Antagonistic potential of Gliocladium virens and Trichoderma longibrachiatum to phytopathogenic fungi

Three isolates of Gliocladium virens (G1, G2 and G3) and two of Trichoderma longibrachiatum (T1 and T2) were screened against isolates of three soilborne plant pathogens namely Rhizoctonia solani, Sclerotium rolfsii and Pythium aphanidermatum. G. virens exhibited stronger hyperparasitism and wider biological spectrum than T. longibrachiatum. Further, similarities as well as variation was observed in the ability of the various isolates to invade the test pathogens in dual culture. For the hyperparasites, acidic pH range (5.0 to 5.5) favoured both growth and spore germination. The hyperparasites made direct contact with the pathogens followed by varied modes of attack invariably leading to cell disruption. Antagonists, G1 and G3 revealed strong antibiosis while T2 showed moderate effect. All the isolates produced enhanced levels of lytic enzymes adaptively and there were marked differences among them. However, no correlation was observed between these attributes and the hyperparasitic potential of the various isolates in dual culture. The relevance and the role of enzymes and toxic metabolite(s) in the antagonism of G. virens and T. longibrachiatum to these pathogens are discussed.     

Fusarium lateritium Nees ex Link as a Parasite and Host in Interfungal Hyphal Interactions

In dual cultures Fusarium lateritium Nees ex Link coiled, penetrated and destroyed young actively growing hyphae of Botrytis cinerea Pers., Cytospora rubescens Fr., Eutypa armeniacae Hansf. et Carter, Sphaeropsis malorum Peck, Rhizoctonia solani Kühn, Trichoderma koningii Oud. (Tk-1) and Trichoderma hamatum (Bon.) Bain. (Tha-2), and conidiophores and conidia of Botrytis cinerea Pers. The isolates of Trichoderma koningii Oud. (Tk-3) and Trichoderma pseudokoningii Rifai (Tp-1) parasited and destroyed the hyphae of Fusarium lateritium. There was no hyphal interaction between Fusarium lateritium and an isolate of Trichoderma longibrachiatum Rifai. Hyphal, interactions occurred mostly on the aerial hyphae of test fungi which grew over the inhibition zone formed between the colonies of Fusarium lateritium and the test fungus, and contacted the hyphae of Fusarium lateritium. Details of dual culture studies are given.     

Characterization of the strain <Emphasis Type="Italic">Monascus floridanus</Emphasis> P. F. Cannon &; E. L. Barnard,isolated from aviation fuel

A fungus was isolated from aviation fuel and identified as Monascus floridanus P.F. Cannon & E.L. Barnard (FR827895) according to its morphological and genetic properties. The isolate has some properties that are unusual for the type strain, including a prominent stripe on one of the sides of the ascospores and occurrence, along with the known Basipetospora-type thallic conidia, of the phialophora-like spore formation. The isolated strain Monascus floridanus, like the known kerosene fungus Hormoconis resinae (Lindau) Arx & G.A. de Vries, is capable of active growth in aviation fuel.     

Isolation of novel benzo[a]anthracene-degrading microorganisms and continuous bioremediation in an expanded-bed bioreactor

In the present work, several samples from lab waste containers polluted with polycyclic aromatic hydrocarbons (PAHs) and heavy metals were investigated as potential sources of PAH-degrading microorganisms. After isolating, two fungal strains were selected as the best degrading microorganisms. Genetic identification by sequencing was carried out and they were identified as Trichoderma longibrachiatum and Byssochlamys spectabilis. Their degradation ability was determined in liquid cultures with 100 μM of benzo[a]anthracene. T. longibrachiatum cultures showed highest degradation values (around 97%) after 9 days, furthermore in a second batch the time was reduced to 6 days. To analyse the viability of industrial application, a continuous treatment in an expanded-bed bioreactor was developed operating at different residence times with T. longibrachiatum immobilised on cubes of nylon sponge. It is noticeable that the bioreactor working in continuous mode was able to operate without operational problems and attaining high degradation levels depending on the residence time.     

