Production of Chitinases and β-1,3-Glucanases by Stachybotrys elegans, a Mycoparasite of Rhizoctonia solani

The in vitro production of chitinases and β-1,3-glucanases by Stachybotrys elegans, a mycoparasite of Rhizoctonia solani, was examined under various culture conditions, such as carbon and nitrogen sources, pH, and incubation period. Production of both enzymes was influenced by the carbon source incorporated into the medium and was stimulated by acidic pH and NaNO₃. The activity of both enzymes was very low in culture filtrates from cells grown on glucose and sucrose compared with that detected on chitin (for chitinases) and cell wall fragments (for β-1,3-glucanases). Protein electrophoresis revealed that, depending on the carbon source used, different isoforms of chitinases and β-1,3-glucanases were detected. S. elegans culture filtrates, possessing β-1,3-glucanase and chitinase activities, were capable of degrading R. solani mycelium.     

Induced hydrolytic enzymes of ectomycorrhizal fungi against pathogen Rhizoctonia solani

Interactions between ectomycorrhizal fungi (Suillus laricinus, S. tomentosus, Amanita vaginata and Gomphidius viscidus) and the pathogen Rhizoctonia solani in co-culture were studied using both light and scanning electron microscopy. S. laricinus, S. tomentosus and A. vaginata inhibited the growth of the pathogen. Moreover, A. vaginata exhibited coiling around and penetration of the hyphae into R. solani was observed in the interaction zone. Furthermore, the production of chitinases, beta-1,3-glucanases and beta-glucosidases by these ectomycorrhizal fungi on colloidal chitin or cell walls of R. solani was evaluated: chitinases were not induced by colloidal chitin but all three enzymes were induced by R. solani cell walls. No correlation between inhibition rate and production of lytic enzymes was found.     

Role of chitinase and beta-1,3-glucanase activities produced by a fluorescent pseudomonad and in vitro inhibition of Phytophthora capsici and Rhizoctonia solani

A study was conducted to investigate the possibility of involvement of chitinase and beta-1,3-glucanase of an antagonistic fluorescent Pseudomonas in growth suppression of phytopathogenic fungi, Phytophthora capsici and Rhizoctonia solani. Fluorescent Pseudomonas isolates GRC(3) and GRC(4) were screened for their antifungal potential against phytopathogenic fungi by using dual culture technique both on solid and liquid media. The percent inhibition was calculated. Various parameters were monitored for optimization of enzyme activities by fluorescent Pseudomonas GRC(3). The involvement of chitinases, beta-1,3-glucanases, and antifungal metabolites of nonenzymatic nature was correlated with the inhibition of P. capsici and R. solani. The results provide evidence for antibiosis as a mechanism for antagonism. The study also confirms that multiple mechanisms are involved in suppressing phytopathogens as evidenced by the involvement of chitinase and beta-1,3-glucanase in inhibition of R. solani but not P. capsici by isolate GRC3.     

Regulation of 36-kDa beta-1,3-glucanase synthesis in Trichoderma harzianum

The effect of carbon sources on the level of beta-1,3-glucanases in the culture filtrates of Trichoderma harzianum (Tc) was investigated. Enzyme activity was detected in all carbon sources, but highest levels were found when laminarin and purified cell walls were used. Three isoforms of beta-1,3-glucanase were produced during growth of the fungus on purified cell walls. Two isoforms were produced on chitin, chitosan, N-acetylglucosamine and laminarin, while only one was detected when the fungus was grown on cellulose and glucose. A 36-kDa beta-1,3-glucanase (GLU36) was secreted from T. harzianum (Tc) grown on all carbon sources tested as demonstrated by Western blot analysis. We found that a significant increase in the level of GLU36 in the culture filtrate follows glucose exhaustion, suggesting that this enzyme is controlled by carbon catabolite repression.     

Purification and characterization of an endo-beta-1,6-glucanase from Trichoderma harzianum that is related to its mycoparasitism.

