Management of palm oil mill effluent through production of cellulases by filamentous fungi

A laboratory scale study to evaluate the potentiality of filamentous fungi for the production of cellulolytic enzymes using palm oil mill effluent (POME) as a basal medium was initiated. A total of 25 filamentous fungi in which 16 filamentous fungi were isolated and purified from oil palm industrial residues and 9 strains from laboratory stock were screened using POME with 1% total suspended solids. Trichoderma reesei RUT C-30 was identified as a potential strain for cellulolytic enzyme production as compared to other genera of Aspergillus, Penicillum, Rhizopus, Phanerochaete, Trichoderma and basidiomycete groups. The results showed that T. reesei RUT C-30 gave the highest filter paper cellulase and carboxy methyl cellulase activity of 0.917 and 2.51 U/ml respectively at day 5 of fermentation. Other parameters such as growth formation, pH, filterability and total biosolids were observed to evaluate the bioconversion process.     

In vitro evaluation of dew-retting of flax by fungi from southern Europe

Flax dew-retting is widely adopted in most flax-growing countries, but it does not represent a practical solution where dry weather conditions occur after harvest. A study of the local microbiological aspects was undertaken as a contribution to improve field-retting of flax under southern European climates. Fungi were isolated from soil and dew-retted flax in northern Italy, and 23 representative strains were chosen to test their ability to ret flax stems. Experiments were performed in vitro on flax stem pieces artificially inoculated with single fungal strains. Retting degree was assessed with a mechanical test, to evaluate the ease with which the bast was detached from the wood core, and by the analysis of the residual fibre pectins using uronic acid. Uronic acid dosage provided a better differentiation of the strains than the mechanical test. There was a large variability in retting ability among the species assayed and even among strains of the same species. The best results were obtained with all Aspergillus and Penicillium strains, while Mucor and Rhizopus strains showed a variable retting ability. Fusarium, Trichoderma strains and Epicoccum nigrum had the poorest retting abilities among all the fungal strains assayed.     

Nematicidal activity of Trichoderma spp. and isolation of an active compound

Trichoderma spp. play an important role in biotic control, and several are efficacious against nematodes. To study the potential of Trichoderma species in controlling nematodes, fungal filtrates of 329 Trichoderma strains were evaluated for their nematicidal activity against Panagrellus redivivus and Caenorhabditis elegans. Fifteen strains exhibited nematicidal activity against P. redivivus, and 14 strains showed activity against C. elegans. The strain YMF1.02647 showed strong nematicidal activity against both nematodes, and the culture broth could cause more than 90% mortality to the tested nematodes within 48 h. A nematicidal compound was isolated from ethyl acetate extract of Trichoderma YMF1.02647 based on bioassay-guided fractionation. The compound was identified as trichodermin according to the spectroscopic data, which could kill more than 95% both P. redivivus and C. elegans in 72 h at 0.4 g l⁻¹.     

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.     

Changes in lignocellulolytic enzyme activites in six <Emphasis Type="Italic">Pleurotus</Emphasis> spp. strains cultivated on coffee pulp in confrontation with <Emphasis Type="Italic">Trichoderma</Emphasis> spp.

Summary The antagonistic effect of six Pleurotus spp. strains was studied in confrontation with three strains of Trichoderma spp. Pleurotus strains were cultivated on sterile coffee pulp, with and without a Trichoderma inoculant. Laccase, Mn peroxidase and endoglucanase activities were determined during incubation. Laccase production was also studied by PAGE analysis to detect enzymatic isoforms. Results show that the presence of Trichoderma induced a significant increase in oxidase production by the Pleurotus strains. Nevertheless, Trichoderma was not observed to induce laccase isoforms.     

