eryngii enzymatic complex demonstrated the contribution of glucanases in the degradation of the hyphal cell walls of F. oxysporum. The lack of cellulase and xylanase activities (acting on plant cell wall polysaccharides) in T. versicolor makes this species a better alternative for the potential control of diseases caused by Fusarium spp.     

Fungal pathogens secrete an inhibitor protein that distinguishes isoforms of plant pathogenesis-related endo-β-1,3-glucanases

Plant endo-β-1,3-glucanases and chitinases inhibit the growth of some fungi and generate elicitor-active oligosaccharides while depolymerizing polysaccharides of mycelial walls. Overexpression of the endo-β-1,3-glucanases and/ or chitinases in transgenic plants provides, in some cases, increased protection against fungal pathogens. However, most of the phytopathogenic fungi that have been tested in vitro are resistant to endo-β-1,3-glucanases and chitinases. Furthermore, some phytopathogenic fungi whose growth is inhibited by these enzymes are able to overcome the effect of these enzymes over a period of hours, indicating an ability of those fungi to adapt to the enzymes. Evidence is presented indicating that fungal pathogens secrete proteins that inhibit selective plant endo-β-1,3-glucanases.A glucanase inhibitor protein (GIP-1) has been purified to homogeneity from the culture fluid of the fungal pathogen of soybeans, Phytophthora sojae f. sp. glycines (Psg), and two basic pathogenesis-related endo-β-1,3-glucanases (EnGL_soy-A and EnGL_soy-B) have been purified from soybean seedlings. GIP-1 inhibits EnGL_soy-A but not EnGL_soy-B. Moreover, GIP-1 does not inhibit endo-β-1,3-glucanases secreted by Psg itself nor does GIP-1 inhibit PR-2c, a pathogenesis-related endo-β-1,3-glucanase of tobacco. Evidence is presented that Psg secretes other GIPs that inhibit other endo-β-1,3-glucanase(s) of soybean. Furthermore, GIP-1 does not exhibit proteolytic activity but does appear to physically bind to EnGL_soy-A. The results reported herein demonstrate specific interactions between gene products of the host and pathogen and establish the need to consider fungal proteins that inhibit plant endo-β-1,3-glucanases when attempting to use the genes encoding endo-β-1,3-glucanases to engineer resistance to fungi in transgenic plants.     

Gene Cloning and Heterologous Expression of Glycoside Hydrolase Family 55 β-1,3-Glucanase from the Basidiomycete Phanerochaete Chrysosporium

The basidiomycete Phanerochaete chrysosporium produces several β-1,3-glucanases when grown on laminarin, a β-1,3/1,6-glucan, as the sole carbon source. To characterize one of the major unknown β-1, 3-glucanases with a molecular mass of 83 kDa, identification, cloning, and heterologous over-expression were carried out using the total genomic information of P. chrysosporium. The cDNA encoding this enzyme included an ORF of 2337 bp and the deduced amino acid sequence contains a predicted signal peptide of 26 amino acids and the mature protein of 752 amino acids. The amino acid sequence showed a significant similarity with glycoside hydrolase family 55 enzymes from filamentous fungi and was named Lam55A. Since the recombinant Lam55A expressed in the methylotrophic yeast Pichia pastoris degraded branched β-1,3/1,6-glucan as well as linear β-1,3-glucan, the kinetic features of the enzyme were compared with those of other β-1,3-glucanases.     

In vitro antagonism of Thielaviopsis paradoxa by Trichoderma longibrachiatum

Seventy-nine Trichoderma strains were isolated from soil taken from 28 commercial plantations of Agave tequilana cv. ‘Azul’ in the State of Jalisco, Mexico. Nine of these isolates produced nonvolatile metabolites that completely inhibited the growth of Thielaviopsis paradoxa on potato dextrose agar plates. These isolates were identified as Trichoderma longibrachiatum on the basis of their morphology and DNA sequence analysis of two genes (ITS rDNA and translation elongation factor EF-1α). Mycoparasitism of Th. paradoxa by T. longibrachiatum strains in dual cultures was examined by scanning electron microscopy. The Trichoderma hyphae grew alongside the Th. paradoxa hyphae, but penetration of Thielaviopsis hyphae by Trichoderma was no apparent. Aleurioconidia of Th. paradoxa were parasitized by Trichoderma. Both hyphae and aleurioconidia of Th. paradoxa lost turgor pressure, wrinkled, collapsed and finally disintegrated. In liquid cultures, all nine Trichoderma isolates produced proteases, β-1,3-glucanases and chitinases that would be responsible for the degradation of Thielaviopsis hyphae. These results demonstrate that the modes of action of T. longibrachiatum involved against Th. paradoxa in vitro experiments are mycoparasitism and the production of nonvolatile toxic metabolites.     

