Colony morphology mutants of Trichoderma harzianum with increased beta-1,4-N-acetyl-glucosaminidase production

A total of 36 UV-induced mutants with altered colony morphology were isolated from strain Trichoderma harzianum T334, a potential biocontrol agent against plant pathogenic fungi with the ability to produce constitutively low levels of chitinases. The level of constitutive beta-1,4-N-acetyl-glucosaminidase production in standing and shaken cultures under non-inductive conditions was tested in mutants and compared to that of the parental strain. About 30% of the mutants showed significantly increased levels of enzyme production, with strain T334 col26a being the best producer. This mutant and the parental strain were subjected to in vitro confrontation assays with plant pathogenic Fusarium culmorum, Pythium debaryanum and Rhizoctonia solani strains. The mutant derivative could be characterized by significantly higher biocontrol index values than the parental strain in each experiment, suggesting, that mutants with improved constitutive extracellular chitinase secretion could be applied for biocontrol purposes against fungal plant pathogens.     

Factors affecting the production of Trichoderma harzianum secondary metabolites during the interaction with different plant pathogens

Aims: Strains of Trichoderma spp. produce numerous bioactive secondary metabolites. The in vitro production and antibiotic activities of the major compounds synthesized by Trichoderma harzianum strains T22 and T39 against Leptosphaeria maculans, Phytophthora cinnamomi and Botrytis cinerea were evaluated. Moreover, the eliciting effect of viable or nonviable biomasses of Rhizoctonia solani, Pythium ultimum or B. cinerea on the in vitro production of these metabolites was also investigated. Methods and Results: T22azaphilone, 1‐hydroxy‐3‐methyl‐anthraquinone, 1,8‐dihydroxy‐3‐methyl‐anthraquinone, T39butenolide, harzianolide, harzianopyridone were purified, characterized and used as standards. In antifungal assays, T22azaphilone and harzianopyridone inhibited the growth of the pathogens tested even at low doses (1–10 μg per plug), while high concentrations of T39butenolide and harzianolide were needed (>100 μg per plug) for inhibition. The in vitro accumulation of these metabolites was quantified by LC/MS. T22azaphilone production was not enhanced by the presence of the tested pathogens, despite its antibiotic activity. On the other hand, the anthraquinones, which showed no pathogen inhibition, were stimulated by the presence of P. ultimum. The production of T39butenolide was significantly enhanced by co‐cultivation with R. solani or B. cinerea. Similarly, viable and nonviable biomasses of R. solani or B. cinerea increased the accumulation of harzianopyridone. Finally, harzianolide was not detected in any of the interactions examined. Conclusions: The secondary metabolites analysed in this study showed different levels of antibiotic activity. Their production in vitro varied in relation to: (i) the specific compound; (ii) the phytopathogen used for the elicitation; (iii) the viability of the elicitor; and (iv) the balance between elicited biosynthesis and biotransformation rates. Significance and Impact of the Study: The use of cultures of phytopathogens to enhance yields of Trichoderma metabolites could improve the production and application of novel biopesticides and biofertilizers based on the active compounds instead of the living microbe. This could have a significant beneficial impact on the management of diseases in crop plants.     

Saccharomyces cerevisiae mutants selected for increased production of Trichoderma reesei cellulases

 Trichoderma reesei endoglucanase I (EGI) was used as a reporter enzyme for screening mutagenized yeast strains for increased ability to produce protein. Sixteen haploid Saccharomyces cerevisiae strains, transformed with a yeast multicopy vector pALK222, containing the EGI cDNA under the ADH1 promoter, produced EGI activity of 10^-5–10^-4 g/l. On the average 93% of the total activity was secreted into the culture medium. Two strains with opposite mating types were mutagenized, and several mutants were isolated possessing up to 45-fold higher EGI activity. The best mutants were remutagenized and a second-generation mutant, strain 2804, with an additional twofold increase in EGI activity was selected. The mutant strain 2804 grew more slowly and reached a lower final cell density than the parental strain. In the selective minimal medium, the 2804 strain produced 40 mg/l immunoreactive EGI protein, but only 2% was active enzyme. In the rich medium the secreted EGI enzyme stayed active, but without selection pressure the EGI production ceased after 2 days of cultivation, when the strain 2804 had produced 10 mg/l of EGI. A sevenfold difference was found between the parental and the 2804 strain in their total EGI production relative to cell density. The difference in favour of the mutant strain was also detected on the mRNA level. The 2804 mutant was found to be more active than the parental strain also in the production of T. reesei cellulases, cellobiohydrolase I, and cellobiohydrolase II. Received: 22 December 1995/Received revision: 26 February 1996/Accepted: 17 March 1996     

