First report of anthracnose fruit rot of papaya caused by Colletotrichum gigasporum in China.

Papaya (Carica papaya L.) belonging to the family Caricaceae is well known for its economic and nutritional value. Anthraconse caused by Colletotrichum spp. is a main postharvest disease of papaya fruit during storage (Cia et al., 2007). In July 2022, papaya fruits with anthracnose symptoms were collected in Changjiang County (108.996180E, 19.246560N), Hainan Province, China. The disease incidence of fruit rot reached 6.3%. Initial symptoms appeared as the watery lesions with tiny black spots, turning to dark brown, sunken necrotic lesions. The diseased tissues were cut into 18 pieces (5×5 mm) from 6 papaya fruits, disinfected with 2% sodium hypochlorite for 60 s, and rinsed three times with sterilized water. The pieces were air-dried and then placed on potato dextrose agar (PDA) at 28 ℃ for five days. Twelve isolates with similar morphology were obtained from 18 tissue pieces. Three isolates (FMG01, FMG02 and FMG03) were selected for morphological identification, molecular identification, and pathogenicity tests. Colonies were initially white, then gradually became dark grey on PDA. The ascospores were hyaline, fusoid, rounded at both ends, 37.43-84.32 (55.79±7.61) µm × 4.30-6.55 (5.36±0.60) µm (n=50). The conidia were hyaline, unicellular, long cylindrical, bluntly rounded at both ends, 11.59-25.54 (18.62±2.33) µm × 5.12-8.44 (7.19±0.62) µm (n=100). Appressoria were gray to dark brown, irregular, pyriform, or ovoid, 10.14-21.40 (13.81±2.25) µm × 6.05-11.85 (9.16±1.29) µm (n=50). Morphological features are similar to Colletotrichum gigasporum identified and described by Rakotoniriana et al (Rakotoniriana et al., 2013). In order to accurately identify the isolates, the internal transcribed spacer region (ITS) of the rDNA, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the partial actin (ACT), the beta-tubulin (TUB2) and the calmodulin (CAL) genes were amplified and sequenced (Cannon et al., 2012). The nucleotide sequences were deposited into GenBank (accession numbers, ITS: OR017446 to OR017448, GAPDH: OR042810 to OR042812, ACT: OR042813 to OR042815, TUB2: OR042816 to OR042818, CAL: OR042819 to OR042821). Based on the BLASTn analysis, these sequences were more than 99% with the reference strain CBS 125476 of C. gigasporum sequences (ITS: MH863698, GAPDH: KF687833, ACT: KF687790, TUB2: KF687875, CAL: KF687814), respectively. The results of the multilocal phylogenetic analysis showed that the three isolates were C. gigasporum based on the Maximum Likelihood and Bayesian inference method. The pathogenicity test was performed by wounded with a sterile needle on the surface-sterilized papaya fruits. The mycelial discs (5 mm diameter) of three isolates were inoculated orderly on the same fruit, and the same inoculation was applied to non-wound papaya fruits. The control group were inoculated with sterilized PDA. Each treatment carried out with 9 fruits. The inoculated fruits were placed at 28 ℃ in plastic preservation boxes (32×22×11 cm) with sterilized distilled water to maintain high humidity. After 5 d of inoculation, typical anthracnose symptoms were observed on wound fruits and the non-wound fruits developed symptoms at 7 dpi, control fruits were symptomless. The fungi re-isolated from the inoculated fruits lesions after inoculation and identified by morphological characterization and molecular identification, fulfilling Koch's postulates. C. gigasporum has been reported causing leaf rot of Dalbergia odorifera in China (Wan et al., 2018). To our knowledge, this is the first report of anthracnose fruit rot of papaya caused by C. gigasporum in China.

First report of anthracnose caused by Colletotrichum karstii on Dalbergia odorifera in China.

