Nigrospora musae Causing Taxus chinensis var. mairei stem blight in Sichuan, China.

Taxus chinensis var. mairei is the endemic, endangered, and first-class protected tree species in China. This species is considered as an important resource plant because it can produce Taxol which is an effective medicinal compound against various cancers (Zhang et al., 2010). Stem blight was observed in two plant nurseries in Ya'an (102°44'E,30°42'N), Sichuan province in April 2021. The symptoms first appeared as round brown spots on the stem. As the disease progressed, the damaged area gradually expanded into an oval or irregular shape, which was dark brown. About 800 square meters of planting area were investigated and the disease incidence was up to approximately 64.8%. Twenty obviously symptomatic stems which exhibited the same symptoms as above were collected from 5 different trees in the nursery. To isolate the pathogen, the symptom margin was cut into small blocks (5 x 5 mm), and the blocks were surface sterilized in 75% ethanol for 90 s and 3% NaClO solution for 60 s . Finally incubated on Potato Dextrose Agar (PDA) at 28℃ for 5 days. Ten pure cultures were isolated by transferring hyphal and the three strains (HDS06, HDS07 and HDS08) were selected as representative isolates for further study. Initially, colonies on the PDA of three isolates were white and cotton-like, and then gradually turned gray-black from the center. After 21 days, conidia were produced and were smooth-walled, single-celled, black, oblate, or spherical, measuring 9.3 to 13.6 × 10.1 to 14.5 µm in size (n = 50). Conidia were present at the tip of conidiophores on hyaline vesicles. These morphological features were generally consistent with those of N. musae (Wang et al., 2017). To validate the identification, DNA were extracted from the three isolates, followed by the amplification of transcribed spacer region of rDNA (ITS), the translation elongation factor EF-1 (TEF-1), and the Beta-tubulin (TUB2) sequences with the respective primer pairs ITS1/ITS4 (White et al., 1990), EF-728F/EF-986R (Vieira et al., 2014) and Bt2a/Bt2b (O'Donnell et al., 1997) .The sequences were deposited in GenBank with the accession numbers ON965533, OP028064, OP028068, OP060349, OP060353, OP060354, OP060350, OP060351 and OP060352, respectively. Phylogenetic analysis of combined ITS, TUB2, and TEF genes using the Mrbayes inference method showed that the three isolates clustered with Nigrospora musae as a distinct clade (Fig. 2). Combine with morphological characteristics and phylogenetic analysis, three isolates were identified as N. musae. 30 2-year-old healthy potted plants of T. chinensis were used for pathogenicity test. 25 of these plants were inoculated by injecting 10 µL of the conidia suspension (1 × 106 conidia/mL) into stems and then wrap around the seal to moisturize. The remaining 5 plants were injected with the same amount of sterilized distilled water as a control. Finally, all potted plants were placed in a greenhouse at 25°C and 80% relative humidity. After 2 weeks, the inoculated stems developed lesions similar to those observed in the field, whereas controls were asymptomatic. N. musae was re-isolated from the infected stem and identified by both morphological characteristics and DNA sequence analysis. The experiments repeated three times showed similar results. As far as we know, this is the first report of N. musae causing T. chinensis stem blight in the world. The identification of N. musae could provide a certain theoretical basis for field management and further research of T. chinensis.

Changes in the Histology of Walnut (Juglans regia L.) Infected with Phomopsis capsici and Transcriptome and Metabolome Analysis.

Phomopsis capsici (P. capsici) causes branch blight of walnuts, which leads to significant economic loss. The molecular mechanism behind the response of walnuts remains unknown. Paraffin sectioning and transcriptome and metabolome analyses were performed to explore the changes in tissue structure, gene expression, and metabolic processes in walnut after infection with P. capsici. We found that P. capsici caused serious damage to xylem vessels during the infestation of walnut branches, destroying the structure and function of the vessels and creating obstacles to the transport of nutrients and water to the branches. The transcriptome results showed that differentially expressed genes (DEGs) were mainly annotated in carbon metabolism and ribosomes. Further metabolome analyses verified the specific induction of carbohydrate and amino acid biosynthesis by P. capsici. Finally, association analysis was performed for DEGs and differentially expressed metabolites (DEMs), which focused on the synthesis and metabolic pathways of amino acids, carbon metabolism, and secondary metabolites and cofactors. Three significant metabolites were identified: succinic semialdehyde acid, fumaric acid, and phosphoenolpyruvic acid. In conclusion, this study provides data reference on the pathogenesis of walnut branch blight and direction for breeding walnut to enhance its disease resistance.

Colletotrichum fructicola Causal agent of Shot-Hole Symptoms on Leaves of Prunus sibirica in China.

Prunus sibirica L. (Siberian apricot) is a member of the Rosaceae family and an ecologically important tree species in China (Buer et al., 2022). Shot hole symptoms on the leaves were observed in five Siberian apricot groves in Chengdu (103.81 E, 30.97 N), Sichuan province in July 2020. The symptoms first appeared as small purplish-brown spots with yellow rings around them. As the disease progressed, the damaged area (diameter 1.5-3.0 cm) became necrotic and fell off. The disease incidence was about 60% and the disease index was 28.6 of leaves in the grove. in most severe cases. Fifteen symptomatic leaves were collected from 5 different trees in an orchard. Pathogen isolation was performed from symptomatic leaf tissue (5 × 5 mm) though surface disinfection (in 70% ethanol and 2% NaClO) and incubation on Potato Dextrose Agar (PDA) at 28℃ for 3 days. Overall 10 isolates with similar colony morphology were obtained from the 15 infected tissue pieces, and three representative isolates (XCK 2-4) were selected for further study. Colonies of the isolates on PDA were initially cottony, pale white to grayish-green with abundant aerial hyphae and produced conidial masses after 7 days. Conidiogenous cells were clavate and aggregated in acervuli. Conidia were smooth-walled, single-celled, straight, and slightly obtusely rounded at both ends, 12.8 to 18.7 × 4.3 to 5.7 µm in size (Fig. 1). The morphological characteristics of the three isolates were consistent with the description of species in the Colletotrichum gloeosporioides complex. DNA was amplified using the following primers pairs for the internal transcribed spacer (ITS) region of rDNA and partial sequences of beta-tubulin (TUB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHS-1), and translation elongation factor (TEF-1), respectively: ITS1/ITS4, T1/Bt2b, GDF/GDR, CHS-F/CHS-R, and EF-F/EF-R (Vieira et al., 2014). Accession numbers (MW228049, MW284974, MW284976, MW284975 and MW284977, respectively) were obtained afterepositing all the resulting sequences in GenBank. Nucleotide blast showed 99 to 100% identities with Colletotrichum fructicola (GenBank accessions nos. MZ961683, MW284974, MN525881, MN525860, MF627961). Phylogenetic analysis of combined ITS-TUB-GAPDH genes using the Mrbayes inference method showed that the three isolates clustered with three reference isolates of C. fructicola as a distinct clade (Fig. 2). To verify Koch's postulates, ten 3-year-old healthy potted plants of P. sibirica were inoculated by spraying a conidial suspension (6 × 105 conidia/mL) of isolate XCK2 on both sides of leaves, and the control leaves were sprayed with sterile water. Then, all treatments were placed in a moist environment (25±2°C, 80% relative humidity, natural light). The inoculated plants showed typical symptoms of plants with natural infections, while the controls remained asymptomatic after 14 days. The pathogen C. fructicola was re-isolated from all inoculated plants, and the culture and fungus characteristics were the same as those of the original isolate. Colletotrichum fructicola was not isolated from the control plants. The results indicated that C. fructicola is the causal agent of the disease. Colletotrichum fructicola was reported as a leaf pathogen on Camellia chrysantha in China (Zhao et al., 2021). This is the first report of C. fructicola causing P. sibirica leaf shot-hole in the world. The identification of C. fructicola could provide relevant information for applying management strategies and research on the Siberian apricot disease.

