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.     

Biological Control Activity of Three Trichoderma Isolates Against Fusarium Wilts of Cotton and Muskmelon and Lack of Correlation with their Lytic Enzymes

The enzymatic activity and the biocontrol ability of two new isolates of Trichoderma spp. (T-68 and Gh-2) were compared in laboratory and glasshouse experiments with a previously studied T. harzianum strain (T-35). In dual culture tests with Fusarium oxysporum f. sp. melonis and F. oxysporum f. sp. vasinfectum, isolates T-68 and Gh-2 overgrew the colonies of Fusarium, whereas T-35 failed to parasitize both wilt pathogens. Under glasshouse conditions, the three isolates of Trichoderma were effective in controlling Fusarium wilt of cotton but only T-35 was effective against F. oxysporum f. sp. melonis on muskmelon. When the three Trichoderma isolates were grown on liquid media containing laminarin, colloidal chitin or F. oxysporum f. sp. melonis cell walls as sole carbon sources, maximum β-1,3-glucanase and chitinase specific activity in the culture filtrates of all fungi was reached after 72h of incubation. When culture filtrates of the three Trichoderma isolates were incubated with freeze-dried mycelium of F. oxysporum f. sp. melonis or F. oxysporum f. sp. vasinfectum, different concentrations of glucose and N-acetyl-D-glucosamine were released. Overall no correlation was found between enzymatic activity and the biocontrol capability against Fusarium wilt on muskmelon and cotton.     

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.     

Decay of Sclerotia of Botrytis cinerea by Trichoderma spp. at Low Temperatures

In vitro, tests were conducted at 10°C and 5°C against sclerotia of Botrytis cinerea with 58 isolates of Trichoderma spp., highly antagonistic at 24°C but differing in their cold tolerance. Some isolates macerated and colonized sclerotia even at 5 °C. With 19 isolates of Trichoderma spp. less than 10 % of the sclerotia remained viable after 42 d at 5 °C. Conidia ol some Trichoderma spp. germinated at 5 °C within a few days and reached germination rates higher than 80 %. It seems to be feasible to use selected isolates of Trichoderma spp. for biological control of sclerotia of ß. cinerea also during the colder season.     

Biocontrol activity of salt tolerant Streptomyces isolates against phytopathogens causing root rot of sugar beet

Biological control of fungi causing root rot on sugar beet by native Streptomyces isolates (C and S2) was evaluated in this study. The dry weight and colony forming unit (CFU) of S2 and C increased when 300 mM NaCl was added to medium. The in vitro antagonism assays showed that both isolates had inhibitory effect against Rhizoctonia solani AG-2, Fusarium solani and Phytophthora drechsleri. In dual culture, Streptomyces isolate C inhibited mycelial growth of R. solani, F. solani and P. drechsleri 45%, 53% and 26%, respectively. NaCl treatment of medium increased biocontrol activity of soluble and volatile compounds of isolate C and S2. After salt treatment, growth inhibition of R. solani, F. solani and P. drechsleri by isolate C increased up to 59%, 70% and 79%, respectively. To elucidate the mode of antagonism, protease, chitinase, beta glucanase, cellulase, lipase and α-amylase activity and siderophore and salicylic acid (SA) production were evaluated. Both isolates showed protease, chitinase and α-amylase activity. Also, biosynthesis of siderophore was detectable for both isolates. Production of siderophore and activity of protease and α-amylase increased after adding salt for both isolates. In contrast, chitinase activity decreased significantly. Production of SA, beta glucanase and lipase by isolate S2 and biosynthesis of cellulase by isolate C were observed in presence and absence of NaCl. Soil treatment with Streptomyces isolate C inhibited root rot of sugar beet caused by P. drechsleri, R. solani and F. solani. Results of this study showed that these two Streptomyces isolates had potential to be utilized as biocontrol agent against fungal diseases especially in saline soils.     

Differential expression of cellulases and xylanases by <Emphasis Type="Italic">Cellulomonas flavigena</Emphasis> grown on different carbon sources

The diversity of cellulases and xylanases secreted by Cellulomonas flavigena cultured on sugar cane bagasse, Solka-floc, xylan, or glucose was explored by two-dimensional gel electrophoresis. C. flavigena produced the largest variety of cellulases and xylanases on sugar cane bagasse. Multiple extracellular proteins were expressed with these growth substrates, and a limited set of them coincided in all substrates. Thirteen proteins with carboxymethyl cellulase or xylanase activity were liquid chromatography/mass spectrometry sequenced. Proteins SP4 and SP18 were identified as products of celA and celB genes, respectively, while SP20 and SP33 were isoforms of the bifunctional cellulase/xylanase Cxo recently sequenced and characterized in C. flavigena. The rest of the detected proteins were unknown enzymes with either carboxymethyl cellulase or xylanase activities. All proteins aligned with glycosyl hydrolases listed in National Center for Biotechnology Information database, mainly with cellulase and xylanase enzymes. One of these unknown enzymes, protein SP6, was cross-induced by sugar cane bagasse, Solka-floc, and xylan. The differences in the expression maps of the presently induced cultures revealed that C. flavigena produces and secretes multiple enzymes to use a wide range of lignocellulosic substrates as carbon sources. The expression of these proteins depends on the nature of the cellulosic substrate.     

