Drought stress-mediated differences in phyllosphere microbiome and associated pathogen resistance between male and female poplars.

Phyllosphere-associated microbes play a crucial role in plant-pathogen interactions while their composition and diversity are strongly influenced by drought stress. As dioecious plant species exhibited secondary dimorphism between the two sexes in response to drought stress, whether such difference will lead to sex-specific differences in phyllosphere microbiome and associated pathogen resistance between male and female conspecifics is still unknown. In this study, we subjected female and male full siblings of a dioecious poplar species to a short period of drought treatment followed by artificial infection of a leaf pathogenic fungus. Our results showed that male plants grew better than females with or without drought stress. Female control plants had more leaf lesion area than males after pathogen infection, whereas drought stress reversed such a difference. Further correlation and in vitro toxicity tests suggested that drought-mediated sexual differences in pathogen resistance between the two plant sexes could be attributed to the shifts in structure and function of phyllosphere-associated microbiome rather than the amount of leaf main defensive chemicals contained in plant leaves. Supportively, the microbiome analysis through high-throughput sequencing indicated that female phyllosphere enriched a higher abundance of ecologically beneficial microbes that serve as biological plant protectants, while males harbored abundant phytopathogens under drought-stressed conditions. The results could provide potential implications for the selection of suitable poplar sex to plants in drought or semi-drought habitats.

First Report of Leaf Blight of Ginkgo biloba L. Caused by Neopestalotiopsis clavispora G.F. Atk. in Sichuan, China.

Ginkgo (Ginkgo biloba L.), the oldest existing tree species in the world, is an important ornamental and medicinal plant, widely planted in China. In October 2022, a new leaf blight disease was observed in Chengdu city (30°05'to 31°26'N, 102°54'to 104°53'E). Disease incidence averaged 82.5% across five foci. The typical symptomatology begins when leaf margins turn yellow and small black spots appear at the edge of the leaf, chlorotic areas turn brown, dry and deformed. Gradually, the necrotic lesions spreads to the middle of the leaf and eventually the whole leaf falls off. Infected tissues from ten leaves were cut into small pieces (5 × 5 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) amended with streptomycin sulfate (50 µg/mL); and incubated at 25°C for 3 to 8 days. A hyphae was removed from the edge of the fungal colony and placed onto potato dextrose agar (PDA) plates. After incubation at 25℃ with a 12-hour light/dark cycle for 8 days, the colony diameter reached 77.5 to 81.5 mm. Colonies grown on PDA were white, cotton, flocculent, undulating on the surface, dense in aerial hyphae and light yellow on the back. Black pycnidia formed superficially, scattered over the PDA, following two weeks of incubation. Pycnidia contained sticky black conidia. The spores were were spindle shaped, with five cells, and four septations measuring 20.9 to 34.8 µm × 6.8 to 8.8 µm (avg. 28.4 × 7.6 µm; n=40). The three median cells were versicolored, typically two dark brown cells and one light brown cell, whereas the basal and apical cells were hyaline. Conidia had a single basal appendage (2.87 to 4.1 µm long; n = 40) and two to three apical appendages (18.3 to 29.1 µm long; n = 40). Based on colony and conidial morphology, the isolate was identified as N. clavispora (Maharachchikumbura et al. 2014). The partial sequence of the internal transcribed spacer (ITS), ß-tubulin gene (TUB2), and translation elongation factor subunit 1-a gene (TEF1) were amplified and sequenced using the universal primer pairs ITS1/ITS4(Zhang et al. 2022), BT2A/BT2B (Li Yuan et al. 2022), and EF1-526F/EF1-1567R (Maharachchikumbura et al. 2012), respectively. Sequences of representative isolate LQYX were deposited in GenBank (ITS: OQ152504, TUB: OQ168328, and TEF1: OQ168329). BLAST results indicated that the ITS, TUB, and TEF1-α sequences showed 99 to 100% identity with N. clavispora sequences at NCBI (GenBank MG729689, MG740735, and MG740758). Identification was confirmed by Bayesian inference using Mr. Bayes. Next, inoculations were conducted on leaves of ten G. biloba in the field to verify the pathogenicity of LQYX. Ten healthy leaves of each plant were surface sterilized with 75% ethanol, and the wound was rubbed out on the leaf edge on the sterilized sanding paper. A conidia suspension (1 × 107 ml-1) was sprayed on the leaves, aseptic water was used as the control, and the transparent plastic bag was used to maintain relative humidity. After 14 days (26 ℃, 14 hours light / 10 hours dark), the inoculated leaves had similar symptoms as the original diseased plants, whereas controls were asymptomatic. The N. clavispora 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. To our knowledge, this is the first report of leaf blight of G. biloba caused by N. clavispora in China, which has greatly affected the appearance of the city and should be further studied. This report can help identify this disease and further develop effective control measures.