PCR with degenerate primers amplifies a subgenomic DNA fragment from the endoglucanase gene(s) of Torula thermophila, a thermophilic fungus

The aim of this study was to enable the polymerase chain reaction (PCR) amplification of DNA fragments within endoglucanase gene(s) of Torula thermophila, by using degenerate primers so that the amplified fragment(s) could be used as homologous probe(s) for cloning of full-length endoglucanase gene(s). The design of the degenerate PCR primers was mainly based on the endoglucanase sequences of other fungi. The endoglucanase gene sequence of Humicola insolens was the only sequence from a thermophilic fungus publicly available in the literature. Therefore, the endoglucanase sequences of the two Trichoderma species, Trichoderma reesei and Trichoderma longibrachiatum, were used to generalize the primers. PCR amplification of T. thermophila genomic DNA with these primers resilied in a specific amplification. The specificity of the amplified fragment was shown by Southern hybridization analysis using egl3 gene of T. reesei as probe. This result suggested that the degenerate primers used in this study may be of value for studies aimed at cloning of endoglucanase genes from a range of related fungi.     

In vitro and in vivo antagonism of biocontrol agents against Colletotrichum lindemuthianum causing bean anthracnose

Bean anthracnose caused by Colletotrichum lindemuthianum is a serious seed borne disease. For devising an effective management strategy, the efficacy of different bioagents, viz. Trichoderma viride, Trichoderma harzianum, Trichoderma hamatum and Gliocladium virens conducted under in vitro and in vivo conditions revealed maximum inhibition of mycelial growth in dual culture (59.48%) and inverted plate (55.98%) with T. viride. All the bioagents overgrew the pathogen and the principal mechanism of mycoparisitism observed was coiling, brusting and disintegration of pathogen hyphae. Culture filtrate from T. viride was found best as it completely inhibited radial growth at 25 and 50% concentration and reduced the spore germination of test fungus significantly. However, lower concentrations of culture filtrate from all bioagents showed little effect on spore germination. Seed application of bioagents was found better as compared to soil application. A maximum increase in seed germination and inhibition of seed borne infection was observed with T. viride followed by T. harzianum under pot culture conditions. T. viride has the maximum potentiality to suppress the spore germination, mycelial growth, seed borne infection of C. lindemuthianum and increased seed germination when compared with the other biocontrol agents.     

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...     

Interaction of the β-Transglycosylase of Trichoderma longibrachiatum with Cellulose

The effects of cellulose on the production and stimulation of β-transglycosylase were studied. The β-transglycosylase of Trichoderma longibrachiatum was produced specifically in the presence of cellulose in Czapeck-Dox medium containing sucrose as a sole carbon source. The enzyme activity was stimulated by the addition of cellulose in the reaction mixture, where the transfer reaction product (a water-insoluble glucan) was apparently synthesized on the surface of the added cellulose fibers.

The hyphal wall fraction of the fungus had the same stimulatory effect on β-transglycosylase as the cellulose fibers. A cellulose-like material in this fraction was found by partial acid hydrolysis and gas chromatography. Cellotriose was the smallest substrate effective for the synthesis of a water-insoluble glucan in the presence of cellulose by the β-transglycosylase, though a significant amount of glucan could not be synthesized without the addition of cellulose.     

Establishment and persistence of the entomopathogenic fungus Metarhizium anisopliae in maize fields

The entomopathogenic fungus Metarhizium anisopliae (Metsch.) Sorokin (Hypocreales: Clavicipitaceae) was applied in maize fields to control the Western Corn Rootworm Diabrotica virgifera virgifera Le Conte (Coleoptera: Chrysomelidae). Establishment and persistence of two strains of M. anisopliae were investigated after application as ‘fungal colonized barley kernels’ (FCBK) into the soil and as a spore suspension on maize leaves and on the soil surface in 2006 and 2007 at two locations in Hungary. The applied fungal strains were able to establish at both locations and a long‐term persistence of at least 15 months could be recorded in the soil. A positive correlation between density of colony forming units (CFU) in the soil and the soil inhabiting stages of the host insect D. v. virgifera could be found. M. anisopliae spores applied on maize leaves were able to survive for no longer than 3 days after application, whereas on the soil surface a noticeably increase of fungus densities were found after treatments. Molecular markers were used to identify the applied M. anisopliae strains before and after application of FCBK into the soil of the maize field.     