Leaf spot on Alocasia macrorrhizos caused by Fusarium asiaticum in Sichuan, China.

Alocasia macrorrhizos (Giant elephant's ear), a perennial herb in the Araceae family, is native to South Asia and the Asia-Pacific (Takano, et al. 2012). It is cultivated as a medicinal and ornamental plant, and has a considerable economic importance in China. In September 2020, a severe infection of unknown leaf spot disease was observed on these plants at the Sichuan Agricultural University, Sichuan, China. The leaf spots first appeared as yellow dots. As these lesions expanded, they became circular to oval and light brown with darker brown edges. Around the lesions, the leaf tissue was chlorotic, thereby creating a yellow halo. When the infection became severe, spots merged into larger irregular lesions. Eventually, the diseased leaves senesced and dried. To identify the pathogen, five leaf samples of diseased plants were collected, and symptomatic tissues were surface-disinfected with 75% ethanol for 30 s followed by 3% NaCl solution for 30 s. Samples were rinsed three times in sterilized water, placed on potato dextrose agar (PDA), and incubated at 25°C ± 1°C in the dark. The colony grown on PDA was white (3 days), the center was brown (5 days), turned pink to dark red (8 days) with fluffy aerial mycelium and pigmentation with age. Ten pure cultures were inoculated into carnation leaf agar (CLA) medium and incubated at 25°C in an incubator (12 h for one light-dark cycle). In CLA medium, pathogen produced hyaline, sickle-shaped, macroconidia with 3 to 5 septa, and an average size of 30 to 50 × 4 to 5 µm (n = 30) macroconidia but no microconidia in 10 days. Chlamydospores were spherical to subspherical (5.4 to 13.8 µm). Morphological characteristics of the all isolates were consistent with the description of the Fusarium asiaticum (Leslie and Summerell 2006). To validate this identification, RNA polymerase II (RPB2) (Liu et al. 1999), translation elongation factor (EF-1) (Geiser et al. 2004), and ß-tubulin (TUB2) gene region of five isolates were amplified and sequenced (O' Donnell et al. 2015; White et al. 1990). The sequence of one representative isolate (ZL10) sequence was submitted to GenBank (ON215729, ON215730, and ON215731). The NCBI BLAST identified the top hits, 100%, 100%, and 99.87% for RPB2, EF, and TUB gene sequences, respectively, all indicating to Fusarium asiaticum. Pairwise matched of RPB2 and EF genes by MycoBank Fusarium MSIL showed the top hit rate of 100% for F. asiaticum (MH582120 and MH582249). For Koch's postulate and pathogenicity test, spore suspensions (1 × 10^7 conidia/ml) collected from PDA and CLA cultures with 0.05% Tween 80 buffer were used to inoculate with a spray bottle on leaves of a one year old A. macrorrhizos plants. Two leaves of each plant (20 pots in total) were inoculated with the spore suspension (approximately 2000 µl per leaf). An equal number of control leaves were applied with water and 0.05% Tween 80 buffer. Twenty days later, the inoculated plants showed similar symptoms to those of the original diseased plants while the controls remained asymptomatic. Fusarium asiaticum was reisolated from the infected leaves and confirmed using morphological characteristics and DNA sequence analysis. The pathogenicity test was repeated three times with similar results. This first report raises awareness of a new leaf spot disease infecting a commercial A. macrorrhizos in China. It provides an insight for a need of systematic survey identifying current spread, disease origin, and ultimately developing disease management strategies. Funding: Funding was provided by Sichuan Agricultural University Subject Dual Support Program (Grant No. 2121993055). Funding was provided by Deyang Science and Technology Bureau (Sichuan Province) for key R&D projects in agriculture and rural areas (Grant No. 2021NZ048). Funding was provided by the Sichuan Provincial Department of science and technology for the Sichuan Provincial Science and technology project for connecting and Promoting Rural Revitalization (Grant No, 2022ZHXC0007) References： Geiser, D. M., et al. 2004. Eur. J. Plant Pathol. 110:473. https://doi.org/10.1023/B:EJPP.0000032386.75915.a0 Crossref, ISI, Google Scholar Leslie, J. F., and Summerall, B. A., eds. 2006. Page 176 in The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA. https://doi.org/10.1002/9780470278376 Liu, Y. J., et al. 1999. Mol. Biol. Evol. 16:1799. https://doi.org/10.1093/oxfordjournals.molbev.a026092 O'Donnell, K., and Cigelnik, E. 1997. Mol. Phylogenet. Evol. 7:103. https://doi.org/10.1006/mpev.1996.0376 Takano K T, et al. 2012, Plant Bio., 14(4). https://doi.org/10.1111/j.1438-8677.2011.00541.x.

Comparative Transcriptomics and Gene Knockout Reveal Virulence Factors of Neofusicoccum parvum in Walnut.

Neofusicoccum parvum can cause stem and branch blight of walnut (Juglans spp.), resulting in great economic losses and ecological damage. A total of two strains of N. parvum were subjected to RNA-sequencing after being fed on different substrates, sterile water (K1/K2), and walnut (T1/T2), and the function of ABC1 was verified by gene knockout. There were 1,834, 338, and 878 differentially expressed genes (DEGs) between the K1 vs. K2, T1 vs. K1, and T2 vs. K2 comparison groups, respectively. The expression changes in thirty DEGs were verified by fluorescent quantitative PCR. These thirty DEGs showed the same expression patterns under both RNA-seq and PCR. In addition, ΔNpABC1 showed weaker virulence due to gene knockout, and the complementary strain NpABC1c showed the same virulence as the wild-type strain. Compared to the wild-type and complemented strains, the relative growth of ΔNpABC1 was significantly decreased when grown with H2O2, NaCl, Congo red, chloramphenicol, MnSO4, and CuSO4. The disease index of walnuts infected by the mutants was significantly lower than those infected by the wild-type and complementary strains. This result indicates that ABC1 gene is required for the stress response and virulence of N. parvum and may be involved in heavy metal resistance.

First Report of Colletotrichum endophyticum, a Causal Agent of Leaf Spot of Bauhinia blakeana in Southwest China.