Soil cadmium stress affects the phyllosphere microbiome and associated pathogen resistance differently in male and female poplars.

Microorganisms associated with the phyllosphere play a crucial role in protecting plants from diseases, and their composition and diversity are strongly influenced by heavy metal contaminants. Dioecious plants are known to exhibit sexual dimorphism in metal accumulation and tolerance between male and female individuals. Hence, in this study we used male and female full-siblings of Populus deltoides to investigate whether the two sexes present differences in their phyllosphere microbiome structures and in their associated resistance to the leaf pathogenic fungus Pestalotiopsis microspora after exposure to excess soil cadmium (Cd). We found that Cd-treated male plants grew better and accumulated more leaf Cd than females. Cd stress reduced the lesion areas on leaves of both sexes after pathogen infection, but male plants exhibited better resistance than females. More importantly, Cd exposure differentially altered the structure and function of the phyllosphere microbiomes between the male and female plants, with more abundant ecologically beneficial microbes and decreased pathogenic fungal taxa harbored by male plants. In vitro toxicity tests suggested that the sexual difference in pathogen resistance could be attribute to both direct Cd toxicity and indirect shifts in the phyllosphere microbiome. This study provides new information relevant for understanding the underlying mechanisms of the effects of heavy metals involved in plant-pathogen interactions.

Procyanidin B2 alleviates uterine toxicity induced by cadmium exposure in rats: The effect of oxidative stress, inflammation, and gut microbiota.

Environmental exposure to hazardous materials causes enormous socioeconomic problems due to its deleterious impacts on human beings, agriculture and animal husbandry. As an important hazardous material, cadmium can promote uterine oxidative stress and inflammation, leading to reproductive toxicity. Antioxidants have been reported to attenuate the reproductive toxicity associated with cadmium exposure. In this study, we investigated the potential protective effect of procyanidin oligosaccharide B2 (PC-B2) and gut microbiota on uterine toxicity induced by cadmium exposure in rats. The results showed that the expression levels of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) were reduced in utero. Proinflammatory cytokines (including tumor necrosis factor-α, interleukin-1ß and interleukin-6), the NLRP3 inflammasome, Caspase-1 and pro-IL-1ß were all involved in inflammatory-mediated uterine injury. PC-B2 prevented CdCl2-induced oxidative stress and inflammation in uterine tissue by increasing antioxidant enzymes and reducing proinflammatory cytokines. Additionally, PC-B2 significantly reduced cadmium deposition in the uterus, possibly through its significant increase in MT1, MT2, and MT3 mRNA expression. Interestingly, PC-B2 protected the uterus from CdCl2 damage by increasing the abundance of intestinal microbiota, promoting beneficial microbiota, and inhibiting harmful microbiota. This study provides novel mechanistic insights into the toxicity of environmental cadmium exposure and indicates that PC-B2 could be used in the prevention of cadmium exposure-induced uterine toxicity.

Brown leaf spot of Cunninghamia lanceolata Caused by Colletotrichum kahawae in Sichuan province, China.

