Identification and Characterization of Colletotrichum Species Associated with Anthracnose Disease of Plum.

Plum (Prunus salicina Lindl.) is commercially cultivated worldwide for the high levels of nutrients in the fruit. In recent years, anthracnose has been severe in some plum planting areas in China, resulting in a large number of necrotic leaves, blight and pre-mature leaf fall. In this study, anthracnose samples of plum leaves were collected from Hezhou, Guilin and Lipu, Guangxi Province, and Meishan city, Abe Tibetan and Qiang autonomous prefecture of Sichuan Province. Characteristics of mycelia on PDA, morphology of appressoria and conidia, and analysis of sequences of several marker regions (internal transcribed spacer [ITS] region, glyceraldehyde-3-phosphate dehydrogenase [GAPDH], chitin synthase [CHS-1], histone H3 [HIS3], actin [ACT], ß-tubulin [TUB2], and the intergenic region between apn2 and MAT1-2-1 [ApMat]). The resulting 101 Colletotrichum isolates obtained were identified as eight species: C. fructicola (50.5%), C. siamense (24.8%), C. karsti (8.9%), C. plurivorum (7.9%), C. aeschynomenes (3.9%), C. gloeosporioides (2%), C. celtidis (1%) and C. phyllanthi (1%). Representatives of all eight Colletotrichum species were found to cause disease on wounded leaves of plum seedlings in pathogenicity assays. As far as we are aware, this is the first report of anthracnose of plum caused by C. celtidis and C. phyllanthi in China.

Fruit Anthracnose on Cavendish bananas Caused by Colletotrichum fructicola in Guangxi, China.

Cavendish banana (Musa spp. AAA group) is one of the main fruit crops worldwide. It is widely planted in Guangdong, Hainan, Guangxi, Fujian and Yunnan provinces in southern China. In November 2020, banana fruits with anthracnose symptoms were collected from Dayu Town (N 23.17°, E 109.80°), Guigang City, and Chengjun Town (N 22.60°, E 110.00°), Yulin City, Guangxi Province, China, where the disease was found on about 70% of the banana plants, and on individual fruit, up to 10% of the surface was covered with symptoms. The symptoms initially began with rust-colored spots on the surface of the immature fruit, which gradually became sunken and cracked as the disease progressed. Small tissues (5×5 mm) from the pericarp at the junction of disease and health were surface-disinfected in 75% ethanol for 10 s, 2% sodium hypochlorite (NaClO) for 1 min, and washed three times in sterile water. Tissue pieces were placed on potato dextrose ager (PDA) and incubated at 25°C. Fifty-nine morphologically similar colonies were obtained after 5 days of incubation, with 100% isolation frequency. Of 59 isolates, GG1-3 isolated from Guigang City and YL4-2 isolated from Yulin City were selected as representative strains for intensive study. Mycelia were off-white for both isolates and conidia obtained from PDA were cylindrical, unicellular, hyaline and obtuse ends, with sizes of 11.5 ± 1.8×3.9 ± 0.8 µm (n=60) and 11.5 ± 1.6×4.1 ± 0.6 µm (n=60) for GG1-3 and YL4-2, respectively (Prihastuti et al. 2009). Genomic DNA was extracted from 7-day-old aerial mycelia using a DNAsecure Plant Kit (Tiangen Biotech, China). The internal transcribed spacer (ITS), the intergenic region of apn2 and MAT1-2-1 (ApMAT) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified and sequenced (White et al. 1990; Silva et al.2012; Templeton et al. 1992). Sequences were deposited in GenBank (ITS, OR596961 to OR596962; GAPDH, OR661771 to OR661772; APMAT, OR661773 to OR661774) and showed 100% identities with the corresponding type strains sequences of C. fructicola. Phylogenetic tree was constructed with software raxmlGUI v.2.0.0 based on sequences of multiple loci (ITS, GAPDH and ApMAT) and Maximum Likelihood method. Phylogenetic analysis revealed that the two isolates and C. fructicola were clustered in the same clade, with 94% bootstrap support. According to morphology and phylogenetic analysis, the two isolates GG1-3 and YL4-2 were identified as C. fructicola. For pathogenicity tests, healthy fruits were surface sterilized with 75% ethanol followed by a wash with sterilized water. Five adjacent needle punctures in a 5-mm-diameter circle were made with a sterilized needle on healthy fruits, followed by inoculation with 20 µL of conidial suspension (106 spores/ml), and sterilized water was used as controls. All banana fruit were incubated in a humid chamber at 28°C. After 4 days, all inoculated fruits showed visible symptoms and had rust-colored spots on the margins, while control banana fruits remained symptomless. The fungus was isolated from the inoculated fruit and the isolates were found to match the morphological and molecular characteristics of the original isolates, confirming Koch's hypothesis. To our knowledge, this is the first report of fruit anthracnose on Cavendish bananas caused by C. fructicola in China. This study will provide valuable information for prevention and management of anthracnose on banana fruit.

Screening and characterization of biocontrol bacteria isolated from Ageratum conyzoides against Collectotrichum fructicola causing Chinese plum (Prunus salicina Lindl.) anthracnose.