Effect of Trichoderma harzianum on perithecial production of Gibberella zeae on wheat straw

Conidial suspensions and cell-free filtrates of Trichoderma harzianum isolates were evaluated for their effectiveness in reducing perithecial and ascospore production of Gibberella zeae on wheat straw. Isolate T-22, which is registered in the US as a biological control agent (Plant Shield™), was included in the study as a positive control. When co-inoculated with G. zeae all 11 isolates of T. harzianum significantly reduced perithecial numbers on wheat straw. Five T. harzianum isolates, including T-22, reduced perithecial formation by 70% or greater. All isolates of G. zeae, varied in their ability to produce perithecia. Isolate 192132 produced the greatest number of perithecia and was used to further evaluate the effect of application time of the T. harzianum isolates. Perithecial reduction was highest (96-99%) when T. harzianum conidial suspension or cell-free filtrate was applied to straw 24 h prior to inoculation with G. zeae. Control was less effective when T. harzianum was applied at the same time (co-inoculated) or 24 h after G. zeae. Treatments which reduced perithecial numbers also reduced ascospore numbers; however, the average numbers of ascospores per perithecia were not significantly lowered. Field trials showed significant reduction of perithecia on residues treated with T. harzianum prior to placement on the soil surface. Both T. harzianum and G. zeae were re-isolated from residues sampled in July and August after 30 and 60 days of exposure to the environment.     

Isolation and characterization of protease overproducing mutants of Trichoderma harzianum

Several Trichoderma strains have been reported to be effective in controlling plant diseases, and the action of fungal hydrolytic enzymes is considered as the main mechanism involved in the antagonistic process. Strain Trichoderma harzianum T334 is a potential biocontrol agent against plant pathogenic fungi with the ability to produce low levels of proteases constitutively. To improve its fungal antagonistic capacity, mutagenetic program was undertaken for the construction of protease overproducing derivates. The mutant strains were obtained by means of UV-irradiation and were selected for p-fluorophenyl-alanine resistance or altered colony morphology. It was revealed by means of specific chromogenic protease substrates that both trypsin-like and chymotrypsin-like protease secretion was elevated in most of the mutant strains. The profiles of isoenzymes were different between the mutants and the wild-type strain, when examined by gel filtration chromatography. Certain mutants proved to be better antagonists against plant pathogens in in vitro antagonism experiments. This study suggests the possibility of using mutants with improved constitutive extracellular protease secretion against plant pathogenic fungi.     

Effect of tunicamycin on N-acetyl-beta-D-glucosaminidase produced by Trichoderma harzianum

The effect of tunicamycin, an inhibitor of protein N-glycosylation, was studied in non-growing mycelium of Trichoderma harzianum induced to secrete N-acetyl-beta-D-glucosaminidase by the addition of N-acetylglucosamine. Tunicamycin (30 microg ml(-1)) had no significant effect on growth of the fungus, or on the total protein secreted or specific activity of N-acetyl-beta-D-glucosaminidase. However, in the presence of the inhibitor an underglycosylated form of the enzyme was produced. The apparent molecular masses for this and the native enzyme were 110 and 124 kDa, respectively. Both forms of the enzyme showed the same optimum pH and temperature, but the underglycosylated form was more sensitive to inactivation by both high temperature (60 degrees C) and the proteolytic enzyme trypsin.     

