First Report of Cladobotryum mycophilum Causing Cobweb on Cultivated King Oyster Mushroom in Spain.

In 2010, symptoms of cobweb were observed on cultivated king oyster mushroom (Pleurotus eryngii) in Castilla-La Mancha (Spain) affecting 16% of the blocks of substrate cultivated. Cobweb appeared at the end of the crop cycle, first as small, white patches on the casing soil, subsequently spreading to the nearest king oyster mushroom by means of a fine gray-white mycelium, and eventually sporulating to produce masses of dry spores. The mycelium can quickly cover pinheads, stalks, pileus, and gills, eventually resulting in decomposition of the entire fruit body. Infected tissues of P. eryngii were plated onto potato dextrose agar (PDA) and the parasitic fungus was isolated. Fungal colonies consisted of abundant and cottony aerial mycelium spreading rapidly on PDA and red pigment spreading in the agar. Conidiogenous cells were 24 to 35 μm long, 3.5 to 5 μm wide basally, and tapered slightly to the tip. Conidia were cylindrical to narrowly ellipsoidal, 17 to 25 (-28) × 8 to 10 μm, and zero to three septate. Total DNA was extracted and the internal transcribed spacer (ITS) region of rDNA was amplified for one isolate using ITS1F/ITS4 primers (1,3). The amplicon was sequenced (GenBank Accession No. JF505112). BLAST analysis showed 100% similarity of the obtained ITS sequence with two sequences of Cladobotryum mycophilum (teleomorph Hypomyces odoratus) (GenBank Accession Nos. Y17096 and Y17095) (2). Pathogenicity tests were performed using 24 blocks containing sterilized, spawned, and incubated P. eryngii substrate (3.6 kg, 352 cm in area). The blocks were placed in a mushroom-growing room and cased with a 40-mm layer of a casing soil (0.7 liter block) made with mineral soil + Sphagnum peat 4:1 (vol/vol). Five days after casing, a conidial suspension (7 × 10 conidia ml) of one isolate of C. mycophilum was sprayed (5 ml per block) onto the surface of the casing layer at a rate of 10 conidia m. Twenty-two blocks were sprayed with sterile distilled water as a control. A temperature of 17 to 18°C and 85 to 90% relative humidity were maintained throughout cropping. The first cobweb symptoms developed 23 days after inoculation and C. mycophilum was consistently reisolated from nine (37.5%) of the inoculated blocks. Noninoculated blocks remained healthy. In a second test, conidial suspensions (3.4 × 10 conidia ml) of one isolate of C. mycophilum were inoculated onto 20 P. eryngii fruit bodies. Ten fruit bodies were inoculated externally while the other 10 fruit bodies were cut in half and inoculated internally with 50 μl of conidial suspension per fruit body. Sterilized distilled water was used as a control. All fruit bodies were then incubated at 22°C in a moist chamber. Assays were conducted twice and the results were recorded after 7 days. C. mycophilum grew on 85% of the internally inoculated fruit bodies and on 40% of those inoculated superficially, while the control mushrooms remained symptomless. To our knowledge, this is the first report of C. mycophilum causing cobweb in king oyster mushroom in Spain. This finding will have a potentially significant impact on button mushroom farms where cobweb is one of the most common diseases. References: (1) M. Gardes and T. D. Bruns. Mol. Ecol. 2:113, 1993. (2) G. J. McKay et al. Appl. Environ. Microbiol. 65:606, 1999. (3) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.