Gray mold of Echeveria plants caused by Botrytis cinerea in Japan

Echeveria plants are a large genus of succulents belonging to the Crassulaceae family. Echeveria plants are suitable for urban living and are therefore highly popular in Japan. Since 2015, symptoms of disease have been observed on E. laui and E. agavoides cultivar Evony in commercial greenhouses (34°57'58.3"N, 136°28'37.6"E) in Mie Prefecture, Japan. First, water-soaked symptoms appeared at the leaf base, the plant then softened, and the leaves dropped off, or abundant gray mold and conidia were formed on the rotted plants. Approximately 5–6% of the plants were affected, which appeared indiscriminately on species or hybrids and was more prevalent from late March to early July. The fungus was isolated from the cultivars Blood Maria × Lindsayana and Monroe Chanel. The affected leaves were surface-sterilized with 1% sodium hypochlorite for 10 s, followed by 70% ethanol for 60 s, rinsed three times with sterile water, and excised into 10 mm 2 squares. Samples were placed on a Botrytis selective medium (Edwards and Seddon 2001). After subculturing the fungal colonies on potato dextrose agar (PDA), 16 isolates were obtained through single-conidial isolation. Fungal colonies of all isolates on PDA were cottony flocculent texture with a color ranging from gray-white to light brown. Strain Ec23_1 was selected as a representative isolate for further studies. The conidia were colorless or gray, oval, and elliptical, measuring 9.0–19.1 × 6.3–15.8 μm (n=131), and were produced in gapelike clusters on the terminal end of the branch conidiophore. Many small sclerotia (0.8–2.3 × 0.7–1.6 mm, n=76) were irregularly produced on PDA at a later stage of growth. The morphology of the isolate closely resembled that of Botrytis (Ellis 1971). For molecular analysis, genomic DNA was extracted using a commercial DNA extraction kit (Qiagen, Hilden, Germany). Nuclear protein-coding genes of glyceraldehyde 3-phosphate dehydrogenase (G3PDH), heat shock protein 60 (HSP60), RNA polymerase II (RPB2), and internal transcribed spacer (ITS) were amplified and sequenced using the specific primers (Staats et al. 2005). All sequences were deposited in GenBank under accession numbers LC890466 (ITS), LC890460 (G3PDH), LC890461 (HSP60), and LC890462 (RPB2). BLAST analyses revealed that the ITS, G3PDH, HSP60, and RPB2 sequences exhibited 99–100% (562/562, 922/925, 1030/1030, 1129/1134 bp) identity to B. cinerea (GenBank: OQ257130, PV470194, MN159921, and MN448501, respectively). Based on morphological and molecular features, the isolate Ec23_1 was identified as B. cinerea. To confirm pathogenicity, young plants of the Sarang cultivar were sprayed with conidial suspensions (10 5 conidia ml -1 , n=9). The inoculated plants were placed in a plastic box and maintained at 24°C under a 12-h photoperiod with high humidity (>90%) overnight, followed by 40%. Symptoms appeared on the lower leaves within 1 week after inoculation, and the plants were finally covered with gray mold. The re-isolated fungus showed the same morphological features and pathogenicity as the original isolates, fulfilling Koch’s postulates. In Crassulaceae, gray mold or rot diseases have been reported on Sedum sarmentosum in Korea caused by B. cinerea and E. gigantea in Mexico by Sclerotinia sclerotiorum. To our knowledge, this is the first report of B. cinerea on Echeveria plants in Japan. This study provides useful information on plant disease management to maintain stable seedling sales and value in the Echeveria market.