In vitro antagonism of Thielaviopsis paradoxa by Trichoderma longibrachiatum

Seventy-nine Trichoderma strains were isolated from soil taken from 28 commercial plantations of Agave tequilana cv. ‘Azul’ in the State of Jalisco, Mexico. Nine of these isolates produced nonvolatile metabolites that completely inhibited the growth of Thielaviopsis paradoxa on potato dextrose agar plates. These isolates were identified as Trichoderma longibrachiatum on the basis of their morphology and DNA sequence analysis of two genes (ITS rDNA and translation elongation factor EF-1α). Mycoparasitism of Th. paradoxa by T. longibrachiatum strains in dual cultures was examined by scanning electron microscopy. The Trichoderma hyphae grew alongside the Th. paradoxa hyphae, but penetration of Thielaviopsis hyphae by Trichoderma was no apparent. Aleurioconidia of Th. paradoxa were parasitized by Trichoderma. Both hyphae and aleurioconidia of Th. paradoxa lost turgor pressure, wrinkled, collapsed and finally disintegrated. In liquid cultures, all nine Trichoderma isolates produced proteases, β-1,3-glucanases and chitinases that would be responsible for the degradation of Thielaviopsis hyphae. These results demonstrate that the modes of action of T. longibrachiatum involved against Th. paradoxa in vitro experiments are mycoparasitism and the production of nonvolatile toxic metabolites.     

Fusant Trichoderma HF9 with enhanced extracellular chitinase and protein content

Strain improvement was carried out to obtain higher chitinase and protein by inter-specific protoplast fusion between Trichoderma harzianum and Trichoderma viride. Fusant HF9 and parental strains of Trichoderma were compared for chitinase and protein production. 1% of glucose, sucrose and fungal cell wall (Rhizoctonia solani), were used as carbon source for cultivation of Trichoderma and fungal cell wall was the best to induce chitinase and protein. Usage of 0.5% colloidal chitin for the fungal growth under aerated conditions at pH 6.5 and 28°C led to higher chitinase and protein production. In these conditions fusant Trichoderma HF9 in comparison with parent strains had 3-, 2.5- and 1.5-fold increase of total chitinase, specific chitinase and protein, respectively. SDS-PAGE analysis revealed that it had 9 major protein bands with up-regulation compared to parent strains. Amino acid analysis showed that protein of culture filtrate of T. harzianum, T. viride and fusant Trichoderma HF9 had 8, 6 and 10 amino acids, respectively. The results obtained suggested that fusant HF9 could be an integration of T. harzianum and T. viride through protoplast fusion.     

In vitro and greenhouse evaluations of Trichoderma isolates for biological control of potato wilt disease (Fusarium solani)

The biological efficacy of Trichoderma species may differ due to variations in ecosystems. This study was conducted to assess the biocontrol efficacy of some native Trichoderma isolates against Fusarium solani, an important causal agent of potato wilt disease under laboratory and greenhouse conditions at Shahrood Agricultural Research Centre, Shahrood, Iran, during 2006–2007. Fourteen isolates were collected among which eight showed promising ability in inhibiting the growth of the pathogen through dual culture and production of volatile and non-volatile inhibitors but T. brevicompactum (T1), T. longibrachiatum (T5) and T. asperellum (T2) were almost better than other isolates in inhibiting the mycelial growth of the pathogen in comparison to control in the above three tests (p ≤ 0.01). Isolates performing mycoparasitism under in vitro condition were evaluated against the disease in pot culture under greenhouse condition. In all treatments in which Trichoderma isolates + F. solani were involved lower disease incidence was noticed in comparison to Fusarium-infested control (p ≤ 0.05). Best disease control was observed in potted plants treated with F. solani + T. longibrachiatum (T5) with 6.25% disease incidence in comparison to Fusarium-infested control, in which disease incidence was observed to be 75%. Interaction of T. brevicompactum (T1) and F. solani also indicated good control of the disease by 12.50% of disease incidence.     

Formation and regeneration of protoplasts of wild-typeNeurospora crassa

Trichoderma reesei was grown using purified cell walls ofNeurospora crassa as a primary source of carbon. The resulting culture medium contained an undefined mixture ofN. crassa cell-wall digesting enzymes. Protoplasts (cell lacking wall) were formed when youngN. crassa hyphae were treated withTrichoderma mixture. The vast majority of protoplasts resynthesized cell-wall material when washed free of cell-wall digesting enzyme; of these, about 40% regenerated a mycelium.     

