First Report of Chocolate Spot Caused by Botrytis eucalypti on Faba bean in China.

Faba bean ( Vicia faba ) is one of the characteristic economic crops in Qinghai Province of China, which has multiple uses as grain, vegetable, fodder, fertilizer and medicine. Chocolate spot is a critical disease of faba bean in the world, and it is widely spread in all production areas of Qinghai. In August 2021, a severe occurrence of chocolate spot was found in a faba bean field in Xunhua County, Qinghai Province (35°52'N, 102°22'E, alt. 1890m). All plants in the field were affected by this disease. A voucher specimen was deposited in the Herbarium of Plant Pathology, College of Agricultural and Forestry Sciences at Qinghai University under accession No. PY015. The pathogen infected the leaves and stems, causing small irregular red spots to appear, which later coalesce into larger spots and faded green lesions appear around the spots. Diseased leaf pieces 5 mm2 were surface sterilized with 75% ethyl alcohol for 30s, 1.2% NaOCl for 30s, and rinsed three times with sterile water. They were then plated on potato dextrose agar (PDA) at 22℃ for 10 days in the dark. Fungal colonies are initially white, then gray, and have produced spores by 5 days. Conidia are clusters, ellipsoidal or ovoid, 9-14 × 6-9 μm. The conidiophore is straight, terminally enlarged, septate, 300-1500 μm long, 8-13 μm wide. No sclerotia were observed during culture. DNA of the strain PY015 was extracted by CTAB method. Molecular identification was first performed using the universal region of ITS (ITS1/ITS4). The PCR product was sequenced, the sequence was deposited in GenBank under the accession number OR739575. The results showed 100% similarity to Botrytis spp. (KX301016, MT250940, LC519322) in BLAST search. Molecular characterization was continued using five specific primer pairs: RPB2 (DNA-dependent RNA polymerase subunit II, RPB2-5F/RPB2-7cR), NEP1 and NEP2 (necrosis and ethylene-inducing proteins, NEP1for/ NEP1revB and NEP2forD/NEP2revD), HSP60 (heat-shock protein 60, HSP60for/HSP60rev), G3PDH (glyceraldehyde-3-phosphate dehydrogenase, G3PDHfor/G3PDHrev). The sequences of PY015 were deposited in GenBank (accession numbers: OR731179, OR731180, OR731181, OR731182, OR731183), and all five sequences showed 100% similarity to Botrytis eucalypti YZU171088 (accession numbers: MH614610 MH614611, MH614612, MH614613 MH614614). A phylogenetic tree based on these five genes was constructed using Mega7.0 (1000 bootstrap replicates, neighbor-joining method), and PY015 was placed in the same clade as YZU171088 with 100% bootstrap values. Morphological and molecular biological results confirmed that isolate PY015 was B. eucalypti . To fulfill Koch's postulates, the spore suspension (2 × 105 conidia/ml) was sprayed on healthy faba bean (Yun-122) plants at the 10-leaf stage, while an equal amount of sterile distilled water was applied to controls. After 7 days, the inoculated plants showed symptoms consistent with field infection and B. eucalypti was re-isolated using the same protocol, while the control remained asymptomatic. The pathogenicity test was repeated twice. The same isolates were recovered from symptomatic leaves and identified by NEP1 sequence. B. eucalypti was morphologically and molecularly identical to the original isolates, completing Koch's postulates. Currently, Botrytis fabae , Botrytis fabiopsis , and Botrytis cinerea are the main pathogens of chocolate spot on faba bean that have been identified and reported nationally and internationally. B. eucalypti is a new species discovered from eucalyptus in southern China in 2016, and its current hosts are only eucalyptus and citrus. To our knowledge, the present study is the first report of chocolate spot caused by B. eucalypti on faba bean in China.