Fruit Rot on Persimmon Caused by Neopestalotiopsis saprophytica and Neopestalotiopsis ellipsospora in Guangxi, China.

Persimmon (Diospyros kaki Thunb.) is widely cultivated in China. On October 15, 2019, about 10% of persimmon fruits showed fruit rot in the orchards of Guilin, Guangxi, China (24°45' N, 110°24' E), which could cause more than 15% of yield losses. The initial symptoms of fruit rot exhibited irregular brown to black spots (range from 2 to 4 cm in diameter), the areas surrounding the blackened spots would be soft and rotten, and three diseased fruit samples were collected from three orchards, respectively. Tissues (5×5 mm) were cut from infected margins, surface-disinfected in 75% ethanol for 10 s, 2% NaClO for 2 min, rinsed three times in sterilized distilled water, and incubated on potato dextrose agar (PDA) at 25°C under 12/12 h light/darkness for a week. Forty-one tissues yielded morphologically similar cultures, and three representative isolates LPG1-1, LPG1-2, and YSG-1 were selected from three samples for further study, respectively. Their colonies showed wavy edges, white surfaces, and dense aerial hyphae on PDA after two weeks. Conidia were fusiform, straight to slightly curved, and 4-septate; basal cells were conical, hyaline, thin, and verruculose with two or three long and hyaline apical appendages and one short apical appendage; three median cells of LPG1-1 with length 14.06 to 17.69 µm (n=100), and LPG1-2 with length 14.03 to 17.61 µm (n=100) were dark brown to olivaceous, while three median cells of YSG-1 with length 12.54 to 15.58 µm (n=100) were dark brown. The conidial sizes of LPG1-1, LPG1-2, and YSG-1 were 17.41 to 27.68 × 4.63 to 8.55 µm (n=100), 18.06 to 27.41 × 4.33 to 8.21 µm (n=100), and 16.58 to 27.73 × 4.99 to 8.39 µm (n=100), respectively. The morphological characteristics were consistent with Neopestalotiopsis spp. (Maharachchikumbura et al. 2012; Maharachchikumbura et al. 2014). Primer pairs ITS4/ITS5, BT2a/BT2b, and EF1-526F/EF-1567R were used to amplify internal transcribed spacer (ITS), beta-tubulin (TUB2), and translation elongation factor 1 alpha (TEF1-α), respectively (Shu et al., 2020). All DNA fragments were sequenced by Sangon Biotech Co., Ltd. (Shanghai, China). Sequences have been deposited in GenBank (ITS: OM349120 to OM349122, TUB2: OM688188 to OM688190, TEF1-α: OM688191 to OM688193). Based on BLASTn analysis of ITS, TUB2, and TEF1-α sequences, the LPG1-1 and LPG1-2 showed over 99% similarity to N. saprophytica, and YSG-1 showed over 99% similarity to N. ellipsospora. Phylogenetic analysis of the three isolates was performed with MEGA10 (version 10.0) based on sequences of ITS, TUB2, and TEF1-α using maximum parsimony analysis. The results revealed that LPG1-1 and LPG1-2 were clustered with N. saprophytica, and YSG-1 was clustered with N. ellipsospora. Pathogenicity tests of three isolates were conducted on 72 healthy persimmon fruits with and without wounds, and 9 fruits are for each treatment. The wound was made by a sterilized needle. Fruits were pre-processed with 75% ethanol for 10 s, 1% NaClO for 2 min and rinsed three times in sterile water. Conidial suspensions (10 µL, 106 conidia/mL in 0.1% sterile Tween 20) were inoculated on each site (4 sites/fruit). Control group was treated with 0.1% sterile Tween 20. All inoculated sites were covered with wet cotton. The inoculated fruits were placed in a plastic box to maintain humidity at 28℃. After 5 days, all wounded fruits showed fruit rot, whereas unwounded and control fruits remained asymptomatic, there were significant differences (P<0.05) in aggressiveness between N. saprophytica (average lesion diameter 13.1 mm) and N. ellipsospora (average lesion diameter 14.9 mm). Koch's postulates were fulfilled by re-isolating the causal agents from inoculated fruits. N. ellipsospora was previously reported as an endophyte in D. montana in southern India (Reddy et al. 2016). N. saprophytica could cause leaf spot of Erythropalum scandens and Magnolia sp., and fruit rot of Litsea rotundifolia in China and leaf spot of Elaeis guineensis in Malaysia (Yang et al. 2021, Ismail et al. 2017). To our knowledge, this is the first report of N. ellipsospora and N. saprophytica causing fruit rot on persimmon in the world. The results will provide a foundation for controlling fruit rot caused by pestalotioid fungi on persimmon.