Control of the bean rust fungus <Emphasis Type="Italic">Uromyces appendiculatus</Emphasis> by means of <Emphasis Type="Italic">Trichoderma harzianum</Emphasis>: leaf disc assays on the antibiotic effect of spore suspensions and culture filtrates

Several species of the fungal genus Trichoderma act as antagonists of other fungi. A number of strains from the Trichoderma species T. harzianum Rifai are used as biological control agents for the control of soilborne as well as foliar plant pathogens. Six T. harzianum strains, five of them isolated from commercial preparations, were evaluated for their capability to control the bean rust fungus Uromyces appendiculatus (Pers. ex Pers.) Unger. Different kinds of leaf disc assays were performed with conidial spore suspensions and sterile culture filtrates of the T. harzianum strains. Great differences were observed concerning the efficacy of the Trichoderma strains to reduce the number of the uredial pustules developing after rust inoculation which followed the application of the particular Trichoderma strains. Efficacy values ranged from 1 to over 50%. Increasing spore or culture filtrate concentrations of the two most effective isolates T12 and T_U led to decreases in the number of developing uredial pustules. Culture filtrate applications had a protective but no curative effect. T12 spore suspensions maintained their disease reducing activity even when autoclaved. This and some other evidence for an antibiotic interaction between T. harzianum and U. appendiculatus are discussed. Handling Editor: Reijo Karjalainen.     

Trichoderma species form endophytic associations within Theobroma cacao trichomes

Trichoderma species are usually considered soil organisms that colonize plant roots, sometimes forming a symbiotic relationship. Recent studies demonstrate that Trichoderma species are also capable of colonizing the above ground tissues of Theobroma cacao (cacao) in what has been characterized as an endophytic relationship. Trichoderma species can be re-isolated from surface sterilized cacao stem tissue, including the bark and xylem, the apical meristem, and to a lesser degree from leaves. SEM analysis of cacao stems colonized by strains of four Trichoderma species (Trichoderma ovalisporum-DIS 70a, Trichoderma hamatum-DIS 219b, Trichoderma koningiopsis-DIS 172ai, or Trichoderma harzianum-DIS 219f) showed a preference for surface colonization of glandular trichomes versus non-glandular trichomes. The Trichoderma strains colonized the glandular trichome tips and formed swellings resembling appresoria. Hyphae were observed emerging from the glandular trichomes on surface sterilized stems from cacao seedlings that had been inoculated with each of the four Trichoderma strains. Fungal hyphae were observed under the microscope emerging from the trichomes as soon as 6 h after their isolation from surface sterilized cacao seedling stems. Hyphae were also observed, in some cases, emerging from stalk cells opposite the trichome head. Repeated single trichome/hyphae isolations verified that the emerging hyphae were the Trichoderma strains with which the cacao seedlings had been inoculated. Strains of four Trichoderma species were able to enter glandular trichomes during the colonization of cacao stems where they survived surface sterilization and could be re-isolated. The penetration of cacao trichomes may provide the entry point for Trichoderma species into the cacao stem allowing systemic colonization of this tissue.     

Effect of pH and Temperature on Enzyme Activity of Chitosanase Produced Under Solid Stated Fermentation by <Emphasis Type="Italic">Trichoderma</Emphasis> spp<Emphasis Type="Italic">.</Emphasis>

Trichoderma strains were extensively studied as biocontrol agents due to their ability of producing hydrolytic enzymes, which are considered key enzymes because they attack the insect exoskeleton allowing the fungi infection. The present work aimed to evaluate the ability of chitosanase production by four Trichoderma strains (T. harzianum, T. koningii, T. viride and T. polysporum) under solid stated fermentation and to evaluate the effect of pH and temperature on enzyme activity. pH strongly affected the enzyme activity from all tested strains. Chitosanase from T. harzianum and T. viride presented optimum activity at pH 5.0 and chitosanase from T. koningii and T. polysporum presented optimum activity at pH 5.5. Temperature in the range of 40–50°C did not affect enzyme activity. T. polysporum was found as the most promising strain to produce chitosanase with maximal enzyme activity of about 1.4 IU/gds, followed by T. viride (~1.2 IU/gds) and T. harzianum (1.06 IU/gds).     