The enzymes from Trichoderma species that degrade fungal cell walls have been suggested to play an important role in mycoparasitic action against fungal plant pathogens. The mycoparasite Trichoderma harzianum produces at least two extracellular beta-1,6-glucanases, among other hydrolases, when it is grown on chitin as the sole carbon source. One of these extracellular enzymes was purified to homogeneity after adsorption to its substrate, pustulan, chromatofocusing, and, finally, gel filtration. The apparent molecular mass was 43,000, and the isoelectric point was 5.8. The first 15 amino acids from the N terminus of the purified protein have been sequenced. The enzyme was specific for beta-1,6 linkages and showed an endolytic mode of action on pustulan. Further characterization indicated that the enzyme by itself releases soluble sugars and produces hydrolytic halli on yeast cell walls. When combined with other T. harzianum cell wall-degrading enzymes such as beta-1,3-glucanases and chitinases, it hydrolyzes filamentous fungal cell walls. The enzyme acts cooperatively with the latter enzymes, inhibiting the growth of the fungi tested. Antibodies against the purified protein also indicated that the two identified beta-1,6-glucanases are not immunologically related and are probably encoded by two different genes.     

Isolation of extracellular 28- and 42-kilodalton beta-1,3-glucanases and comparison of three beta-1,3-glucanases produced by Bacillus circulans IAM1165.

Bacillus circulans IAM1165 produces three major extracellular beta-1,3-glucanases (molecular masses, 28, 42, and 91 kDa) during the stationary phase of growth. The 28- and 42-kDa enzymes were purified to homogeneity from the culture supernatant in this study. The properties of these two enzymes were examined, together with those of the 91-kDa enzyme previously isolated. The enzymatic properties of the 28- and 42-kDa beta-1,3-glucanases closely resemble each other. The enzymes belong to a category of endo type 1,3-beta-D-glucan glucanohydrolases. The enzymes were active at pH 4.0 to 7.0. The optimum temperature of the reactions was 60 degrees C when laminarin (a soluble beta-1,3-glucan) was used as the substrate at pH 7.0. The enzymes hydrolyzed barley glucan and lichenan (beta-1,3-1,4-glucans) more effectively than laminarin. Of the three enzymes, the 42-kDa enzyme lysed fungal cell walls the most effectively.     

Enzymes of carbohydrate metabolism of mycelial fungi from marine environments. beta-1,3-glucanase of the marine fungus Chaetomium indicum

Thirty samples of fungi belonging to 17 species living in marine environments were studied for their ability to produce extracellular enzymes. In the culture fluids, a variety of glycosidases (beta-glucosidases, N-acetyl-beta-glucosaminidase, beta-galactosidases, and alpha-mannosidases) and glucanases (amylases and beta-1,3-glucanases) were found. Several cultures were found that could be used as efficient producers of either individual enzymes or a whole complement of enzymes degrading carbohydrate-containing compounds. Optimal growth conditions for the fungus Chaetomium indicum and beta-1,3-glucanase biosynthesis were developed. beta-1,3-Glucanase was isolated by a combination of ion-exchange chromatography, ultrafiltration, and gel chromatography. The molecular mass of the enzyme determined by gel-filtration was 54 kD. The enzyme was stable at temperatures below 50 degrees C, had a temperature optimum for activity at 60 degrees C, and retained activity between pH 4.5 and 7.5. The pH dependence of the beta-1, 3-glucanase activity showed two maxima, at pH 4.4 and 5.6; this suggested the existence of two forms of the enzyme. Analysis of the products of enzymatic hydrolysis of laminaran, transglycosylating ability, and the effect of a specific natural inhibitor indicates that both forms are exo-beta-1,3-glucanases.     

Extracellular hydrolases of strain Bacillus sp. 739 and their involvement in the lysis of micromycete cell walls

The mycolytic bacterial strain Bacillus sp. 739 produces extracellular enzymes which degrade in vitro the cell walls of a number of phytopathogenic and saprophytic fungi. When Bacillus sp. 739 was cultivated with Bipolaris sorokiniana, a cereal root-rot pathogen, the fungus degradation process correlated with the levels of the beta-1,3-glucanase and protease activity. The comparative characteristic of Bacillus sp. 739 enzymatic preparations showed that efficient hydrolysis of the fungus cell walls was the result of the action of the complex of enzymes produced by the strain when grown on chitin-containing media. Among the enzymes of this complex, chitinases and beta-1,3-glucanases hydrolyzed most actively the disintegrated cell walls of B. sorokiniana. However, only beta-1,3-glucanases were able to degrade the cell walls of native fungal mycelium in the absence of other hydrolases, which is indicative of their key role in the mycolytic activity of Bacillus sp. 739.     

