Effect of biocontrol strains of Trichoderma on plant growth, Pythium ultimum polulations, soil microbial communities and soil enzyme activities

Five strains of Trichoderma with known biocontrol activities were assessed for their effect upon pea growth and their antagonistic activity against large Pythium ultimum inocula. The effect of Trichoderma inocula upon the indigenous soil microflora and soil enzyme activities in the presence and absence of Pythium is assessed. In the absence of Pythium, Trichoderma strain N47 significantly increased the wet shoot weight by 15% but did not significantly affect the dry weight, whilst strains T4 and N47 significantly increased the root weights by 22% and 80%) respectively. Strains TH1 and N47 resulted in significantly greater root lengths. Pythium inoculation significantly reduced the root length and the number of lateral roots and nodules, and significantly increased the root and rhizosphere soil fungal populations. Pythium inoculation significantly reduced the plant wet and dry shoot weights and significantly increased the wet and the dry shoot/root ratio. All the Trichoderma strains reduced the number of lesions caused by Pythium and increased the number of lateral roots. The effect of the Pythium on emergence and shoot growth was significantly reduced by all the Trichoderma strains except strain To10. Inoculation with Trichoderma strains TH1 and T4 resulted in significantly greater wet root weights (62% and 57%, respectively) in the presence of Pythium compared to the Pythium control. Strain N47 significantly increased the shoot/root ratio compared to the Pythium control. Inoculation with Trichoderma strains T4, T12 and N47 significantly reduced Pythium populations. Pythium increased the activity of C, N and P cycle enzymes, whilst four Trichoderma strains reduced this effect, indicating reduced plant damage and C leakage. Overall, strains T4 and N47 had the greatest beneficial characteristics, as both these strains improved plant growth in the absence of Pythium and reduced plant damage in the presence of Pythium. The dual properties of these strains improve the commercial application, giving them an advantage over single action inocula, especially in the absence of plant pathogens.     

Effects of temperature,pH and water potential on growth of four fungi with disease biocontrol potential

Effects of temperature, pH and water potential on blomass production or hyphal extension of Gliocladium virens (G20) and three Trichoderma isolates were determined in vitro. Optimum blomass production occurred between 20 and 30°C and at pH ranges between 4.6 and 6.8. Two isolates of T. viride grew at 5°C and G. virens grew at 35°C but no isolates grew at 40°C. Hyphal extension rates and conidial germination of all fungi declined with decreasing water potential over the range -0.7 to -14.0 MPa. In general, growth rates for each isolate were lower on potato/dextrose agar with water potential adjusted with polyethylene glycol than when adjusted with NaCl or glycerol. No mycelial growth or spore germination occurred on agar at-14.0 MPa.The authors are with the Microbiology and Crop Protection Department, Horticulture Research International, Littlehampton, West Sussex BN17 6LP, UK. J.M. Whipps is the corresponding author     

Changes in selected enzyme activities during growth of pure and mixed cultures of the white-rot decay fungus Trametes versicolor and the potential biocontrol fungus Trichoderma harzianum.

Two filamentous fungi, the white-rot fungus Trametes versicolor and the soil fungus and potential biocontrol organism Trichoderma harzianum, have been grown in pure and mixed cultures on low-N (0.4 mM) and high-N (4 mM) defined synthetic media to determine the activities of selected wood-degrading enzymes such as cellobiase, cellulase, laccase, and peroxidases. Growth characteristics and enzyme activities were examined for potential correlations. Such correlations would allow the use of simple enzyme assays for measuring biomass development and would facilitate predictions about competitiveness of species in mixed fungal cultures. Our results show that while laccase and Poly Red-478 peroxidase activities indicate survival of the decay fungus, none of the monitored extracellular enzymes can serve as a quantitative indicator for biomass accumulation. As expected, the level of available nitrogen affected the production of the enzymes monitored: in low-N media, specific cellobiase, specific cellulase, and peroxidase activities were enhanced, while laccase activities were reduced. Most importantly, laccase activities of Trametes versicolor, and to a smaller extent, cellobiase activities of both fungi, were significantly induced in mixed cultures of Trametes versicolor and Trichoderma harzianum.     

