The most common biological control agents (BCAs) of the genus Trichoderma have been reported to be strains of Trichoderma virens, T. harzianum, and T. viride. Since Trichoderma BCAs use different mechanisms of biocontrol, it is very important to explore the synergistic effects expressed by different genotypes for their practical use in agriculture. Characterization of 16 biocontrol strains, previously identified as "Trichoderma harzianum" Rifai and one biocontrol strain recognized as T. viride, was carried out using several molecular techniques. A certain degree of polymorphism was detected in hybridizations using a probe of mitochondrial DNA. Sequencing of internal transcribed spacers 1 and 2 (ITS1 and ITS2) revealed three different ITS lengths and four different sequence types. Phylogenetic analysis based on ITS1 sequences, including type strains of different species, clustered the 17 biocontrol strains into four groups: T. harzianum-T. inhamatum complex, T. longibrachiatum, T. asperellum, and T. atroviride-T. koningii complex. ITS2 sequences were also useful for locating the biocontrol strains in T. atroviride within the complex T. atroviride-T. koningii. None of the biocontrol strains studied corresponded to biotypes Th2 or Th4 of T. harzianum, which cause mushroom green mold. Correlation between different genotypes and potential biocontrol activity was studied under dual culturing of 17 BCAs in the presence of the phytopathogenic fungi Phoma betae, Rosellinia necatrix, Botrytis cinerea, and Fusarium oxysporum f. sp. dianthi in three different media.
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.
The genus Trichoderma includes biocontrol agents (BCAs) effective against soilborne plant pathogenic fungi. Several potentially useful strains for biological control are difficult to distinguish from other strains of Trichoderma found in the field. So, there is a need to find ways to monitor these strains when applied to natural pathosystems. We have used random amplified polymorphic DNA (RAPD) markers to estimate genetic variation among sixteen strains of the species T. asperellum, T. atroviride, T. harzianum, T. inhamatum and T. longibrachiatum previously selected as BCAs, and to obtain fingerprinting patterns. Analysis of these polymorphisms revealed four distinct groups, in agreement with previous studies. Some of the RAPD products generated were used to design specific primers. Diagnostic PCR performed using these primers specifically identify the strain T. atroviride 11, showing that DNA markers may be successfully used for identification purposes. This SCAR (sequence-characterised amplified region) marker can clearly distinguish strain 11 from other closely related Trichoderma strains.
Trichoderma is known for being the most frequently used biocontrol agent in agriculture. A fundamental part of the Trichoderma antifungal system relies on a series of genes coding for a variety of extracellular lytic enzymes. Characterization of the polymorphism between five putative isoenzymatic activities [beta-1,3-glucanase (EC 3.2.1.39, EC 3.2.1.58), beta-1,6-glucanase (EC 3.2.1.75), cellulase (EC 3.2.1.4; EC 3.2.1.21, EC 3.2.1.91), chitinase (EC 3.2.1.30, EC 3.2.1.52), protease (EC 3.4.11; EC 3.4.13-19; EC 3.4.21-24, EC 3.4.99)] was carried out using 18 strains from three sections of Trichoderma. Of these, seven strains were from T. sect. Pachybasium, nine from T. sect. Trichoderma and two from T. sect. Longibrachiatum. Thirty-seven different alleles in total were identified: 13 for beta-1,3-glucanase, four for beta-1,6-glucanase, three for cellulase, eight for chitinase and nine for protease activity. A dendrogram (constructed by the unweighted pair group method with arithmetic averages) based on isoenzymatic data separated the 18 strains into three main enzymatic groups: T. harzianum, T. atroviride/T. viride/T. koningii and T. asperellum/T. hamatum/T. longibrachiatum. Isoenzymatic groupings obtained from biocontrol strains are discussed in relation to their phylogenetic location, based on their sequence of internal transcribed spacer 1 in ribosomal DNA and their antifungal activities.
Trichoderma spp. are associated with green mold of mushrooms. This fungal disease has caused severe losses in mushroom production in countries such as Ireland, the United Kingdom, Canada, and the United States. This disease is caused by two biotypes of T. harzianum (Th2, Europe; Th4, North America) (1,2). Both biotypes have not been detected in mushrooms or other material in Spain previously. However, during 1998, green mold was detected at facilities dedicated to produce compost, as well as in facilities used to produce Agaricus bisporus (Lange) Imbach. Three compost samples were isolated from commercial bags with mushroom substrate and three more samples were taken from mushroom yards. Several spores were isolated by the dilution plate method. Initial identification of the pathogenic fungi was made by examining cultures grown on potato dextrose agar. Morphological characteristics of all isolates coincided with the description of T. harzianum (3). The following amounts of CFU per g were found in commercial compost samples: 1.2 × 108, 5.5 × 107, and 1.4 × 107 per g; whereas 3 × 108, 12.4 × 107, and 2.2 × 106 were obtained from mushroom yards. The fragment containing the internal transcribed spacer (ITS1) was amplified and sequenced for each of the six samples obtained. The ITS1 sequence (201 bp) was identical in all samples, and the sequence was aligned, with Clustal W, with Th2 and Th4 biotype sequences of the EMBL data base. The ITS1 sequence showed 0.55% divergence from Th2 isolates and more distance, 3.3%, with Th4 isolates. The ITS1 sequence obtained with all Spanish samples studied, EMBL accession number AJ1321550, was identical to that described for the Irish isolate Th2I (#63), with accession number U78880 in the EMBL data base (1). This is the first description of the Th2 biotype in Spain. References: (1) M. D. Ospina-Giraldo et al. Mycologia 90:76, 1998. (2) D. L. Rinker et al. Mushroom World 8:71, 1997. (3) D. A. Seaby. Plant Pathol. 45:905, 1996.
Cryptococcus adeliensis was initially described as a psycrophilic species containing a single strain CBS 8351(T) isolated from decayed algae in Terre Adelie (Antartida). Later, a second strain of this species was isolated from an immunosuppressed patient affected by leukaemia in Germany and recently several strains from this species have been found in human patients and pigeon droppings of the same country. In this study, we isolated from sheep droppings in Spain a xylanolytic strain named LEVX01 that was phenotypically related to the strain CBS 8351(T) and showed a 100% similarity in the D1/D2 domain and 5.8S-ITS region sequences with respect to the remaining described strains of C. adeliensis. These findings suggest that this species has a wide geographical distribution and that the animal faeces are a common habitat for C. adeliensis. The chemotaxonomic analyses showed the absence of detectable amounts of xylose in the cell walls of the strains LEVX01 and CBS8351(T) in contrast to other Cryptococcus species. Interestingly, the ultrastructural study showed the presence of fimbriae in these two strains that could be involved in the attachment to the host cells and, as occurs in Candida albicans, they could also be a pathogenicity factor for the man.
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