Dalbergia odorifera (Family: Fabaceae) is a national second-grade protected tree in China with high medicinal and economic value (Zhao et al., 2020). In July, 2022, a leaves spot disease on D. odorifera with typical anthracnose symptoms was observed in plantations in Haikou (110.319153°E, 19.072900°N), Dongfang (108.630297°E, 19.103838°N) and Qiongzhong (109.704460°E, 19.088440°N), Hainan Province, China. Disease incidence was 7.5% (n = 50 plants). Early symptoms of infected leaves were small and round dark brown spots, which developed into larger irregular necrotic lesions and leaves withered. Leaf tissues (5×5 mm) at the disease-health junction of spots from 19 leaves were sterilized with 2.5% sodium hypochlorite for 1 min, and rinsed with sterile distilled water three times. These sterilized tissues were placed on potato dextrose agar (PDA) and incubated at 28 ℃ for 5 d. 7 strains of fungi with similar morphology were isolated, and 3 single-hyphal isolates (HHL01, HHL02 and HHL03) from each location were selected for further study. Colonies on PDA were fluffy orange-yellow mycelium. Conidia were aseptate, cylindrical, smooth-walled, straight, hyaline with both ends bluntly rounded, 11.82 to 15.77 × 3.87 to 6.71 µm (n = 100; average = 13.75 × 5.52 µm). Appressoria formed on slides, measured 5.54 to 10.64 × 4.19 to 7.41 µm (n = 30; average = 8.06 × 5.97 µm) were brown to black, elliptical to irregular. For molecular biological identification, the genomic DNA of three isolates was extracted by fungal genomic DNA extraction kit (Tiangen Biotech (Beijing) Co., Ltd.). The partial sequences of internal transcribed spacer region (ITS; ITS1/ITS4), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; GDF1/GDR1), actin (ACT; ACT512F/ACT783R), ß-tubulin (TUB2; TI/Bt2b) and calmodulin (CAL; CL1C/CL2C) were amplified and sequenced by Sangon Biotech (Shanghai) Co., Ltd (Carbone and Kohn, 1999; Weir et al., 2012). The sequences were deposited as GenBank Accession Nos. OR018110-OR018112 (ITS); OR050529-OR050537 (GAPDH, ACT and CAL) and OR192168-OR192170 (TUB). BLASTn results showed these sequences were more than 99% identity with the strain of C. karstii CORCK1 (GenBank Accession Nos. HM585406, HM585387, HM581991, HM585424 and HM582010, respectively). Multi-locus phylogenetic tree of Colletotrichum spp. showed that those three isolates were sister to C. karstii based on the maximum likelihood and bayesian inference methods. To verify pathogenicity, 2 mL spore suspension (1 × 106conidia/ml) of the isolates was sprayed on each leaves of 1-year-old D. odorifera plants, and sterile distilled water was similarly sprayed on other leaves as a negative control. The plants were incubated in a greenhouse under 90% ± 5% RH at 28 °C. Light brown small round necrotic patches developed 3 days after inoculation, while the control was asymptomatic. Photographs were taken on the fifth day after inoculation. The fungi were re-isolated from the diseased leaves and identified by morphological characterization and molecular identification, fulfilling Koch's postulates. C. karstii has been reported causing leaf rot of Carissa grandiflora in Spain (Garcia-Lopez et al., 2021), and anthracnose caused by C.tropicale was reported on D. odorifera (Yi et al., 2023). To our knowledge, this is the first report of Dalbergia odorifera leaf spot disease caused by Colletotrichum karstii. This finding provides an important basis for further research on the control of the disease.

First report of Neofusicoccum sinoeucalypti causing leaf spot on Terminalia catappa in China.