Influence of water activity on the enzyme biosynthesis and enzyme activities produced by Trichoderma viride TS in solid-state fermentation

Influence of water activity (a_w) on biosynthesis of polygalacturonase, d-xylanase and β-glucosidase in solid culture system of Trichoderma viride TS was studied. It was found that the production of enzymes was strongly affected by water activity of substrate and nature of a_w depressor used. The polygalacturonase and d-xylanase production were maximized at a_w = 0.995 whereas β-glucosidase formation was favored at a_w = 0.96–0.98. The influence of water activity on catalytic effect of enzymes using sodium chloride, glycerol and sorbitol as a_w depressor was also investigated. It was observed that sorbitol improved the thermal stability of polygalacturonase and d-xylanase.     

Effect of temperature and water activity on spore germination and mycelial growth of three fungal biocontrol agents against water hyacinth (Eichhornia crassipes)

Aims: To determine the effect of water activity (a_w = 0·880–0·960) and temperature (15–35°C) on the percentage of viable conidia and mycelial growth of three biocontrol agents effective against water hyacinth in Mali: Alternaria sp. isolate Mlb684, Fusarium sacchari isolate Mln799 and Cadophora malorum isolate Mln715. Methods and Results: The fungi were grown in vitro on plates containing potato dextrose agar medium at different a_w values (glycerol being added to adjust the a_w). The percentage of viable conidia and radial growth rate decreased with decreasing water activity. Statistical analysis showed a significant effect of a_w, temperature and the a_w × temperature interaction on mycelial growth (P < 0·0001). Water activity emerged as the factor exerting the greatest influence. Differences were observed between the fungi tested, the C. malorum appearing more tolerant to low a_w and the F. sacchari more tolerant to high temperature (35°C). Growth models predicting the combined effect of a_w and temperature were developed and response surfaces generated, showing fairly good agreement with the experimental values. Conclusions: Our results confirm the previous finding that a_w has a greater influence than temperature on fungal growth. Under most conditions, variation of environmental factors has a detrimental influence on the percentage of viable conidia and mycelial growth rate of fungal isolates. Significance and Impact of the Study: The developed models may contribute to predicting the best environmental conditions for use of these fungi as effective biocontrol agents against water hyacinth.     

A graphical method of analyzing pH dependence of enzyme activity

A method for graphically analyzing the pH dependence of enzyme activity is presented. Linear plots can be made to test the applicability of the assumed models. For three simple cases, if the pH dependence curves are measured at two substrate concentrations, it is possible to evaluate ion dissociation constants from the linear plots. The method would be useful for studies of pH effects on enzyme activities if applied complementarily to other methods.     

Nutrient availability and pH jointly constrain microbial extracellular enzyme activities in nutrient-poor tundra soils

Background and aims

Tundra soils, which usually contain low concentrations of soil nutrients and have a low pH, store a large proportion of the global soil carbon (C) pool. The importance of soil nitrogen (N) availability for microbial activity in the tundra has received a great deal of attention; however, although soil pH is known to exert a considerable impact on microbial activities across ecosystems, the importance of soil pH in the tundra has not been experimentally investigated.

Methods

We tested a hypothesis that low nutrient availability and pH may limit microbial biomass and microbial capacity for organic matter degradation in acidic tundra heaths by analyzing potential extracellular enzyme activities and microbial biomass after 6 years of factorial treatments of fertilization and liming.

Results

Increasing nutrients enhanced the potential activity of β-glucosidase (synthesized for cellulose degradation). Increasing soil pH, in contrast, reduced the potential activity of β-glucosidase. The soil phospholipid fatty acid concentrations (PLFAs; indicative of the amount of microbial biomass) increased in response to fertilization but were not influenced by liming.

Conclusions

Our results show that soil nutrient availability and pH together control extracellular enzyme activities but with largely differing or even opposing effects. When nutrient limitation was alleviated by fertilization, microbial biomass and enzymatic capacity for cellulose decomposition increased, which likely facilitates greater decomposition of soil organic matter. Increased soil pH, in contrast, reduced enzymatic capacity for cellulose decomposition, which could be related with the bioavailability of organic substrates.     

In vitro PYOCIN activity ofPseudomonas aeruginosa strains pretreated with antibiotics

Fifty-six selected strains of Pseudomonas aeruginosa belonging to 8 different pyocin types (H, I, 15, 6, PTI-1, PTI-2, PTI-3, PTI-4) were treated with subinhibitory concentrations (MIC/2) of either gentamicin or carbenicillin. Both treatments induced changes in pyocin patterns for all types but at different levels. The percentage of strains that retained their pyocin pattern were more or less equal in both treatments. In treated and untreated producers, the growth inhibition ability for 5 different strains of Enterobacteriaceae (Escherichia coli K12, E. coli EB, Proteus vulgaris, Salmonella typhi, Shigella flexneri) was also investigated. In all pyocin patterns the number of producers that inhibit the growth of these strains was lower after treatment with gentamicin or with carbenicillin, a smaller decrease was detected in the latter treatment. It appeared that the subinhibitory concentrations of these antibiotics are capable of protecting the Enterobacteriaceae strains from the action of the pyocins.     