Apparent cellulase activity of purified xylanase is due to contamination of assay substrate with xylan

Summary Purified xylanase A ofTrichoderma longibrachiatum was active on one of two carboxymethyl cellulose (CMC) preparations used as cellulase assay substrates. The pattern of enzyme activity, and analysis of the substrate by acid hydrolysis and thin-layer chromatography (TLC) suggested that the enzyme had acted on xylan present in the CMC.     

Transmission of the mycoparasite Coniothyrium minitans by collembolan Folsomia Candida (Collembola: Entomobryidae) and glasshouse sciarid Bradysia sp. (Diptera: Sciaridae)

Folsomia Candida was maintained on potato dextrose agar (PDA) plates precolonised by the mycoparasite Coniothyrium minitans for 3 yr but the sciarid Bradysia sp. survived for a maximum of only three generations. Collembolans and sciarid larvae from these cultures were able to transmit C. minitans to uninoculated PDA plates through the survival of spores in faecal pellets. Adult and larval sciarids also transmitted C. minitans from PDA culture to uninoculated PDA plates by contamination on the cuticle. In soil and sand both sciarids and collembolans were able to transmit C. minitans from C. m/m'tans-inoculated to uninoculated sclerotia of Sclerotinia sclerotiorum. Inoculation of sclerotia with C. minitans enabled greater populations of larger collembolans to develop. In the glasshouse where C. minitans had been applied to the soil, one adult sciarid and four collembolans out of 70 and 101 insects collected respectively yielded C. minitans on placement onto PDA + Aureomycin.     

Cellulolytic Microbes in the Yanbaru,a Subtropical Rainforest with an Endemic Biota on Okinawa Island,Japan

Cellulolytic microbes in the soil of the Yanbaru, a subtropical forest with an endemic biota, on Okinawa Island, were isolated and characterized in a search for novel microbial strains with biotechnological potential. Soil samples of the Yanbaru were suspended in sterilized water, inoculated on mineral salt agar overlaid with a filter paper as carbon source, and cultivated aerobically at 30 °C. After 2 weeks of cultivation, emerging colonies were isolated and subjected to phylogenetic and enzyme analyses. The phylogenetic analyses revealed bacterial and fungal isolates belonging to nine and three genera respectively. All isolates possessed cellulase activity, and several strains showed strong activity comparable to Trichoderma cellulase. Many isolates also exhibited xylanase activity.     

Potential use of Trichoderma asperellum (Samuels,Liechfeldt et Nirenberg) T8a as a biological control agent against anthracnose in mango (Mangifera indica L.)

Twenty isolates of Trichoderma were obtained from orchards located in three main mango-producing States in Mexico: Chiapas, Oaxaca, and Michoacan, which represent different agronomical management practices and levels of soil fertility. Phylogenetic analysis showed that Trichoderma isolates belong to the following taxa: Hypocrea lixii (10 isolates), Hypocrea jecorina (four isolates), Trichoderma asperellum (three isolates), Trichoderma spirale (two isolates), and Trichoderma brevicompactum (one isolate). The genus Hypocrea is the teleomorph (sexual) stage of the genus Trichoderma, anamorph stage. Seventeen Trichoderma isolates showed at least 67% growth inhibition against the phytopathogenic fungus Colletotrichum gloeosporioides ATCC MYA 456 and three Trichoderma isolates showed complete overgrowth of this pathogen. One member of this group, identified as T. asperellum T8a, was able to control C. gloeosporioides ATCC MYA 456 in vitro and in vivo, as well as five C. gloeosporioides isolates obtained from mango orchards from the State of Oaxaca. Assay of the lytic enzymes involved suggest that cellulases of T. asperellum T8a play a role in biological control against C. gloeosporioides ATCC MYA 456 more than chitinase or glucanase. Thus, native T. asperellum T8a associated with mango trees can be used to enhance mango production, controlling anthracnose through cellulase activity.     