The Bauhinia blakeana is originated in South Asia and is widely planted in Chinese cities. It is distributed in Guangdong, Fujian, Hainan, Guangxi, Sichuan, and other places in China (Gu S et al. 2019). It is applied to urban greening as the street trees, garden trees, and scenic forest trees, and is an excellent landscaping tree species in South China. In August 2021, the new leaf spot disease was found in Chengdu (30°42'N, 103°51'E), and the incidence rate was about 70%. The symptoms began to appear from April to May, the worst from June to August. Firstly, the typical symptom is that round, oval, or irregular, brown and slightly concave necrotic spots begin to appear at the edge of the leaves, and the color of the spots changes from light brown to dark brown. Gradually, the number of necrotic spots increases and the spots spread from the edge of the leaf to the middle of the leaf. There is an obvious dark brown boundary between the diseased part and the healthy part, and their yellow-green halos around the spots. Finally, the leaves turn yellow and fall off. On September 1, 2021, infected tissue from samples was cut into small pieces 5 × 5 mm, surface sterilized for 30 seconds in 3% NaClO, 60seconds in 75% ethanol, rinsed three times in sterile water, placed on potato dextrose agar (PDA) amended with streptomycin sulfate (50 µg/mL), and incubated at 25°C in a dark. Finally, 10 typical isolates exhibited the morphology described as Colletotrichum endophyticum (De Silva et al. 2019). After 6 days, the colony diameter reached 63.4 to 67.7mm and had white to pale orange aerial mycelium, but was grey-green at the base. Black conidia formed after 10 days, which were round, oval, elongated spindle-shaped, with sharp ends, measuring 3.25 to 5.85 x 1.95 to 2.60µm (average: 6.18 x 2.28µm). Since the 10 isolated strains were consistent in morphology, a representative strain was selected from the 10 isolated strains to continue the next test. For molecular identification, DNA was extracted from 10 fungal colonies (the 10 fungal colonies used to isolate genomic DNA were derived from single isolates) using a plant genomic DNA extraction kit (Solarbio, Beijing). The 5.8S nuclear ribosomal genes with the two flanking internal transcribed spacer (ITS), the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), partial sequences of the actin (ACT) and beta-tubulin (TUB2) genes were amplified and sequenced using the primer pairs ITS4/ITS5 (White et al. 1990), ACT-512F/ACT-783R (Carbone and Kohn. 1999), GDF1/GDR1 (Guerber et al. 2003) and T1/Btub4R (O'Donnell and Cigelink. 1997; Aveskamp et al. 2009), respectively (Fang Qiu et al. 2021). Sequences were deposited in GenBank (ITS:OK560626; ACT:OK562583; GAPDH:OK562584; TUB2:OK562585). BLAST analysis showed >98% identity with several reference sequences of C. endophyticum previously deposited in GenBank. To confirm pathogenicity and fulfill Koch's postulates, the pathogenic fungal cakes were inoculated on the leaves of 5-year-old B. blakeana, and the sterile medium was used as a control. Three fungal cakes were placed on each leaf and repeated three times. Five days later, the inoculated plants showed the similar symptoms observed in diseased plants; controls remained asymptomatic. The C. endophyticum was re-isolated from the infected leaves and identified by morphological characteristics and DNA sequence analysis. The pathogenicity test was repeated three times with similar results, confirming Koch's postulates. This is the first report of B. blakeana leaf spot caused by C. endophyticum in China.

First report of Coniella koreana causing leaf blight on Loropetalum chinense var. rubrum (Chinese Fringe Flower) in Sichuan, China.

Loropetalum chinense var. rubrum (Chinese Fringe Flower) is widely distributed in the middle and lower reaches of the Yangtze River, as well as northern India. It is a popular landscape plant for its red evergreen foliage and its showy red flowers in the spring. In July 2020, This leaf blight was discovered in Chengdu city (30°42＇41＂N, 103°51＇58＂E). In June 2021, the disease incidence rate at two places in Wenjiang District of Chengdu was 76% and 64%, respectively. The symptoms began to appear from May to June, worsened from July to August, and then disappeared gradually in November. Initially, brown-edged irregular necrotic patches appeared at the leaf margins. Progressively, the patches increased in number, expanded to leaf middle, and turned grayish-white. The scattered black fruiting bodies (conidia) were appeared at patches under humid conditions. Eventually, the leaves tended to dry up and fall off. Infected tissues from five samples and collected were cut into small pieces 2×2 mm, surface sterilized for 30 s in 3% sodium hypochlorite, 60 s in 75% ethanol, rinsed three times in sterile water, placed onto potato dextrose agar (PDA), and incubated at 25℃ in the dark. A total of eight isolates were collected, five isolates exhibited similar culture characteristics while two were Nigrospora sp. and one was a Fusarium sp.. The five similar isolates produced sparse, grayish-withe mycelia with a flat elevation and curled margin. Abundant globose and yellow pycnidia were formed on the PDA surface and arranged in irregular concentric zones. Conidia were 18.20 to 22.36 × 2.64 to 3.05 µm (average 20.36 × 2.82 µm, n=50) in size, fusiform, sickle-shaped, aseptate. DNA was extracted from the representative strain (HMcj B03), and the internal transcribed spacer (ITS) region, the large subunit of the nuclear ribosomal DNA (LSU), translation elongation factor 1-alpha (tef1-α), and the DNA-directed RNA polymerase II second largest subunit (rpb2), were amplified by polymerase chain reaction and sequenced with primers ITS1/ITS4 (White et al. 1990), LR0R/LR7 (Rehner and Samuels 1994; Vilaglys and Hester 1990), 728F/986R (Carbome and Kohn 1999), and 5F2/7cR (Alvarez et al. 2016), respectively. The sequences were deposited in GenBank, viz. OL468959, OL469170, OL489770, and OL855833, respectively. BLAST analysis showed >98.7% identity with several reference sequences of Coniella koreana strain CBS 143.97 and Coniella quercicola strain CBS 904.69, deposited in GenBank. A conidial suspension (1 × 107 conidia/mL) having 0.05% Tween 80 buffer was used for foliar inoculation of 6-year-old Loropetalum chinense var. rubrum plants for pathogenicity test. Ten leaves of each plant (10 pots in total) were inoculated with spore suspensions (20 µL onto the wounded sites). An equal number of control leaves were sprayed with 0.05% Tween 80 buffer to serve as a control. The experiment was repeated three times, and all plants were incubated in a growth chamber (a 12h light and 12h dark period, 25°C, RH > 80%). Twenty days later, all the inoculated leaves showed similar symptoms as the original diseased plants, however, the controls remained asymptomatic. The C. koreana was re-isolated from the infected leaves. To our knowledge, this is the first report of L. chinense var. rubrum caused by C. koreana in China. The discovery of this new disease will provide useful information for developing effective control strategies, and prove beneficial in reducing economic losses in floral product.

Fusarium proliferatum Associated with Basal Rot Disease of Bambusa pervariabilis × Dendrocalamopsis grandis in China.