Changes in activity of extracellular enzymes in dual cultures of Lentinula edodes and mycoparasitic Trichoderma strains

AIMS: The main problem that arises during the cultivation of Lentinula edodes, the Asian Shiitake mushroom, is that the logs on which the cultivation is performed are contaminated by competing micro-organisms, especially Trichoderma spp. The aim of this study was to examine the changes in activity of extracellular enzymes in dual cultures of Trichoderma spp. and L. edodes. METHODS AND RESULTS: Extracellular enzyme activities were determined spectrophotometrically. Trichoderma enzymes important for the degradation of fungal cell walls (N-acetyl-beta-glucosaminidase and laminarinase) were shown to be induced by inactive L. edodes mycelia in liquid culture. The changes that occurred in the extracellular enzyme activities of L. edodes and mycoparasitic Trichoderma spp. (T. aureoviride, T. harzianum and T. viride) were examined during antagonistic interactions on solid medium. The extracellular enzyme patterns of both partners proved to be altered. Trichoderma spp. were induced to produce N-acetyl-beta-glucosaminidase and laminarinase in the presence of active L. edodes mycelia, similarly as observed in liquid culture. The activities of both laccase and manganese peroxidase of L. edodes decreased after physical contact with active Trichoderma mycelia, possibly in consequence of the beginning of degradation of L. edodes by the Trichoderma enzymes. However, besides a decrease in manganese peroxidase activity, an enhancement of L. edodes laccase activity was observed on solid media containing crude culture fluids from Trichoderma liquid cultures. The metabolites responsible for these effects proved to be heat stable. CONCLUSIONS: Induction and inhibition of several extracellular enzymes of both partners were shown in dual cultures of L. edodes and Trichoderma strains, indicating the important role of these enzymes in the antagonistic interaction between the two species. SIGNIFICANCE AND IMPACT OF THE STUDY: As the main problem during the large-scale cultivation of L. edodes is the contamination of the growth substrate by Trichoderma mycelia, the particular knowledge of the mechanism of this competition might be relevant.     

洞庭湖湿地木霉多样性及生防活性

【目的】了解湖南省洞庭湖湿地木霉种类及分布,丰富我国的木霉种质资源,为功能菌株筛选应用奠定基础。【方法】利用ITS序列比对分析结合形态学特征对分离到的木霉菌株进行种类鉴定,构建系统发育进化树分析其亲缘关系。通过菌丝生长速率法测定菌株的平板抑菌能力,根据水解带大小检测菌株的水解酶活性,利用灰色关联度分析筛选综合生防效果较好的木霉菌株。【结果】从52个土样和18个水样中分离得到114株木霉菌株,经鉴定分属16个木霉种类:哈茨木霉、绿木霉、刺孢木霉、土星孢木霉、钩状木霉、拟康宁木霉、短密木霉、深绿木霉、猥木霉、毛细木霉(中国新记录种)、长枝木霉、卵孢木霉、侧耳木霉、加纳木霉、厚木霉及一个疑似新种;哈茨木霉为洞庭湖湿地中的优势种,占总菌株数量的19.30%;16种木霉在系统发育树中归于7个进化支:Harzianum Clade、Virens Clade、Longibrachiatum Clade、Lutea Clade、Viride Clade、Hamatum Clade、Unknown Clade。灰色关联度分析表明,菌株TW21990、QT22040和QT22094的灰色关联度较高,分别为0.849 5、0.798 6和0.732 6,综合生防性状较好。【结论】洞庭湖湿地木霉具有种类多样性和分布多样性,发现了一个中国新记录种毛细木霉和一个疑似新种,哈茨木霉TW21990、长枝木霉QT22040和卵孢木霉QT22094是潜在的优良生防菌株。     

In vitro conversion of proapoprotein A-I to apoprotein A-I. Partial characterization of an extracellular enzyme activity