Some environmental factors affect growth and antibiotic production by the mycoparasite Coniothyrium minitans

The effects of temperature and pH on growth and antibiotic production by three isolates of Coniothyrium minitans (Conio, Contans and IVT1), known to produce the macrolide antibiotic macrosphelide A, were examined in modified Czapek Dox broth (MCD). Antibiotic production was determined by incorporating heated (60°C for 5 min) C. minitans spent culture filtrates of MCD (10%, v/v) into potato dextrose broth and assessing the ability of the filtrates to inhibit growth of S. sclerotiorum. All isolates grew over the temperature range of 10–30°C, with the optimum at approximately 15–20°C. Antibiotics were produced by all isolates at 10–30°C. Culture filtrates of MCD from all isolates incorporated into PDB inhibited growth of S. sclerotiorum by >50%, whereas there was a reduction in inhibition at 30°C for Conio and IVT1 but not Contans. All three isolates grew over the pH range of 3–7, with greater biomass production in buffered pH 3–5 than the unbuffered control (pH 4.8) media. Antibiotics were produced by all isolates at pH 3–5. Culture filtrates of MCD from all three isolates grown at pH 3–5 inhibited growth of S. sclerotiorum, with the greatest effect on inhibition observed at pH 3. There were no differences in growth inhibition between isolates at pH 3 and 4, but culture filtrates from Conio grown at pH 5 inhibited S. sclerotiorum more than those of IVT1 grown at the same pH. The significance of these results for biocontrol and optimizing antibiotic production by C. minitans is discussed.     

A new xylanase from a Trichoderma harzianum strain

A new xylanase (XYL2) was purified from solid-state cultures of Trichoderma harzianum strain C by ultrafiltration and gel filtration. SDS-PAGE of the xylanase showed an apparent homogeneity and molecular weight of 18 kDa. It had the highest activity at pH 5.0 and 45°C and was stable at 50°C and pH 5.0 up to 4 h xylanase. XYL2 had a low K _m with insoluble oat spelt xylan as substrate. Compared to the amino acid composition of xylanases from Trichoderma spp, xylanase XYL2 presented a high content of glutamate/glutamine, phenylalanine and cysteine, and a low content of serine. Xylanase XYL2 improved the delignification and selectivity of unbleached hardwood kraft pulp. Received 02 February 1999/ Accepted in revised form 17 April 1999     

Inheritance of antagonistic properties and lytic enzyme activities in sexual crosses of Talaromyces flavus

Two wild-type strains and three benomyl-resistant mutants of the antagonistic ascomycete Talaromyces flavus were crossed in six combinations, two of which yielded hybrid cleistothecia. Parental strains and their ascospore progenies varied in several traits considered to play an important role in the capacity to control soilborne fungal pathogens: extracellular activities of glucose oxidase and cell-wall degrading enzymes, antibiosis towards Verticillium dahliae, and parasitism and biocontrol of Sclerotium rolfsii. A non-Mendelian quantitative mode of inheritance was found for β-1, 3-glucanase and chitinase activities but only the latter exhibited a normal frequency distribution. Some of the progenies exhibited higher glucanase and chitinase activities than those found in the parental strains. Progeny analysis for chitinase, glucanase, cellulase, and glucose oxidase activities revealed no genetic association between any two of these enzymes. Antibiosis was correlated with glucose-oxidase activity in one cross, but not in the other. The ability to reduce bean root rot caused by S. rolfsii was correlated with mycoparasitic activity against S. rolfsii sclerotia in one cross, but not in the other. One out of the 20 progenies tested was able to reduce bean root rot more effectively than its parent strains, thus demonstrating the feasibility of improving a biocontrol agent by conventional breeding.     

Degradation of oxalic acid by the mycoparasite Coniothyrium minitans plays an important role in interacting with Sclerotinia sclerotiorum

Coniothyrium minitans (Cm) is a mycoparasite of the phytopathogenic fungus Sclerotinia sclerotiorum (Ss). Ss produces a virulence factor oxalic acid (OA) which is toxic to plants and also to Cm, and Cm detoxifies OA by degradation. In this study, two oxalate decarboxylase genes, Cmoxdc1 and Cmoxdc2, were cloned from Cm strain Chy‐1. OA and low pH induced expression of Cmoxdc1, but not Cmoxdc2. Cmoxdc1 was partially responsible for OA degradation, whereas Cmoxdc2 had no effect on OA degradation. Disruption of Cmoxdc1 in Cm reduced its ability to infect Ss in dual cultures where OA accumulated. Compared with Chy‐1, the Cmoxdc1‐disrupted mutants had reduced expression levels of two mycoparasitism‐related genes chitinase (Cmch1) and β‐1,3‐glucanase (Cmg1), and had no detectable activity of extracellular proteases in the presence of OA. On the other hand, the cultural filtrates of the Cmoxdc1‐disrupted mutants in OA‐amended media showed enhanced antifungal activity, possibly because of increased production of antifungal substances under acidic pH condition resulted from reduced Cmoxdc1‐mediated OA degradation. This study provides direct genetic evidence of OA degradation regulating mycoparasitism and antibiosis of Cm against Ss, and sheds light on the sophisticated strategies of Cm in interacting with metabolically active mycelia and dormant sclerotia of Ss.     