Chinese fir (Cunninghamia lanceolata) is an important timber species that has been widely cultivated in southern China. It is extensively applied in medicine, environmental monitoring, furniture, urban (e.g., street trees) and rural landscaping, windbreak forest, soil and water conservation. In January 2022, distinct leaf spot symptoms were observed in Chinese fir in Hongya Forestry (29°45'N, 103°11'E) Meishan City, Sichuan Province, China. Field surveys showed that the disease was widespread, with around 70% disease incidence. The typical symptoms initially appeared as yellowish-brown necrotic lesions on the margin of the leaves. Subsequently, lesions gradually expanded and developed into larger necrotic areas with red-brown irregular shape. The lesions later expanded throughout the leaf. Infected leaves turned dark brown and wilted, leading to seeding's death. Diseased leaves with typical symptoms were collected for pathogen isolation and identification. Infected tissues from ten samples were cut into small pieces of 2 × 2 mm. Infected tissues were surface disinfected with 3% sodium hypochlorite and 75% ethanol for 30s and 60s, respectively, and rinsed with sterile water 3 times. They were blotted dry with autoclaved paper towels and incubated on potato dextrose agar (PDA) with streptomycin sulfate (50 µg/mL) for 5 ~ 8 days at 25°C. and 12 h light/dark period. The diameter of the colonies reached 65.7 to 75.9 mm, with a gray to black center, and white edges while the reverse sides were gray to orange. Conidia were single-celled, colorless, straight, cylindrical, bluntly rounded at both ends, Conidia dimensions varied from, 7.3 µm to 15.7 µm in length and 3.3 µm to 6.1 µm in width (n = 100). For molecular identification, the genomic DNA of isolate SM2290708, SM229070801 and SM229070802 were extracted using a fungal genomic DNA extraction kit (Beijing Solarbio Science & Technology Co., Ltd., City, China). The internal transcribed spacers of the ribosomal RNA (ITS) [ITS1/ITS4 (White et al., 1990), calmodulin (CAL) (Weir et al., 2012), ß-tubulin (TUB2) (O'Donnell et al., 1997), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Templeton et al. 1992) were amplified. Sequences were deposited in GenBank (ITS: ON564877, OQ535027 and OQ535028; CAL: ON583827, OQ538101 and OQ538102; TUB2: ON583830, OQ538104 and OQ538105; and GAPDH: ON583831, OQ538108 and OQ538109). BLAST results showed that our ITS, CAL, TUB2 and GAPDH sequences were >99% identical to the corresponding sequences of Colletotrichum kahawae deposited at NCBI (GenBank JX010231, JX009642, JX010444, and JX010012). Identification was confirmed by Bayesian inference using MrBayes (Fig 2). The conidial suspension (1 × 106 conidia/ml) was used for inoculation by spraying leaves of ten 3-year-old Chinese fir plants for pathogenicity test. Fifteen leaves of each plant were inoculated. An equal number of control leaves was sprayed with sterilized distilled water as a control. Finally, all potted plants were placed in a greenhouse at 28°C under a 16 h/8 h photoperiod and in 73% to 79% relative humidity. After fifteen days, the symptoms observed on the inoculated plants were similar to those of the original diseased plants, but the controls remained asymptomatic. Colletotrichum kahawae was re-isolated from the infected leaves and identified by both morphological characteristics and DNA sequence analysis. The pathogenicity test was repeated three times, which showed similar results, confirming Koch's postulates. To our knowledge, this is the first report of brown leaf spot on C. lanceolata caused by C. kahawae in China. The results of this study provide basic information for diagnosis of the pathogen and developing prevention strategies to manage C. lanceolata leaf spot disease.

Leaf spot of Alnus cremastogyne Caused by Colletotrichum gloeosporioides in Sichuan, China.