Effect of Carbon Source on Production of Lytic Enzymes by the Sclerotial Parasites Trichoderma atroviride and Coniothyrium minitans

Three isolates of Trichoderma atroviride and two isolates of Coniothyrium minitans known to efficiently degrade sclerotia of Sclerotinia sclerotiorum were cultured on minimal medium with sucrose, carboxymethyl cellulose (CMC), xylan, laminarin, colloidal chitin or powdered sclerotia as carbon source. The activity of endochitinase, endo‐β‐1,3‐glucanase, endoxylanase and endocellulase in culture filtrates was determined after 7 and 15 days of culture using dye‐labelled substrates. The strongest inducers of chitinase were colloidal chitin and sclerotia powder. Chitinase activity appeared to be faster induced in the isolates of T. atroviride than in the isolates of C. minitans, but the maximum level of activity present in culture filtrates of the two species was similar. With CMC and xylan as carbon source, concurrent production of the corresponding enzymes was observed for the Trichoderma isolates. The isolates of C. minitans produced cellulase on xylan but not on CMC, whereas xylanase was produced on both carbon sources. Laminarin induced the formation of glucanases in the three isolates of T. atroviride but not the isolates of C. minitans. However, in the sclerotia‐containing cultures of C. minitans glucanase activity was even higher than in the respective cultures of the Trichoderma isolates. During the 31‐day study period, the pattern of enzyme production in shake cultures containing sclerotia powder was very similar for the isolates of T. atroviride and C. minitans. Glucanase activity generally reached a peak 24 days after inoculation of the flasks, whereas the activity of chitinase, cellulase and xylanase remained fairly constant throughout the experiment.     

Production of Antifungal Chitinase by Aspergillus niger LOCK 62 and Its Potential Role in the Biological Control

Aspergillus niger LOCK 62 produces an antifungal chitinase. Different sources of chitin in the medium were used to test the production of the chitinase. Chitinase production was most effective when colloidal chitin and shrimp shell were used as substrates. The optimum incubation period for chitinase production by Aspergillus niger LOCK 62 was 6?days. The chitinase was purified from the culture medium by fractionation with ammonium sulfate and affinity chromatography. The molecular mass of the purified enzyme was 43?kDa. The highest activity was obtained at 40?°C for both crude and purified enzymes. The crude chitinase activity was stable during 180?min incubation at 40?°C, but purified chitinase lost about 25?% of its activity under these conditions. Optimal pH for chitinase activity was pH 6–6.5. The activity of crude and purified enzyme was stabilized by Mg²⁺ and Ca²⁺ ions, but inhibited by Hg²⁺ and Pb²⁺ ions. Chitinase isolated from Aspergillus niger LOCK 62 inhibited the growth of the fungal phytopathogens: Fusarium culmorum, Fusarium solani and Rhizoctonia solani. The growth of Botrytis cinerea, Alternaria alternata, and Fusarium oxysporum was not affected.     

Antifungal Activity of Chitinases from <Emphasis Type="Italic">Trichoderma aureoviride</Emphasis> DY-59 and <Emphasis Type="Italic">Rhizopus microsporus</Emphasis> VS-9

Two chitinolytic fungal strains, Trichoderma aureoviride DY-59 and Rhizopus microsporus VS-9, were isolated from soil samples of Korea and Vietnam, respectively. DY-59 and VS-9 crude chitinases secreted by these fungi in the 0.5% swollen chitin culture medium had an optimal pH of 4 and the optimal temperatures of 40°C and 60°C, respectively. Enzymatic hydrolysis products from crab swollen chitin were N-acetyl-β-D-glucosamine (GlcNAc) by DY-59 chitinase, and GlcNAc and N, N′-diacetylchitobiose (GlcNAc)₂ by VS-9 chitinases. The chitinases degraded the cell wall of Fusarium solani hyphae to produce oligosaccharides, among which GlcNAc, (GlcNAc)₂, and pentamer (GlcNAc)₅ were identified by high-pressure liquid chromatography. DY-59 and VS-9 chitinases inhibited F. solani microconidial germination by more than 70% and 60% at final protein concentrations of 5 and 27 μg mL⁻¹, respectively, at 30°C for 20 h treatment.     

Protein Engineering of Chit42 Towards Improvement of Chitinase and Antifungal Activities

The antagonism of Trichoderma strains usually correlates with the secretion of fungal cell wall degrading enzymes such as chitinases. Chitinase Chit42 is believed to play an important role in the biocontrol activity of Trichoderma strains as a biocontrol agent against phytopathogenic fungi. Chit42 lacks a chitin-binding domain (ChBD) which is involved in its binding activity to insoluble chitin. In this study, a chimeric chitinase with improved enzyme activity was produced by fusing a ChBD from T. atroviride chitinase 18–10 to Chit42. The improved chitinase containing a ChBD displayed a 1.7-fold higher specific activity than chit42. This increase suggests that the ChBD provides a strong binding capacity to insoluble chitin. Moreover, Chit42-ChBD transformants showed higher antifungal activity towards seven phytopathogenic fungal species.     