Production of thermostable xylanase by a thermophilic fungus,Thermoascus aurantiacus

Twenty-one strains of thermophilic fungi in the Forintek culture collection were screened for their production of xylanolytic (and cellulolytic) enzymes in both solid and aqueous media containing various hemicellulosic and cellulosic substrates. Thermoascus aurantiacus strain C436 was selected as the best producer of extracellular xylanase (1,4-β-d-xylan xylanohydrolase, EC 3.2.1.8) enzymes. High xylanase activity was detected in fungal culture filtrates even when realistic lignocellulosic residues (including steam-exploded aspenwood and untreated aspenwood sawdust) were used as substrates. Maximum xylanase activity (575.9 U ml⁻¹) was detected in cultures grown in Vogel's medium containing oat-spelt xylan. The xylanase activity exhibited a temperature optimum of 75°C and pH optimum around 5.0. The half-lives of the xylanase activity at 70 and 60°C were 1.5 h and 4 days, respectively. Over 90% of the xylanase activity was retained after 12 weeks at 50°C. Crude culture filtrates concentrated by membrane ultrafiltration could effectively hydrolyse xylan and steam-exploded aspenwood hemicellulose to release near theoretical yields of low molecular weight pentose oligomers.     

Xylanase production by Trichoderma harzianum E58

Summary Growth of Trichoderma harzianum E58 on hemicellulose-rich media, both in batch and fermentor cultures, resulted in independent profiles for the production of xylanase and endoglucanase enzymes. Dramatic differences in the ratio of xylanase to endoglucanase activities were observed among cultures grown on cellulose-rich Solka Floc and xylan. These results indicated that the induction of xylanases and cellulases was likely to be under separate regulatory control. The specific activity and amount of xylanases produced were found to be dependent on the concentration of xylan in the growth media. Growth on oat spelts xylan or the hemicellulose-rich, water-soluble fraction from steam-treated aspenwood (SEA-WS) greatly enhanced the production of xylanases and xylosidase in the culture filtrates. Constitutive levels of xylanase and endoglucanase enzymes were detected during growth of the fungus on glucose.Offprint requests to: D. J. Senior     

The Lignicolous Fungus <Emphasis Type="Italic">Coniochaeta pulveracea</Emphasis> and Its Interactions with Syntrophic Yeasts from the Woody Phylloplane

The yeast-like fungus Coniochaeta pulveracea was studied with regard to its novel lignocellulolytic activities and the possible effect thereof on yeasts from the woody phylloplane. An enrichment procedure was used to isolate C. pulveracea from a decaying Acacia tree, and the identity of the isolate was confirmed using morphology, as well as molecular and phylogenetic techniques. This isolate, as well as strains representing C. pulveracea from different geographical regions, were compared with regard to optimum growth temperature and enzyme activity to representatives of closely related species. These include strains of Coniochaeta boothii, Coniochaeta rhopalochaeta, and Coniochaeta subcorticalis. Plate assays for cellulase and xylanase activity indicated that all representatives of the above-mentioned species were able to produce extracellular hydrolytic enzymes and were also able to degrade birchwood toothpicks during a 50-day incubation period at 30°C. To test the ability of these fungi and their enzymes to release simple sugars from complex cellulosic substrates, filtrates obtained from liquid cultures of Coniochaeta, cultivated on carboxymethyl cellulose (CMC) as sole carbon source, were analyzed using high-performance liquid chromatography analysis. Consequently, the presence of mono- and disaccharides such as glucose and cellobiose was confirmed in these culture filtrates. Two subsequent experiments were conducted to determine whether these simple sugars released from woody material by Coniochaeta may enhance growth of phylloplane yeasts. In the first experiment, representatives of Coniochaeta were co-cultured with selected yeasts suspended in agar plates containing birchwood toothpicks, followed by examination of plates for colony formation. Results indicated that Coniochaeta growth on the toothpicks enhanced growth of nearby yeast colonies in the agar plates. In the second experiment, representatives of selected yeasts and Coniochaeta species were co-cultured on CMC and xylan-containing plates where after yeast colony formation was recorded on the plates. Saccharomyces cerevisiae strains, engineered to utilize specific wood degradation products, i.e., cellobiose or xylose, as sole carbon source were used as positive controls. While it was found that cellobiose released from CMC was assimilated by the yeasts, no evidence could be obtained that xylose released from xylan was used as carbon source by the yeasts. These ambiguous results could be ascribed to secretion of nutritious metabolic end products, other than the products of fungal xylanases.