Bambusa pervariabilis × Dendrocalamopsis grandis is the main cultivated bamboo species used for ecological construction in the Yangtze River basin. This species has the advantages of easy reproduction, wide adaptability and strong resistance and has high economic, ecological and social benefits (Peng et al. 2020). One area of B. pervariabilis × D. grandis with basal rot disease was discovered in Renshou County, Sichuan Province, China (29°41'N, 104°11'E) in June 2020. The disease occurrence area was 68 hm2 in Renshou County, with an incidence rate of 34.8%, and 5% of the B. pervariabilis × D. grandis with basal rot disease died. The pathogen initially invaded from the first section of the base of the bamboo stalk, appearing as black to yellowish brown strips or lumps of disease spots, and rapidly developed horizontally and vertically, which caused the whole plant to wither in severe cases. Diseased tissues were collected from the base of a 4-year-old bamboo stalk with a sterile blade. 100 pieces (5 × 5 × 2 mm) of diseased tissues were sterilized with 3% NaClO for 30 s and in 75% ethanol for 90 s, rinsed three times with sterile distilled water, dried with sterile surface water on sterile filter paper, plated onto potato dextrose agar amended with streptomycin sulfate (Solarbio, 50 µg/ml), and incubated at 25 °C for 7 days with light. A total of five isolates were obtained, of which four isolates were similar in morphology. Using the method of monospore isolation (Leslie and Summerell 2006) and culturing it on PDA, the fungus produced round colonies with a diameter of approximately 8.4 mm and a surface color ranging from white to purple within 7 days at 25 °C. For identification by typical spores, the fungus was cultured on carnation leaf agar (CLA) medium at 25 °C for 7 days. The microconidia by the isolates BD2002, BD2004, BD2008 and BD2010 cultured on CLA medium were elliptical, ovoid, without septum, and measured 4.56 to 15.53 µm long × 1.36 to 6.98 µm wide (n=100). The macroconidia were rod-shaped or slightly curved, tapering apically with three to five septa, and measured 18.86 to 52.99 × 1.56 to 6.42 µm in size (n=100). According to the morphological characteristics of macroconidia and microconidia, the isolates were identified as Fusarium sp. (Leslie and Summerell 2006). For molecular identification, fungal DNA of isolates BD2002, BD2004, BD2008 and BD2010 was extracted by a fungal genomic DNA extraction kit. Polymerase chain reactions (PCRs) were performed with primers ITS1/ITS4 for the internal transcribed spacer (ITS) rDNA region (White et al. 1990), primers Bt2a/Bt2b for the ß-tubulin (TUB) region (Glass and Donaldson 1995), primers EF1F/EF2R for the translation elongation factor 1α (TEF) region (Carbone et al. 1999), primers 5f2/7cr for the RNA polymerase II genes (RPB2) region (O'Donnell et al. 2010), primers H3-1a/H3-1b for the histone H3 (HIS) region (Jacobs et al. 2010), and primers NMS1/NMS2 for the mitochondrial small subunit (mtSSU) rDNA region (Stenglein et al. 2010). Using BLASTn to search GenBank for ITS, TUB, TEF, RPB2, HIS and mtSSU sequences, all isolates showed the highest similarity with Fusarium proliferatum (Matsushima) Nirenberg. The representative isolate BD2010 showed that ITS had 99.61% similarity to F. proliferatum Z23-28 (FJ648201.1); HIS had 99.57% similarity to F. proliferatum M06A_4G_4 (KX681532.1); and the TUB, TEF, RPB2, and mtSSU sequences showed 99.67%, 99.10%, 99.06%, and 99.57% similarity, respectively, to F. proliferatum ITEM2287 (accession numbers LT841243.1, LT841245.1, LT841252.1, and LT841247.1 in GenBank). The GenBank numbers of the representative isolate BD2010 were ITS, OK325614; TUB, OK377026; TEF, OK377027; RPB2, OK377028; HIS, OK377029; and mtSSU, OK338638. To confirm the pathogenicity, thirty 4-year-old healthy bamboo plants were grown in 30 pots. Each five plants were inoculated with one isolate, and a total of twenty-five plants were inoculated with five isolates. A conidia suspension (1 × 106 conidia/ml) of the fungus was inoculated (100 µl each) into plants that had been acupunctured at the base by a sterile syringe. Five control plants were inoculated only with the same amount of sterile distilled water. The inoculation site was wrapped with wet gauze to maintain moisture. All bamboo plants were watered every seven days. The illumination conditions were 12 h light and 12 h dark. All plants were cultured in a greenhouse at 25-28 °C and 70-80% relative humidity. One month later, twenty plants inoculated with conidial suspensions of BD2002, BD2004, BD2008 and BD2010 showed the same symptoms as those observed in the field, whereas plants inoculated with the other fungus and the control treatment remained asymptomatic. The pathogenicity test was conducted three times, and the experimental results were consistent. Furthermore, the fungi were reisolated from the diseased part and were identified as F. proliferatum by morphological and molecular comparison. To our knowledge, this is the first report of basal rot disease caused by F. proliferatum on B. pervariabilis × D. grandis in China. This research is conducive to laying the foundation for the development of effective control strategies for basal rot disease in this species.

Comparative Transcriptomics and Gene Knockout Reveal Virulence Factors of Arthrinium phaeospermum in Bambusa pervariabilis × Dendrocalamopsis grandis.

Arthrinium phaeospermum can cause branch wilting of Bambusa pervariabilis × Dendrocalamopsis grandis, causing great economic losses and ecological damage. A. phaeospermum was sequenced in sterile deionized water (CK), rice tissue (T1) and B. pervariabilis × D. grandis (T2) fluid by RNA-Seq, and the function of Ctf1ß 1 and Ctf1ß 2 was verified by gene knockout. There were 424, 471 and 396 differentially expressed genes between the T2 and CK, T2 and T1, and CK and T1 groups, respectively. Thirty DEGs had verified the change in expression by fluorescent quantitative PCR. Twenty-nine DEGs were the same as the expression level in RNA-Seq. In addition, ΔApCtf1ß 1 and ΔApCtf1ß 2 showed weaker virulence by gene knockout, and the complementary strains Ctf1ß 1 and Ctf1ß 2 showed the same virulence as the wild-type strains. Relative growth inhibition of ΔApCtf1ß 1 and ΔApCtf1ß was significantly decreased by 21.4% and 19.2%, respectively, by adding H2O2 compared to the estimates from the wild-type strain and decreased by 25% and 19.4%, respectively, by adding Congo red. The disease index of B. pervariabilis × D. grandis infected by two mutants was significantly lower than that of wild type. This suggested that Ctf1ß genes are required for the stress response and virulence of A. phaeospermum.

Brown Culm Rot of Dendrocalamus latiflorus Munro Caused by Diaporthe guangxiensis in Sichuan Province, China.

Dendrocalamus latiflorus Munro, the most widely cultivated bamboo species in southern China, has high ornamental value used in gardens, while culms are also used for buildings and as fibers and edibles (Gao et al. 2011). In June 2020, brown culm rot of bamboo was observed in Yibin city, Sichuan Province, in an area of approximately 1000 hectares. Disease incidence was approximately 60%, of which 30% of the plants had died. At the end of June, the lesions expanded but did not surround the base of the culm. From the end of June to the beginning of September, the lesions expanded upward and formed a streak, of which the color gradually deepened to purple-brown and black-brown. At the same time, the disease spots at the base of the culm also expanded horizontally. After the spots surrounded the base of the culm, the diseased bamboo died. Ten culms showing typical symptoms were collected and cut into 5×5 mm pieces at the junction of infected and healthy tissues. The tissues were sterilized for 1 to 2 min in 3% sodium hypochlorite, decontaminated in 75% alcohol for 3 to 5 min, placed on modified potato glucose agar (PDA) with streptomycin sulfate (50 µg/ml), and incubated at 26°C. Two isolates were obtained by the single-spore method (Sivan et al. 1992). The isolates both produced white round colonies similar to Diaporthe guangxiensis and two types of conidia: one was α type (5.5 to 8.2×1.0 to 2.8 µm, n=30), colourless, single-celled, undivided, and oval, containing two oil droplets; and ß type (21.1 to 30.2×0.8 to 1.4 µm, n=30), colourless, single celled and hook shaped. Genomic DNA was extracted from the two isolates by using a fungal genomic DNA extraction kit (Solarbio, Beijing). The products were amplified by polymerase chain reaction (PCR) with primers for the internal transcribed spacer 1 (ITS) region (White et al. 1990), calmodulin (CAL) gene (Carbone and Kohn 1999), translation elongation factor 1-alpha (TEF) gene (Glass and Donaldson 1995) and beta-tubulin (TUB) gene (Soares et al. 2018). The amplified products were sequenced and blasted in GenBank (accession numbers MW380383, MW431318, MW431317 and MW431316 for ITS, CAL, TEF, and TUB, respectively). The ITS, CAL, TEF, and TUB sequences showed 100%, 99.33%, 100%, and 99.80% identity to D. guangxiensis JZB320094 (accession numbers MK335772.1, MK736727.1, MK523566.1, MK500168.1 in GenBank), respectively. To evaluate the pathogenicity of the isolates, five plants were each inoculated with two isolates. The cortex of potted bamboo were injured locally with sterilized needle, and the bamboo culms were inoculated with 100 µl of conidial suspension (105 cfu/ml). The surface of the inoculation wound was covered with gauze soaked with sterilized water. Five plants inoculated with sterile water were used as controls. The treated plants were maintained in a greenhouse at a temperature of 22 to 29°C and relative humidity of 70 to 80%. One month later, of all inoculated plants showed similar symptoms as those observed in the field. D. guangxiensis was re-isolated from all inoculated plants. The pathogenicity test was repeated three times with similar results. This is the first report of D. guangxiensis causing brown culm rot of D. latiflorus in China. These results will facilitate an enhanced understanding of factors affecting bamboo and the design of effective management strategies of the pathogenic species on bamboo and thus to develop corresponding control measures.