Only Specific Tobacco (Nicotiana tabacum) Chitinases and [beta]-1,3-Glucanases Exhibit Antifungal Activity

Different isoforms of chitinases and [beta]-1,3-glucanases of tobacco (Nicotiana tabacum cv Samsun NN) were tested for their antifungal activities. The class I, vacuolar chitinase and [beta]-1,3-glucanase isoforms were the most active against Fusarium solani germlings, resulting in lysis of the hyphal tips and in growth inhibition. In additon, we observed that the class I chitinase and [beta]-1,3-glucanase acted synergistically. The class II isoforms of the two hydrolases exhibited no antifungal activity. However, the class II chitinases showed limited growth inhibitory activity in combination with higher amounts of class I [beta]-1,3-glucanase. The class II [beta]-1,3-glucanases showed no inhibitory activity in any combination. In transgenic tobacco plants producing modified forms of either a class I chitinase or a class I [beta]-1,3-glucanase, or both, these proteins were targeted extracellularly. Both modified proteins lack their C-terminal propeptide, which functions as a vacuolar targeting signal. Extracellular targeting had no effect on the specific activities of the chitinase and [beta]-1,3-glucanase enzymes. Furthermore, the extracellular washing fluid (EF) from leaves of transgenic plants expressing either of the secreted class I enzymes exhibited antifungal activity on F. solani germlings in vitro comparable to that of the purified vacuolar class I proteins. Mixing EF fractions from these plants revealed synergism in inhibitory activity against F. solani; the mixed fractions exhibited inhibitory activity similar to that of EF from plants expressing both secreted enzymes.     

Molecular characterization and expression in Escherichia coli of three beta-1,3-glucanase genes from Lysobacter enzymogenes strain N4-7

Lysobacter enzymogenes strain N4-7 produces multiple biochemically distinct extracellular beta-1,3-glucanase activities. The gluA, gluB, and gluC genes, encoding enzymes with beta-1,3-glucanase activity, were identified by a reverse-genetics approach following internal amino acid sequence determination of beta-1,3-glucanase-active proteins partially purified from culture filtrates of strain N4-7. Analysis of gluA and gluC gene products indicates that they are members of family 16 glycoside hydrolases that have significant sequence identity to each other throughout the catalytic domain but that differ structurally by the presence of a family 6 carbohydrate-binding domain within the gluC product. Analysis of the gluB gene product indicates that it is a member of family 64 glycoside hydrolases. Expression of each gene in Escherichia coli resulted in the production of proteins with beta-1,3-glucanase activity. Biochemical analyses of the recombinant enzymes indicate that GluA and GluC exhibit maximal activity at pH 4.5 and 45 degrees C and that GluB is most active between pH 4.5 and 5.0 at 41 degrees C. Activity of recombinant proteins against various beta-1,3 glucan substrates indicates that GluA and GluC are most active against linear beta-1,3 glucans, while GluB is most active against the insoluble beta-1,3 glucan substrate zymosan A. These data suggest that the contribution of beta-1,3-glucanases to the biocontrol activity of L. enzymogenes may be due to complementary activities of these enzymes in the hydrolysis of beta-1,3 glucans from fungal cell walls.     

Cell-lytic activity of tobacco BY-2 induced by a fungal elicitor from alternaria alternata attributed to the expression of a class I beta-1,3-glucanase gene

Stress-induced cell-lytic activity was found in tobacco BY-2 cells treated with various stresses. Among 14 stresses, an elicitor fraction isolated from Alternaria alternata showed the highest inducing activity. Cell-lytic activity increased for 72 h even in the control sample, treated with distilled water, and several isozymes of beta-1,3-glucanases and chitinases were found to be involved in it. In contrast, cell-lytic activity in BY-2 cells treated with a fungal elicitor reached a higher level after 60 h. The principal enzymes specifically involved in this stress-induced portion are speculated to be basic beta-1,3-glucanases. A class I beta-1,3-glucanase gene (glu1) was found to be the specific gene for the stress-induced cell-lytic activity. Its expression became observable at 24 h, and the intensity reached a maximum at about 60-72 h. The glu1 was thus assigned as a late gene. Its role in the stress response is discussed in conjunction with earlier genes such as chitinases.     