Philodendron bipinnatifidum anthracnose Caused by Colletotrichum karsti and C. endophytica in Guangxi, China.

Philodendron bipinnatifidum Schott ex Endl (Araceae) is native to South America. It was introduced in Guangdong around the 1980s, and then gradually promoted for use as a landscape ornamental in South China (You et al. 2013). Previous studies showed that an extract of P. bipinnatifidum displayed antinociceptive and anti-inflammatory activities (Scapinello et al. 2019). In August 2019 and June 2020, leaf spot disease was observed on P. bipinnatifidum leaves in Qingxiushan Park, Nanning, Guangxi province, China, with approximately 80% disease incidence. Symptoms began as small brown spots that extended into large, irregular, dark brown, necrotic, sunken lesions. The leaves eventually became yellow and then withered and died. The symptomatic leaves were sampled from three different places in the park. Leaf pieces (5× 5 mm) of three samples were cut from the junction of diseased and healthy leaf tissue, disinfected in 75% (v/v) alcohol for 10 sec, 2% (v/v) sodium hypochlorite for 1 min, and then rinsed three times in sterile distilled water before pieces were incubated on potato dextrose agar (PDA) at 25°C for 7 days. Eighty-one Colletotrichum isolates were obtained, with an 88% isolation rate, and three of these (GBZ7-1, GBZ7-3 and GBZ8-2) were selected for intensive study. After 7 days, the colonies on PDA showed white-to-gray aerial mycelium. Conidia (n=90) were elliptical, single-celled, hyaline, straight, 14.61 ± 0.08 µm × 6.84 ± 0.04 µm (C. karsti), and 15.15 ± 0.11 µm ×5.04 ± 0.04 µm (C. endophytica). Appressoria (n=90) were melanized, subglobose, irregular, 9.57 ± 0.17 µm × 7.18 ± 0.10 µm (C. karsti), and 7.36 ± 0.18 µm × 5.52 ± 0.13 µm (C. endophytica). To confirm morphological identification, the rDNA internal transcribed spacer region (ITS), actin (ACT), calmodulin (CAL), chitin synthase (CHS-1), ß-tubulin 2 (TUB2) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes (Weir et al. 2012) were amplified and sequenced (GenBank accessions Nos. ITS (MZ962373 ~ MZ962375), ACT (OK040200 ~ OK040202), CAL (OK040205 ~ OK040207), CHS-1 (OK040210 ~ OK040212), TUB2 (OK040220 ~ OK040222) and GAPDH (OK040215 ~ OK040217) of GBZ7-1, GBZ7-3 and GBZ8-2 respectively). Phylogenetic analysis was done using RAXML (Version 2.0) based on sequences of multiple loci (ITS, ACT, CAL, CHS-1, TUB2 and GAPDH). Isolates GBZ7-1 and GBZ7-3 were identified as C. karsti and GBZ8-2 as C. endophytica. Pathogenicity tests were performed with the three isolates on 45 asymptomatic attached leaves of nine one-year-old plants (three plants per isolate). Every leaf was punctured at three points using a sterile needle and inoculated with 10 µl of conidial suspension (106spores/ml) on each wound. Wounded leaves treated with sterilized water under the same conditions served as controls. The experiment was repeated three times. All plants were sprayed with water and covered with plastic bags to maintain high humidity. Sunken necrotic lesions were observed on all inoculated leaves after 15 days at 28 °C, whereas no symptoms were observed on the control leaves. C. karsti and C. endophytica were consistently re-isolated from the inoculated leaves which was confirmed by morphology and sequencing, fulfilling Koch's postulates. C. siamense was previously reported as a pathogen on P. bipinnatifidum in China (Ning et al. 2021). To our knowledge, this is the first report of leaf spot caused by C. karsti and C. endophytica on P. bipinnatifidum worldwide. This research may accelerate the development of future epidemiological studies and management strategies for anthracnose caused by C. karsti and C. endophytica on P. bipinnatifidum.

Identification and Observation of Infection Processes of Colletotrichum Species Associated with Pearl Plum Anthracnose in Guangxi, China.

