RESUMO
Wampee (Clausena lansium [Lour.] Skeels) is a tropical fruit. In July 2022, shoot rot symptom was observed in wampee (cv. JIXIN) in a field ((21°25'N, 110°10'E, about 100 ha ), Guangdong Province, China. The most obvious symptom of the disease was the rotting and withering of the tops. Disease incidence was approximately 90% (n = 500). Twenty diseased samples were randomly collected from the field and cut into 2 mm × 2 mm pieces next to the margins of diseased tissues. These pieces were then sterilized with 75% alcohol for 30 s and 2% sodium hypochlorite for 3 min and subsequently washed with sterile water three times. Tissue pieces were placed onto potato dextrose agar (PDA) and incubated at 25â for 3 days. Pure cultures were obtained by transferring hyphal tips to new PDA plates. Sixty isolates of Fusarium ssp. (60/80 = 75%) were obtained. Three representative single-spore isolates (CLFP-1, CLFP-2, and CLFP-3) were used for further study. Colonies were white to pink on PDA. Conidiogenous cells were monophialidic or polyphialidic. Macroconidia were slightly curved, tapering apically with 3 to 5 septa, and measured from 31.7 to 55.5 µm × 2.5 to 5.0 µm in size (n=50). The morphological features of these fungi were analogous to F. proliferatum (Leslie and Summerell 2006). For molecular identification, a colony PCR method (Lu et al. 2012) was used to amplify the internal transcribed spacer (ITS) and portions of elongation factor 1-α (EF1-α), RNA polymerase II largest subunit (RPB1), and RNA polymerase II second largest subunit (RPB2) genes using primers ITS1/ITS4, EF1-728F/EF1-986R, RPB1-R8/RPB1-F5, and RPB2-7CF/fRPB2-11aR, respectively (O'Donnell et al. 1998; 2010). The sequences were submitted to GenBank under accession numbers OP740961 to OP740963 (ITS), and OP800846 to OP800854 (RPB1, RPB2, EF1-α). The BLAST comparison of the sequences showed the three isolates were 100% similar to F. proliferatum (ITS: MT378328; TEF1: MH582344; RPB1: MN193921; RPB2: MN892349). The sequences of the three isolates were 100% identical (ITS, 537/537 bp; RPB1, 1606/1606 bp; RPB2, 770/770 bp and EF1-α, 683/683 bp) with those of F. proliferatum (accession nos. MT378328, MN193921, MH582196, and MH582344) through BLAST analysis. Analysis of the concatenated sequences revealed a 99.87 to 100% identity with the isolates of the F. proliferatum (F. fujikuroi species complex, Asian clade) by polyphasic identification using the FUSARIUM-ID database (Yilmaz et al. 2021). The sequences were also concatenated for phylogenetic analysis by the maximum likelihood method. The isolates clustered with F. proliferatum. Pathogenicity was tested through in vivo experiments. The inoculated and control plants (n = 5, 3 months old, cv. JIXIN) were sprayed with a spore suspension (1 × 105 per mL) of the three isolates and sterile distilled water, respectively, until run-off (Feng and Li. 2019). The test was performed three times. The plants were grown in pots in a greenhouse at 25â to 28â, with relative humidity of approximately 80%. Symptoms were observed on the inoculated plants with disease incidence 100% after 2 weeks, while the control plants remained healthy. The pathogen re-isolated from all the inoculated plants was identical to the inoculated isolates in morphology and ITS sequences. No pathogen were isolated from the control plants. To the best of our knowledge, this study is the first to report F. proliferatum causing shoot blight symptom in wampee (cv. JIXIN). This disease has caused severe losses and will provide the foundation for management strategies.