Growth stimulation in bean (Phaseolus vulgaris L.) by Trichoderma

Trichoderma species are commonly used as biological control agents against phytopathogenic fungi and some strains are able to produce metabolites that enhance plant growth. In the current study we evaluated the production of potential growth-promoting metabolites, rhizosphere competence and endophytism for 101 isolates of Trichoderma from Colombia, and assessed the relationship of these factors to the enhancement of early stages of growth on bean seedlings. Twenty percent of these Trichoderma strains were able to produce soluble forms of phosphate from phosphoric rock. Only 8% of the assessed strains showed consistent ability to produce siderophores to convert ferric iron to soluble forms by chelation. Sixty percent of isolates produced indole-3-acetic acid (IAA) or auxin analogues. The production of any of these metabolites was a characteristic of specific strains, as the ability to produce these metabolites varied greatly within species. Moreover, the production of these substances did not correlate with enhanced growth on bean seedlings, measured as the combined increase in length of roots and aerial parts in the V3 stage of growth. Seven Trichoderma isolates significantly improved the growth of bean seedlings. However, metabolite production varied widely in these seven strains, and some isolates did not produce any of the assessed growth-promoting metabolites. Results indicated that growth was enhanced in the presence of rhizosphere competent and endophytic strains of Trichoderma, and these characteristics were strain-specific and not characteristic for species.     

拮抗北细辛菌核病木霉菌的分离、鉴定及生防效果

【背景】菌核病是北细辛根部主要病害之一,木霉菌作为目前应用最广泛的生物防治真菌,利用木霉菌防治北细辛菌核病是目前研究的热点。【目的】通过稀释分离法对健康北细辛植株根际土壤进行菌株分离,以期筛选出有效拮抗北细辛菌核病的生防木霉菌。【方法】以北细辛菌核病菌为靶标菌,采用平板对峙培养、挥发性与非挥发性物质抑菌的方法对分离得到的木霉菌进行筛选,采用生长速率法对筛选出的木霉菌的发酵液进行抑菌效果测定,并采用硫代巴比妥酸法测定筛选出的木霉对北细辛菌核病菌的丙二醛(Malondialdehyde,MDA)含量、紫外吸收法测定过氧化氢酶(Catalase,CAT)活性、氮蓝四唑法测定超氧化物歧化酶(Superoxide Dismutase,SOD)活性、愈创木酚法测定过氧化物酶(Peroxidase,POD)活性的影响。【结果】从土壤中分离出木霉菌共14株,通过形态学和ITS-RPB2双基因联合构建系统发育树,鉴定其为哈茨木霉(Trichoderma harzianum)、钩状木霉(Trichoderma hamatum)、拟康氏木霉(Trichoderma koningiopsis)、深绿木霉(Tr...     

Effect of fungi–termite interaction on the chlorophyll content of three rice varieties grown on ultisol soil of Ikenne,south-west Nigeria

The effect of fungi–termite interaction on three rice varieties was conducted in a screen house at the Africa Rice Center (AfricaRice) Ibadan, Nigeria. Of the 10 fungi species (Fusarium verticilloides, Trichoderma sp., Aspergillus niger, Aspergillus flavus, Macrophoma sp., Neurospora sp., Botryodiplodia theobromae, Penicillum sp., Rhizopus sp. and Sclerotium rolfsii) isolated from termites, soil and rice plants, F. verticilloides, Trichoderma sp. and B. theobromae were used for the interaction study. Each fungus was inoculated singly and in combinations with termite into the root of each rice variety in potted soil. Leaf samples were taken to measure the chlorophyll content which is a major parameter to estimate effect of termite–fungi interaction. The chlorophyll content of the inoculated rice plants was significantly reduced when compared with the control. Fungi interaction with termite had significant reduction on the chlorophyll content. The synergistic relationship between the fungi and the termite was discussed.     

粮食上木霉菌的分离鉴定及其生防效果

【背景】粮食在生长和收储期极易受到病原真菌或产毒真菌的污染,造成严重的损失。众多实践证明木霉属(Trichoderma)可以有效防治植物病原真菌。【目的】鉴定和筛选能有效抑制粮食常见危害真菌的木霉生防菌株,开发生防菌剂,保障粮食生产安全。【方法】从粮食上分离筛选出35株木霉,通过多基因系统发育分析和形态学观察方法进行菌种鉴定,利用平板对峙试验筛选出对粮食常见危害真菌有抑制作用的菌株。【结果】35株木霉分属于8个种,分别为非洲哈茨木霉(Trichodermaafroharzianum)、类棘孢木霉(Trichodermaasperelloides)、 Trichoderma amoenum、近深绿木霉(Trichoderma paratroviride)、Trichoderma obovatum、长枝木霉(Trichoderma longibrachiatum)、东方木霉(Trichodermaorientale)和深绿木霉(Trichodermaatroviride)。对峙试验结果表明,这8种木霉对于粮食上分离到的10种危害真菌均具有较好的抑制效果。非洲哈茨木霉(T.afroharzi...     

