LAUSR

Pitaya (Hylocereus costaricensis), belonging to the Cactaceae family, has rich functional substances, such as a variety of amino acids, which are popular with consumers (Wichienchot et al. 2010). In May 2019, flowers showed symptoms of rot, with an incidence of 15% in a plantation (233.3 ha) in Changjiang (19°46'N; 108°93'E) (Hainan province), China. The initial disease symptoms of flower were small scattered purple-red spot (1~2 mm), including circular, long oval or irregular in shape. The spots were gradually expanded and coalesced, forming abundant reddish-brown lesions. Later, this disease resulted in rotting and blackening of the whole flower. Many black mildew layers (conidiophores and conidia) on the surface of the lesions were observed under compound microscopy. Symptomatic flower tissue (4 cm2) from collecting samples was disinfected in 75% ethanol for 25 s, followed by 1 min in 5% sodium hypochlorite, rinsed 3 times with sterile water, plated on potato dextrose agar (PDA) for 3 days, and incubated at 28ºC. A fungus was consistently isolated from symptomatic flower samples with 90% isolation rate. Resultant colony of the fungus was circular, dark green, velvety, hairy, after 7 days, incubated at 28ºC. Hyphae were septate, 6.2-8.9 μm (average 7.6±0.5) in diameter. Conidia were straight, obclavate, pale to mid brown, 2-6 septate, 23.0 to 42.2 μm (average 31.0±3.2) × 6.5 to 9.8 μm (average 8.0±0.6) (n = 100). The conidia were normally produced germ tubes from one end or both ends. The width of conidiophore was 5.1 to 6.6 μm (average 5.8±0.4) (n = 50). Sequences were generated from the isolate using primers for the internal transcribed spacer region (ITS) (ITS1/ITS4) (White et al. 1990), ribosomal large subunit (LSU) (LROR/LR5) (Vilgalys et al. 1990), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (GPD1/GPD2) (Berbee et al. 1999) loci. The resulting sequences were deposited in GenBank with accession numbers MN960109, MN966852, and MT542865. BLAST analysis demonstrated that these sequences were 99% similar to ITS (HM193535), LSU (MH869295), and GAPDH (HM598681) of Bipolaris cactivora. A maximum likelihood phylogenetic analysis based on combined dataset of ITS, LSU, and GAPDH sequences using MEGA7.0 revealed that the isolate was placed in the same clade as B. cactivora with 100% bootstrap support. A conidial suspension (1 × 105 conidia/ml) of the fungal isolate was prepared by harvesting conidia from pure culture of the fungus grown on PDA 25 days. The 10 mL suspension was sprayed onto ten flowers with no wounding. Ten additional flowers sprayed with sterile distilled water were served as controls. All flowers were covered with plastic bags to maintain high humidity and incubated under natural condition. Typical symptoms of purple-red spot were observed on all the inoculated flowers on the third day. Abundant dark-brown to dark lesions were observed on the surface of flowers and were similar to those observed on the naturally infected flowers after 5 days. The control flowers remained asymptomatic. The fungal isolate of B. cactivora was reisolated from lesion of the flowers and reidentified by morphological and molecular characteristics, thus fulfilled Koch's postulates. Pathogenicity tests were repeated thrice with the same results. B. cactivora had been reported causing flowers and fruit rot of pitaya in South Florida (Tarnowski et al. 2010). This is the first report of B. cactivora causing flower rot of pitaya (H. costaricensis) in China. The flower rot may provide inoculum for the fruit rot, which will cause reduction of pitaya yield.