Screening of bioagents against root rot of mung bean caused by Rhizoctonia solani

A laboratory and green house experiment was carried out on the comparative antagonistic performance of four different bioagents (Aspergillus sp., Gliocladium virens, Trichoderma harzianum and T. viride) isolated from soil against Rhizoctonia solani. Under laboratory conditions, T. harzianum exhibited maximum (75.55%) mycelial growth inhibition of R. solani This was followed by T. viride, which showed 65.93 per cent mycelial growth inhibition of the pathogen. Gliocladium virens was also found to be effective antagonists, which exhibited 57.77 per cent mycelial growth inhibition. While Aspergillus sp exhibited minimum growth inhibition (45.74%) in comparison to other bioagents. Under green house conditions, T. harzianum gave maximum protection of the disease (72.72%) followed by T. viride, which exhibited 54.54 per cent disease control. However, G. virens and Aspergillus sp were found least effective in controlling root rot of mungbean.     

An immunological approach to quantifying the saprotrophic growth dynamics of Trichoderma species during antagonistic interactions with Rhizoctonia solani in a soil-less mix

Studies of the saprotrophic growth dynamics of Trichoderma species and their fungal hosts during antagonistic interactions are severely hampered by the absence of methods that allow the unambiguous identification and quantification of individual genera in complex environments such as soil or compost containing mixed populations of fungi. Furthermore, methods are required that allow discrimination between active hyphal growth and other components of fungal biomass such as quiescent spores that are produced in large numbers by Trichoderma species. This study details the use of monoclonal antibodies to quantify the saprotrophic growth dynamics of the soil-borne plant pathogen Rhizoctonia solani and biological control strains of Trichoderma asperellum and Trichoderma harzianum during antagonistic interactions in peat-based microcosms. Quantification was based on the immunological detection of constitutive, extracellular antigens that are secreted from the growing tip of Rhizoctonia and Trichoderma mycelium and, in the case of Trichoderma harzianum, from quiescent phialoconidia also. The Trichoderma-specific monoclonal antibody (MF2) binds to a protein epitope of the enzyme glucoamylase, which was shown by immunofluorescence and immunogold electron gold microscopy studies of Trichoderma virens in vitro to be produced at the origin of germ tube emergence in phialoconidia and from the growing tip of germ tubes. In addition, a non-destructive immunoblotting technique showed that the enzyme was secreted during active growth of Trichoderma asperellum mycelium in peat. The Rhizoctonia solani-specific monoclonal antibody (EH2) similarly binds to a protein epitope of a glycoprotein that is secreted during active mycelial growth. Extracts derived from lyophilized mycelium were used as a quantifiable and repeatable source of antigens for construction of calibration curves. These curves were used to convert the absorbance values obtained in ELISA tests of peat extracts to biomass equivalents, which allowed comparisons of the saprotrophic growth dynamics of the pathogen and antagonists to be made in single or mixed species microcosms. Trichoderma species were able to compete successfully with R. solani for nutrients and to prevent saprotrophic growth of the pathogen. Specificity of the Trichoderma quantitative assay was tested in non-sterile soil-based microcosms artificially inoculated with T. asperellum. The assay was highly specific and only detected T. asperellum population dynamics. No cross-reactivity was found with extracts from soil samples containing contaminant fungi.     

Chitinase gene expression during mycoparasitic interaction of Trichoderma harzianum with its host.

For monitoring chitinase expression during mycoparasitism of Trichoderma harzianum in situ, we constructed strains containing fusions of green fluorescent protein (GFP) to the 5'-regulatory sequences of the T. harzianum nag1 (N-acetyl-beta-d-glucosaminidase-encoding) and ech42 (42-kDa endochitinase-encoding) genes. Confronting these strains with Rhizoctonia solani led to induction of gene expression before (ech42) or after (nag1) physical contact. A 12-kDa cut-off membrane separating the two fungi abolished ech42 expression, indicating that macromolecules are involved in its precontact activation. No ech42 expression was triggered by culture filtrates of R. solani or by placing T. harzianum onto plates previously colonized by R. solani. Instead, high expression occurred upon incubation of T. harzianum with the supernatant of R. solani cell walls digested with culture filtrates or purified endochitinase 42 (CHIT42, encoded by ech42) from T. harzianum. The chitinase inhibitor allosamidin blocked ech42 expression and reduced inhibition of R. solani growth during confrontation. The results indicate that ech42 is expressed before contact of T. harzianum with R. solani and its induction is triggered by soluble chitooligosaccharides produced by constitutive activity of CHIT42 and/or other chitinolytic enzymes.     