Effect of<Emphasis Type="Italic">Trichoderma</Emphasis> species on growth of Fusarium<Emphasis Type="Italic">proliferatiom</Emphasis> and production of fumonisins,fusaproliferin and beauvericin

Trichoderma species has been suggested as potential biocontrol agent forFusarium verticillioides on maize. In this cereal,F. verticillioides and F. proliferatum contributed to fumonisin accumulation. In addition,F. proliferatum could produce beauvericin and fusaproliferin. The aim of this work was to evaluate the effect ofTrichoderma spp. on growth and fumonisin B¹ fusaproliferin and beauvericin production byF. proliferatum. Dual cultures of F.proliferatum andT. harzianum ITEM 3636 andT. longibrachiatum ITEM 3635 on maize meal agar at 0.995 aw were done. The effect ofTrichoderma spp. on the lineal growth ofF. proliferatum was determined. The effect ofTrichoderma species on fumonisin B¹, fusaproliferin and beauvericin production byF. proliferatum was determined on co-inoculated maize kernels by HPLC.T. harzianum suppressedF. proliferatum growth once contact between the colonies occurred.T. longibrachiatum showed a less antagonistic effect againstF. proliferatum. A reduction on fumonisin B¹ production of 98% and 88% was observed in the co-incubation ofF. proliferatum withT. harzianum andT. longibrachiatum, respectively. The decrease of FB¹ production was significant even in maize kernels on whichF. proliferatum had been growing 7 days prior to the addition ofTrichoderma spp. The concentration of beauvericin and fusaproliferin produced during 30 days coincubation ofF. proliferatum with bothTrichoderma spp. did not differ to those produced byF. proliferatum alone. These mycotoxins might enter the food chain causing so far unknown consequences to the health of domestic animals and humans. For this reason it is important, when a potential biocontrol agent is under study, to test the effect on the fungal growth and on the putative mycotoxin produced. Part of the information was presented at the Mycotoxin Prevention Cluster Dissemination Day and Mycoglobe Launch Conference, Brussels, Belgium, Oct 20–21, 2004 Financial support: Agenda Córdoba Ciencia, grant N^o 0279–000431/00     

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 fungal colonization on mechanical performance of cork

The industrialization of traditional processes relies on the scientific ability to understand the empirical evidence associated with traditional knowledge. Cork manufacturing includes one operation known as stabilization, where humid cork slabs are extensively colonized by fungi. The implications of fungal growth on the chemical quality of cork through the analysis of putative fungal metabolites have already been investigated. However, the effect of fungal growth on the mechanical properties of cork remains unexplored. This study investigated the effect of cork colonization on the integrity of the cork cell walls and their mechanical performance. Fungal colonization of cork by Chrysonilia sitophila, Mucor plumbeus Penicillium glabrum, P. olsonii, and Trichoderma longibrachiatum was investigated by microscopy. Growth occurred primarily on the surface of the cork pieces, but mycelium extended deeper into the cork layers, mostly via lenticular channels and by hyphal penetration of the cork cell wall.In this first report on cork decay in which specific correlation between fungal colonization and mechanical proprieties of the cork has been investigated, all colonizing fungi except C. sitophila, reduced cork strength, markedly altering its viscoelastic behaviour and reducing its Young's modulus.     

Trichoderma longibrachiatum as an antagonist of Botrydiplodia theobromae

The antagonistic potential of Trichoderma longibrachiatum against the pathogen Botrydiplodia theobromae was examined in vitro. Both fungi were paired on 9 cm Petri plates of acidified potato dextrose agar (APDA). Three pairing methods were employed, viz., ‘inoculating antagonist before pathogen’, ‘inoculating pathogen before antagonist’ and ‘simultaneous inoculation of pathogen and antagonist’. Radial growth (cm) of both fungi were later analysed using the GLM Procedure of SAS. Growth inhibition of B. theobromae by T. longibrachiatum in all pairing methods was significantly different from control (P = 0.05, R ² = 0.89). Growth inhibition of the pathogen was best when using ‘inoculation of antagonist before pathogen’. Duration of pairing (DAP) and pairing method are both critical to significant antagonistic impact of T. longibrachiatum on B. theobromae (P > 0.0001). Inhibition mechanism includes competition for space and nutrients. T. longibrachiatum could thus be a promising antagonist of B. theobromae.     