Neofusicoccum parvum Causing Stem and Branch Blight Disease of Zanthoxylum bungeanum in Sichuan, China.

The Chinese pepper Zanthoxylum bungeanum Maxim is a special economically important species and a traditional spice in China. It is widely used in medicine, food, timber, tourism, soil and water conservation. In April 2019, A stem and branch blight disease of Z. bungeanum was discovered in Muli, Puge and Yanyuan counties, in Liangshan Prefecture (27°15'20″-27°19'38″N, 101°44'58″-102°04'10″E), causing approximately 15% yield loss in the three counties. Among all fields in Muli County, approximately 41.38%, 10.79% and 2% of Chinese peppers exhibited mild, moderate and severe branch blight, respectively. The symptoms started to occur from March to April. First, red-brown spots on the base of the stem, branches or main trunks of young trees observed but were not obvious. In May, the spots became gray-brown to dark brown ovals and gradually expanded into long strips (Figure 1a, b). When the spots surrounded the branches, the branches above them withered and died, and the spots gradually expanded downward. Around June or July, scattered black dot-shaped fruiting bodies were observed on the lesion. The branches of infected trees were sampled systematically by cutting the branch at the junction of infected and healthy areas in 5×5 mm sections. Each sample was surface-sterilized with 3% NaClO and 75% alcohol for 60 s before being rinsed three times with sterilized distilled water. The sterile filter paper was used to dry the tissue, and the samples were cultured on potato dextrose agar (PDA) amended with streptomycin sulfate (50 µg/ml). Plates were incubated at 25°C in the dark. From the five isolates obtained, four exhibited the morphology described by Yu et al. (2015) for Neofusicoccum parvum. The colonies were white fluffy at first and grew fast (Figure 1c). After five days, the colony diameter reached 75.2-84.8 mm, produced yellow pigment and the mycelium in the middle of the colony began to turn gray (Figure 1d). and the entire colony turned dark gray 7-8 days post culturing as observed previously (Javier-Alva et al. 2009) and formed a black fruiting body at 20 days (Figure 1e). The width of the mycelium measured 2.3-4.8 µm, and with the diaphragm (Figure 1f). The spores were round or fusiform, colorless, transparent, smooth, thin-walled, and measured 6.3-10.6×3.1-5.2 µm (Figure 1g, h), similar to N. parvum (Yu et al. 2013). For molecular identification, DNA was extracted from the mycelia of four fungal isolates using a plant genomic DNA extraction kit (Solarbio, Beijing). Polymerase Chain Reaction (PCR) was performed with the primers ITS1/ITS4 (White et al. 1990), EF446F/EF1035R (Inderbitzin et al. 2005), BTF/BTR2 and HspF3/HspR (Inderbitzin et al. 2010) for the ribosomal internal transcribed spacer region (ITS), elongation factor-1alpha (EF1-alpha), beta-tubulin (TUB) and heat shock protein (HSP) genes, respectively. BLAST searches in the GenBank database indicated that the ITS, TUB, HSP and EF-1α sequences had 100%, 99.0%, 99.7% and 99.7% identity to N. parvum, respectively. Representative sequences were deposited in GenBank (ITS: MT355871; TUB: MT409397; EF-1α: MT409399; HSP: MT460413). A pathogenicity test was performed using N. parvum on ten 2-year-old potted Z. bungeanum plants at 22-28°C and 60% humidity indoors. The conidial suspension (1×107 conidia/ml) collected 25 days old PDA cultures with 0.05% tween buffer was used for inoculation by brushing the wounded area of branch scratched by epidermis with a piece of sandpaper. Ten plants in pots were inoculated with sterile water and served as controls. Thirty days post-inoculation, the plants showed the same symptoms as the original diseased plants, and the controls remained asymptomatic. N. parvum was re-isolated from the infected tissues and identified by morphological characteristics and DNA sequence analysis. The pathogenicity test was repeated three times with similar results, confirming Koch's postulates. This fungus is an important pathogen on a variety of woody hosts, and represents a serious problem in the vineyards worldwide (Mélanie, et al. 2017). To our knowledge, this is the first report of N. parvum causing stem and branch blight of Z. bungeanum trees in China.

First Report of Round Spot Disease of Zanthoxylum armatum DC Caused by Fusarium fujikuroi in China.

Zanthoxylum armatum DC, a deciduous tree in Rutaceae, has significant economic value as an important food condiment, spice, and medicine (Cao et al. 2019). Recently, an unknown round leaf spot disease has been found on Z. armatum in Meishan and surroundings areas of Sichuan province. The disease mainly affected the leaves, mostly on seedlings, with incidence of approximately 50%. Isolate HJYB-4 was isolated from typical diseased leaves and purified on potato dextrose agar (PDA). The isolate produced floccose white, magenta, or grey aerial mycelium. On the reverse side of the culture, the colony had the pigment of pale gray or magenta, with concentric rings of dark red and pale brown in the center. Morphological characteristics were recorded using a pure culture grown on PDA and Synthetic low nutrient agar (SNA). The hyphae of the isolate were colorless and septate. There were two types of conidia on SNA, microconidia, and macroconidia. Macroconidia were long and slender with parallel dorso-ventral sides, usually 3 to 5 septate, 21.89 to 47.21 × 3.33 to 4.73 µm. Microconidia were oval, melon-shaped, ovate, 0-1 septate, mostly no septate, 5.35 to 11.22 × 2.24 to 3.79 µm. A few pyriform microconidia and chlamydospores were observed. DNA of the isolate was extracted using the Column Fungal DNAout 2.0 (Tiandz Inc., Irvine, Beijing, China). PCR was performed using the following primers, ITS1/ITS4, TEF1/TEF2, TUBT1/TUBT2, fRPB2-5f2/fRPB2-7cr, LR0R/LR5, and NL1/NL4 to amplify the loci of the representative isolate in the ribosomal internal transcribed spacers (ITS), translation elongation factor EF-1α (TEF1), ß-tubulin 2 (TUB2), RNA polymerase II largest-subunit (RPB2), the Large subunit (LSU) region of rDNA, 26S rDNA D1/D2 domain. The products were sequenced and Blasted. Blast analysisf the ITS, TFE, TUB2, RPB2, LSU, and NL amplicon revealed more than 99% of sequence identify with Fusarium fujikuroi. These sequences were submitted to GenBank and the GenBank accession numbers were as follows MT864359 (ITS), MT864358 (LSU), MT877222 (NL), MT902141 (RPB2), MT902140 (TEF), and MT902139 (TUB2). The phylogenetic tree was inferred from the combined datasets (TEF, TUB, and PRB2) from members of the F. fujikuroi species complex analyzed in this study (Jayawardena et al. 2019). The Phylogenetic tree revealed isolate HJYB-4 matched F. fujikuroi with a clade credibility value of 100%. According to the morphological characteristics and multi-gene phylogenetic analysis, the isolate was identified as F. fujikuroi. To complete pathogenicity tests, healthy leaves were needle-wound inoculated with mycelial plugs. The leaves that were inoculated with PDA-only plugs served as the control. After 3 days incubation at 26±2°C and 100% relative humidity, brown lesions that developed from inoculated leaves were similar to those in the field. No symptoms developed in the controlled leaves. Typical fungal cultures consistently isolated from symptomatic leaves, indicating that the fungus was responsible for the development of the disease. F. fujikuroi has been reported to cause root rot on Reineckia carnea (Sun et al. 2018), black rot on Macleaya cordata (Yu et al. 2019), and a wilt disease on sugarcane (Bao et al. 2020). This is the first report of F. fujikuroi attacking leaf of Z. armatum in China. The identification of this disease could provide the basis for the prevention and control of the disease at the seedling stage of Z. armatum.

