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 Mucor irregularis Causing Flower Rot on Selenicereus undatus in China.

Dragon fruit (Selenicereus undatus (Haw.) D.R.Hunt is a famous tropical fruit (Korotkova et al. 2017). In May 2021, a flower rot disease was found on Dragon fruit in a field (21˚19'42''N, 110˚28'32''E), Zhanjiang, Guangdong Province, China. The incidence rate was approximately 30% (n=500 investigated plants from about 30 hectares). Flower rot was evident, and was light brown, watery, soft, and covered with white mycelia. The pathogen could continue to infect the fruit during the fruit ripening stage with about 20% rot rate. Ten samples of symptomatic flowers were collected in the field. Margins of the diseased tissue were cut into 2 mm × 2 mm pieces. The surfaces were disinfected with 75% ethanol for 30 s and 2% sodium hypochlorite for 60 s. Pure cultures were obtained by transferring hyphal tips to new PDA plates. Three representative isolates (HUM-1,HUM-2, and HUM-3) by single-spore isolation were randomly selected for further study. Colonies on PDA were circular with massive aerial hyphae, white to ochraceous in color. Nonseptate hyphae were hyaline. Sporangiophores arose from hyphae. Sporangiospores were hyaline, smooth-walled, mostly subspherical to ellipsoidal, and measured 3.15 to 6.55 µm × 1.35 to 2.85 µm (n =50). Morphological characteristics of isolates were consistent with the description of Mucor irregularis (Lima et al. 2018). Molecular identification was done using the colony PCR method with MightyAmp DNA Polymerase (Takara-Bio, Dalian, China) (Lu et al. 2012) used to amplify the internal transcribed spacer (ITS) region and large subunit (LSU) with ITS1/ITS4 and LR0R1/LR5 (Vilgalys et al. 1990). The amplicons were sequenced and the sequences were deposited in GenBank with accession numbers ITS, OL376751-OL376753, and LSU, OM672239-OM672241. BLAST analysis of these sequences revealed a 100% identity with M. irregularis in GenBank. The sequences were also concatenated for phylogenetic analysis by the maximum likelihood method. The isolates clustered with M. irregularis (the type strain CBS 103.93).The pathogenicity was tested through in vivo experiments. Nine healthy flowers of Dragon fruit were inoculated with 3-day-old mycelial plugs (5 × 5 mm) of isolates, while another five healthy flowers were treated with PDA plugs (controls). Those plugs were embedded inside the calyxes, and each flower was inoculated with one plug in one calyx. Besides, the inoculated and control flowers (n = 5) were sprayed with a spore suspension (1 × 105 per mL) of the three isolates individually and sterile distilled water, respectively, until run-off (Feng and Li. 2019). The plants were grown in pots in a greenhouse at 28°C, with relative humility approximately 80%. The test was repeated three times. After 3 days of incubation, rot symptoms developed on the inoculated flowers, which were similar to those observed on the naturally samples in the field. The control flowers remained healthy. The fungus was reisolated from the inoculated flowers and confirmed as M. irregularis by morphology and ITS analysis. M. irregularis was reported as a pathogen causing human skin diseases and post-harvest diseases of crop (Álvarez et al. 2011; Lima et al. 2018; Wang et al. 2022). This is the first report of M. irregularis causing flower rot of Dragon fruit and reduce yield in China. This research can provide a theoretical basis for the fruit industry to maintain yield.

First report of Chaetomium pseudocochliodes causing flower rot disease of Camellia reticulata in Yunnan, China.

Camellia reticulata is the world-famous ornamental flower (Wang et al. 2021). In February 2021, the infected flowers of C. reticulata 'Shizitou' were collected in Zixi Mountain, Chuxiong city, Yunnan province, China (24°9'95″ N, 101°42'53″ E). Flower rot disease incidence ranged from 40% to 75% in the garden. The infected flowers showed symptoms of varying degrees of yellow-browning, dry or wet rot to the whole flower wilted and even dropped (Figure 1A, B, C). Three symptomatic flowers were randomly collected in the garden. Tissues from the infected flowers (cut to 5×5 mm size) were surface-disinfected by 75% ethyl alcohol for 30s, rinsed in sterile water for 3 times to air dry, and cultured in Potato Dextrose Agar medium (PDA) at 25℃±2 in the normal light for 5-7 days (Fang, 1998). Similar fungal colonies were isolated from 50%-75% of the infected flowers. Three isolates from different flowers showed similar colony morphology. After sub-culturing of hyphal tips on PDA for 5-7 days, the initially yellow colored colonies showed abundant white aerial mycelium, with sporulation (Figure 1E, F). The asci (Figure 1G) sporulation site is 24(-37) ×7(-14) µm, and the stalk length is 17-42 µm, with 8 biseriate acuminate ascospores. The mature ascospores (Figure 1H) are olive-brown or brown, lemon-like, double-pointed, with slightly umbilical protrusions at both ends, flat on both sides, 9(-11.5) × 7(-9) × 5.5(-7) µm in size, with germ holes on the top (Wang et al. 2016). These morphological features are consistent with Chaetomium pseudocochliodes. The genomic DNA was extracted from the isolated strains. To identified this fungal pathogen genetically, sequence analyses were conducted using the ITS1/ITS4 (Henson et al. 1993), NL1/NL4 (Liu et al. 2011), EF1-938F /EF1-2218R (Tan et al. 2016) primers for the internal transcribed spacer (ITS), large ribosomal subunit (LSU), and elongation factor 1-α (EF1-α) genes. The obtained sequences were deposited in GenBank with accession numbers MZ817067 (ITS), MZ817072 (LSU), MZ820167 (EF1-α). The phylogenetic trees (Figure 2) were constructed to determine the phylogenetic relationships based on MEGA 6.0 maximum likelihood method. In order to confirm the pathogenicity, the tests were conducted with fungus plug (5 mm) from a 7-day-old colony placed onto the surface of healthy petals. The sterile water-absorbent cottons place onto healthy petal surface near fungus plug and plastic wraps cover in petri dish were used for moisturizing. A total of 3 replicates in each of 3 groups were included (3 healthy petals for a group, 1 for wounded inoculation, 1 for unwounded inoculation, and 1 for sterile PDA plug). A sterile PDA plug was placed onto the surface of healthy petals as a control. After incubation at room temperature for 5 days (Ren. 2019), 3 replicates in each of 2 groups of treated healthy petals for wounded inoculation showed obvious symptoms (Figure 1D), and no symptoms appeared in the control, and healthy petals unwounded showed symptoms for 7-10 days. The fungus was re-isolated from the lesions of inoculated tissues. The re-isolated fungal colonies showed identical morphology and high sequence similarity with ITS, LSU and EF-1α of the initial isolate. No fungus was isolated from the controls. The first extraction of C. pseudocochliodes was also obtained from the roots of the Panax notoginseng in Wenshan, Yunnan (Wang et al. 2016). To our knowledge, this is the first report of flower rot caused by C. pseudocochliodes on C. reticulata in China.

First Report of Bipolaris cactivora Causing Flower Rot of Pitaya (Hylocereus costaricensis) in China.

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.