Two new species of trichoderma associated with green mold of oyster mushroom cultivation in Korea.

This paper describes and illustrates two new species, Trichoderma pleurotum and T. pleuroticola, associated with green mold disease of oyster mushroom in Korea.

Oyster mushroom (Pleurotus ostreatus) production in Korea has increased rapidly since the early 1980s. It is now one of the most valuable horticultural enterprises in Korea. During the last 10 years, however, commercial production of oyster mushroom has been seriously affected by green mold epidemics. The typical symptoms of the green mold are the appearance of green fungal sporulation on oyster mushroom substrates. In severe outbreaks, no mushrooms are produced from the contaminated beds (Park et al., 2005a; Yu, 2001).

To identify the Trichoderma species associated with the green mold epidemics of commercially grown oyster mushroom in Korea, we collected more than one hundred isolates of Trichoderma from oyster mushroom substrates throughout Korea over a period of 6 years from 1997 to 2002 (Park et al., 2004, 2005a; Yu, 2001). The isolates were classified into 7 distinct species based on morphological and cultural characteristics, and two of the 7 species were revealed as new taxa (Park et al., 2003, 2004, 2005a). Phylogenetic analysis of the ITS, the translating elongation factor 1 alpha gene (EF-1α) and RNA polymerase II gene (RPB2) sequences of the two taxa also supported their status as new to science (Park et al., 2003, 2004, 2005b). However, we have not reported new epithets and Latin diagnosis for the two fungal species. In this paper the two new species are described as Trichoderma pleurotum and T. pleuroticola, respectively.

Materials and Methods

Trichoderma strains, CNUMH 501 and CNUMH 601, were isolated from green molded waste cotton substrates at oyster mushroom farms in Chuncheon, Gangwon Province, Korea, April, 1998, and in Paju, Gyonggi Province, Korea, May, 1999, respectively. Colony characteristics and growth rates of each species were examined from their cultures grown in darkness at 25℃ for 7 days on PDA. Micromorphological data were examined on the cultures grown on cornmeal dextrose agar (CMDA) and PDA for 7 days at 25℃. The examination and measurements of conidiophores and conidia were made from slide preparations stained with 3% KOH. Differential contrast microscopy was used for observation and 50 units of morphological characters were measured. The morphology of each species was also illustrated with the aid of a drawing tube.

Taxonomy

Trichoderma pleurotum S. H. Yu & M. S. Park, sp. nov. Fig. 1

Coloniae in agaro succroso solanaceo sat rapide crescentes ad, 52~60 mm diametro post 3 dies (25 C). Conidiophorae pyramidae verticillata (typo Gliocladium), Phialides plerumque ampulliformes raro lageniformes, plerumque 4~7 subverticillatae, 4.2~7.0 × 2.0~4.0 µm. Conidia globosa vel ellipsoidea, laevia, 2.8~4.2 × 1.6~2.2 µm, in medio 3.4 × 2.0 µm. Chlamydosporae subglobosae vel ellipsoideae, laeves, 5.0~10.0 µm diam. Simile Trichodermati virenso, sed conidia magis parva et per sequentem "ITS" "EF-1α" et "RPB2" gene distinguible.

Holotype

dried culture specimen on PDA, isolated from the waste cotton substrate of oyster mushroom, Chuncheon, Gangwon Province, Korea, April 1998, S. H. Yu, CNUMH 501, deposited in the Mycological Herbarium, Chungnam National University (CNUMH), Korea, culture ex-type KCTC 26314.

Additional material examined

dried culture specimen on PDA, isolated from the waste cotton substrate of oyster mushroom, Boeun, Chungbuk Province, Korea, Oct. 1998, S. H. Yu, CNUMH 523.

Etymology

from the host oyster mushroom Pleurotus.