Mycoparasitism studies of Trichoderma harzianum strains against Rhizoctonia solani: evaluation of coiling and hydrolytic enzyme production

The genus Trichoderma is a potential biocontrol agent against several phytopathogenic fungi. One parameter for its successful use is an efficient coiling process followed by a substantial production of hydrolytic enzymes. The interaction between fifteen isolates of Trichoderma harzianum and the soil-borne plant pathogen, Rhizoctonia solani, was studied by light microscopy and transmission electron microscopy (TEM). Macroscopic observations of fungal growth in dual cultures revealed that growth inhibition of the pathogen occurred soon after contact with the antagonist. All T. harzianum isolates tested exhibited coiling around the hyphae of R. solani. The strains ALL23, ALL40, ALL41, ALL43 and ALL49 did not differ in coiling frequency and gave equal coiling performances. No correlation between coiling frequency and the production of cell wall-degrading chitinases, N-acetyl-β-d-glucosaminidase and β-1,3-glucanases, was found.     

Metabolite profiling can assist variability analysis in Trichoderma species

Variability in 41 isolates of Trichoderma belonging to 21 species was observed in the phenolic acid profile of their culture filtrates. The phenolic acid profiles were observed to be very stable in the culture filtrate of Trichoderma species. The similarity in phenolic acid profile was recorded and based on it the species were grouped into three distinct groups, viz. highly similar, moderately similar and least similar. Of the 21 species, seven species showed highly similar trend, whereas two and four species showed moderate and least similarity in their phenolic acid profiles, respectively. Looking into the stability of phenolic acid profile in the culture filtrate of the Trichoderma species the present tool may help in diversity analysis in Trichoderma species originating from different geographical areas.     

Mycoparasitism ofFusarium SPP. onRhizoctonia solani Kühn

Summary Experiments on nutrient and staled agar were carried out to investigate the mycoparasitic activity of some fusaria againstRhizoctonia solani Kühn. Penetration and coiling byFusarium oxysporum Sch.,F. semitectum Berk & Rav. andF. udum Butler in and around theR. solani hyphae was observed. Lysis ofF. udum hyphae was observed inside theR. solani hyphae showing the reverse of the normal direction of necrotrophic mycoparasitic relationships. The mycoparasitic activity ofFusarium spp. was much affected in staled agar plates.     

The growth form of the inducing microorganism and chitin addition affect mycolytic enzyme production byTrichoderma harzianum

The effect of the growth form of the inducing microorganism on specificTrichoderma harzianum mycolytic enzyme production was studied. The pelleted form ofRhizopus nigricans gave a better product concerning protoplast formation ability. The maximum yield of protoplasts from the target fungusCochliobolus lunatus was 1×10⁸ ml^–1. Analysis of individual specific enzyme activities inTrichoderma mycolytic enzyme preparations confirms the importance of high chitinase and low protease activity for high protoplast yields. Supplementation of the production medium with chitin increased the chitinase activity in theTrichoderma exoenzyme mixture.     

Effect of peat-bran inoculum ofTrichoderma species on biological control ofRhizoctonia solani in lettuce

A range of known biocontrol or plant growth-stimulating species ofTrichoderma orGliocladium were grown on peat-bran substrate to yield between 5×10⁷–3×10¹⁰ colony forming units (cfu's)g^–1 substrate after 14 days growth. Inocula were incorporated into peat:sand potting compost infested withRhizoctonia solani to give 7–8 × 10⁴ cfu's of antagonist g^–1 compost and assessed for biological control activity using lettuce seedlings. Six of the eight antagonists decreased daming-off and three of these consistently increased yield in comparison withR. solani treatment alone.Subsequently, peat-bran inoculum ofT. harzianum isolate TH1 was incorporated at 0.5% w/v intoR. solani infested potting compost. Both autoclaved and nonautoclaved inoculum ofT. harzianum TH1 decreased disease and increased yield. Incorporation of ethyl acetate-extracted autoclaved inoculum ofT. harzianum TH1 resulted in similar levels of biocontrol and improved plant growth as did incorporation of nonautoclaved and autoclavedT. harzianum TH1 inoculum. The need to standardize inocula and controls is emphasized.     

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.     

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.     

Synthesis of aTrichoderma chitinase which affects theSclerotium rolfsii andRhizoctonia solani cell walls

ATrichoderma sp. isolate, hereafter called T₆, produces a 46-kDa endochitinase (CHIT 46) which had been shown to drastically affectin vitro the cell walls of the phytopathogensSclerotium rolfsii andRhizoctonia solani. We attempted to gain insight into its properties. The CHIT 46 N-terminal amino acid sequence shares a very high homology with other fungal chitinases. Western blot analysis using polyclonal antibodies anti-CHIT 46 revealed that this enzyme is immunologically distinct from other proteins produced by the sameTrichoderma isolate T₆, but is immunologically identical with proteins having equivalent molar mass, probably chitinases, produced by otherTrichoderma spp. isolates. In addition, the antibodies revealed also that a substantial amount of this enzyme is secreted into the culture medium 2 d after theTrichoderma isolate T₆ comes into contact with chitin.     