Chinese plum (Prunus salicina Lindl.) is a nutritionally and economically important stone fruit widely grown around the world. Anthracnose, caused by Collectotrichum spp., is one of the primary biotic stress factors limiting plum production. Medicinal plants may harbor rhizospheric or endophytic microorganisms that produce bioactive metabolites that can be used as anthracnose biocontrol agents. Here, 27 bacterial isolates from the medicinal plant A. conyzoides with diverse antagonistic activities against C. fructicola were screened. Based on morphological, physiological, biochemical, and molecular characterization, 25 of these isolates belong to different species of genus Bacillus, one to Pseudomonas monsensis, and one more to Microbacterium phyllosphaerae. Eight representative strains showed high biocontrol efficacy against plum anthracnose in a pot experiment. In addition, several Bacillus isolates showed a broad spectrum of inhibitory activity against a variety of fungal phytopathogens. Analysis of the volatile organic compound profile of these eight representative strains revealed a total of 47 compounds, most of which were ketones, while the others included alkanes, alkenes, alcohols, pyrazines, and phenols. Overall, this study confirmed the potential value of eight bacterial isolates for development as anthracnose biocontrol agents.

Leaf Spot Caused by Colletotrichum siamense, C. fructicola, and C. aeschynomenes on Ixora chinensis in Guangxi, China.

Ixora chinensis Lam., an important ornamental flower, has become more and more popular in the southwest and southeast regions of China for its bright and abundant flowers (Li et al. 2019). In March 2022, 100% I. chinensis plants showed typical anthracnose symptoms on leaf in Nanning, Guangxi, China (108°22' N, 22°48' E). The central areas of lesions were grayish white with small black particles arranged in a wheel pattern, and the edges of lesions were light red to brown. Three diseased leaf samples were collected from three gardens, respectively. 5×5 mm tissues 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 PDA at 25°C under 12/12 h light/darkness. Eighty-three morphologically similar colonies were observed on PDA after 5 days, and eight isolates G1-3, G2-1, G3-3, W-1, W-2, LCH2-1, LCH3-3, and LCH4-1 were selected for further study. Genomic DNA of these isolates were extracted from 7-day-old mycelia. Primer pairs ITS1/ITS4, GDF1/GDR1, T1/ßt2b, CHS Ⅰ-79F/CHS Ⅰ-354R, CL1/CL2, ACT-512F/ACT-783R, and MAT1-2-1/apn2 were used to amplify ITS loci and GAPDH, CHS-Ⅰ, CAL, ACT, ApMAT genes, respectively (Yang et al. 2009; Silva et al. 2012; Liu et al. 2015). Sequences have been deposited in GenBank (ITS: OQ771884 to OQ771891, GAPDH: OQ759576 to OQ759583, TUB2: OQ759584 to OQ759591, CHS-1: OQ759568 to OQ759575, CAL: OQ759560 to OQ759567, ACT: OQ759552 to OQ759559, ApMat: OQ759544 to OQ759551). Phylogenetic analysis was performed with raxmlGUI v.2.0.0. based on combined sequences of ITS, GAPDH, TUB2, CHS-1, CAL, ACT, and ApMAT using maximum parsimony analysis. The results revealed that isolates G2-1 and W-2 were clustered with Collectrichum fructicola, G3-3, W-1, G1-3, LCH2-1, and LCH3-3 were clustered with C. siamense, and LCH4-1 was clustered with C. aeschynomenes. Three representative isolates W-2, G3-3, and LCH4-1 were selected for morphology and pathogenicity observation. On PDA, the colonies of three isolates presented white to grey cottony mycelia，from the margin to the center, W-2 was white, grey, and light gray, G3-3 showed light gray, white, and grey, LCH4-1 was white and light gray, respectively. Conidia were all hyaline, one-celled, cylindrical, and straight. The conidial sizes of W-2, G3-3, and LCH4-1 were 11.03 to 17.53 × 4.93 to 8.42 µm (n=100), 10.63 to 19.06 × 3.73 to 6.92 µm (n=100), and 11.61 to 20.39 × 3.65 to 6.67 µm (n=100), respectively. Pathogenicity tests of three isolates were conducted on leaves of 1-year-old I. chinensis plants with and without wounds, three plants for each treatment, and five leaves inoculated for each plant. Conidial suspensions (10 µL, 106 conidia/mL in 0.1% sterile Tween 20) were inoculated on each site. Control group was treated with 0.1% sterile Tween 20. All inoculated sites were covered with wet cotton, and all plants were bagged and placed in the greenhouse to maintain humidity at 25℃. After 10 days, all wounded and inoculated leaves showed leaf spot, whereas unwounded and control leaves remained asymptomatic. Koch's postulates were fulfilled by re-isolating the causal agents from diseased leaves. C. siamense and C. aeschynomenes could cause anthracnose of I. chinensis in China (Liu et al. 2016, Li et al. 2021). However, to our knowledge, this is the first report of C. fructicola infecting I. chinensis in China. This study may provide reference for further epidemiological study and prevention of anthracnose on I. chinensis.

Leaf Spot of Sanhua Plum caused by Botryosphaeria wangensis in China.