Isolation and evaluation of bacteria and fungi as biological control agents against Rhizoctonia solani

Rhizoctonia solani is one of the most important limiting factors for potato production and storage in Belgium and worldwide. Its management is still strongly dependent on chemical treatments. The aim of this work was to evaluate the possibility of exploiting bacteria and fungi in order to control this pathogen. Among a collection of 220 bacterial strains isolated from different organs of healthy potato plants and rhizospheric soils, 25 isolates were selected using screening methods based on in vitro dual culture assays. The mycelial growth inhibition rate of the pathogen was ranged from 59.4 to 95.0%. Also seven fungal strains isolated from the rhizospheric soil and potato roots showed a highly mycelial growth inhibition of R. solani. The mycelial growth inhibition rate obtained with these fungi was included between 60.0 and 99.4%. From this preliminary study, the further investigations will be planned to determine the bacterial isolates systematic, species of fungal strains by using molecular tools and to assess their efficacy against R. solani in greenhouse trials.     

Physiological and biochemical characterization of Trichoderma harzianum, a biological control agent against soilborne fungal plant pathogens.

Monoconidial cultures of 15 isolates of Trichoderma harzianum were characterized on the basis of 82 morphological, physiological, and biochemical features and 99 isoenzyme bands from seven enzyme systems. The results were subjected to numerical analysis which revealed four distinct groups. Representative sequences of the internal transcribed spacer 1 (ITS 1)-ITS 2 region in the ribosomal DNA gene cluster were compared between groups confirming this distribution. The utility of the groupings generated from the morphological, physiological, and biochemical data was assessed by including an additional environmental isolate in the electrophoretic analysis. The in vitro antibiotic activity of the T. harzianum isolates was assayed against 10 isolates of five different soilborne fungal plant pathogens: Aphanomyces cochlioides, Rhizoctonia solani, Phoma betae, Acremonium cucurbitacearum, and Fusarium oxysporum f. sp. radicis lycopersici. Similarities between levels and specificities of biological activity and the numerical characterization groupings are both discussed in relation to antagonist-specific populations in known and potential biocontrol species.     

Major secondary metabolites produced by two commercial Trichoderma strains active against different phytopathogens

AIMS: Trichoderma harzianum strains T22 and T39 are two micro-organisms used as active agents in a variety of commercial biopesticides and biofertilizers and widely applied amongst field and greenhouse crops. The production, isolation, biological and chemical characterization of the main secondary metabolites produced by these strains are investigated. METHODS AND RESULTS: Of the three major compounds produced by strain T22, one is a new azaphilone that shows marked in vitro inhibition of Rhizoctonia solani, Pythium ultimum and Gaeumannomyces graminis var. tritici. In turn, filtrates from strain T39 were demonstrated to contain two compounds previously isolated from other T. harzianum strains and a new butenolide. The production of the isolated metabolites was also monitored by liquid chromatography/mass spectrometry during in vitro interaction with R. solani. CONCLUSIONS: This paper reports the isolation and characterization of the main secondary metabolites obtained from culture filtrates of two T. harzianum strains and their production during antagonistic interaction with the pathogen R. solani. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first work on secondary metabolites produced by the commercially applied strains T22 and T39. Our results provide a better understanding of the metabolism of these fungi, which are both widely used as biopesticides and/or biofertilizers in biocontrol.     