Purification and characterization of an exo-beta-1,3-glucanase produced by Trichoderma asperellum

Trichoderma asperellum produces at least two extracellular beta-1,3-glucanases upon induction with cell walls from Rhizoctonia solani. A beta-1,3-glucanase was purified by gel filtration and ion exchange chromatography. A typical procedure provided 35.7-fold purification with 9.5% yield. The molecular mass of the purified exo-beta-1,3-glucanases was 83.1 kDa as estimated using a 12% (w/v) SDS-electrophoresis slab gel. The enzyme was only active toward glucans containing beta-1,3-linkages and hydrolyzed laminarin in an exo-like fashion to form glucose. The K(m) and V(max) values for exo-beta-1,3-glucanase, using laminarin as substrate, were 0.087 mg ml(-1) and 0.246 U min(-1), respectively. The pH optimum for the enzyme was pH 5.1 and maximum activity was obtained at 55 degrees C. Hg(2+) strongly inhibited the purified enzyme.     

Analysis of the beta-1,3-Glucanolytic System of the Biocontrol Agent Trichoderma harzianum

The biocontrol agent Trichoderma harzianum IMI206040 secretes beta-1,3-glucanases in the presence of different glucose polymers and fungal cell walls. The level of beta-1,3-glucanase activity secreted was found to be proportional to the amount of glucan present in the inducer. The fungus produces at least seven extracellular beta-1,3-glucanases upon induction with laminarin, a soluble beta-1,3-glucan. The molecular weights of five of these enzymes fall in the range from 60,000 to 80,000, and their pIs are 5.0 to 6.8. In addition, a 35-kDa protein with a pI of 5.5 and a 39-kDa protein are also secreted. Glucose appears to inhibit the formation of all of the inducible beta-1,3-glucanases detected. A 77-kDa glucanase was partially purified from the laminarin culture filtrate. This enzyme is glycosylated and belongs to the exo-beta-1,3-glucanase group. The properties of this complex group of enzymes suggest that the enzymes might play different roles in host cell wall lysis during mycoparasitism.     

Purification and characterization of two basic beta-1,3-glucanases induced in Colletotrichum lindemuthianum-infected bean seedlings.

Two beta-1,3-glucanases which are rapidly induced in the incompatible interaction between bean (cv. Processor) and Colletotrichum lindemuthianum race beta were purified to homogeneity. Characterization of the two enzymes, GE1 and GE2, showed that they both had a basic isolectric point and a similar molecular weight (36,500 for GE1 and 36,000 for GE2), but differed in their pH optimum, thermal stability, and specific activity. GE2 was present in higher amounts but was shown to be less active than GE1 against laminarin and fungal cell walls isolated from race beta of the fungus. Both enzymes were specific for beta-1,3 linkages and showed a strict endolytic mode of action. Further characterization of GE2 was achieved by amino acid sequence analysis of tryptic peptides; the degree of homology shared with other basic beta-1,3-glucanases depended on the plant source. A time-course study showed that GE1 and GE2 were increased during infection. They were also induced by fungal elicitors, thereby indicating that they originate from the host.     

Derepression of beta-1,3-glucanases in Penicillium italicum: localization of the various enzymes and correlation with cell wall glucan mobilization and autolysis.