Nigrospora oryzae Causing Black Leaf Spot Disease of Hibiscus mutabilis in China.

Cotton rose (Hibiscus mutabilis Linn.) is a deciduous shrub native to China. It has been widely cultivated in many provinces in China for its ornamental and ecological value (Shang et al., 2020). In May 2017, an unknown leaf spot symptom was first observed on H. mutabilis at the Chengdu Campus of Sichuan Agricultural University (30°42'31″ N, 103°51'28″ E). The disease occurred from May to September with approximately 81% incidence by field sample survey of 300 plants in Chengdu Greenway. The symptoms at first appeared as irregular black spots on the leaves. Then the lesions grew and coalesced into large, black necrotic areas, which later produced leaf chlorosis and abscission (Fig. 1-A). This disease seriously reduced the ornamental value of H. mutabilis. Forty diseased lesions (4 × 5 mm) were surface sterilized with 75% alcohol for 60 s and 3% NaClO for 45 s, rinsed three times in sterile water, placed onto potato dextrose agar (PDA), and then incubated in a dark at 25°C. From the 7 obtained isolates, 4 isolates exhibited the morphology described as Nigrospora oryzae (Hao et al., 2020). The fungus produced initially circular white colonies, and then the centers turned dark gray or black with age on the PDA. Hyphae were smooth, branched, septate, hyaline, or pale brown. Conidia (N = 100 spores) were abundant, and were solitary, dark-brown to black, smooth, aseptate, and measured 11 to 15 µm in diameter (Fig. 1). DNA was extracted from the fungal colonies using a DNeasyTM Plant Mini Kit (Qiagen). The internally transcribed spacer (ITS), ß-tubulin gene (TUB), and translation elongation factor 1-alpha (TEF1) were amplified with primers ITS1/ITS4 (White et al., 1990), BT2A/BT2B (Glass and Donaldson 1995), and EF1-728F/EF1-986R (O'Donnell et al., 1998; Carbone and Kohn 1999), respectively. BLAST results indicated that the ITS, TUB, and TEF1 sequences (GenBank accession Nos. MN515070, MN733956, and MN635723, respectively) had 99% identity with N. oryzae sequences (GenBank accession Nos. KX986031, KY019553, and KY019358). The result was confirmed by multilocus phylogenetic analysis (Fig. 2). The morphological characteristics and molecular analyses of the isolate matched the description of N. oryzae. To confirm pathogenicity, Koch's postulates were fulfilled under controlle conditions. The seedlings of 20 two-year-old potted H. mutabilis plants were inoculated by spraying conidial suspension at the concentration of 1 × 106 conidia/ml on both sides of leaves. Sterilized distilled water (20 seedlings) were used as negative controls. The experiment was performed three times. All plants were incubated at 25°C ± 2°C under a 16 h/8 h photoperiod and 70%-75% relative humidity (RH) after inoculation, and observed daily for disease development. Two weeks later, the inoculated plants showed the same symptoms as the original diseased plants and the controls remained asymptomatic. The pathogen N. oryzae was re-isolated from all ioculated plants, and the culture and fungus characteristics were the same as those of the original isolate. But N. oryzae was not isolated from the control plants. The results indicated that N. oryzae is a causal agent of the disease. N. oryzae was reported as a leaf pathogen on cotton (Zhang et al., 2012), but this is the first report of N. oryzae causing leaf black spot on H. mutabilis in the world. The identification could provide relevant information for adopting appropriate management strategies to control the disease.

Stem Rot Disease of Juglans sigillata Dode Caused by Fusarium fujikuroi in China.

Juglans sigillata Dode is an endemic species in the southwest China, and is an important nut and woody oil tree. The shell of its fruit is hard and can be used to make various crafts. From 216 to 2019, typical stem rot symptoms of 8-year-old J. sigillata were observed in cultivated fields in a 600-ha orchard in Zigong, Sichuan province, China. At this orchard, approximately 35% of the trees have been seriously damaged over the past few years. The typical symptoms were water-soaking on the stem, rotting, wilting, and blighting, eventually leading to the death of the plant. In June, ten diseased tissues were collected and surface-sterilized by 3% NaClO and 75% alcohol. Morphological observations were made from the isolates grown on Potato dextrose agar (PDA) and incubated at 25°C for 3 to 9 days. Morphological characteristics were made on pure cultures grown on Synthetic low nutrient agar (SNA). Five isolates with similar morphology were isolated from single spores. Colonies on PDA reached 8.3 cm in diameter after 6 days at 25 °C, aerial mycelia were white to cream and wol-like, later turning violet and dark purple with age. The hyphae of the strain were colorless and septate. There were two types of conidia on SNA, microconidia and macroconidia. Microconidia (n = 50) were oval, elliptic or clavate, no septate, 2.2 to 3.8 × 7.6 to11.7 µm. Conidiophores were branched or unbranched, solitary or in groups, phialides cylindrical to flask-shaped, monophialidic and polyphialidic. Macroconidia (n = 50) were long slender with a curved apical cell and foot-like basal cell, 3 to 4 septate and 2.1 to 3.9 × 26.2 to 53.4 µm. For molecular identification, the internal transcribed spacer (ITS), ß-tubulin (TUB2), translation elongation factor (TEF1) and large subunit (LSU) were amplified with the corresponding primer pairs ITS1/ITS4 (White et al. 1990), BT2A/BT2B, EF1/EF2 (O'Donnell et al. 1997), and LROR/LR5 (Rehner and Samuels 1994), respectively. BLAST search results indicated that the ITS, TUB2, TEF1, LSU sequences (GenBank acc. nos. MT791384, MT786729, MN853324, and MT705246) showed 99 to100% identity with Fusarium fujikuroi sequences at NCBI (GenBank acc. nos. MG798789, MH398245, MK604519 and KJ954504). The results were confirmed by multilocus phylogenetic analysis. Based on the morphological characteristics and molecular analysis of the isolates, the fungus was identified as F. fujikuroi (Leslie and Summerell 2006). Koch's postulates were checked under controlled conditions. Fifteen 2-year-old healthy potted J. sigillata were inoculated by pricking the epidermis of stem with a needle and applying 150 µl of a microconidial suspension (1 × 106 spores/ml) to the wounded surface with a brush. Sterilizd distilled water was used as the control. The experiment was repeated three times. All the plants were incubated at 25 ± 2°C after inoculation for daily observation of disease development. After 12 days, the inoculated plants showed the same symptoms as observed in the original diseased plants, while the control plants were asymptomatic. The fungus was re-isolated from the symptomatic stems and was completely identical to the isolates used to inoculate the plants. Thus, we confirmed that F. fujikuroi caused the stem rot of J. sigillata. To our knowledge, this is the first report of this fungus causing stem rot in J. sigillata in China. Our results can help identify stem rot disease of J. sigillata and develop control measures for the disease.