Sanhua plum (Prunus salicina L.) is planted widely in Babu district of Hezhou, Guangxi with a planting history of more than 70 years (Zhou et al., 2021). In August 2021, leaf spot disease was observed with approximately 50% incidence on Sanhua plum leaves in Babu district in Hezhou, Guangxi (N23°49'-24°48', E111°12'-112°03'). The symptoms initially appeared as small, round, and chlorotic spots. As the disease progressed, the lesions enlarged and margins became dark brown. To isolate the pathogen, small pieces (5 × 5 mm) of the infected tissue margins were sterilized by exposure to 75% ethanol for 10 sec, 2% sodium hypochlorite for 1 min and rinsed three times in sterile water. Pieces were incubated on potato dextrose agar (PDA) at 28℃. In total, 75 isolates were obtained from leaves which were collected from three trees. Fifty of them were morphologically identical with a 67% average isolation frequency. Three representative isolates (HZ13-1, HZ26-3 and HZ47-1) were selected for further study. The cultures on PDA were initially white, fluffy with uneven margins and turned smoky gray to olivaceous at the surface. The reverse sides were olivaceous gray to iron gray after seven days. The growth rate of mycelium was 2.5 cm/day. Conidia were produced after two weeks by exposure to near-fluorescent light for 10 hours per day. Conidia were fusiform, hyaline, thin-walled, smooth with granular contents unicellular, and 19.7 ± 0.13 × 5.8 ± 0.06 µm (n=90), 19.8 ± 0.09 × 6.5 ± 0.23 µm (n=90), and 20.6 ± 0.20 × 6.7 ± 0.12 µm (n=90) for HZ13-1, HZ26-3 and HZ47-1, respectively. These characteristics were consistent with the descriptions of the Botryosphaeria wangensis (Hattori et al. 2021). The DNA was extracted from mycelia, and the internal transcribed spacer (ITS), elongation factor 1-alpha gene (EF1-α) and ß-tubulin (TUB2) were amplified using the primer pairs ITS1/ITS4, EF1-728F/EF1-986R and T1/BT2b (White et al. 1990, Carbone et al. 1999, Yu et al. 2021), respectively. The sequences were compared with GenBank and they all showed over 99% identity to the type strain of B. wangensis CERC 2298 (Li et al. 2020). Sequences of the three isolates were deposited in GenBank (Accession Nos.: ITS, OP804110-OP804112; EF1-α, OP821748-OP821750; TUB2, OP821745-OP821747). The three isolates were identified as B. wangensis based on the maximum likelihood phylogenetic tree of concatenated sequences of ITS, EF1-α, and TUB2 with RAxML version 2.0. Pathogenicity tests were performed on healthy leaves of 2-year-old Sanhua plum, which were wounded by a sterilized needle in a greenhouse. A 5-mm-diam hyphal plug was placed on the wound. Each isolate was used to inoculate three plants, with 20 leaves per plant. Control plants were inoculated with sterile PDA plugs. All the plants were sprayed with distilled water and covered with plastic bags. After four days of incubation at 28℃ with constant light, lesion began to develop in the inoculated leaves. After ten days, the average diameter of lesions was up to 1.5 cm but controls remained symptom-free. The fungi were reisolated from inoculated symptomatic leaves and were identical to the inoculated isolates, thus completing Koch's postulates. To our knowledge, this is the first report of B. wangensis associated with leaf spot of Sanhua plum in China. The results will contribute to accelerating the development of future epidemiological studies of B. wangensis on Sanhua plum.

The evolution of mini-chromosomes in the fungal genus Colletotrichum.

Anthracnose diseases caused by Colletotrichum species are among the most common fungal diseases. These symptoms typically manifest as dark, sunken lesions on leaves, stems, and fruit. In China, mango anthracnose seriously affects fruit yield and quality. Genome sequencing of several species shows the presence of mini-chromosomes. These are thought to contribute to virulence, but their formation and activity remain to be fully elucidated. Here, we assembled 17 Colletotrichum genomes (16 isolated from mango plus one from persimmon) through PacBio long-read sequencing. Half of the assembled scaffolds had telomeric repeats at both ends indicating full-length chromosomes. Based on comparative genomics analysis at interspecies and intraspecies levels, we identified extensive chromosomal rearrangements events. We analyzed mini-chromosomes of Colletotrichum spp. and found large variation among close relatives. In C. fructicola, homology between core chromosomes and mini-chromosomes suggested that some mini-chromosomes were generated by recombination of core chromosomes. In C. musae GZ23-3, we found 26 horizontally transferred genes arranged in clusters on mini-chromosomes. In C. asianum FJ11-1, several potential pathogenesis-related genes on mini-chromosomes were upregulated, especially in strains with highly pathogenic phenotypes. Mutants of these upregulated genes showed obvious defects in virulence. Our findings provide insights into the evolution and potential relationships to virulence associated with mini-chromosomes. IMPORTANCE Colletotrichum is a cosmopolitan fungal genus that seriously affects fruit yield and quality of many plant species. Mini-chromosomes have been found to be related to virulence in Colletotrichum. Further examination of mini-chromosomes can help us elucidate some pathogenic mechanisms of Colletotrichum. In this study, we generated novel assemblies of several Colletotrichum strains. Comparative genomic analyses within and between Colletotrichum species were conducted. We then identified mini-chromosomes in our sequenced strains systematically. The characteristics and generation of mini-chromosomes were investigated. Transcriptome analysis and gene knockout revealed pathogenesis-related genes located on mini-chromosomes of C. asianum FJ11-1. This study represents the most comprehensive investigation of chromosome evolution and potential pathogenicity of mini-chromosomes in the Colletotrichum genus.