Antifungal Activities of Four Fatty Acids against Plant Pathogenic Fungi

The effect of the fatty acids linolenic acid, linoleic acid, erucic acid and oleic acid on the growth of the plant pathogenic fungi Rhizoctonia solani, Pythium ultimum, Pyrenophora avenae and Crinipellis perniciosa were examined in in vitro studies. Linolenic and linoleic acids exhibited activity against all of the fungi. However, whereas linolenic acid reduced mycelial growth of R. solani and C. perniciosa at 100 microM, the concentration had to be increased to 1000 microM before any effect on mycelial growth of P. ultimum and P. avenae was observed. Linoleic acid only reduced mycelial growth of R. solani, P. ultimum and P. avenae at 1000 microM, but led to a significant reduction in growth of C. perniciosa at 100 microM. In contrast, oleic acid had no significant effect on growth of R. solani or P. avenae, but gave significant reductions in mycelial growth of P. ultimum at 100 microM and reduced growth of C. perniciosa significantly at 1000 microM. All of the fatty acids reduced biomass production by all of the fungi significantly in liquid culture when added to the media at 100 microM. Erucic acid had no effect on fungal growth at any concentration examined. The antifungal activities exhibited by linolenic, linoleic and oleic acids may be useful in the search for alternative approaches to controlling important plant pathogens, such as those examined in this study.     

Processed Manure as Carrier to Introduce Trichoderma harzianum: Population Dynamics and Biocontrol Effect on Rhizoctonia solani

Manure pellets produced from processed swine faeces can be used as carrier material for the biocontrol fungus Trichoderma harzianum. The antagonist can grow and sporulate on the processed manure powder as the sole source of carbon and nutrients. The incorporation of conidia in pellets of the processed manure was shown to be feasible on a laboratory scale. Survival of the fungus in the pellets during storage was satisfactory. The population dynamics of T. harzianum were studied using a benomyl-resistance marker after introduction of conidia into soil. The antagonist could colonize and spread through a number of non-sterile soils and was able to establish a stable population over a period exceeding 125 days. Under sterile conditions, the propagation of T. harzianum in soil was much greater than under non-sterile conditions. The incorporation of antagonist conidia in pellets was found to be essential for the successful colonization of non-sterile soil. In growth chamber experiments, application of T. harzianum via processed manure pellets reduced damping-off of sugar beet seedlings caused by Rhizoctonia solani in artificially and naturally infested soil. In artificially infested soil, T. harzianum reduced the population of R. solani and protected beet seedlings from damping-off 3 weeks after introduction. The application of T. harzianum to naturally infested soil increased the number of healthy beet seedlings more than two-fold.     

Amendment with peony root bark improves the biocontrol efficacy of Trichoderma harzianum against Rhizoctonia solani

We tested Trichoderma harzianum as a biocontrol agent for Rhizoctonia solani AG2-1, using six natural antifungal materials to improve its efficacy. Among the six materials tested, peony (Paeonia suffruticosa) root bark (PRB) showed the strongest antifungal activity against R. solani AG2-1, and was not antagonistic to T. harzianum. Scanning electron microscopy showed that treatment with PRB extract resulted in shortened and deformed R. solani AG2-1 hyphal cells. The control of radish damping-off caused by R. solani AG2-1 was greatly increased by combined treatments of T. harzianum and PRB, as compared with either of the two treatments alone, with the control effect increased from 42.3-51.5% to 71.4-87.6%. The antifungal compound in PRB, which was isolated in chloroform and identified as paeonol by mass spectrometry, 1H NMR, and 13C NMR analyses, inhibited the growth of R. solani AG2-1 but not that of T. harzianum. Thus, PRB powder or extract may be used as a safe additive to T. harzianum to improve the control of the soil borne diseases caused by R. solani AG2-1.     

Effect of ultraviolet-induced mutants of Trichoderma harzianum with altered antibiotic production on selected pathogens in vitro

Mutants of Trichoderma harzianum with altered antibiotic production were isolated using ultraviolet light mutagenesis. These included strains whose activity in a Fusarium oxysporum spore germination assay was greater than twice that of the parental strain and one that had no detectable antifungal activity. Characterisation of extracellular metabolites of these strains using thin-layer chromatography and gas-liquid chromatography showed that the strains with high activity produced only elevated levels of a 6-n-pentyl pyrone, the antibiotic produced by the parental strain, but two new antifungal compounds. One of these has been identified as an isonitrile antibiotic. The nature of the interactions of the mutants with Fusarium oxysporum, Rhizoctonia solani, and Pythium ultimum was examined in an in vitro dual-plating assay using two media. High antibiotic production by two T. harzianum strains, BC10 and BC63, did increase inhibition of hyphal growth of R. solani and P. ultimum, but there was no correlation between increased antibiotic production and colonisation ability. In some cases the increased antibiotic levels appeared to impede colonisation of F. oxysporum and R. solani by the mutants. Slow growth rate also affected colonising ability. The types of interactions showed great variability depending on the nature of the T. harzianum isolate and on the test fungus.     