Brown leaf spot of Cycas debaoensis Caused by Colletotrichum siamense in Sichuan, China.

Cycas debaoensis Y. C. Zhong et C. J. Chen is an endemic species in China that is listed among China's national key preserved wild plants (Class I) (Xie et al. 2005). It is mainly distributed in south China (Guangxi, Guizhou, and other regions). In April 2017, a new leaf disease of C. debaoensis was found in Chengdu (30°35'32″ N; 104°05'11″E) in China with an incidence over 40%. Symptoms on C. debaoensis initially appeared as brown necrotic lesions on the margin or in the center of leaves. The lesions then enlarged gradually and developed into brown spots, necrotic lesions with dark brown margins. Many small and black dots were observed on necrotic lesions. Eventually, the diseased leaves withered and died. Ten samples were collected and surface-sterilized by 3% NaClO and 75% ehanol respectively for 60s and 90s, rinsed with autoclaved distilled water and then blot-dried with autoclaved paper towels. Five isolates from diseased leaves with similar morphology were isolated from single spores. Morphological characteristics were recorded from pure cultures grown on potato dextrose agar (PDA) incubated at 25°C for 3-9 days. Initially, the colonies grown on PDA were white, then, became pale gray with concentric zones and greenish black beneath. Conidia were single-celled, smooth-walled, straight, colorless, cylindrical with both ends bluntly rounded,13.0-16.5 × 4.7-5.8 µm in size (n = 100 spores). For molecular identification, the genomic DNA of the isolates was extracted using a DNeasyTM Plant Mini Kit (Qiagen). The internal transcribed spacer (ITS) (ITS1/ITS4 White et al., 1990), ß-tubulin (TUB2) (BT2A/BT2B (O'Donnell et al., 1997)), actin (ACT) (ACT512F/ACT (Carbone & Kohn, 1999)), calmodulin (CAL) (CL1C/CL2C (Weir et al., 2012)), mating type protein and chitin synthase (CHS-1) (CHS-1) (CHS-9 79F/CHS-345R (Carbone & Kohn, 1999)) were amplified. BLAST results indicated that the ITS, TUB2, ACT, CAL, CHS-1 sequences (GenBank MN305712, MN605072, MT478663, MT465591 and MT478664) showed 99-100% identity with C. siamense sequences at NCBI (GenBank JF710564, MK341542, MK855094, MH351155 and MK471373). The Phylogenetic tree inferred from the combined dataesets (TEF, TUB and ACT) show that the isolate belongs to C. siamense clade with a credibility value of 99%. Two-year-old potted plants of C. debaoensis (10 plants) were used for pathogenicity test. On each plant, 5 leaves were sprayed with a conidial suspension (1 × 106 conidia/ml) on both sides of the leaves. Autoclaved distilled water was used as negative control (10 plants). Plants were kept in the greenhouse at 25 °C under 16h/8h photoperiod and 70-75% relative humidity (RH). The symptoms observed on the inoculated plants were similar to those observed in the field, while the controls remained asymptomatic. C. siamense was re-isolated from all diseased inoculated plants, and the culture and fungus characteristics were the same as the original isolate. The morphological characteristics and molecular analyses of the isolate matched the description of C. siamense (Prihastuti et al., 2009). C. siamense was previously reported infecting Citrus reticulata (Cheng et al. 2013), but this is the first report of brown leaf spot on C. debaoensis caused by C. siamense in China. This finding provides important basis for further research on the control of the disease.

First Report of Fusarium fujikuroi Causing Black Stem Rot of Zanthoxylum bungeanum in China.

Chinese prickly ash (Zanthoxylum bungeanum Maxim.), native to China, is an important tree species for soil and water conservation, barren mountain afforestation, and garden greening. Its fruit is commonly used for seasoning and medicine. In August 2016, black stem rot of Z. bungeanum was first observed in Hanyuan County, Ya'an City. In June 2019, the symptoms were observed on > 60% of 10,000 plants in Hanyuan County. At its early stage, the bark was wet and rotten, slightly concave, and accompanied by gummosis. The lesions were dark brown and long oval, peeling off the rotten bark covered with white hyphae. At the later stage, the lesions shrunk and cracked, with many orange-red particles (conidia) and dense black particles (ascospores). Larger lesions often caused large-scale bark necrosis. After the lesions girdled the trunk, the plants rapidly died. A total of 36 isolates were isolated from 320 infested tissue fragments (5 × 5 mm) that were surface sterilized for 60 s in 3% sodium hypochlorite, and 60 s in 75% ethanol, rinsed three times in sterilized water, placed onto potato dextrose agar (PDA) amended with streptomycin sulfate (50 µg/ml), and incubated in the dark at 25°C. Among them, 28 exhibited morphological characteristics described as Fusarium fujikuroi Nirenberg. On PDA, the fungus produced white to grey orange, circular colonies. On carnation leaf agar (CLA), the microconidia were club shaped with a flattened base and 0 to 1 septum, 5 to 15 × 2 to 4 µm in size, whereas macroconidia were relatively slender, medium length with no significant curvature, and had a tapered apical cell and a poorly developed, notched basal cell, 3 to 5 septa, 20 to 50 × 3 to 5 µm in size. For molecular identification, DNA was extracted from a representative isolate using a fungus genomic DNA extraction kit (Solarbio, Beijing). PCRs were performed with primers EF1f/EF2r for translation elongation factor 1α (EF-1α) region, and primers 5f2/7cr for RNA polymerase II genes (RPB2) region. PCRs were amplified and their products (GenBank accession nos. MT448248 and MT448247) were sequenced and blasted, showing 99 to 100% sequence homology with known F. fujikuroi isolates (GenBank accession nos. MN102101.1 and MN193888.1). To conduct pathogenicity test, twigs of 42 three-year-old potted Z. bungeanum plants were superficially wounded with a needle on the cortex, and each twig was inoculated by dropping a 100 µl conidial suspension (1×106 conidia/ml) of the fungus onto its wound surface. The inoculated areas were bandaged with gauze moistened with sterilized water. The wounded wigs that were inoculated with sterilized water served as the controls. Treated plants were maintained in a greenhouse with temperature ranging from 22 to 29°C and RH from 70% to 80%. One month later, 100% of inoculated plants showed the symptoms similar to those observed in the field, with the size of lesions ranging from 0.5 to 1.0 cm2. The fungus was re-isolated from the branch cortexes and confirmed morphologically and molecularly. To our knowledge, this is the first report of F. fujikuroi as a causal agent of black stem rot disease on Z. bungeanum in China. These results will help correctly identify this disease and develop proper strategies to manage the disease.