Terminalia catappa belonging to the family Combretaceae, spreads in tropical and subtropical coastal areas. It mainly serves as shading and decorative tree (Anand et al, 2015). It is planted as roadside tree in Southern China. A leaf spot disease of T. catappa was observed at Wencheng Town (110.805323°E, 19.524567°N), Wenchang City, Hainan province, China in June, 2022. The disease incidence of leaves reached 10%. The occurrence of this leaf spot would reduce the ornamental value of T. catappa. The early symptoms of infected leaves were small, round, dark brown spots surrounded by irregular light halos, developing to larger irregular necrotic lesions and leaves withered. Twelve diseased leaves were collected from three survey trees. Symptomatic leaf samples were collected and cut into small pieces (3×3 mm). The pieces were surface sterilized with 2.5% sodium hypochlorite for 1 min, rinsed with sterile distilled water three times, placed on potato dextrose agar (PDA) medium and incubated at 28 ℃ in the dark for 3 days. Three hyphal tip isolates (DYLR-1, DYLR-2 and DYLR-3) were cultured on PDA. Colonies on PDA reached the edge of the 90 mm plates after 3 d and had fluffy mycelia with an uneven margin, initially creamy white, becoming light grey (5 d) to mouse grey (10 d) at the surface with the black globular cavity. To induce sporulation, the isolates were transferred to 2% water agar media with sterilised pine needles placed on the surface of the media. Conidia was hyaline, unicellular, thin-walled, smooth with granular contents, aseptate, narrowly fusiform, base subtruncate to bluntly rounded, 11.1 to 16.7 (14.5±1.4) × 4.6 to 7.6 (6.2±0.7) µm (n=50). Spermatia was hyaline, unicellular, aseptate, allantoid to rod-shaped, 3.2 to 6.9 (5.1±0.9) µm × 2.0 to 3.8 (2.5±0.4) µm (n=50). Pathogenicity tests were performed both in vitro and in vivo, and replicated twice. All three isolates were used for pathogenicity tests, with 18 detached leaves used for pathogenicity tests in vitro and 3 seedlings used for pathogenicity tests in vivo. A 5-mm-diameter agar plug containing mycelia were placed on the leaves both without and with wound. Sterile PDA plugs were used as controls. The leaves were moisturized with a clear plastic bag for 24 hours in a greenhouse under 90% ± 5% RH at 25 ℃. Brown spot symptoms were observed at 1 day post-inoculation (dpi) in vitro and 3 dpi in vivo. The same strains were reisolated from lesions of inoculated leaves. Control plants were symptomless. For molecular identification, internal transcribed spacer region and intervening 5.8S nrRNA gene (ITS; ITS1/ITS4 primers; White et al. 1990), translation elongation factor 1-alpha gene (tef1-α; EF1-728F/EF1-986R primers; Carbone and Kohn 1999), beta-tubulin gene (tub2; Bt2a/Bt2b primers; Glass and Donaldson 1995) and DNA directed RNA polymerase II second largest subunit gene (rpb2; RPB2bot6F/RPB2bot7R; Sakalidis et al. 2011) regions were PCR amplified from genomic DNA. The sequences (GenBank accessions numbers: OP435357 to OP435359 of ITS; OP535354 to OP535356 of tef1-α; OP535351 to OP535353 of tub2; OP535348 to OP535350 of rpb2) had 100%, 99.7%, 100%, 100% similar to the type strain of Neofusicoccum sinoeucalypti CERC2005 (GenBank accessions numbers: KX278061, KX278166, KX278270 and KX278290), respectively. Multi-locus phylogenetic tree (ITS, tef1-α, tub2 and rpb2) of Neofusicoccum spp. (Zhang et al. 2021) showed that those three isolates were sister to N. sinoeucalypti based on the maximum likelihood and bayesian inference methods. N. sinoeucalypti was first reported pathogen causing from Eucalyptus plantations and adjacent plants in China (Li et al. 2018). To our knowledge, this is the first report of Neofusicoccum sinoeucalypti causing leaf spot disease on Terminalia catappa in China. Neofusicoccum species, commonly cause diseases in woody plants worldwide, and identification of this pathogen is important for effective disease management and control.