Alnus cremastogyne Burk, a broad-leaved tree endemic to south-western China, has both ecological and economic value. The tree is widely used in furniture, timber, windbreaks and sand fixation, and soil and water conservation (Tariq et al. 2018). In December 2020, a new leaf spot disease was discovered on A. cremastogyne in two plant nurseries in Bazhong City (31°15' to 32°45N, 106°21' to 107°45'E), with 77.53% disease incidence. Among the infected trees, 69.54% of the leaves were covered with symptoms of the disease. The typical symptoms initially appeared as irregular brown necrotic lesions, while some lesions were surrounded by a light yellow halo. As the disease progressed, the number of necrotic lesions increased, and lesions gradually expanded and coalesced (Fig. 1). Finally, the disease caused the leaves of A. cremastogyne to wither, curl, die, and fall off. Ten symptomatic leaves were collected from 5 different trees in the two plant nurseries. The leaves with symptoms of leaf spot disease were collected and cut from the junction between the diseased and the healthy tissues. The infected tissues from 10 samples were cut into small 2.5 × 2.5 mm pieces. Infected tissues was sterilized in 3% NaClO solution for 60 s followed by 75% ethanol for 90 s, rinsed three times in sterile water, blot-dried with autoclaved paper towels, and then cultured on potato dextrose agar (PDA) at 25℃ for 4 to 8 days in 12 h/12 h light/dark conditions. After 8 days, the colony diameter reached 71.2 to 79.8 mm. The colonies were initially light pink, and then turned white with pale orange beneath. The conidia were single-celled, aseptate, colorless, cylindrical, straight, bluntly rounded at both ends, and measured 11.6 to 15.9 × 4.3 to 6.1 µm (n = 100). These morphological characteristics were consistent with the description of Colletotrichum gloeosporioides (Pan et al. 2021). For molecular identification, the genomic DNA of a representative isolate, QM202012, was extracted using a fungal genomic DNA extraction kit (Solarbio, Beijing). The internal transcribed spacer (ITS), actin (ACT), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified with primers ITS1/ITS4 (White et al. 1990), ACT-512F/ACT-783R (Carbone & Kohn, 1999) and GDF/GDR (Templeton et al. 1992), respectively. Sequences were deposited in GenBank (ITS: OL744612, ACT: OL763390, and GAPDH: OL799166). BLAST results indicated that the ITS, ACT, and GAPDH sequences showed >99% identity with C. gloeosporioides sequences in NCBI (GenBank NR160754, MG561657, and KP145407). Identification was confirmed by Bayesian inference using Mr Bayer (Fig 2) A conidial suspension (1 × 106 conidia/ml) was used to test pathogenicity on the leaves of 4-year-old A. cremastogyne plants (10 plants). Fifteen leaves of each plant (10 pots in total) were inoculated with the spore suspension on the leaves. The same number of control leaves was sprayed with sterilized distilled water as a control. Finally, all potted plants were placed in a greenhouse at 25°C under 16 h/8 h photoperiod and 67 to 78% relative humidity. The symptoms observed on the inoculated plants were similar to those of the original diseased plants, with 100% of the inoculated plants being infested with brown leaf spots, but the controls remained symptom-free. C. gloeosporioides was re-isolated from the infected leaves and identified by both morphological characteristics and DNA sequence analysis. The pathogenicity test was repeated three times, showing similar results each time, confirming Koch's postulates. To our knowledge, this is the first report of leaf spot on A. cremastogyne caused by C. gloeosporioides in China. This finding indicates that C. gloeosporioides may become a serious threat to A. cremastogyne production in Bazhong City and helps to further examine and prevent leaf spot disease in A. cremastogyne growing areas in Bazhong City.

Root rot Caused by Setophoma terrestris on Illicium verum in Yunnan Province, China.

Star anise (Illicium verum Hook. f.), a genus of star anise in the family Magnoliaceae, is an important cash crop of "medicinal and food" origin, mainly from China. In August 2021, root rot of I. verum was first observed on more than 80% of the plants grown within a 500 hectares area in Wenshan city, Yunnan Province. At the early stage of the disease, the phloem of the root was dark yellow-brown, and the leaves turn yellow. With further disease development, the whole root became black (Fig. 1a, 1b), and the leaves gradually fall off, affecting the growth, yield and eventually caused death of the whole plant. A total of 20 root samples were collected from typical symptomatic plant roots with 20 years old in Wenshan City (23°18'12″N, 103°56'98″E) and were cut into 2 × 2 mm pieces at the junction of infected and healthy tissue. Each sample was surface-sterilized with 3% NaClO and 75% alcohol for 60 s before rinsing three times with distilled water. The sterile filter paper (5×5 cm) was used to dry the tissue, and samples were cultured on potato dextrose agar (PDA) amended with streptomycin sulfate (50 µg/ml). Plates were incubated at 25°C in the dark in the incubator. From 9 isolates obtained in culture, 7 exhibited the morphology described by Boerema et al. (Boerema et al. 2004) for Setophoma sp. The hyphae were hyaline and septate (Fig.1c). After 14 days of culture on V8 juice agar, white round colonies are formed, but there is no groove in the middle of the colonies (Fig.1d), and transparent, oval, or cylindrical conidia were produced, 6.0-8.0 x 2.5 to 4.0 um (Fig.1e). DNA was extracted from a representative isolate BJGF-04 for molecular identification using a fungal genomic DNA extraction kit (Solarbio, Beijing, China). Polymerase chain reactions (PCRs) were performed with primers ITS1/ITS4 for the internal transcribed spacer (ITS) region (White et al. 1990) and primers T1/ß-Sandy-R for the ß-tubulin gene (TUB) region (Yang et al. 2017) and primers NL3/ LR5 for 28S large subunit rDNA (LSU) region (Hu et al. 2021) and NS1/ NS4 for 5.8S large subunit rDNA (SSU) region (Mahesha et al. 2021). Newly generated representative sequences were deposited in GenBank: ITS sequence (ON645256), TUB sequence (ON854484), and LSU sequence (ON644445), SSU sequence (ON644451). were sequenced and blasted, showing 99 to 100% sequence homology with known S. terrestris. Pathogenicity was performed using one-year asymptomatic plants of I. verum. A conidial suspension (1 x 106 conidia/ml) collected from V8 juice cultures with 0.05% Tween buffer was poured at a volume of 10 ml/plant. Three individual seedlings were used as replicates for each treatment, and sterile water was used as the negative control. All plants were placed in an artificial climate incubator at 25°C under 90% relative humidity. After 20 days, all inoculated plants showed symptoms identical to those described above, whereas controls remained healthy. Setophoma terrestris was reisolated from the infected roots, which was confirmed by morphological and molecular identification, which completed Koch's postulates. To our knowledge, this is the first report of S. terrestris as a causal agent of root rot on I. verum in China.