Previous studies have established that human hepatocellular carcinoma cells (Hep G2) secrete into serum-free medium the pro form of apolipoprotein A-I (proapo-A-I) suggesting that its conversion to mature apo-A-I occurs after secretion. In order to assess the mode and site of proapo-A-I to apo-A-I conversion, we incubated the medium from [3H]proline-labeled Hep G2 cells with either human plasma, serum, lymph, or fractions thereof obtained by density gradient ultracentrifugation. The conversion was monitored by two-dimensional gel electrophoresis and by Edman degradation. Human plasma, serum, or mesenteric lymph all induced proapo-A-I to apo-A-I conversion; this was time dependent, unaffected by the serine protease inhibitor phenylmethylsulfonyl fluoride and inhibited by EDTA. Purified radiolabeled proapo-A-I bound to lymph chylomicrons and plasma high density lipoproteins. The converting enzyme was associated with both of these particles. Activity was also found in the d greater than 1.21-g/ml fraction and may have been derived from high density lipoprotein after displacement by high salts and/or ultracentrifugal force. We conclude that the conversion of proapo-A-I to apo-A-I occurs extracellularly and is probably effected by a metallo-enzyme which may act at the amphiphilic surface of either chylomicrons or high density lipoproteins.     

Regulation of vascular endothelial growth factor binding and activity by extracellular pH

Angiogenesis, the growth of new blood vessels, is regulated by a number of factors, including hypoxia and vascular endothelial growth factor (VEGF). Although the effects of hypoxia have been studied intensely, less attention has been given to other extracellular parameters such as pH. Thus, the present study investigates the consequences of acidic pH on VEGF binding and activity in endothelial cell cultures. We found that the binding of VEGF165 and VEGF121 to endothelial cells increased as the extracellular pH was decreased from 7.5 to 5.5. Binding of VEGF165 and VEGF121 to endothelial extracellular matrix was also increased at acidic pH. These effects were, in part, a reflection of increased heparin binding, because VEGF165 and VEGF121 showed increased retention on heparin-Sepharose at pH 5.5 compared with pH 7.5. Consistent with these findings, soluble heparin competed for VEGF binding to endothelial cells under acidic conditions. However, at neutral pH (7.5) low concentrations of heparin (0.1-1.0 microg/ml) potentiated VEGF binding. Extracellular pH also regulated VEGF activation of the extracellular signal-regulated kinases 1 and 2 (Erk1/2). VEGF165 and VEGF121 activation of Erk1/2 at pH 7.5 peaked after 5 min, whereas at pH 6.5 the peak was shifted to 10 min. At pH 5.5, neither VEGF isoform was able to activate Erk1/2, suggesting that the increased VEGF bound to the cells at low pH was sequestered in a stored state. Therefore, extracellular pH might play an important role in regulating VEGF interactions with cells and the extracellular matrix, which can modulate VEGF activity.     

Soil microbial biomass influence on growth and biocontrol efficacy of Trichoderma harzianum

Hyphal growth and biocontrol efficacy of Trichoderma harzianum may depend on its interactions with biotic components of the soil environment. Effects of soil microbial biomass on growth and biocontrol efficacy of the green fluorescent protein transformant T. harzianum ThzID1-M3 were investigated using different levels of soil microbial biomass (153, 328, or 517 μg biomass carbon/g of dry soil). Hyphal growth of T. harzianum was significantly inhibited in soil containing 328 or 517 μg biomass carbon/g of dry soil compared with soil containing 153 μg biomass carbon/g. However, when ThzID1-M3 was added to soil as an alginate pellet formulation, recoverable populations of ThzID1-M3 varied, with the highest populations in soil containing 517 μg biomass carbon/g. When sclerotia of Sclerotinia sclerotiorum were added to soils (10 sclerotia per 150 g soil) with ThzID1-M3 (20 pellets per 150 g soil), colonization of sclerotia by ThzID1-M3 was significantly lower in the soil containing the highest level of biomass. Addition of alginate pellets of ThzID1-M3 to soils (10 pellets per 50 g) resulted in increased indigenous microbial populations (total fungi, bacteria, fluorescent Pseudomonas spp., and actinomycetes). Our results suggest that higher levels of microbial soil biomass result in increased interactions between introduced T. harzianum and soil microorganisms, and further that microbial competition in soil favors a shift from hyphal growth to sporulation in T. harzianum, potentially reducing its biocontrol efficacy.     

In vitro efficacy of some fungicides on mycelial growth of Alternaria alternata and Mucor pyriformis

Various chemical fungicides, systemic and non-systemic, were tested against fruit rot pathogens viz. Alternaria alternata and Mucor pyriformis for the evaluation of inhibition of mycelial growth. In A. alternata, among the systemic fungicides used, hexaconozole showed highest inhibition of mycelial growth followed by carbendazim and least effective was myclobutanil. While in M. pyriformis, hexaconozole showed highest inhibition and least effective was bitertanol. Among the non-systemic fungicides tested in both A. alternata and M. pyriformis, mancozeb showed highest inhibition of mycelial growth followed by capton and the least inhibition was shown by zineb.     