Optimization of cultural conditions for the production of antifungal chitinase by Streptomyces sporovirgulis

Actinomycetes were screened from soil in the centre of Poland on chitin medium. Amongst 30 isolated strains one with high activity of chitinase was selected. It was identified as Streptomyces sporovirgulis. Chitinase activity was detected from the second day of cultivation, then increased gradually and reached maximum after 4 days. The maximum chitinase production was observed at pH 8.0 and 25–30°C in the medium with sodium caseinate and asparagine as carbon and nitrogen sources and with shrimp shell waste as inducer of enzyme. Chitinase of S. sporovirgulis was purified from a culture medium by fractionation with ammonium sulphate as well as by chitin affinity chromatography. The molecular weight of the enzyme was 27 kDa. The optimum temperature and pH for the chitinase were 40°C and pH 8.0. The enzyme activity was characterised by high stability at the temperatures between 35 and 40°C after 240 min of preincubation. The activity of the enzyme was strongly inhibited in the presence of Pb²⁺, Hg²⁺ and stabilized by the ions Mg²⁺. Purified chitinase from S. sporovirgulis inhibited growth of fungal phytopathogen Alternaria alternata. Additionally, the crude chitinase inhibited the growth of potential phytopathogens such as Penicillium purpurogenum and Penillium sp.     

Characterization of field isolates of Trichoderma antagonistic against Rhizoctonia solani

The aim of the present study was to characterize sixteen isolates of Trichoderma originating from a field of sugar beet where disease patches caused by Rhizoctonia solani were observed. Use of both molecular and morphological characteristics gave consistent identification of the isolates. Production of water-soluble and volatile inhibitors, mycoparasitism and induced systemic resistance in plant host were investigated using in vitro and in vivo tests in both sterilized and natural soils. This functional approach revealed the intra-specific diversity as well as biocontrol potential of the different isolates. Different antagonistic mechanisms were evident for different strains. The most antagonistic strain, T30 was identified as Trichoderma gamsii. This is the first report of an efficient antagonistic strain of T. gamsii being able to reduce the disease in different conditions. The ability to produce water-soluble inhibitors or coil around the hyphae of the pathogen in vitro was not related to the disease reduction in vivo. Additionally, the strains collected from the high disease areas in the field were better antagonists. The antagonistic activity was not characteristic of a species but that of a population.     

The in vitro screening of 118 Trichoderma isolates for antagonism to Rhizoctonia solani and an evaluation of different environmental sites of Trichoderma as sources of aggressive strains

Trichoderma isolates were collected from different sources and screened for their in vitro parasitism of Rhizoctonia solani. They were grouped according to the different sources and each group compared statistically. 74% of the total isolates collected were regarded as antagonistic to R. solani in vitro. Isolates associated with pine bark source were very aggressive. The most aggressive strains were isolated from soil samples collected under the Speedling^® trays of a commercial seedling nursery.     

Isolation of Trichoderma and Evaluation of their Antagonistic Potential against Alternaria porri

Nine isolates of Trichoderma were collected from Assiut Governorate, Egypt, as leaf surface and endophytic fungi associated with onion flora stalks. Four isolates were identified as Trichoderma harzianum, while five isolates were belonging to Trichoderma longibrachiatum. The antagonistic activity of these isolates against onion purple blotch pathogen Alternaria porri was studied in vitro using dual culture assay. All tested Trichoderma isolates showed mycoparasitic activity and competitive capability against the mycelial growth of A. porri. Mycoparastic activity of Trichoderma was manifested morphologically by the overgrowth upon the mycelial growth of the pathogen and microscopically by production of coiling hyphae around pathogen hyphae. Isolates of T. harzianum exhibited high ability to compete on potato dextrose agar (PDA) medium causing the maximum rate of pathogen inhibition (73.12%), while isolates of T. longibrachiatum showed inhibition rate equalling 70.3%. Chitinase activity of Trichoderma was assayed, and T. harzianum Th‐3013 showed the maximum value contributing 2.69 U/min. Application of T. harzianum Th‐3013 to control purple blotch disease in vivo under greenhouse conditions caused disease reduction up to 52.3 and 79.9% before and after 48 h of pathogen inoculation, respectively, while the fungicide Ridomil Gold Plus caused disease reduction comprising 56.5 and 71.7%, respectively. This study proved that T. harzianum Th‐3013 as a biocontrol agent showed significant reduction in onion purple blotch disease compared with the tested fungicide.