Trichoderma aggressivum f. aggressivum (T.a.f.a.) and T. aggressivum f. europeanum (T.a.f.e.) cause green mold disease in the cultivated mushroom (Agaricus bisporus) in North America and Europe, respectively. T.a.f.a. and T.a.f.e. were previously reported as T. harzianum biotypes Th4 and Th2 (Samuels et al., 2002). The disease is characterized by a rapid infestation of the compost by the pathogen and inhibition of A. bisporus fructification, resulting in serious yield losses worldwide in mushroom production (Ospino-Giraldo et al., 1999). Various molecular approaches were successfully used to characterize these isolates and distinguish them from non-aggressive Th1 (T. harzianum) and Th3 (T. atroviride) biotypes (Miyazaki et al., 2009; Ospino-Giraldo et al., 1999; Samuels et al., 2002). Based on Th4 genomic DNA, the polymerase chain reaction (PCR) marker of 444 bp was developed, which was amplified form both aggressive biotypes (Chen et al., 1999). To distinguish T.a.f.a. and T.a.f.e., the random amplified polymorphic DNA technique was successfully used (Chen et al., 1999; Szczech et al., 2008). However, this technique often suffers from poor reproducibility. Hatvani et al. (2007) used restriction fragment length polymorphism of mitochondrial DNA to distinguish Trichoderma strains. We report a simple, reliable approach to recognize T.a.f.a. and T.a.f.e. by endonuclease restriction of the amplicon Th444. Six isolates, CBS 466.94 (Th 1), CBS 693.94 (Th 3), CBS 100.528 (T.a.f.a.), CBS 450.95 (T.a.f.a.), CBS 689.96 (T.a.f.e.), and CBS 100.526 (T.a.f.e.), were obtained from the Centraalbureau voor Schimmelcultures (CBS) collection (Utrecht, The Netherlands). Twenty-eight Polish mushroom isolates of T.a.f.e. were received from the Research Institute of Vegetable Crops collection, Skierniewice, Poland (Table 1). Morphological and molecular studies of the T.a.f.e isolates are described in Szczech et al. (2008).
Polish T.a.f.e isolates used in this study.
DNA was extracted from mycelium grown on malt extract agar according to Aljanabi and Martinez (1997). The PCR marker Th444 was visible as a strong band on ethidium bromide-stained agarose gels both in T.a.f.a. and T.a.f.e. and was not generated in T. harzianum biotypes Th1 and Th3 (data not shown), because it has been reported by Chen et al. (1999). Polymorphism in T.a.f.a. and T.a.f.e. was revealed by digestion of Th444 with the restriction enzyme BseGI (Fermentas, UAB, Lithuania). The specific restriction fragment of ≈260 bp was informative for the T.a.f.a. reference isolates CBS 100.528 and CBS 450.95 (Fig. 1, Lanes 1 and 17, respectively), whereas the restriction product of 300 bp was visible in the reference isolates T.a.f.e. CBS 689.96 and CBS 100.526 (Fig. 1, Lanes 2 and 18, respectively) and all 28 T.a.f.e. Polish isolates (Fig. 1, Lanes 3 to 16 and 19 to 32). To our knowledge, it is the first example of the reproducible molecular markers that can considerably simplify detection of aggressive biotypes of Trichoderma in compost used in mushroom production.
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