Bacterial Leaf Spot of Plum Caused by Sphingomonas spermidinifaciens in Guangxi, China.

Plum (Prunus salicina L.) is a traditional fruit in Southern China and is ubiquitous throughout the world. In August 2021, leaves of plum trees showed water-soaking spots and light yellow-green halos with incidence exceeding 50% in Babu district in Hezhou, Guangxi (N23°49'-24°48', E111°12'-112°03'). To isolate the causal agent, three diseased leaves collected from three different trees growing in different orchards were cut into 5 mm × 5 mm pieces, disinfected with 75% ethanol for 10 sec, 2% sodium hypochlorite for 1 min and rinsed three times in sterile water. The diseased pieces were ground in sterile water and then kept static for about 10 min. Ten-fold serial dilutions in water were prepared and 100 µL of each dilution from 10-1 to 10-6 were plated on Luria-Bertani (LB) Agar. After incubation at 28℃ for 48 h, the proportion of isolates with similar morphology was 73%. Three representative isolates (GY11-1, GY12-1 and GY15-1) were selected for further study. The colonies were non-spore-forming, yellow, round, opaque, rod shaped, convex with smooth and bright neat edges. Biochemical test results showed that the colonies were strictly aerobic and gram-negative. The isolates were able to grow on LB agar containing 0-2% (w/v) NaCl and could utilize glucose, lactose, galactose, mannose, sucrose, maltose and rhamnose as a carbon source. They displayed a positive reaction for H2S production, oxidase, catalase and gelatin, but negative for starch. Genomic DNA of the three isolates was extracted for amplification of the 16S rDNA with primers 27F and 1492R. The resulting amplicons were sequenced. Additionally, five housekeeping genes atpD, dnaK, gap, recA, and rpoB of the three isolates were amplified using the corresponding primer pairs and sequenced. The sequences were deposited in GenBank (16S rDNA, OP861004-OP861006; atpD, OQ703328-OQ703330; dnaK, OQ703331-OQ703333; gap, OQ703334-OQ703336; recA, OQ703337-OQ703339; and rpoB, OQ703340-OQ703342). The isolates were identified as Sphingomonas spermidinifaciens based on the phylogenetic tree inferred by maximum-likelihood using MegaX 7.0 of the concatenated six sequences (multilocus sequence analysis, MLSA) compared with sequences from different Sphingomonas type strains . Pathogenicity of the isolates was tested on healthy leaves of the two-year-old plum plants in a greenhouse. The leaves were wounded by a sterilized needle and sprayed with bacterial suspensions prepared in PBS (Phosphate buffer saline) at OD600=0.5. PBS buffer solution was used as negative control. Each isolate was used to inoculate on 20 leaves per plum tree. The plants were covered with plastic bags to maintain high humidity. Dark brown-to-black lesions were observed on leaves 3 days post incubation at 28℃ with constant light. The average diameter of lesions was 1 cm after seven days, but the negative controls were symptomless. Bacteria reisolated from the diseased leaves were the same as the ones used for inoculation on the basis of morphological and molecular identification, fulfilling Koch's postulates. Plant disease caused by a Sphingomonas species has been reported on mango, pomeand Spanish melon. However, this is the first report of S. spermidinifaciens causing leaf spot disease of plum in China. This report will help to develop effective disease control strategies in the future.

Fruit Rot Caused by Fusarium sulawesiense on Chilli in Guangxi, China.