Isolation of uracil auxotrophic mutants of Trichoderma harzianum and their transformation with heterologous vectors

Abstract The growth of Lactobacillus hilgardii X₁B and Pediococcus pentosaceus 12p, isolated from Argentinian wines, were studied in pure and mixed cultures. In the mixed culture, an amensalistic growth response was observed: Pediococcus pentosaceus growth was inhibited until 24 h; after this time, no viable cells were detected. In pure and mixed cultures, Lactobacillus hilgardii produced hydrogen peroxide early in the growth cycle, reaching the maximum at 24 h. Hydrogen peroxide and increased acidity were responsible for Pediococcus pentosaceus inhibition in the mixed culture.     

In vitro antagonism of bioluminescent fungi by Trichoderma harzianum

Two species of bioluminescent fungi, Panellus stypticus and Omphalotus olearius were placed in contact with three different strains of interfungal pathogenic Trichoderma harzianum. Subsequent light emission by the luminous fungi and advance of the interfungal pathogens were compared. Relative differences among the pathogens were reflected in their rate of mycelial advance, the total area over which they produced spores upon the host fungi, and decreases in host bioluminescence. After ten days differences in the total surface areas of spore production varied from 1 to 53 per cent. Differences in the reduction of bioluminescence of the same material ranged over 2 orders of magnitude. Final reduction in luminescence ranged over 6 orders of magnitude. A marked reduction in bioluminescence was observed to precede the advance of spore production. The greatest reduction in luminescence was correlated with the presence of T. harzianum hyphae. Two strains of T. harzianum, NRRL 1698 and ATCC 58674, were effective against both bioluminescent fungi within the study period while a third strain, NRRL 13019, was only effective against Omphalotus olearius.     

Effect of carbon and nitrogen sources on xylanase production by Trichoderma harzianum 1073 D3

In order to determine the effect of different carbon and nitrogen sources on xylanase production by Trichoderma harzianum 1073 D3, xylan in the xylanase production medium was replaced with different carbon sources. In order to reduce production time, glucose was added to the production media containing xylan. The effects of sucrose, maltose and lactose were investigated and maximum xylanase activity was observed in the presence of sucrose. Ammonium sulphate was the most appropriate inorganic nitrogen source for xylanase production and urea increased xylanase activity slightly.     

Antagonistic potentiality of Trichoderma harzianum towards seed-borne fungal pathogens of winter wheat cv. Protiva in vitro and in vivo

The antagonistic effect of Trichoderma harzianum on a range of seed-borne fungal pathogens of wheat (viz. Fusarium graminearum, Bipolaris sorokiniana, Aspergillus spp., and Penicillium spp.) was assessed. The potential of T. harzianum as a biocontrol agent was tested in vitro and under field conditions. Coculture of the pathogens and Trichoderma under laboratory conditions clearly showed dominance of T. harzianum. Under natural conditions, biocontrol effects were also obtained against the test fungi. One month after sowing, field emergence (plant stand) was increased by 15.93% over that obtained with the control treatment, and seedling infection was reduced significantly. Leaf blight severity was decreased from 22 to 11 at the heading stage, 35 to 31 at the flowering stage, and 86 to 74 at the grain filling stage. At harvest, the number of tillers per plant was increased by 50%, the yield was increased by 31.58%, and the 1,000-seed weight was increased by 21%.     

Combined effects of Brassica napus seed meal and Trichoderma harzianum on two soilborne plant pathogens