Pseudomonas aeruginosa inducing rice resistance againstRhizoctonia solani: Production of salicylic acid and peroxidases

Three isolates of Pseudomonas aeruginosa were used for seed treatment of rice; all showed plant growth promoting activity and induced systemic resistance in rice against Rhizoctonia solani G5 and increased seed yield. Production of salicylic acid (Sal) by P. aeruginosa both in vitro and in vivo was quantified with high performance liquid chromatography. All three isolates produced more Sal in King's B broth than in induced roots. Using a split root system, more Sal accumulated in root tissues of bacterized site than in distant roots on the opposite site of the root system after 1 d, but this difference decreased after 3 d. Sal concentration 0-200 g/L showed no inhibition of mycelial growth of R. solani in vitro, while at > or =300 g/L it inhibited it. P. aeruginosa-pretreated rice plants challenged inoculation with R. solani (as pathogen), an additional increase in the accumulation of peroxidase was observed. Three pathogenesis-related peroxidases in induced rice plants were detected; molar mass of these purified peroxidases was 28, 36 and 47 kDa. Purified peroxidase showed antifungal activity against phytopathogenic fungi R. solani, Pyricularia oryzae and Helminthosporium oryzae.     

Trichoderma harzianum antagonistic activity and competition for seed colonization against seedborne pathogenic fungi of sunflower

This study investigated the antagonistic effects of Trichoderma harzianum isolate (TRIC8) on mycelial growth, hyphal alteration, conidial germination, germ tube length and seed colonization by the seedborne fungal pathogens Alternaria alternata, Bipolaris cynodontis, Fusarium culmorum and F. oxysporum, the causes of seedling rot in over 30% of sunflowers. The antagonistic effect of TRIC8 on mycelial growth of pathogens was evaluated on dual culture that included two inoculation assays: inoculation of antagonist at 48 h before pathogen (deferred inoculation) and inoculation at the same time with pathogen (simultaneous inoculation). TRIC8 inhibited mycelial growth of the fungal pathogens between 70·67 and 76·87% with the strongest inhibition seen with deferred inoculation. Alterations in hyphae were observed in all pathogens. Conidial germination of F. culmorum was inhibited by most of the fungal pathogens (38·28%) by TRIC8. Inhibition of germ tube length by the antagonist varied from 31·83 to 37·67%. In seed colonization experiments, TRIC8 was applied in combination with each pathogen to seeds of a sunflower genotype that is highly tolerant to downy mildew. Seed death was inhibited by TRIC8 and the antagonist did not allow growth of A. alternata, B. cynodontis and F. culmorum on seeds and inhibited the growth of F. oxysporum at the rate of 58·32%.     

Interaction between proteinases secreted by the fungal plant pathogen Rhizoctonia solani and natural proteinase inhibitors produced by plants

The fungal plant pathogen Rhizoctonia solani Kuhn. grown in a medium containing thermostable potato tuber proteins produced proteinases active at moderately alkaline pH values. Electrophoretic analysis in polyacrylamide gel with SDS and copolymerized gelatin showed that the extracellular proteinase complex contained four components that differed in molecular weight. Studies on the action of the exoenzymes on various synthetic substrates indicated that the culture liquid of R. solani contained mainly trypsin-like proteinases. The exoproteinase activity was virtually completely suppressed by trypsin inhibitor proteins isolated from potato tubers and seeds of various legume species. The results suggest that the extracellular proteinases produced by R. solani play a significant role in attacking plant tissue, and natural inhibitors contribute to the protection of Solanaceae and Leguminosae from this fungal pathogen.     