Chilli (Capsicum annuum) is considered as one of the most important vegetables and spice crops throughout the world which is widely cultivated in China. In October 2019, fruit rot symptoms were observed on chilli in Guilin, Guangxi, China (N24°18', E109°45'). Irregular dark green spots initially appeared on the middle or bottom of the fruit, then extended to larger grayish brown lesions and started to rot. In the late stage, the whole fruit dried up after water loss. Three disease samples were collected from three towns of different counties in Guilin where the disease incidence of chilli fruits was 15-30%. The margin of diseased fruits was cut into small pieces (3×3 mm), disinfected with 75% ethanol solution for 10 s, 2% NaOCl for 1 min, and rinsed in sterile distilled water three times. Tissue pieces were separately plated on potato dextrose agar (PDA) and incubated at 25°C for seven days. Fifty-four fungal isolates with similar morphology were consistently recovered from diseased tissues of three fruits, with 100% isolation frequency. Three representatives GC1-1, GC2-1 and PLX1-1 were selected for further analysis. The colonies produced abundant whitish to yellowish aerial mycelium on PDA after 7 days incubation at 25°C in the dark. Macroconidia cultured on carnation leaf agar (CLA) for 7 days were long, hyaline, falcate, with dorsal and ventral lines often gradually wider toward apex, curved apical cell and foot-shaped basal cell, mostly 2 to 5 septa, and ranged from 24.16 to 38.88 × 3.36 to 6.55 µm (average 31.39×4.48 µm), from 19.44 to 28.68 × 3.02 to 4.99 µm (average 23.02×3.89 µm), and from 20.96 to 35.05 × 3.30 to 6.06 µm (average 26.24×4.51 µm) for GC1-1, GC2-1, and PLX1-1, respectively. Microconidia were hyaline, fusoid or ovoid, one-septate or nonseptate, and ranged from 4.61 to 10.14 × 2.61 to 4.77 µm (average 8.13×3.58 µm), from 3.55 to 7.85 × 1.95 to 3.04 µm (average 5.79×2.39 µm), and from 6.75 to 18.48 × 3.05 to 9.07 µm (average 14.32×4.31 µm) for GC1-1, GC2-1, and PLX1-1, respectively. Genomic DNA was extracted from 7-day-old aerial mycelia of these isolates. The internal transcribed spacer (ITS), translation elongation factor (TEF1), calmodulin (CAM) and partial RNA polymerase second largest subunit (RPB2) were amplified using primers ITS4/ITS1, EF1/EF2, CL1/CL2A, and 5F2/7cR, respectively (White et al. 1990; O'Donnell et al. 2000, 2010). Sequences were deposited in GenBank (ITS: OQ080044-OQ080046; TEF1: OQ101589-OQ101591; CAM: OQ101586-OQ101588; RPB2: OQ101592-OQ101594). A maximum Likelihood (ML) phylogenetic tree was constructed with RAxML version 8.2.10 based on the concatenated sequences (ITS, CAM, TEF1, RPB2). According to morphology and phylogenetic analysis, the isolates were identified as Fusarium sulawesiense (Maryani et al. 2019). For pathogenicity tests, multiple punctures in a 5-mm-diameter circle were made with a sterilized toothpick on detached young healthy fruits, followed by inoculation with 10 µl of conidial suspension (106 spores/ml in 0.1% sterile Tween 20). Each isolate was inoculated onto eighteen fruits. Controls were inoculated with water containing 0.1% sterile Tween 20 under the same conditions. Symptoms were observed on the inoculated fruits 7 days after incubation at 25°C, whereas non-inoculated controls were asymptomatic. The fungus was re-isolated from inoculated chilli fruits, completing Koch's postulates. To our knowledge, this is the first report of Fusarium sulawesiense causing fruit rot on Chilli in China. These results will provide valuable information for prevention and management of fruit rot on Chilli.

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.

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

Persimmon originated from China where it has a long cultivation history. Anthracnose fruit rot and leaf blight caused by Colletotrichum species are major diseases of persimmon in China and cause severe economic losses. To determine the species causing anthracnose of persimmon in Guilin, Guangxi Province, diseased samples were collected from the four local counties: Gongcheng, Yangshuo, Pingle, and Lipu. Seventy-five isolates were obtained from persimmon samples with anthracnose symptoms and had similar morphological characteristics. Isolates were identified using a BLAST search and phylogenetic analysis of the internal transcribed spacer region, glyceraldehyde-3-phosphate dehydrogenase, partial actin, ß-tubulin, chitin synthase genomic regions, Apn2-Mat1-2 intergenic spacer, and the partial mating type gene and calmodulin genes. Five species (C. fructicola, C. horii, C. karstii, C. cliviicola, and C. siamense) accounted for 54.7, 25.3, 12.0, 5.3, and 2.7%, respectively, of the total isolates. All five Colletotrichum species were pathogenic on attached leaves and detached fruits of persimmon (cultivar Gongcheng Yueshi) in pathogenicity assays. The infection processes of the five Colletotrichum species were observed on persimmon leaves using light microscopy. Conidia of C. fructicola germinated at 12 h post inoculation (hpi) and quickly formed acervuli at 6 days post inoculation (dpi) and were the most aggressive. By contrast, conidia of C. cliviicola germinated at 3 hpi but produced the acervuli at 8 dpi and were the least aggressive. This is the first description of C. fructicola, C. cliviicola, and C. siamense as causal agents of persimmon anthracnose in Guangxi Province, China.

First Report of Bacterial Necrosis Caused by Pantoea vagans in Mango in China.