First report of Neovaginatispora fuckelii causing leaf spot on Phoenix canariensis.

Phoenix canariensis Chabaud is a vital ornamental and widely planted in the urban landscape of China (Lan et al. 2019). In December 2019, six of seven P. canariensis plants exhibited typical symptoms of leaf spot in Chengdu campus of Sichuan Agricultural University in Sichuan, China, with roughly 80% leaves per plant affected. Symptomatic leaves initially showed dark brown spots with a yellow halo at the periphery. As the disease progressed, spots gradually expanded and eventually formed necrotic spots, with the whole leaf dying when severely infected. 10 diseased leaves from different plants were collected in December 2019 and deposited in Sichuan Agricultural University Herbarium. A single spore isolation was performed following Chomnunti et al. (2014) and transferred to potato dextrose agar (PDA). Five fungal isolates were obtained from five different infected leaves respectively and deposited in Sichuan Agricultural University Culture Collection. The fungal colonies were incipiently greyish-white becoming pale brown at the margins and dark brown in the center. The reverse was the same as the obverse with radial cracking at the center. Morphological characteristics on the host were examined by light microscopy. Ascomata were semi-immersed to immersed, black, pyriform to subglobose, and measured 180-310 × 140-260 µm. The peridium comprised several layers of hyaline cells of textura angularis and measured 9-27 µm wide. Pseudoparaphyses were numerous, septate, hyaline, usually longer than asci. Asci were 8-spored, bitunicate, cylindric-clavate, and measured 50-95 × 8-11.5 µm (n=35). Ascospores were 1-septate, straight or slightly curved, hyaline, usually with globose appendages at both ends, and measured 14-18.5 × 4-6.5 µm (n=50). Conidiogenesis was not observed on PDA incubated four months at 25℃ and 95 % relative humidity on a 12-h fluorescent light/dark incubation. Morphology was consistent with the sexual stage of Neovaginatispora fuckelii (Sacc.) A. Hashim. et al. described by Hashimoto et al. 2018, Tennakoon et al. 2018, and Hyde et al. 2020. The rDNA internal transcribed spacer region (ITS), 28S large subunit (LSU), 18S small subunit (SSU), translation elongation factor 1-alpha (TEF 1-α), and RNA polymerase II subunit 2 (RPB2) gene regions were amplified by using the primers ITS5/ITS4, LR0R/LR5, NS1/NS4, EF1-983F/EF1-2218R, and fRPB2-5F/fRPB2-7cR, respectively. The DNA amplification products of the representative isolate SICAUCC 20-0008 isolated from SICAU 20-0008 were sequenced and accessioned in GenBank, viz MT427731, MT427734, MT427738, MT441923, and MT441920, respectively. The sequences were compared with the GenBank database using nucleotide BLAST, and these five sequences were great identical with the sequences of the fungus N. fuckelii (GenBank accession no. ITS, LC001732, 522/524, 99.62%; LSU, AB619009, 839/840, 99.88%; SSU, AB618690, 976/976, 100%; TEF 1-α, LC001750, 895/912, 98.14%; RPB2, MN482130, 935/935, 100%). Using phylogenetic analysis in which reference sequences collected from GenBank were included, the isolate SICAUCC 20-0008 clustered within N. fuckelii with high bootstrap support (Fig. 1). To conduct Koch's postulates, fifteen healthy P. canariensis 2 to 3-year-old plants were inoculated by placing a mycelium plug from the growing margin of 15-day-old colonies upside down directly onto fresh wounds punctured with a fine needle. Five healthy plants were inoculated with a sterile agar plug as the control. Plants were incubated in a growth chamber at 25 ± 1℃ and 95% relative humidity on a 12-h fluorescent light/dark regimen. After ten days, the leaves of inoculated plants turned brown and gradually expanded and became necrotic spots, similar to symptoms observed in the field. The control plants were symptomless. The pathogen was re-isolated from leaf lesions and identified by morphological characteristics, whereas no fungus was recovered from control treatments. N. fuckelii has been often reported as a saprobe on numerous hosts including species of Acer, Carpinus, Carya, Epilobium, Prunus, Quercus, Rhus, Salix, and Vitis, as well as other genera (Thambugala et al. 2015, Hashimoto et al. 2018, Hyde et al. 2020). To our knowledge, this is the first report of N. fuckelii causing leaf spot on P. canariensis. Some proteaceous plants and other palms are known to be infected by this fungus (Hyde et al. 2000, Taylor et al. 2000), which may result in a great threat to ornamental horticulture. Fungicides treatments should be considered to prevent the damage to plants and spread of this fungus.

Whole-genome sequence of Arthrinium phaeospermum, a globally distributed pathogenic fungus.

Arthrinium phaeospermum (Corda) M.B. Ellis is a globally distributed pathogenic fungus with a wide host range; its hosts include not only plants, but also humans and animals. This study aimed to develop genomic resources for A. phaeospermum to provide solid data and a theoretical basis for further studies of its pathogenesis, transcriptomics, proteomics, metabolomics and RNA genomics. The genome was obtained from the mycelia of the strain AP-Z13 using a combination of analyses with the high-throughput Illumina HiSeq 4000 system and PacBio RSII LongRead sequencing platform. Functional annotation was performed by BLASTing protein sequences against those in different publicly available databases to obtain their corresponding annotations. The genome is 48.45 Mb in size, with an N90 scaffold size of 1,931,147 bp, and encodes 19,836 putative predicted genes. This is the first report of the genome-scale assembly and annotation for A. phaeospermum, the first species in the genus Arthrinium to be subjected to whole genome sequencing.

Metabolomics responses of Bambusa pervariabilis × Dendrocalamopsis grandis varieties to Biotic (pathogenic fungus) stress.

Bambusa pervariabilis × Dendrocalamopsis grandis blight, caused by Arthrinium phaeospermum, is one of the most common and serious diseases in bamboo and occurs in the newly born twigs. Bamboo has suffered large dead areas, including more than 3000 hm2, which greatly threatens the process of returning farmlands to forests and the construction of ecological barriers. To identify differential metabolites and metabolic pathways associated with B. pervariabilis × D. grandis to A. phaeospermum, ultra-performance liquid chromatography (UPLC) and quadrupole-time of flight (Q-TOF) Mass Spectrometry (MS) combined with a data-dependent acquisition method was used to analyse the entire sample spectrum. In total, 13223 positive ion peaks and 10616 negative ion peaks were extracted. OPLS-DA and several other analyses were performed using the original data. The OPLS-DA models showed good quality and had strong predictive power, indicating clear trends in the analyses of the treatment and control groups. Clustering and KEGG pathway analyses were used to screen the differential metabolites in the treatment and control groups from the three B. pervariabilis × D. grandis varieties and reflected their metabolic responses induced by A. phaeospermum infection. The results showed that the three B. pervariabilis × D. grandis varieties mode showed significant changes in the following six resistance-related metabolites after A. phaeospermum invasion in positive and negative ion modes: proline, glutamine, dictamnine, apigenin 7-O-neohesperidoside, glutamate, and cis-Aconitate. The following four main metabolic pathways are involved: Arginine and proline metabolism, Glyoxylate and dicarboxylate metabolism, Biosynthesis of alkaloids derived from shikimate pathway, and Flavone and flavonol biosynthesis. This study lays a foundation for the later detection of differential metabolites and metabolic pathways for targeting, and provides a theoretical basis for disease-resistant breeding and the control of B. pervariabilis × D. grandis blight.