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.

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.

Identification and Characterization of the BBX Gene Family in Bambusa pervariabilis × Dendrocalamopsis grandis and Their Potential Role under Adverse Environmental Stresses.

Zinc finger protein (ZFP) transcription factors play a pivotal role in regulating plant growth, development, and response to biotic and abiotic stresses. Although extensively characterized in model organisms, these genes have yet to be reported in bamboo plants, and their expression information is lacking. Therefore, we identified 21 B-box (BBX) genes from a transcriptome analysis of Bambusa pervariabilis × Dendrocalamopsis grandis. Consequently, multiple sequence alignments and an analysis of conserved motifs showed that they all had highly similar structures. The BBX genes were divided into four subgroups according to their phylogenetic relationships and conserved domains. A GO analysis predicted multiple functions of the BBX genes in photomorphogenesis, metabolic processes, and biological regulation. We assessed the expression profiles of 21 BBX genes via qRT-PCR under different adversity conditions. Among them, eight genes were significantly up-regulated under water deficit stress (BBX4, BBX10, BBX11, BBX14, BBX15, BBX16, BBX17, and BBX21), nine under salt stress (BBX2, BBX3, BBX7, BBX9, BBX10, BBX12, BBX15, BBX16, and BBX21), twelve under cold stress (BBX1, BBX2, BBX4, BBX7, BBX10, BBX12, BBX14, BBX15, BBX17, BBX18, BBX19, and BBX21), and twelve under pathogen infestation stress (BBX1, BBX2, BBX4, BBX7, BBX10, BBX12, BBX14, BBX15, BBX17, BBX18, BBX19, and BBX21). Three genes (BBX10, BBX15, and BBX21) were significantly up-regulated under both biotic and abiotic stresses. These results suggest that the BBX gene family is integral to plant growth, development, and response to multivariate stresses. In conclusion, we have comprehensively analyzed the BDBBX genes under various adversity stress conditions, thus providing valuable information for further functional studies of this gene family.

Integrated Transcriptome and Metabolome Analysis Reveals the Molecular Mechanism of Rust Resistance in Resistant (Youkang) and Susceptive (Tengjiao) Zanthoxylum armatum Cultivars.