The effects of soil amendment with rapeseed meal from Brassica napus cv. 'Dwarf Essex' (high glucosinolate concentrations) and 'Stonewall' (low glucosinolate concentrations) on the biological control activity of Trichoderma harzianum towards Sclerotinia sclerotiorum and Aphanomyces euteiches were evaluated. Trichoderma harzianum added to soil reduced myceliogenic germination of S. sclerotiorum by 94%, but did not affect carpogenic germination. In contrast, 100% reduction in carpogenic germination was observed in soil amended with Dwarf Essex meal, along with a 33% reduction in myceliogenic germination. With Stonewall meal as soil amendment, carpogenic germination was reduced by 44% and myceliogenic germination was not affected. Both Dwarf Essex and Stonewall meals inhibited colonization of sclerotia in soil by T. harzianum, from 100% to 0% and 8%, respectively, so that biocontrol activity of T. harzianum was reduced in the presence of either meal. Aphanomyces euteiches root rot of pea was significantly reduced by T. harzianum alone (100%), by amendment with Dwarf Essex meal alone (77%), and by T. harzianum in combination with Dwarf Essex meal (100%). Amendment with Stonewall meal alone did not control root rot, and combination of Stonewall meal with T. harzianum reduced the biocontrol efficacy of T. harzianum.     

Self-fusion of protoplasts enhances chitinase production and biocontrol activity in Trichoderma harzianum

Protoplasts were isolated from Trichoderma harzianum strain PTh18 using lysing enzymes and self-fusion of T. harzianum protoplasts was carried out using polyethylene glycol in STC buffer. The fused protoplasts of T. harzianum were regenerated and 15 self-fusants were selected to study the chitinase production and biocontrol activity. High chitinase activity was measured in the culture filtrates of most of the self-fusants (87%) than the parent. Among the fusants, the strain SFTh8 produced maximum chitinase with a two-fold increase as compared to the parent strain. All the self-fusants exhibited increased antagonistic activity against Rhizoctonia solani than the parent. The crude chitinase preparation of SFTh8 lysed the mycelia of T. harzianum, Trichoderma viride and Trichoderma reesei and released the protoplasts in higher number than the crude chitinase preparation of parent strain PTh18.     

Isolation of Vibrio cholerae mutants with altered toxin production

V. cholerae multiple-labeled mutants 569B with altered toxin production have been obtained by the method of induced mutagenesis with the use of nitrosoguonidine. These mutants can be used for the genetic mapping of tox genes on the chromosome of V. cholerae.     

Preparation of mutants of Trichoderma reesei with enhanced cellulase production.

The development of an agar plate screening technique has allowed the isolation of a range of mutants of Trichoderma reesei capable of synthesizing cellulase under conditions of high catabolite repression. The properties of one of these mutants (NG-14) is described to illustrate the use of this technique. NG-14 produced five times the filter paper-degrading activity per ml of culture medium and twice the specific activity per mg of excreted protein in submerged culture when compared with the best existing mutant, QM9414. NG-14 also showed enhanced endo-beta-glucanase and beta-glucosidase production. Although these mutants were isolated as cellulase producers in the presence of 5% glycerol on agar plates, in similar liquid medium, NG-14 exhibits only partial derepression of the cellulase complex. Since the proportions of filter paper activity, endo-beta-glucanase, and cellobiase were not the same in mutants NG-14 and QM9414, and the yields of each enzyme under conditions repressive for cellulase synthesis were different, differential control of each enzyme of the cellulase complex is implied. These initial results suggest that the selective technique for isolating hyper-cellulase-producing mutants of Trichoderma will be of considerable use in the development of commercially useful cellulolytic strains.     

Effect of the recC gene in Escherichia coli on frequencies of ultraviolet-induced mutants