The relationship between cellular and calcium responses of <Emphasis Type="Italic">Aspergillus awamori</Emphasis> to external influences

The cellular and Ca²⁺ responses to physiological stimuli of different nature were studied in the experiments with the strain Aspergillus awamori 66A containing recombinant aequorin, a Ca²⁺-dependent photosensitive protein. The relationship between the cellular response registered by changes in the development of the mycelial fungus (colony growth, hyphal branching, and the rate of spore formation) and the level and duration of calcium flares in the cytosol was assessed. The physical or chemical stimuli (mechanical effect, osmotic shock) inducing short-time calcium flares in the cytosol did not influence significantly the development of A. awamori grown in liquid or on solid nutrient media. The action on the 24-h A. awamori culture of the Ca²⁺-selective ionophore (A23187) inducing long-term changes in calcium homeostasis caused disorders in the fungus development and morphology (hyperbranching of mycelial hyphae, formation of spherical cells, and inhibition of colony growth and spore formation). Thus, it was established that the development of cellular response in the micromycete correlated with the duration of the calcium flare in the cytosol.     

Xylanase prebleaching of fractions of Douglas-fir kraft pulp of different fibre length

A commercial xylanase from Trichoderma longibrachiatum was used to treat the fractions of Douglas-fir kraft pulp of different fibre length. Enzymatic prebleaching was followed by chelation and peroxide bleaching. An evaluation of both optical and physical properties of the distinct fractions was conducted. A difference in susceptibility of the fractions of different fibre length to xylanase prebleaching was observed. The bleach-boosting effect observed with all fractions appeared to be related to the high-molecular-mass UV-absorbing material solubilized during enzyme treatments. Xylanase treatments resulted in beneficial effects to handsheet density and roughness as well as to some of the strength properties. However, the response to the xylanase treatments exhibited by all fractions of different fibre length was not uniform, indicating that fiber composition played a key role in the effectiveness of xylanase treatments.     

Atkinsiella awabi sp. nov. isolated from stocked abalone,Haliotis sieboldii

A fungal disease in the abalone,Haliotis sieboldii, stocked in Yamaguchi Prefecture, Japan, showed external signs of infection of tubercle-like swelling on the mantle and melanized lesions on the peduncle. The fungus responsible was isolated by inoculating materials taken from the lesions onto PYGS agar with streptomycin sulphate and ampicillin, and incubation at 20°C. For morphological observation and spore formation study, the fungus was transferred respectively into PYGS broth and sterilized artificial seawater and incubated at 20°C. Resulting, hyphae were stout, irregular, branched, 16–140µm diam, sporadically consisting of dense cytoplasmic swollen hyphae. Sporangia were formed through the formation of septa and lateral or terminal discharge tubes which were wavy or coiled. Zoospores were pyriform, biflagellate and diplanetic. The encysted spore generally developed a hairlike filament with globular enlarged tip in PYGS broth. Direct germination without filament formation also occurred occasionally. This fungus was identified as belonging to the genusAtkinsiella, and was designatedAtkinsiella awabi sp. nov. The fungus was exclusively a marine fungus and grew best in shrimp extract medium at 20°C. Five chemicals were tested for their effects against fungal zoospores.     