Production of lytic enzymes by<Emphasis Type="Italic">Trichoderma</Emphasis> spp. and their effect on the growth of phytopathogenic fungi

The production of β-1,3-glucanases and chitinases by three strains ofTrichoderma in submerged cultures was determined. The synthesis of enzymes was induced by cell wall biopolymers of phytopathogenic fungi (Botrytis cinerea, Fusarium culmorum andF. oxysporum). T. hamatum produced the highest β-1,3-glucanase activity; the most effective inducer of enzyme synthesis was the biomass ofF. oxysporum. All examined strains ofTrichoderma inhibited phytopathogen growth in biotic tests. The diffusion tests showed that the lytic enzymes take part in growth inhibition of phytopathogenic fungi.     

Production of Inter- and Intra-strain Hybrids of Trichoderma spp. by Protoplast Fusion and Evaluation of Their Biocontrol Activity Against Soil-borne and Foliar Pathogens

Protoplast fusion techniques were used for the production of new antagonistic strains of Trichoderma spp. Two British and two Italian strains of Trichoderma with different biocontrol potential against Botrytis cinerea, Sclerotinia sclerotiorum and Pythium ultimum, were treated with u.v. radiation and mutants resistant to hygromycin B or propiconzole were selected. Protoplasts were obtained from these mutants after digestion of young hyphae with Novozym 234 and used in inter- and intra-strain protoplast fusion experiments. Hybrids were obtained in 13 crosses isolated on fungicide-amended media by the inheritance of resistance markers from both parental strains. Selected, fast-growing, stable mutants were tested in biocontrol trials against P. ultimum on lettuce seedlings and B. cinera on grape bunches in comparison with their parental strains. Intra-strain hybrids derived from cross XIII were tested in vitro for their mycoparasitic ability on sclerotia of S. sclerotiorum. A high degree of variability in the biocontrol and the mycoparasitic ability of the fusants was observed but no significant increase in the activity was accomplished after fusion, the hybrids being generally less activewas accomplished after fusion, the hybrids being generally less active than their parental strains.     

三株海洋木霉的应用潜力

【目的】探索3株海洋生境木霉的应用潜力。【方法】经过筛选和诱变,获得高抑菌活性及产孢量的木霉突变株;通过优化培养基、温度、初始p H考察其产孢量及最适培养条件;综合抑菌谱、重寄生及抑菌相关基因考察其抑菌活性;采用特殊培养基法考察其产纤维素酶、植酸酶、铁载体以及降解磷钾的能力,高效液相色谱法测定其产吲哚乙酸能力。【结果】3株木霉菌的产孢量分别为3.45×108、3.10×108和2.55×108 CFU/cm2,与野生型相比分别提高了88.52%、63.16%和180.22%;且均可产生厚垣孢子,其中XG20-1厚垣孢子产量最高,达到3.56×108 CFU/m L。3株木霉菌具有较广抑菌谱及对番茄早疫病菌的重寄生作用,同时扩增得到Tex1、Nag1、Eg1基因,生物学测试显示其均具有产纤维素酶、几丁质酶以及铁载体的能力,证明其抑菌活性是多种机制共同作用的结果;菌株可以降解磷钾,且吲哚乙酸产量分别为2.61、1.57和1.92 mg/L,具有促进植物生长的潜力。【结论】本文中3株木霉菌在开发为生防菌与生物肥料方面展现出良好的应用潜力。     

Isolation,characterization and function of laccase from Trichoderma

Of fourteen natural isolates of Trichoderma, no correlation was found between substrate weight loss and phenol oxidase (PO) activity in rice straw cultures. The highest PO producer from these laccase-positive strains was subjected to UV mutagenesis in order to select high and low PO activity mutants. There was no significant difference in substrate weight loss for mutant strains with six times higher and six times lower PO activity than the parent strain. Nor did the enzyme activity result in decreased growth inhibition by inhibitory phenolic compounds. PO enzyme from the parent Trichoderma and one of its high-PO-activity mutants was subsequently purified by ethanol precipitation from liquid cultures optimized by supplementation with copper sulphate and cycloheximide. Protein staining and activity staining of disc electrophoresis gels showed that only one PO enzyme of approximately 71 000 Da was produced. The enzyme could be defined as a laccase (benzenediol: oxygen oxidoreductase E.C. 1.10.3.2) because it catalysed the oxidation of syringaldazine and p-phenylenediamine in the absence of hydrogen peroxide, and because it was inhibited by cetyltrimethylammonium because but not by cinnamic acid. No specific in-vivo function could be assigned to this enzyme.Correspondence to: T. W. Flegel     