Pearl plum (Prunus salicina Lindl.) is mainly cultivated in Tian'e County in Guangxi Province, southern China. Anthracnose is a devastating disease on pearl plum, causing extensive leaf blight. Diseased leaves were sampled from 21 orchards in Tian'e County. Isolates were first screened for ones resembling Colletotrichum, and 21 representative isolates were selected for sequencing of portions of the ribosomal internal transcribed spacer (ITS), the intergenic region of apn2 and MAT1-2-1 genes (ApMAT), actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), calmodulin (CAL), chitin synthase (CHS-1), and ß-tubulin 2 (TUB2). Based on colony, conidial, and appressorial morphology and sequence analyses, the Colletotrichum isolates associated with pearl plum anthracnose were identified as four species: Colletotrichum fructicola (16 isolates), C. gloeosporioides (3 isolates), C. cigarro (1 isolate), and C. siamense (1 isolate). The results of pathogenicity tests showed that isolates of all four species were pathogenic to wounded leaves of pearl plum seedlings. In this study, we microscopically observed the infection processes of isolates of these four species on attached pearl plum leaves. For C. cigarro and C. siamense, the entire infection processes took 120 h; for C. fructicola and C. gloeosporioides, it only took 72 h. This is the first report of C. fructicola and C. cigarro causing anthracnose on pearl plum worldwide, and also the first report of C. siamense causing anthracnose on pearl plum in China.

First Report of Leaf Spot Caused by Colletotrichum citricola on Cavendish bananas in China.

Cavendish banana (Musa spp. AAA group) is an important tropical and subtropical fruit with significant economic value. It is widely planted in Guangxi, Yunnan, Hainan, Fujian and Guangdong provinces in China. In November 2020, leaf spots were observed on nearly 80% of the plants growing in three Cavendish banana plantations in Chongzuo, Guangxi, China. The symptoms on Cavendish banana leaves initially appeared as small black necrosis spots, which gradually expanded and connected, eventually covered the entire leaf. Three diseased leaves from three plantations were collected, sectioned into small pieces (5 ×5 mm), surface sterilized (10 s in 75% ethanol, followed by 1 min in 1% sodium hypochlorite and rinsed three times in sterile water) and placed on potato dextrose agar (PDA) at 28℃ for 5 days for pathogen isolation. The fungal colonies were white, cottony, while the reverse sides were white, concentric circles with yellowish-brown discoloration in 7-day cultures. The conidia were hyaline, aseptate, cylindrical, oval, measuring 10.3 to 17.71 µm (mean 14.06 ± 1.45 µm; n = 200) in length and 4.48 to 9.57 µm (mean 7.46 ± 0.69 µm; n = 200) in width. Three representative isolates (DX1-5, LZ4-5, and FS1-3) were obtained by monosporic isolation. The partial internal transcribed spacer (ITS) regions, actin (ACT), chitin synthase (CHS-1), glyceraldehydes-3-phosphate dehydrogenase (GAPDH), calmodulin (CAL), and ß-tubulin (TUB2) were amplified from genomic DNA for the three isolates (Weir et al. 2012). The sequences of the amplified fragments were deposited in GenBank (accessions OL361844 to OL361858, for GAPDH, CAL, ACT, CHS-1, and TUB2 of isolate DX1-5, LZ4-5 and FS1-3; OL305066 to OL305068 for ITS) and showed over 99% identities with the corresponding sequences of C. citricola. A neighbor-joining phylogenetic tree based on the above six genes of type or ex-type specimens of Colletotrichum (Fu et al. 2019) was constructed with MEGA 5.2 using the concatenation of multiple sequences (Kumar et al. 2016). All three isolates clustered together with the type culture of C. citricola (CBS 134228, CBS 134229, CBS 134230) with 82% bootstrap support in the phylogenetic tree. According to the molecular and morphological characteristics, all three isolates were identified as C. citricola. Pathogenicity tests were conducted on one-month-old primary hardened tissue culture plantlets. Tender, healthy leaves were gently scratched with a sterile needle, and each wound site was inoculated with sterile cotton impregnated with conidial suspension (106 spores/ml) for each isolate. Wounded leaves were treated with sterile cotton impregnated with conidial suspension of C. fructicola as positive controls and sterile water as negative controls. Each isolate was inoculated with three tissue culture plantlets, six inoculated sites on each plantlet, the same as controls. All inoculated tissue culture plantlets were covered with plastic bags to maintain high humidity and placed in a 28℃ growth chamber with constant light. Black necrotic lesions were clearly observed on the inoculated leaves and the positive controls after 7 days, whereas no symptoms appeared on the negative control leaves. The fungus was re-isolated from inoculated leaves, and these isolates matched the morphological and molecular characteristics of the original isolates confirming Koch's postulates. To our knowledge, this is the first report of leaf spot caused by C. citricola on Cavendish banana worldwide.