Mango (Mangifera indica L.) is one of the most important tropical fruits in China. Bacterial black spot is one of the primary factors limiting mango production and thus leads to huge economic losses (Bie et al. 2022). In June 2020, necrotic symptoms similar to bacterial black spot was observed with incidence 30% to 65% on mango cultivar Yuwen, Jinhuang, Tainong and Guifei in Baise, Guangxi, China. Typically, the lesions began as chlorotic spots that coalesced into an irregular shape, becoming black and slightly raised, with a yellow halo. Thirteen diseased samples collected from five orchards were cut into approximately 5-mm pieces, sterilized for 10 s with 75% ethanol, soaked with 2% NaClO for 1 min, and rinsed in sterilized water three times. The samples were then homogenized and a 10-fold serial dilution was made before plating onto Lysogeny broth (LB) agar. After incubation at 28°C for 3 days, one representative colony that was beige to yellow in color, round, convex and smooth with entire margins from each orchard was selected for further study. Genomic DNA was extracted to amplify the 16S rRNA gene (Lane et al. 1991). The resulting 16S rRNA sequences were compared in GenBank using BLASTn and shared at least 99% identity with Pantoea spp.. Furthermore, six housekeeping genes fusA, gyrB, leuS, pyrG, rplB and rpoB partial sequences of five isolates were amplified and sequenced (Delétoile et al. 2009). The sequences were deposited in GenBank (16S rRNA: OL413424 to OL413246, OP225727-OP225728; leuS: OL441796, OL441798 to OL441801; fusA, gyrB, leuS, pyrG, rplB and rpoB: OP272638-OP272662). The five bacterial isolates were classified as P. vagans based on the phylogenetic tree of the concatenated sequences and sequences derived from different Pantoea reference isolates inferred by maximum-likelihood using MegaX software (Kumar et al. 2018). Biochemical tests showed the isolates were Gram-negative, oxidase negative, and hydrogen oxidase positive, and could use D-glucose, D-fructose, L-rhamnose, D-galactose and D-mannitose as a carbon resource (Bradbury, 1986). Pathogenicity tests were performed on mango cv. Yuwen. The representative isolate was inoculated by infiltration with sterile needleless syringes on healthy leaves and spraying onto slightly scratched leaves with bacterial suspensions (OD600=0.1) respectively (Kutschera, et al. 2019). A Xanthomonas citri pv. mangiferaeindicae (Xcm) suspension and sterilized water were used as positive and negative controls, respectively. Inoculated plants were kept with 90 ± 5% relative humidity and 28 ± 1°C in the greenhouse for 1 week. Black to brown necrotic symptoms were observed on all leaves inoculated by infiltration except the negative control. These were observed in plants inoculated by spraying only after 2 weeks. Bacteria re-isolated from diseased tissues were consistent with the inoculated isolates and identified as P. vagans, fulfilling Koch's postulates. To date, P. vagans have been isolated from eucalyptus with bacterial blight and dieback, and maize with brown stalk rot (Brady et al. 2009). However, to our knowledge, this is the first report of P. vagans causing bacterial necrosis on mango in China. It was also found that some of the diseased samples were coinfected with P. vagans and Xcm in our investigation. Therefore, it is necessary to further study the infection mechanisms of this pathogen.

Ferroptosis regulator SLC7A11 is a prognostic marker and correlated with PD-L1 and immune cell infiltration in liver hepatocellular carcinoma.

Background: Liver hepatocellular carcinoma (LIHC) is a complicated disease with poor survival and lack of viable treatment options. The roles of ferroptosis and immunotherapy in LIHC are increasingly prominent, but the interplay of ferroptosis with the tumor microenvironment (TME) in LIHC is currently under-investigated. Methods: In this study, we analyzed normal liver tissues and tumor tissues from the TCGA and GTEx databases to obtain differentially expressed ferroptosis-related genes (FRGs). We then clustered LIHC based on the expression levels of selected FRGs and acquired distinct subtypes with significant heterogeneity regarding survival prognoses, PD-L1 expression, and immune cell infiltration. The correlation of those FRGs with TME in LIHC and pan-cancer analysis was also investigated. GO functional annotations and KEGG pathway analyses were performed to investigate the potential reactions of the obtained differentially expressed genes (DEGs). Further external validation was performed using microarrays on the GEO database and the key ferroptosis regulator SLC7A11 expression between LIHC and normal cells was detected by Western blotting. Results: A large proportion of genes were upregulated in the LIHC group. Among three clusters, cluster 3 had the worst prognosis combined with the highest PD-L1 expression and was positively correlated with various immune cells. Subsequently, survival analysis and Cox regression analysis screened out SLC7A11 as an independent prognostic factor in LIHC featured strong PD-L1 expression and unfavorable survival time. We filter out SLC7A11 as an independent prognostic signature in LIHC patients with strongly associated PD-L1 expression and unfavorable survival probability. In the pan-cancer analysis, high expression of SLC7A11 showed poor overall survival in seven cancers, while the correlation between immune checkpoints (ICs) and SLC7A11 varied by cancer type, indicating the potential therapeutic effects of SLC7A11 in cancers other than LIHC. Western blot was further employed to verify the expression of SLC7A11 in LIHC in vitro. Conclusion: Ferroptosis and TME synergistically play key roles in oncogenesis and progression of LIHC, and SLC7A11 can be used as a predictive biomarker for customized immunotherapy.

Multi-omics analysis reveals the metabolic regulators of duodenal low-grade inflammation in a functional dyspepsia model.

Several gastrointestinal phenotypes and impairment of duodenal mucosal barrier have been reported in clinical studies in patients with functional dyspepsia (FD). Due to the preferential colonization of the mucosa, intestinal microbes and their metabolites are commonly involved in host metabolism and immune responses. However, there are no studies on the intertwined correlation among multi-level data. For more comprehensive illustrating, a multi-omics analysis focusing on the duodenum was performed in the FD rat model. We found that differential microbiomes in the duodenum were significantly correlated with the biosynthesis of lipopolysaccharide and peptidoglycan. The innate immune response-related genes, which were upregulated in the duodenum, were associated with the TLR2/TLR4-NFκB signaling pathway. More importantly, arachidonyl ethanolamide (anandamide, AEA) and endocannabinoid analogues showed linear relationships with the FD phenotypes. Taken together, multi-level data from microbiome, transcriptome and metabolome reveal that AEA may regulate duodenal low-grade inflammation in FD. These results suggest an important cue of gut microbiome-endocannabinoid system axis in the pathogenesis of FD.