Fumonisin B1, a toxin produced by <Emphasis Type="Italic">Fusarium verticillioides</Emphasis>, modulates maize β-1,3-glucanase activities involved in defense response

Fusarium verticillioides is an important pathogen in maize that causes various diseases affecting all stages of plant development worldwide. The fungal pathogen could be seed borne or survive in soil and penetrate the germinating seed. Most F. verticillioides strains produce fumonisins, which are of concern because of their toxicity to animals and possibly humans, and because they enhance virulence against seedlings of some maize genotypes. In this work, we studied the action of fumonisin B1 (FB1) on the activity of maize β-1,3-glucanases involved in plant defense response. In maize embryos, FB1 induced an acidic isoform while suppressing the activity of two basic isoforms. This acidic isoform was induced also with 2,6-dichloroisonicotinic acid, an analog of salicylic acid. Repression of the basic isoforms suggested a direct interaction of the enzymes with the mycotoxin as in vitro experiments showed that pure FB1 inhibited the basic β-1,3-glucanases with an IC₅₀ of 53 μM. When germinating maize embryos were inoculated with F. verticillioides the same dual effect on β-1,3-glucanase activities that we observed with the pure toxin was reproduced. Similar levels of FB1 were recovered at 24 h germination in maize tissue when they were treated with pure FB1 or inoculated with an FB1-producing strain. These results suggest that β-1,3-glucanases are a relevant physiological target and their modulation by FB1 might contribute to F. verticillioides colonization.     

<Emphasis Type="Italic">Mucor rouxii</Emphasis> Rho1 protein; characterization and possible role in polarized growth

We have previously shown that protein kinase A of the medically important zygomycete Mucor rouxii participates in fungal morphology through cytoskeletal organization. As a first step towards finding the link between protein kinase A and cytoskeletal organization we here demonstrate the cloning of the Rho1 gene and the characterization of its protein product. The RHO1 protein primary sequence shows 70–85% identity with fungal RHO1 or mammalian RhoA. Two protein kinase A phosphorylation sequences in adequate context are predicted, Ser73 and Ser135. The peptide IRRNSQKFV, containing Ser135 proved to be a good substrate for M. rouxii protein kinase A catalytic subunit. The over-expressed Rho1 fully complements a Saccharomyces cerevisiae null mutant. The endogenous protein was identified by western blot against a developed antibody and by ADP-ribosylation. Localization in germlings was visualized by immunofluorescence; the protein was localized in patches in the mother cell surface and excluded from the germ tube. Measurement of Rho1 expression during germination indicates that Rho1, at both the mRNA and protein levels, correlates with differentiation and not with growth. Rho1 has been shown to be the regulatory protein of the β-1,3-glucan synthase complex in fungi in which β-1,3-glucans are major components of the cell wall. Even though glucans have not been detected in zygomycetes, caspofungin, an echinochandin known to be an inhibitor of β-1,3-glucan synthase complex, is shown here to have a negative effect on growth and to produce an alteration on morphology when added to M. rouxii growth culture medium. This result has an important impact on the possible participation of β-1,3-glucans on the regulation of morphology of zygomycetes.     

A simple method for protoplast formation and improvement of protoplast regeneration from various fungi using an enzyme from Trichoderma harzianum

Summary An enzyme from Trichoderma harzianum dissolved the cell walls of a wide range of filamentous fungi belonging to Basidiomycotina, Ascomycotina, Deuteromycotina, and Zygomycotina and so could be used to make protoplasts. A lyophilized preparation of the Trichoderma enzyme had about 0.3 units/mg -1,3-glucanase activity and 0.36 units/mg chitinase activity. About twice as many protoplasts were produced from different species of fungi by a single treatment with this enzyme than with combined commercial enzymes. The greatest number of protoplasts could be produced from most of the fungi by incubation for about 2 h t 30°C, but the number was decreased by incubation for more than 4 h or by use of a higher dose of the enzyme. An enzyme prepared by bentonite treatment from the original Trichoderma enzyme had less proteinase activity and protoplasts were fairly stable with this product during incubation for 8 h. Protoplasts produced by the proteinase-reduced preparation of the Trichoderma enzyme from three fungi regenerated at about 1.8 times the rate of those produced by the original enzyme.     

Purification and characterization of a β-1,3-glucanase from the novel mycoparasite <Emphasis Type="Italic">Periconia byssoides</Emphasis>

An extracellular β-1,3-glucanase with antifungal properties was secreted by the novel mycoparasite, Periconia byssoides. The glucanase has a molecular mass of 35 kDa estimated by SDS-PAGE. Its optimum activity was at pH 6.0 and 50°C (over 2 h). The purified β-1,3-glucanase was capable of degrading cell walls, and inhibiting mycelia growth and spore germination of plant pathogenic fungi including Fulvia fulva, Fusarium sp. and Rhizoctonia solani. The N-terminal amino acid residues of the purified β-1,3-glucanase are LKNGGPSFGA, which do not have any homology with previously described glucanases, suggesting it may be a novel member of the fungal β-1,3-glucanases. Chao Lin and Jinkui Yang contributed equally to this work.