Proteomic analysis of secreted proteins from Trichoderma harzianum. Identification of a fungal cell wall-induced aspartic protease

Trichoderma mycoparasitic activity depends on the secretion of complex mixtures of hydrolytic enzymes able to degrade the host cell wall. We have analysed the extracellular proteome secreted by T. harzianum CECT 2413 in the presence of different fungal cell walls. Significant differences were detected in 2DE maps, depending on the use of specific cell walls or chitin. A combination of MALDI-TOF and liquid chromatography mass spectrometry allowed the identification of a novel aspartic protease (P6281: MW 33 and pI 4.3) highly induced by fungal cell walls. A broad EST library from T. harzianum CECT 2413 was used to obtain the full-length sequence. The protein showed 44% identity with the polyporopepsin (EC 3.4.23.29) from the basidiomycete Irpex lacteus. Lower identity percentages were found with other pepsin-like proteases from filamentous fungi (<31%) and animals (<29%). Northern blot and promoter sequence analyses support the implication of the protease P6281 in mycoparasitism.     

Suppression of the biocontrol agent trichoderma harzianum by mycelium of the arbuscular mycorrhizal fungus glomus intraradices in root-free soil

Trichoderma harzianum is an effective biocontrol agent against several fungal soilborne plant pathogens. However, possible adverse effects of this fungus on arbuscular mycorrhizal fungi might be a drawback in its use in plant protection. The objective of the present work was to examine the interaction between Glomus intraradices and T. harzianum in soil. The use of a compartmented growth system with root-free soil compartments enabled us to study fungal interactions without the interfering effects of roots. Growth of the fungi was monitored by measuring hyphal length and population densities, while specific fatty acid signatures were used as indicators of living fungal biomass. Hyphal 33P transport and beta-glucuronidase (GUS) activity were used to monitor activity of G. intraradices and a GUS-transformed strain of T. harzianum, respectively. As growth and metabolism of T. harzianum are requirements for antagonism, the impact of wheat bran, added as an organic nutrient source for T. harzianum, was investigated. The presence of T. harzianum in root-free soil reduced root colonization by G. intraradices. The external hyphal length density of G. intraradices was reduced by the presence of T. harzianum in combination with wheat bran, but the living hyphal biomass, measured as the content of a membrane fatty acid, was not reduced. Hyphal 33P transport by G. intraradices also was not affected by T. harzianum. This suggests that T. harzianum exploited the dead mycelium but not the living biomass of G. intraradices. The presence of external mycelium of G. intraradices suppressed T. harzianum population development and GUS activity. Stimulation of the hyphal biomass of G. intraradices by organic amendment suggests that nutrient competition is a likely means of interaction. In conclusion, it seemed that growth of and phosphorus uptake by the external mycelium of G. intraradices were not affected by the antagonistic fungus T. harzianum; in contrast, T. harzianum was adversely affected by G. intraradices.     

绒毛栓孔菌液体培养过程中胞外酶活性的研究

本研究以绒毛栓孔菌为材料,采用液体培养的方法分析其在发酵过程中胞外酶的活性变化,并对其菌丝体生物量和发酵液pH值进行了测定。结果表明:胞外酶活性与菌丝体生长状况密切相关。菌丝体生物量增长呈"S"型,6～8d增长最快,第12天达到最大值,在此过程中漆酶、锰过氧化物酶、淀粉酶、羧甲基纤维素酶、果胶酶和蛋白酶活性均出现高峰。酶活性的变化表明,在液体培养过程中绒毛栓孔菌首先分解木质素,其次利用淀粉和纤维素作为碳源,蛋白质作为氮源。若要获得最大菌丝体生物量,缩短培养时间,就必须在培养过程中保证碳氮源的均衡供给。本试验说明不同的酶其分泌高峰期可以作为判断菌丝体营养利用情况和培养周期的依据,以此获取最大菌丝体生物量,为工业生产利用奠定基础。     

Growth of Trichoderma harzianum and Myceliophthora thermophila in palm oil sludge

Uncentrifuged palm oil mill sludge (POS) diluted to about 50% (v/v in tap water) supports good mycelial growth of Myceliophthora thermophila and Trichoderma harzianum. Both of the selected fungi are non-toxic to mice. After 24 h M. thermophila grown in batch culture in POS yielded 28.6 g/l of mycelial biomass with biological oxygen demand (BOD) and chemical oxidation demand (COD) reductions of 72% and 74% respectively. T. harzianum yielded 24.4 g/l of mycelial biomass with BOD and COD reductions of 67% and 68% respectively. The crude protein of the mycelial biomass of M. thermophila and T. harzianum was twice that of untreated POS. T. harzianum showed amylolytic activity while M. thermophila was cellulolytic and lipolytic.     

