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The Longibrachiatum Clade of the genus Trichoderma in Japan was examined, among which two new species and three new records are herewith reported. The new species, T. tsugarense and T. kunigamense were isolated from a bed log (cultivation of Lentinula edodes ) and volcanic ash soil, respectively. These species are distinguished from closely related species by growth and morphological characteristics and in phylogenetic analysis. Additional species new to Japan were T. ghanense , T. parareesei and T. sinense . The significance of their distribution is discussed. Most species of the Longibrachiatum Clade are tropical rather than temperate in distribution. Their in vitro optimum growth tends to be >35 °C but the optimum temperature for some Japanese species was lower. Some species are endophytes of temperate plant species, some of which are endemic in Japan.

We have developed a polymerase chain reaction (PCR)-based detection method for Trichoderma harzianum, which causes green mold disease in mushroom cultivation fields and facilities. Based on the sequence data of the internal transcribed spacer (ITS) region of T. harzianum strains and several other species, six primers consisting of three forward and three reverse primers were designed. Among the nine possible combinations of these primers, PCR with the pair THITS-F2 and THITS-R3 distinguished most T. harzianum strains from other Trichoderma species. The optimal annealing temperature for detection of T. harzianum strains was from 62° to 63°C with this primer combination. We designed new primers derived from THITS-F2 and THITS-R3. Annealing temperatures to detect T. harzianum ranged from 64° to 67°C using the new primers. The detection limit of T. harzianum DNA was 50 fg by nested PCR with THITS-F1 and LR1-1 for the first PCR and the new primers for the second PCR. T. harzianum was readily detectable in contaminated cultures of Lentinula edodes by this method.