Generation,annotation, and analysis of ESTs from four different <Emphasis Type="Italic">Trichoderma</Emphasis> strains grown under conditions related to biocontrol

The functional genomics project “TrichoEST” was developed focused on different taxonomic groups of Trichoderma with biocontrol potential. Four cDNA libraries were constructed, using similar growth conditions, from four different Trichoderma strains: Trichoderma longibrachiatum T52, Trichoderma asperellum T53, Trichoderma virens T59, and Trichoderma sp. T78. In this study, we present the analysis of the 8,160 expressed sequence tags (ESTs) generated. Each EST library was independently assembled and 1,000–1,300 unique sequences were identified in each strain. First, we queried our collection of ESTs against the NCBI nonredundant database using the BLASTX algorithm. Moreover, using the Gene Ontology hierarchy, we performed the annotation of 40.9% of the unique sequences. Later, based on the EST abundance, we examined the highly expressed genes in the four strains. A hydrophobin was found as the gene expressed at the highest level in two of the strains, but we also found that other unique sequences similar to the HEX1, QID3, and NMT1 proteins were highly represented in at least two of the Trichoderma strains. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effect of fungicides on the mycelial growth of soft fruit spoilage fungi

The in vitro activity of a range of fungicides was tested on strains of Botrytis cinerea, Mucor mucedo, Rhizopus stolonifer, and R. sexualis. Di-chlofluanid and dichloran were more active than the other fungicides against all of the strains of B. cinerea, while dichloran, dichlorophen and the dithiocarbamate fungicides were the most active against the Rhizopus species. Only dichlorophen and thiram markedly inhibited the growth of M. mucedo.     

Changes in hyphal morphology and activity of phenoloxidases during interactions between selected ectomycorrhizal fungi and two species of <Emphasis Type="Italic">Trichoderma</Emphasis>

Patterns of phenoloxidase activity can be used to characterize fungi of different life styles, and changes in phenoloxidase synthesis were suspected to play a role in the interaction between ectomycorrhizal and two species of Trichoderma. Confrontation between the ectomycorrhizal fungi Amanita muscaria and Laccaria laccata with species of Trichoderma resulted in induction of laccase synthesis, and the laccase enzyme was bound to mycelia of ectomycorrhizal fungi. Tyrosinase release was noted only during interaction of L. laccata strains with Trichoderma harzianum and T. virens. Ectomycorrhizal fungi, especially strains of Suillus bovinus and S. luteus, inhibited growth of Trichoderma species and caused morphological changes in its colonies in the zone of interaction. In contrast, hyphal changes occurred less often in the ectomycorrhizal fungi tested. Species of Suillus are suggested to present a different mechanism in their interaction with other fungi than A. muscaria and L. laccata.     

Interaction between Colletotrichum falcatum pathotypes and biocontrol agents

Abstract

To select efficient antagonistic strain(s) of biocontrol agents against most of the existing pathotypes of Colletotrichum falcatum, an in vitro interaction study was carried out with 13 pathotypes, 12 isolates of Pseudomonas spp. and 6 isolates of Trichoderma spp. Antagonistic pseudomonad strains exhibited greater variation in their activity depending on the virulence of the pathotype. The lower the pathogen virulence, the higher was the antagonistic activity noticed. In general, sub-tropical pathotypes were suppressed at a comparatively higher level than the tropical pathotypes. Among the four efficient P. fluorescens strains selected based on their inhibitory effect against various pathotypes, ARR1G and VPT4 were effective against tropical pathotypes and FP7 showed moderate effect against all the pathotypes. The strain KKM2 was effective against sub-tropical and weaker tropical pathotypes. Strains of Trichoderma spp. did not show much variation in antagonism, but varied in their mode of action in suppressing the pathogen growth. However, based on higher rate of hyperparasitism, T. harzianum strains T5 and T62 were selected against all the pathotypes.