LAUSR

Cassava (Manihot esculenta Crantz) is an important tropical and subtropical crop that feeds nearly 600 million people worldwide and is widely grown in Hainan Province, China (Vanderschuren et al., 2014). In November 2021, leaf blight symptoms were observed on South China 6 (SC6) cassava plants in Haikou City, Hainan Province, China. The disease was presented in almost every cassava plant we observed. The rotten leaves were shown to be infected but not the root or stem. The lesions started on the plant's lower leaves and gradually developed on the upper leaves of the entire cassava plant. The infected leaves gradually withered. Microscopic observation showed that the infected leaves exhibited necrotic lesions with pycnidial structures all over their surface. Diseased leaf segments (4 × 4 mm) were disinfected for 30 seconds with sodium hypochlorite 1% solution and then rinsed with sterile water for 30 seconds before being placed on potato dextrose agar (PDA) medium. Plates were incubated at 28°C in complete darkness. Marginal hyphae were picked and placed on a new PDA medium, and pure cultures were obtained after multiple transfers. The hyphae started white and gradually changed to a fluffy black-gray color as it grew on the PDA. Microscopic observation showed that there were a large number of ellipsoidal microsclerotia between the hyphae. Microsclerotia were sub fusiform, and hyaline, with a length of about 40 μm. The ribosomal internal transcribed spacer (ITS) region, ribosomal small subunit (SSU) region, and ribosomal large subunit (LSU) region of the isolate were amplified and sequenced using primers ITS1 and ITS4, NS1 and NS4 (White et al., 1990), and LROR and LR5 (Moriya et al., 2005), respectively. The obtained ITS (GenBank accession no. OP185242), SSU (GenBank accession no. OQ165195), and LSU (GenBank accession no. OQ118350.1) had 99.8% (100% coverage), 100% (100% coverage), and 100% (100% coverage) identities with the references ITS (GenBank accession no. KF951698), SSU (GenBank accession no. KF766281.1), and LSU (GenBank accession no. KF766364.1) in Macrophomina phaseolina, respectively. A phylogenetic tree was constructed with software MEGA7 using the maximum likelihood method, showing that the isolate was grouped in the same clade as M. phaseolina. To prove Koch's postulates, five healthy SC6 cassava plants (2-month-old) with 4-6 leaves were wounded with a small pin and inoculated with PDA blocks (3 × 3 mm) excised from the margin of a 7-day-cultured colony (Hu et al., 2022). Healthy plants treated with sterile PDA plugs served as controls. All plants were grown at 25°C with a 12-h light/dark rotation. After 7 days, typical blight symptoms developed on leaves inoculated with M. phaseolina, but not on the controls. The fungus was isolated from infected leaves. Based on molecular identification, M. phaseolina was re-isolated from leaves with leaf blight symptoms. Macrophomina is typically found to cause root and lower stem rot on cassava in Africa (Msikita et al., 1998). To the best of our knowledge, this is the first report of M. phaseolina causing leaf blight on cassava in China. Our finding provides a foundation to management of this disease.