UV induction of Lac^? mutations was compared with UV induction of Mal⁺ mutations in E. coli B/r strains differing in the recC gene. The frequency of Lac^? mutants per survivor induced by the same dose was not significantly affected by the recC gene but the percentage of pure rather than sectored Lac^? colonies was greater when the recC gene was present. On the other hand, as reported previously, frequencies of Mal⁺ mutants induced by the same UV dose were lower when the strain was recC. The reduction factor was the same as for spontaneous Mal⁺ mutants. The difference in the effect of the recC gene on the yields of Lac^? and Mal⁺ mutants can be explained by taking into account the influence of lethal sectoring, which introduces an artifact when mutants arising in the recC strain are scored selectively as in the case for Mal⁺ mutants, but not when the scoring is non-selective as for Lac^? mutants. Lethal sectoring as indicated by a discrepancy between total cell counts and numbers of colony-formers, was observed for the recC strain growing in liquid minimal medium corresponding to the agar medium used to score Mal⁺ mutants but was not observed for the rec⁺ strain. Both strains showed lethal sectoring in the liquid medium corresponding to the agar medium to score Lac^? mutants. The hypothesis concerning the role of lethal sectoring in the selective scoring of mutants arising in a recC background is supported by evidence concerning the UV induction of mutants in a polA1 background. Like the recC gene, the polA1 gene did not affect yields of Lac^? mutants. However, unlike the recC gene, the polA1 gene has previously been shown not to influence UV yields of prototrophic mutations (scored selectively) and not to cause lethal sectoring except under irrelevant conditions.     

Preparation of mutants of Trichoderma reesei with enhanced cellulase production.

The development of an agar plate screening technique has allowed the isolation of a range of mutants of Trichoderma reesei capable of synthesizing cellulase under conditions of high catabolite repression. The properties of one of these mutants (NG-14) is described to illustrate the use of this technique. NG-14 produced five times the filter paper-degrading activity per ml of culture medium and twice the specific activity per mg of excreted protein in submerged culture when compared with the best existing mutant, QM9414. NG-14 also showed enhanced endo-beta-glucanase and beta-glucosidase production. Although these mutants were isolated as cellulase producers in the presence of 5% glycerol on agar plates, in similar liquid medium, NG-14 exhibits only partial derepression of the cellulase complex. Since the proportions of filter paper activity, endo-beta-glucanase, and cellobiase were not the same in mutants NG-14 and QM9414, and the yields of each enzyme under conditions repressive for cellulase synthesis were different, differential control of each enzyme of the cellulase complex is implied. These initial results suggest that the selective technique for isolating hyper-cellulase-producing mutants of Trichoderma will be of considerable use in the development of commercially useful cellulolytic strains.     

Influence of growth substrate on production of cellulase enzymes by Trichoderma harzianum E58

Cellulase production by Trichoderma harzianum E58 grown on lactose and various cellulosic substrates such as Solka Floe, Avicel, and steamed aspenwood was investigated. The culture filtrates of T. harzianum E58 obtained after growth on these substrates were assayed for their cellulase activities and overall hydrolytic activities. The severity of the steaming conditions used for the aspenwood had a pronounced effect on the cellulolytic activity of the produced culture filtrates. Those substrates that were more readily hydrolyzed by the cellulase complex were the poorest substrates for inducing an active cellulase complex. Substrates such as acid-impregnated aspenwood and lactose induced a less hydrolytic efficient cellulase complex than more recalcitrant substrates such as microcrystalline cellulose.     

Screening of Bacillus subtilis transposon mutants with altered riboflavin production

To identify novel targets for metabolic engineering of riboflavin production, we generated about 10,000 random, transposon-tagged mutants of an industrial, riboflavin-producing strain of Bacillus subtilis. Process-relevant screening conditions were established by developing a 96-deep-well plate method with raffinose as the carbon source, which mimics, to some extent, carbon limitation in fed batch cultures. Screening in raffinose and complex LB medium identified more efficiently riboflavin overproducing and underproducing mutants, respectively. As expected for a "loss of function" analysis, most identified mutants were underproducers. Insertion mutants in two genes with yet unknown function, however, were found to attain significantly improved riboflavin titers and yields. These genes and possibly further ones that are related to them are promising candidates for metabolic engineering. While causal links to riboflavin production were not obvious for most underproducers, we demonstrated for the gluconeogenic glyceraldehyde-3-phosphate dehydrogenase GapB how a novel, non-obvious metabolic engineering strategy can be derived from such underproduction mutations. Specifically, we improved riboflavin production on various substrates significantly by deregulating expression of the gluconeogenic genes gapB and pckA through knockout of their genetic repressor CcpN. This improvement was also verified under the more process-relevant conditions of a glucose-limited fed-batch culture.