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.     

A mixed mechanistic-electrostatic model to explain pH dependence of glycosyl hydrolase enzyme activity

Glycosyl hydrolases are a vast group of enzymes that share a common topology at their active site with two acidic residues that are responsible for activity. In spite of their similarity, they exhibit a wide range of pH optima that must depend on other factors. Using structural and mechanistic knowledge about glycosyl hydrolases from families 7, 10, and 16, we have formulated a new mathematical model that can include not only the ionization behavior of the catalytic residues but also as many ionizable residues as desired in the active site. In addition, the model can incorporate electrostatic influences via acid dissociation equilibrium constants and chemical relationships such as hydrogen bonds. The results of the simulations indicate a clear shift in the pH dependence of activity for the enzymes only when a close interrelation (hydrogen bond) between the catalytic and auxiliary residues in the active site is taken into account. This explains the observations from mutagenesis studies that show this type of shift and cannot be explained by a purely electrostatic interaction theory. Moreover, the presence of the kind of chemical interaction proposed could provide stabilization of the activity in the presence of environmental, structural, pH and electrostatic variations. These findings and the implications for the design of new mutagenesis strategies are discussed. The results suggest a way to modify, via site-directed mutagenesis, the acid dissociation of one of the catalytic residues in the active site independently of the other, which could have clear advantages over the purely electrostatic modifications that usually affect both residues simultaneously.     

The importance of water potential range tolerance as a limiting factor on Trichoderma spp. biocontrol of Sclerotinia sclerotiorum

Biological control of fungal phytopathogens is often more variable in efficacy compared with disease suppression achieved by conventional pesticide use. Matching the environmental range of a potential biocontrol agent with that of the target phytopathogen is necessary if consistent disease suppression is to be achieved under field conditions. Strains of Trichoderma that could parasitise sclerotia of Sclerotinia sclerotiorum had their spore germination and mycelial growth (five strains) and ability to parasitise sclerotia (two strains) tested under a range of water potentials under laboratory conditions. Relative mycelial growth and germination of all strains decreased with decreasing osmotic and matric potentials, with matric potential having a greater impact on growth and germination over the range examined. Trichoderma harzianum LU698 mycelial growth was the least affected by decreasing osmotic potential than the other isolates, and Trichoderma atroviride LU141 growth least affected by decreasing matric potential. The germination of LU698 and LU144 was also generally less affected by decreasing osmotic potential, although generally decreasing matric potential had the greatest affect on the germination of LU698 along with T. atroviride LU132. Soil treatments of LU698 and Trichoderma asperellum LU697 reduced sclerotial viability in all but the lowest soil water potential tested, with LU698 being most effective at ?0.1 and ?0.3 MPa after 28 days and LU697 most effective at ?0.01 and ?1.5 MPa after 28 days. We conclude that differences in the tolerance of potential biocontrol agents to changing water potential is an important experimental factor to consider when assaying biocontrol or making predictions of biocontrol efficacy in the field.     

In vitro antagonism of cotton seedlings fungi and characterization of chitinase isozyme activities in Trichoderma harzianum

The antagonistic fungus Trichoderma harzianum is widely recognized as a potential biocontrol agent against several soil-borne plant pathogens. T. harzinum is rich source of chitinoltic enzymes. In vitro screening of 5 isolates of T. harzinum, one isolate of Chaetomium globosum and one isolate of Conetherium mentance, revealed that all of them had reduced growth area of Macrophomina phaseolina, Fusarium solaniand Rhizoctonia solani on PDA medium, significantly. The inhibition percentage ranged from 77.9 % to 55.9% for M. phaseolina and 59.2% to 40.4% for R. solani by T. harzinum and C. mentance, respectively. Inhibition for F. solani ranged from 76.5% to 55.7% by T. harzinum and C. globosum, respectively. Isozyme gel electrophoresis was used to assess chitinase activity secreted by selected isolates of T. harzinum under different pH degrees and temperatures. Obtained results indicated that activity of chitinase isozyme produced at 30 °C was higher than 15–20 °C for all tested isolates and activity of chitinase produced by isolates No. 4 and 5 of T. harzinum at pH (7–7.5) was higher than at pH 6, respectively.