Colonies (on PDA at 25℃ after 3 days) growing rapidly, 52~60 mm in diam. Conidiation sparse and powdery; greenish white or grayish green, becomimg darker at age. Conidiophores usually simply branched; primary branches tended to be held at nearly right angles, apics of conidiophores and primary branches usually with a Gliocladium-like head comprised of 2~4 branches terminated by crowded whorls of philides; relatively broad. Phialides mostly ampulliform, infrequently lageniform, mostly arising in crowded whorls (verticils) of 4~7, 4.2~7.0 × 2.0~4.0 µm. Conidia ellipsoidal to obovoid, 2.8~4.2 × 1.6~2.2 µm (av. 3.4 × 2.0 µm), smooth-walled, pale green. Chlamydospores subglobose to ellipsoidal, smooth-walled, 5.0~10.0 µm diam.

Comments

T. pleurotum resembles T. virens in penicillate branching patterns of conidiophores and phialids, but can be distinguished readily from the latter by its predominant conidiation, many divided branches, more gathering of the fingers to top and more phialids to the apex. Additionally, the conidia of the new species are smaller (2.8~4.2 × 1.6~2.2 µm) compared to the conidia of T. virens (4.3~5.6 × 3.2~4.4 µm) and the conidiophores of the new species are broader (5~8 µm in diameter) than those of T. virens (3~6 µm in diameter). The molecular trees supporting circumscription of the taxa as new was also provided by the authors (Park et al., 2004, 2005b). The species was the most predominant species and the virulence of the species was significantly greater than that of the other Trichoderma species found in oyster mushroom environment (Park et al., 2005a).

Trichoderma pleuroticola S. H. Yu & M. S. Park sp. nov. Fig. 2

Coloniae in agaro succroso solanaceo sat rapide crescentes ad, 52~60 µm diametro post 3 dies (25 C). Conidiophorae pyramidae verticillata. Phialides ampulliformes vel lageniformes, plerumque 2-4 subverticillatae raro solitariae, 5.5~11.0 × 2.6~4.0 µm. Conidia continua, subglobosae vel lato ellipsoideae, laevia, 2.9~4.5 × 2.4~3.5 µm. in medio 3.3 × 2.7 µm. Chlamydosporae subglobosae vel ellipsoideae, 5.0~10.0 µm diam. Simile Trichodermati harziani, sed phialides magis longae et per sequentem "ITS" "EF-1α" et "RPB2" gene distinguible.

dried culture specimen on PDA, isolated from the waste cotton substrate of oyster mushroom, Paju, Gyonggi Province, Korea, May 1999, S. H. Yu, CNUMH 601, deposited in the Mycological Herbarium, Chungnam National University, Korea, culture ex-type KCTC 26315.

dried culture specimen on PDA, isolated from rice straw substrate of oyster mushroom, Inje, Gangwon Province, Korea, May 1999, S. H. Yu, CNUMH 571.

from the Latin Pleurotus, the epithet of the host oyster mushroom.

Colonies (on PDA at 25℃ after 3 days) growing rapidly, 60-65 mm in diam. Conidiation effuse, fasciculate, small pustules coalesced and forming broad concentric rings; initially greenish gray turning grayish-green to dark green. Conidiophores tending to be regularly verticillate and forming a more or less pyramidal structure. Phialides ampulliform to lageniform, mostly 3~4 verticillate or paired, occasionally arising singly, 5.5~11.0 × 2.6~4.0 µm. Conidia one celled, subglobose to broadly ellipsoidal, often pointed at the base, 2.9~4.5 × 2.4~3.5 µm (av. 3.3 × 2.7 µm), smooth-walled, pale green. Chlamydospores subglobose to ellipsoidal, 5.0~10.0 µm diam.

T. pleuroticola resembles T. harzianum in the morphology of conidiophores and branching patterns but differs in the length of phialides and conidial shape; in T. pleuroticola, the phialides are longer (5.5~11.0 × 2.6~4.0 µm) compared to 3.5~7.6 × 1.8~3.4 µm in T. harzianum; and T. pleuroticola has subglobose to broadly ellipsoidal conidia, while T. harzianum has globose to broadly ellipsoidal conidia. Phylogenetic analysis of ITS, EF-1α and RPB2 gene sequences also revealed that T. pleuroticola was phylogenitically distinct not only from T. harzianum but also from other previously reported Trichoderma species (Park et al., 2004, 2005b).