Enhanced growth and nutrition of micropropagated Ficus benjamina to Glomus mosseae co-inoculated with Trichoderma harzianum and Bacillus coagulans

A greenhouse investigation was conducted to study the influence of the arbuscular mycorrhizal (AM) fungus Glomus mosseae and the plant growth-promoting rhizomicroorganisms (PGPRs) Bacillus coagulans and Trichoderma harzianum on the growth and nutrition of micropropagated Ficus benjamina plantlets. The AM fungus was inoculated either singly or in combination with the PGPRs. Plants showed maximum plant height, biomass, P content, mycorrhizal root colonization, spore numbers and populations of T. harzianum and B. coagulans in root zone soil when all the three organisms were inoculated together. Thus, when G. mosseae co-inoculated with PGPRs enhances growth and nutrition of Ficus benjamina. T. harzianum and B. coagulans are thus designated as mycorrhizal helper organisms.     

Sensitivity of Clonostachys rosea and Trichoderma spp. as Potential Biocontrol Agents to Pesticides

Clonostachys rosea 47 (CR47), Trichoderma atroviride 59 (TA59), T. atroviride 312 (TA312), Trichoderma harzianum 24 (TH24), Trichoderma longibrachiatum 9 (TL9), T. longibrachiatum 144 (TL144) and Trichoderma viride 15 (TV15) were tested to evaluate their in vitro sensitivity towards five fungicides (carboxin, guazatine, prochloraz, thiram and triticonazole) and four herbicides (chlorsulfuron, chlorotoluron, flufenacet and pendimethalin). All antagonists showed low sensitivity to carboxin and thiram and high sensitivity to prochloraz. For mycelial radial growth, TV15 was highly sensitive to guazatine, prochloraz and triticonazole and TH24 moderately insensitive to carboxin, guazatine and thiram. For conidial germination TL144 was the most sensitive to the fungicides, for mycelial radial growth and conidial germination CR47 was the least sensitive. None of the antagonists showed any mycelial radial growth inhibition in presence of the herbicides at field dose, except for TL144. Most antagonists did not show any conidial germination inhibition by the herbicides. The in vitro toxicity of prochloraz, guazatine and triticonazole towards the antagonists was confirmed by light and scanning electron microscope showing hyphal disruptions and extrusion of cytoplasmic content. A mixture of CR47 and/or TA312 with carboxin, thiram and triticonazole, applied to wheat seeds, was able to control Fusarium culmorum artificially inoculated to wheat seedlings in growth chambers. In the field, the antagonists applied along with triticonazole or thiram, at 1/10 of the field dose to seeds naturally infected by F. culmorum, gave a disease control comparable to that induced by triticonazole at full field dose. Our results demonstrate how an integration of microorganisms with pesticides makes the control of wheat foot rot possible.     

Effects of prometryn and acetochlor on arbuscular mycorrhizal fungi and symbiotic system

Prometryn and acetochlor are common herbicides widely used to control weeds in agricultural systems. The impacts of the two herbicides on spore germination, hyphal elongation, the biomass and malondialdehyde content of carrot hairy roots were investigated using a strict in vitro cultivation system associating the Ri T‐DNA‐transferred carrot hairy roots with Glomus etunicatum. Alternatively, root colonization, daughter spore production and the proportion of hyphae with succinate dehydrogenase (SDH) and alkaline phosphatase (ALP) activities were also investigated. No significant impact on spore germination was noted in the presence of acetochlor at all three concentrations tested, while a significant decrease was observed with prometryn only at the highest concentration. Moreover, an inverse correlation was identified between herbicides concentrations and G. etunicatum root colonization and spore production as well as hyphal SDH and ALP activity, with a positive correlation identified among these four factors. Both herbicides exerted negative effects on the arbuscular mycorrhizal (AM) fungus and symbiosis at increasing concentrations, with prometryn apparently more toxic than acetochlor. Furthermore, the AM symbiotic system was shown to improve biomass, reduce malondialdehyde accumulation and ease lipid peroxidation in carrot hairy roots and decrease damage in host plants, thus enhancing plant tolerance to adverse conditions.

Significance and Impact of the Study

In this study, the effect of prometryn and acetochlor on the physiology and metabolic activities of the AM fungus Glomus etunicatum were investigated. Our findings demonstrate for the first time, the impact of the two herbicides at three concentrations (0·1, 1 and 10 mg l^?1) on transformed carrot hairy roots/AM fungus association under strict in vitro culture conditions, which may guide the application of the two herbicides in modern agriculture.