INHBB is a novel prognostic biomarker and correlated with immune infiltrates in gastric cancer.

Inhibin subunit beta B (INHBB) is a potential prognostic biomarker for a variety of cancers. However, its role in gastric cancer (GC) remains elusive. The differential expression data of INHBB in tumor and normal tissues were extracted from several databases and genetic alterations of INHBB were assessed by cBioPortal. Kaplan-Meier analysis was used to evaluate the survival rate of patients with GC with INHBB and association with clinical features in GC. Cox regression analysis was used to explore the prognostic value of clinical indicators and INHBB in GC, and a nomogram prognostic model was established. In addition, the predictive validity of the nomogram model was assessed by time-depended receiver operating characteristic (ROC) and calibration curves. Functional enrichment analyses were conducted to functionally annotate INHBB. Notably, we found that the quantitative assessment of immune cell subpopulation infiltration correlated with INHBB expression. INHBB expression is upregulated in GC and is correlated with several clinical features including prognostic indicators and a histological type. Genetic alterations were observed in INHBB, its DNA methylation level was negatively correlated with INHBB expression. High INHBB expression is associated with a poor prognosis and is an independent risk factor for prognosis in GC, along with age and residual tumor. The nomogram model showed a good prediction ability and was validated by time-depended ROC and calibration curves. Functional enrichment analysis indicated that INHBB-associated genes were enriched in tumor microenvironment Gene Ontology (GO) terms and were correlated with tumor-associated pathways. INHBB has a regulatory function in immune cell infiltration, especially macrophage infiltration in GC. Specifically, patients with GC with high INHBB expression and high macrophage infiltration have a worse prognosis. INHBB expression was negatively correlated with the expression of chemokines/chemokine receptors and plays a regulatory role in immunoinhibitor/immunostimulator-involved pathways. INHBB is a potential prognostic biomarker for GC and may drive the abnormal activity of critical cancer-associated pathways, potentially contributing to immune cell infiltration to promote GC development.

First Report of Anthracnose of Sanhua Plum Caused by Colletotrichum aeschynomenes in Guangxi, China.

There is nearly 5,800 ha of Sanhua plum (Prunus salicina Linn) planted in Babu district in Hezhou, Guangxi, with over 67,000 tons of annual output. In August 2021, anthracnose symptoms were observed on Sanhua plum leaves in three different cultivated towns in Babu district in Hezhou, Guangxi (N23°49' - 24°48', E111°12' - 112°03'). The plant disease incidence was over 50% with approximately 20 to 30% of leaves on a plant being symptomatic. The disease outbreak occurred in the warm and damp climate (June to August) in Hezhou. Initially, small chlorotic spots developed on the leaves which gradually enlarged to larger irregular dark brown sunken lesions with yellowish halos, necrotic lesions abscised and formed holes at a later stage. In severe cases, the whole leaf withered and defoliated. Three symptomatic leaf samples were collected from three different cultivated towns in Hezhou. Margins of infected tissues were cut into 3×3 mm pieces, surface disinfected with 75% alcohol for 10 s, 2% NaOCl for 2 min followed by three washes in sterile distilled water and transferred to potato dextrose agar (PDA) plates. In total, forty-one isolates were obtained after 4 days of incubation at 25℃ on PDA, and thirty-one of them were Colletotrichum (average isolation frequency 76%). Three representative isolates (HZ18-1, HZ22-3, and HZ46-3) were selected for further study. After 7 days on PDA at 25℃, isolates had white to light grey cottony aerial mycelium on the obverse and revealed dark grey on the reverse. Conidia were hyaline, cylindroid, tapering slightly near both ends, measuring 16.3 ± 1.2 µm × 5.6 ± 0.4 µm, 16.1 ± 1.4 µm × 6.4 ± 0.7 µm, 16.2 ± 1.1 µm × 6.0 ± 0.4 µm (n=90) for HZ18-1, HZ22-3, and HZ46-3, respectively. Appressoria were brown, elliptic or fusoid, deeply lobed, measuring 10.2 ± 1.6 µm × 6.8 ± 1.0 µm, 10.7 ± 1.3 µm × 6.6 ± 0.8 µm, 9.3± 1.3 µm × 6.9 ± 0.9 µm (n=90) for HZ18-1, HZ22-3, and HZ46-3, respectively. These characteristics were consistent with the descriptions of Colletotrichum aeschynomenes B. Weir & P. R. Johnst (Weir et al. 2012). The internal transcribed spacer (ITS) region and the intergenic region and flanking regions of Apn2 and MAT1-2-1 (ApMAT) were amplified using ITS1/ITS4 and AM-F/AM-R primers, respectively (White et al. 1990; Silva et al. 2012). BLASTn analysis of the sequences showed over 99% identity with the corresponding loci from the culture collection C. aeschynomenes ICMP 17673 (ex-type). Sequences from the three isolates were deposited in GenBank (Accession Nos.: ITS, OM838335, OM838339, OM838370; ApMAT, OM816771, OM816775, OM816806). Phylogenetic maximum likelihood analysis with RAxML version 8.2.10 based on the concatenated sequences of ITS and ApMAT showed that the three isolates clustered with the ex-type specimen of C. aeschynomenes ICMP 17673. Pathogenicity was confirmed on leaves with and without wounds of 24 two-year-old Sanhua plum plants in a greenhouse. The wound was made with a sterilized toothpick. Wounded and unwounded leaves were inoculated with 20 µL of conidial suspension (106 conidia/mL) of the three isolates and control plants were inoculated with sterile distilled water (20 leaves/plant, 3 plants/treatment). All plants were covered with plastic bags to maintain high humidity. After 8 days of incubation at 25℃ with constant light, necrotic lesions were observed on inoculated leaves, whereas control plants showed no symptoms. To fulfill Koch's postulates, all fungi were successfully reisolated from symptomatic leaves. This species has been reported on Aeschynomene virginica in the United States (Weir et al. 2012), Manihot esculenta in Thailand (Sangpueak et al. 2018), Theobroma cacao (Nascimento et al. 2019) and Myrciaria dubia (Matos et al. 2020) in Brazil. To our knowledge, this is the first report of C. aeschynomenes causing Sanhua plum leaf anthracnose in China. The results will provide valuable information for management of anthracnose associated with Sanhua plum.