The localization of the derepressible beta-1,3-glucanases of Penicillium italicum and the cell wall autolysis under conditions of beta-1,3-glucanase derepression (24 h in a low-glucose medium) were studied. About 15% of the total activity was secreted into the culture medium during the 24-h period and consisted of similar amounts of each of the three beta-1,3-glucanases (I, II, III) produced by this species. Treatment of derepressed mycelia with periplasmic enzyme-inactivating agents resulted in a loss of 45% of the mycelium-bound beta-1,3-glucanase. Analysis of periplasmic enzymes solubilized by 2 M NaCl or by autolysis of isolated cell walls revealed that only beta-1,3-glucanases II and III were bound to the cell wall. These two enzymes were capable of releasing in vitro reducing sugars from cell walls, whereas beta-1,3-glucanase I was not. In addition, the autolytic activity of cell walls isolated from derepressed mycelium was greater than that of cell walls isolated from repressed mycelium. The incubation of the fungus in the low-glucose medium also resulted in the in vivo mobilization of 34% of the cell wall beta-1,3-glucan, and this mobilization was fully prevented by cycloheximide, which also blocked derepression of beta-1,3-glucanases. Derepression of beta-1,3-glucanase seems to be coupled to the mobilization of cell wall glucan.     

Regulation of the beta-1,3-glucanase system in Penicillium italicum: glucose repression of the various enzymes.

The microscopic fungus Penicillium italicum when grown in a synthetic liquid medium produced at least three enzymes with beta-1,3-glucanase activity which were separated by diethylaminoethyl-Sephadex column chromatography. These were named beta-1,3-glucanases I, II, and III respective to their order of elution from the column. A tentative characterization of these three enzymes indicated that they have different modes of action; the first one is an endoglucanase, the second is an exoglucanase, and the third probably has both mechanisms of action. Glucose had a repressive effect on all three enzymes. Only small amounts of beta-1,3-glucanases II and III were present in the cells when they were actively growing in the presence of this sugar. However, when the cells were transferred to a medium low in glucose, a significant increase in the specific activity of beta-1,3-glucanase took place; this was due in part to a much more active production of beta-1,3-glucanases II and III and in part to the appearance of beta-1,3-glucanase I, which could only be detected after more than 12 h of incubation in this medium. The results are discussed in the context of possible beta-1,3-glucanase functions in the fungal cells.     

Comparative degradation of oomycete, ascomycete, and basidiomycete cell walls by mycoparasitic and biocontrol fungi

Fourteen fungi (primarily representing mycoparasitic and biocontrol fungi) were tested for their ability to grow on and degrade cell walls (CWs) of an oomycete (Pythium ultimum), ascomycete (Fusarium equisetii), and basidiomycete (Rhizoctonia solani), and their hydrolytic enzymes were characterized. Protein was detected in the cultural medium of eleven of the test isolates, and these fungi significantly degraded CWs over the 14-day duration of the experiment. In general, a greater level of CW degradation occurred for F. equisetii and P. ultimum than for R. solani. Fungi that degraded F. equisetii CWs were Coniothyrium minitans, Gliocladium roseum, Myrothecium verrucaria, Talaromyces flavus, and Trichoderma harzianum. Taxa degrading P ultimum CWs included Chaetomium globosum, Coniothyrium minitans, M. verrucaria, Seimatosporium sp., Talaromyces flavus, Trichoderma hamatum, Trichoderma harzianum, and Trichoderma viride. Production of extracellular protein was highly correlated with CW degradation. Considerable variation in the molecular weights of CW-degrading enzymes were detected among the test fungi and the CW substrates in zymogram electrophoresis. Multivariate analysis between CW degradation and hydrolysis of barley beta-glucan (beta1,3- and beta1,4-glucanases), laminarin (beta1,3- and beta1,6-glucanases), carboxymethyl cellulose (endo-beta1,4-glucanases), colloidal chitin (chitinases), and chitosan (chitosanases) was conducted. For F. equisetii CWs, the regression model accounted for 80% of the variability, and carboxymethyl cellulases acting together with beta-glucanases contributed an R2 of 0.52, whereas chitinases and beta-glucanases alone contributed an R2 of 0.11 and 0.12, respectively. Only 61% of the variability observed in the degradation of P. ultimum CWs was explained by the enzyme classes tested, and primarily beta-glucanases (R2 of 0.53) and carboxymethyl cellulases (R2 of 0.08) alone contributed to CW break down. Too few of the test fungi degraded R. solani CWs to perform multivariate analysis effectively. This study identified several fungi that degraded ascomyceteous and oomyceteous, and to a lesser extent, basidiomycetous CWs. An array of enzymes were implicated in CW degradation.     

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.     

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.