Isolation of two aspartyl proteases from Trichoderma asperellum expressed during colonization of cucumber roots

Trichoderma asperellum and cucumber seedlings were used as a model to study the modulation of Trichoderma gene expression during plant root colonization. Seedlings were grown in an aseptic hydroponics medium and inoculated with Trichoderma spore suspension. Proteins differentially secreted into the medium were isolated. Three major proteins of fungal origin were identified: two arabinofuranosidases (Abf1 and Abf2) and an aspartyl protease. Differential mRNA display was conducted on Trichoderma mycelia interacting and non-interacting, with the plant roots. Among the differentially regulated clones another aspartyl protease was identified. Sequencing of the genes revealed that the first aspartyl protease is a close homologue of PapA from T. harzianum and the other, of AP1 from Botryotinia fuckeliana. RT-PCR analysis confirms that the proteases are induced in response to plant roots attachment and are expressed in planta. papA, but not papB, is also induced in plate confrontation assays with the plant pathogen Rhizoctonia solani. These data suggest that the identified proteases play a role in Trichoderma both as a mycoparasite and as a plant opportunistic symbiont.     

The secreted ribonuclease T2 protein FoRnt2 contributes to Fusarium oxysporum virulence

Secreted RNase proteins have been reported from only a few pathogens, and relatively little is known about their biological functions. Fusarium oxysporum is a soilborne fungal pathogen that causes Fusarium wilt, one of the most important diseases on tomato. During the infection of F. oxysporum, some proteins are secreted that modulate host plant immunity and promote pathogen invasion. In this study, we identify an RNase, FoRnt2, from the F. oxysporum secretome that belongs to the ribonuclease T2 family. FoRnt2 possesses an N-terminal signal peptide and can be secreted from F. oxysporum. FoRnt2 exhibited ribonuclease activity and was able to degrade the host plant total RNA in vitro dependent on the active site residues H80 and H142. Deletion of the FoRnt2 gene reduced fungal virulence but had no obvious effect on mycelial growth and conidial production. The expression of FoRnt2 in tomato significantly enhanced plant susceptibility to pathogens. These data indicate that FoRnt2 is an important contributor to the virulence of F. oxysporum, possibly through the degradation of plant RNA.     

Gene expression profiling of the plant pathogenic basidiomycetous fungus Rhizoctonia solani AG 4 reveals putative virulence factors

Rhizoctonia solani is a ubiquitous basidiomycetous soilborne fungal pathogen causing damping-off of seedlings, aerial blights and postharvest diseases. To gain insight into the molecular mechanisms of pathogenesis a global approach based on analysis of expressed sequence tags (ESTs) was undertaken. To get broad gene-expression coverage, two normalized EST libraries were developed from mycelia grown under high nitrogen-induced virulent and low nitrogen/methylglucose-induced hypovirulent conditions. A pilot-scale assessment of gene diversity was made from the sequence analyses of the two libraries. A total of 2280 cDNA clones was sequenced that corresponded to 220 unique sequence sets or clusters (contigs) and 805 singlets, making up a total of 1025 unique genes identified from the two virulence-differentiated cDNA libraries. From the total sequences, 295 genes (38.7%) exhibited strong similarities with genes in public databases and were categorized into 11 functional groups. Approximately 61.3% of the R. solani ESTs have no apparent homologs in publicly available fungal genome databases and are considered unique genes. We have identified several cDNAs with potential roles in fungal pathogenicity, virulence, signal transduction, vegetative incompatibility and mating, drug resistance, lignin degradation, bioremediation and morphological differentiation. A codon-usage table has been formulated based on 14694 R. solani EST codons. Further analysis of ESTs might provide insights into virulence mechanisms of R. solani AG 4 as well as roles of these genes in development, saprophytic colonization and ecological adaptation of this important fungal plant pathogen.