First report of Rhizopus stolonifer causing flower rot of yellow passion fruit (Passiflora edulis f. flavicarpa Deg.) in China.

The genus Passiflora, commonly known as passion fruit, originated in South America, is an economically important horticulture crop and widely distributed in the tropics and subtropics. Yellow passion fruit (Passiflora edulis f. flavicarpa) and purple passion fruit (Passiflora edulis f. edulis) are the two most planted species (Santos-Jiménez et al., 2022), which have been largely cultivated in southern China. The average annual production reaches 600,000 tons, of which yellow fruit accounts for more than 70% (Zhou et al., 2022). In 2022 to 2023, a disease caused flower rot severely in passion fruit plantations. The incidence rate was generally 10% in purple passion fruit, with an incidence up to 60% in yellow passion fruit 'Qinmi No. 9'. Flower rot occurs mainly in the rainy season, especially during periods of prolonged rain. Infected flowers had black patches that were water-soaked on the interior of the flower bud. The patches covered the entire flower bud, and fluffy mycelium and sporangia developed, which caused the flower bud rotten and abscised easily. Five symptomatic flowers from Wuhua, Guangdong (23°23'N, 115°18'E) and 8 symptomatic flowers from Shangsi, Guangxi (21°15'N, 107°98'E) of 'Qinmi No. 9' were collected during flowering period in 2022 and 2023. Diseased flower pieces were surface-sterilized with 70% ethanol for 2 to 3 min, rinsed with sterile distilled water 3 times, and placed on PDA medium at 25℃ in darkness. Four and 6 fungal isolates with similar morphology were isolated from the infected samples of Wuhua and Shangsi, respectively. Two isolates, PRFJ01 from Wuhua and PRGX02 from Shangsi, were randomly selected for further study. Purified fungal colonies at the age of 3 days accompany with diffuse cottony mycelia, turned white to gray later. The mycelia were hyaline and aseptate. Sporangiophores with 0.56 (0.22~1.10) mm in length and 6.1 (3.18~10.87) µm in width (n=100) were erect, light brown, and had rhizoids and stolons at their bases. Sporangia with 48.0 (23.45~92.85) µm in diameter (n=100) were dark-colored, near spherical and having dark ovoid sporangiospores with 3.56 (2.34~6.39) µm × 2.82 (1.73~4.70) µm (n=100). The morphology of the fungus were identical to Rhizopus stolonifer (Ehrenb.) Vuill (Haque et al. 2023). The two isolates were molecularly identified using genomic regions of 28S large ribosomal subunit (LSU) with NL1 and LR3 primers (Cruz-Lachica et al., 2018). The phylogenetic trees revealed the sequences of PRFJ01 (OR801560.1) and PRGX02 (OR801561.1) were 100% and 99% identical to R. stolonifer (MK705761.1 and KC412868.1), respectively. Pathogenicity tests were conducted on healthy flowers and leaves of 5-month-old grafted 'Qinmi No. 9' plants. Mycelial plugs with 5-mm diameter were placed on the flowers and leaves. Three plants were performed for each of the isolates, and the test was repeated twice. The inoculated plants were moisturized with plastic bags. Healthy flowers and leaves inoculated with sterile PDA plugs were used as control. Typical symptoms were observed on inoculated plants after 2 days. The dark grey mycelia and sporangia covered the entire flower after 4 days inoculation. The flower bud became putrid and the flower stalk split off. Lesions on leaves expanded accompany with numerous aerial mycelium. However, the controls were symptomless. R. stolonifer was reisolated from inoculated tissues. Previously, flower rot on passion fruit caused by R. stolonifer has only been recorded in Brazil (Ploetz, 2003). To our knowledge, this is the first report of R. stolonifer causing flower rot on passion fruit in China.

First report of leaf spot on passion fruit in China, caused by Alternaria alternata.

Passion fruit (Passiflora edulis Sims), which is native to South America, is an important fruit crop in tropical and subtropical countries. Passion fruit growing areas have increased rapidly in southern China. In 2018 to 2019, circular spots on passion fruit were observed in Shangsi, Guangxi, China (21°15'N, 107°98'E). The disease occurred from June to April of the following year. The disease incidence was generally between 10% to 30%, but could reach up to 50% in purple passion fruit 'Tainong No.1'. The initial lesions on the fruits were small, with a brown center and a greasy margin, and then became sunken and lighter brown with a diameter of about 1 cm in later stages. The spots on the leaves were often surrounded by a yellow halo and turned into larger lesions after coalescence.. Five typical symptomatic fruit and leaves were collected from Shangsi county for the presumed pathogen isolation. Section of the samples were surface sterilized to isolate the fungus on potato dextrose agar (PDA) at 28°C. Five fungal isolates with similar morphology on PDA were obtained by single spore isolation. Colonies at the age of 7 days accompany with flourishing aerial hyphae, showed surface color varying from white to grey. Conidia were ovate or elliptic, light brown to brown, with 2 to 5 diaphragms, 0 to 4 longitudinal-oblique diaphragms, and mostly 8.2 to 36.7 µm × 5.4 to 15.8 µm. The morphology of the fungus resembled Alternaria alternata (Fr.) Keissl (Simmons, 2007). Each of the five isolates (SF-001, SF-002, SF-003, SF-004 and SF-005) was molecularly identified using genomic regions of 18S nrDNA (SSU), 28S nrDNA (LSU), RNA polymerase second largest subunit (RPB2), internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and translation elongation factor 1-alpha (TEF1) (Jayawardena et al., 2019). Nucleotide sequences of SSU (MZ275254, ON055696, ON055697, ON055698 and ON055699), LSU (MZ275253, ON062947, ON062948, ON062949, ON062950), RPB2 (MZ275251, ON055377, ON055378, ON055379 and ON055380), ITS (MW866522, MW866523, ON053451, ON053452 and ON053453), GAPDH (MZ286628, ON055381, ON055382, ON055383 and ON055384) and TEF1 (MZ275255, ON055373, ON055374, ON055375 and ON055376) were deposited in GenBank database. The LSU, GAPDH and TEF1 sequences showed 100% identity with A. alternata in NCBI (KX609773, MK683852 and MK637432, respectively). The SSU, RPB2 and ITS sequences showed 99% identity to A. alternata (U05194, MK605898 and MN856409, respectively). In pathogenicity test (Zhang et al., 2020), 3-month-old grafted 'Tainong No.1' seedlings and mature fruit were used. Five-mm-diameter mycelial plugs taken from 7-day-old PDA colonies of each of 5 isolates were placed on the leaves and fruit that were wounded with a sterilized needle to form 3 pinpricks. Sterile PDA plugs were used as control. Three plants and three fruits were used in each treatment, and the test was repeated twice. The inoculated plants and fruit were kept in plastic bags and grown in a chamber at 28℃. Typical lesions were observed on inoculated plants and fruit after 3 days, but the controls remained healthy. A. alternata was consistently reisolated from these typical lesions. Previously, leaf spot on passion fruit caused by A. alternata has only been recorded in New Zealand (Rheinländer, 2010). To our knowledge, this is the first report of A. alternata (Fr.) Keissl. causing leaf spot on passion fruit in China. The identification of the pathogen may help to take effective management strategies of controlling this disease.