First Report of Cobweb on White Button Mushroom (Agaricus bisporus) in Spain Caused by Cladobotryum mycophilum.

Between 2008 and 2011, symptoms of cobweb were observed in commercial white button mushroom (Agaricus bisporus) crops in Castilla-La Mancha (Spain). Typical symptoms started as white, cobweb-like mycelial growth over the surface of the casing soils and fruiting bodies. Later, the mycelium changed to a grayish white, dense powder and the affected fruiting bodies turned pale yellow or reddish brown before rotting. Two types of cap spotting were observed, dark brown spots with a poorly defined edge and light brown spots. The first symptoms were commonly seen in the second or third break (flush) of mushrooms. Infected tissues of A. bisporus were plated onto potato dextrose agar (PDA) and a parasitic fungus was isolated. Fungal colonies consisted of abundant, cottony, aerial mycelium spreading rapidly over the PDA, and red pigment spreading into the agar. The cultures lacked a camphor odor. Conidiogenous cells were 24 to 45 μm long, 3 to 6 μm wide basally, and tapered slightly to the tip. Conidia were cylindrical to narrowly ellipsoidal, 15 to 28 × 8 to 11 μm, and zero- to three-septate. Total DNA was extracted and the internal transcribed spacer (ITS) region of rDNA amplified for one mycelial isolate using ITS1F/ITS4 primers (2,4). The amplicon was sequenced (GenBank Accession No. JQ004732). BLAST analysis showed highest similarity (99 and 100%) of the ITS sequence to four ITS sequences of Cladobotryum mycophilum (teleomorph Hypomyces odoratus) (GenBank Accession Nos. AB527074, JF505112, Y17095, and Y17096) (1,3) among other sequences of the same species. Two pathogenicity trials (A and B) were performed in mushroom-growing rooms, with 24 blocks in each assay containing pasteurized, spawned, and incubated A. bisporus substrate (10 kg, 0.15 m²). The blocks were cased with a 35-mm layer of a peat-based casing soil (5.5 liter/block). Nine days after casing, a conidial suspension (7.5 × 10³ conidia/ml) of one isolate of C. mycophilum was sprayed (20 ml/block) onto the surface of the casing layer of 12 blocks at 10⁶ conidia/m². Twelve blocks were sprayed with sterile distilled water as a control treatment. Blocks were maintained at 17.5°C and 90% relative humidity. The first cobweb symptoms developed 25 days after inoculation, between the second and third breaks in trial A; and after 11 days, between the first and second breaks in trial B. C. mycophilum was consistently reisolated from eight inoculated blocks (67%) in trial A, and 11 inoculated blocks (92%) in trial B. The total area of the crop affected by cobweb was 30% in inoculated blocks in trial A and 45% in trial B. The noninoculated blocks remained healthy. Compared with the noninoculated control blocks, a 10.7% decrease in yield of mushrooms was observed in trial A and 9.1% in trial B. Previously, C. dendroides was the only known causal agent of cobweb in Spain. To our knowledge, this is the first report of C. mycophilum causing cobweb in white button mushroom in Spain, although the disease and causal agent were previously reported on cultivated king oyster mushroom (Pleurotus eryngii) in Spain (3). References: (3) C.-G. Back et al. J. Gen. Plant Pathol. 76:232, 2010. (1) M. Gardes and T. D. Bruns. Mol. Ecol. 2:113, 1993. (4) F. J. Gea et al. Plant Dis. 95:1030, 2011. (2) T. J. White et al. PCR Protocols. A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.