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.

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.

First Report of Leaf Blight Caused by Fusarium pernambucanum and Fusarium sulawesiense on Plum in Sichuan, China.

Plum (Prunus salicina Lindl.) is widely cultivated for its rich nutrients and flavor in China. In August 2020, leaf blight symptoms were observed on plum in Meishan, Sichuan, China (N29°24', E104°30'). Irregular brown spots initially appeared on the edge or tip of the leaf, then extended to larger taupe lesions that were surrounded by a chlorotic halo. In the late stage, grey-brown blighted tissue covered the entire leaf causing leaves to wither, curl and abscise. The leaves with blight were collected from three different towns in Meishan where the disease incidence was found on 15-30% of plum plants. The margin of diseased leaves was cut into small pieces (3×3 mm), surface disinfected with 75% ethanol solution for 10 s, 2% NaOCl for 1 min, and rinsed in sterile distilled water three times. Tissue pieces were plated on potato dextrose agar (PDA) and incubated at 25°C. Forty-nine morphologically similar colonies were observed on PDA plates after 3-5 days and three of these (TEY9-1, TEY12-1, TEY15A-1) were selected for intensive study. The colonies produced abundant whitish to yellowish aerial mycelium after 7 days incubation at 25°C in the dark. Macroconidia on carnation leaf agar (CLA) were falcate, hyaline, straight to slightly curved, smooth to slightly rough with 3 to 6 septa, the apical cell was blunt or hooked, and the basal cell was barely notched, 31.6 ± 2.4 µm × 4.7 ± 0.4 µm, 28.9 ± 3.0 µm × 4.5 ± 0.5 µm, 32.5 ± 3.4 µm × 4.5 ± 0.5 for TEY9-1, TEY12-1, TEY15A-1, respectively. Microconidia were hyaline, fusoid or ovoid, nonseptate or one-septate, 14.4 ± 3.9 µm × 4.3 ± 0.6 µm, 13.0 ± 3.0 µm × 4.0 ± 0.4 µm, 11.0 ± 2.4 µm × 3.7 ± 0.5 for TEY9-1, TEY12-1, TEY15A-1, respectively. Genomic DNA was extracted from 7-day-old aerial mycelia of these isolates. The internal transcribed spacer (ITS), translation elongation factor (TEF1), calmodulin (CAM) and partial RNA polymerase second largest subunit (RPB2) were amplified using primers ITS4/ITS1, EF1/EF2, CL1/CL2A, and 5f2/7cr, respectively (White et al. 1990; O'Donnell et al. 2000, 2010). Sequences were deposited in GenBank (ITS: OK315638-OK315640; TEF1: OK338756-OK338758; CAM: OK338759-OK338761; RPB2: OK338762-OK338764). A maximum Likelihood (ML) phylogenetic tree was constructed with RAxML version 8.2.10 based on the concatenated sequences (ITS, TEF1, CAM, RPB2). According to morphology and phylogenetic analysis, TEY9-1 and TEY15A-1 were identified as Fusarium pernambucanum, and TEY12-1 was identified as Fusarium sulawesiense. Pathogenicity tests were conducted on young healthy leaves of 12 two-year-old plum plants in a 28°C greenhouse in Nanning, Guangxi, China. The epidermis of tested leaves was slightly scratched with sterile toothpick-tips forming a 3-mm-diameter cross-shaped wound, followed by inoculation with a 10 µl conidial suspension (106 spores /ml in 0.1% sterile Tween 20). Control leaves were wounded in the same way and treated with 0.1% sterile Tween 20. Plants were covered with polythene bags to maintain high humidity for 5 days. Inoculated leaves showed light brown to dark brown lesions, whereas control leaves were symptomless. Both species were re-isolated from symptomatic leaves, completing Koch's postulates. To our knowledge, this is the first report of F. pernambucanum and F. sulawesiense causing leaf blight on plum trees in China. These results will provide valuable information for prevention and management of leaf blight on plum trees.

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.