Chinese pepper rust is a live parasitic fungal disease caused by Coleosporium zanthoxyli, which seriously affects the cultivation and industrial development of Z. armatum. Cultivating and planting resistant cultivars is considered the most economical and environmentally friendly strategy to control this disease. Therefore, the mining of excellent genes for rust resistance and the analysis of the mechanism of rust resistance are the key strategies to achieve the targeted breeding of rust resistance. However, there is no relevant report on pepper rust resistance at present. The aim of the present study was to further explore the resistance mechanism of pepper by screening the rust-resistant germplasm resources in the early stage. Combined with the analysis of plant pathology, transcriptomics, and metabolomics, we found that compared with susceptible cultivar TJ, resistant cultivar YK had 2752 differentially expressed genes (DEGs, 1253 up-, and 1499 downregulated) and 321 differentially accumulated metabolites (DAMs, 133 up- and 188 down-accumulated) after pathogen infection. And the genes and metabolites related to phenylpropanoid metabolism were highly enriched in resistant varieties, which indicated that phenylpropanoid metabolism might mediate the resistance of Z. armatum. This finding was further confirmed by a real-time quantitative polymerase chain reaction analysis, which revealed that the expression levels of core genes involved in phenylpropane metabolism in disease-resistant varieties were high. In addition, the difference in flavonoid and MeJA contents in the leaves between resistant and susceptible varieties further supported the conclusion that the flavonoid pathway and methyl jasmonate may be involved in the formation of Chinese pepper resistance. Our research results not only help to better understand the resistance mechanism of Z. armatum rust but also contribute to the breeding and utilization of resistant varieties.

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.

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.

Brown leaf spot of Jacaranda mimosifolia Caused by Colletotrichum karstii in Sichuan, China.

Jacaranda mimosifolia D. Don is widely cultivated in southwest China (Yunnan, Sichuan, and other regions). It is widely applied in papermaking, medicine, environmental monitoring, timber, urban and rural afforestation, and soil and water conservation. In October 2020, a new brown leaf spot disease of J. mimosifolia was discovered in Xichang City (27°49' to 27°56'N, 102°16' to 102°11'E), with approximately 66.23% disease incidence. Firstly, the typical symptoms showed deep yellow necrotic lesions in the center or on the margin of the leaves. Gradually, the necrotic lesions expanded and developed into brown spots. Under humid conditions, the edges of necrotic lesions turned dark brown progressively. Finally, the leaves withered, died, and fell off. Infected tissues from ten samples were cut into small pieces of 2.5 × 2.5 mm. The surfaces of infected tissues were sterilized for 30 s in 3% sodium hypochlorite, 60 s in 75% ethanol, and rinsed three times in sterile water. They were then blot-dried with autoclaved paper towels and cultured on potato dextrose agar (PDA) at 25℃ for 3 to 8 days. After culturing for 8 days at 25℃ and 12 h/12 h light/dark on PDA, the colony diameter reached 78.2 to 82.7 mm. The colonies were light orange, turned pale pink with light orange beneath. The conidia were single-celled, aseptate, cylindrical, smooth-walled, straight, hyaline with both ends bluntly rounded, measuring 12.3 to 16.8 × 4.3 to 5.6 µm (n = 100; average=14.5 × 5.1µm). These morphological characteristics were consistent with the description of C. karstii (Zhao et al. 2021). For molecular identification, the genomic DNA of the representative isolate JM202010 was extracted using a fungal genomic DNA extraction kit (Solarbio, Beijing). The internal transcribed spacer (ITS) [ITS1/ITS4 (White et al., 1990)], calmodulin (CAL) [CL1C/CL2C (Weir et al., 2012)], actin (ACT) [ACT512F/ACT-783R (Carbone & Kohn, 1999)], chitin synthase (CHS-1) [CHS-79F/CHS-345R (Carbone & Kohn, 1999)], ß-tubulin (TUB2) [BT2A/BT2B (O'Donnell et al., 1997)], and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [GDF/GDR (Templeton et al. 1992)] were amplified. Sequences were deposited in GenBank (ITS: OL454787, CAL: OL518966, ACT: OL518967, CHS-1: OL518968, TUB2: OL518969, and GAPDH: OL518970). BLAST results indicated that the ITS, CAL, ACT, CHS-1, TUB2 and GAPDH sequences showed >99% identity with Colletotrichum karstii sequences at NCBI (GenBank MW494453.1, MW495036.1, MG387951.1, MW495038.1, MW495042.1, and MG602034.1). The conidial suspension (1 × 106 conidia/ml) was sprayed on the leaves of 4-year-old J. mimosifolia plants (10 plants) and inoculated for pathogenicity test. Fifteen leaves of each plant (10 pots in total) were inoculated with spore suspensions on both sides of the leaves. An equal number of control leaves was sprayed with sterilized distilled water as a control. Finally, all pots were kept in a greenhouse at 26°C under a 16 h/8 h photoperiod and 60 to 68% relative humidity. The inoculated plants showed symptoms similar to those of the original diseased plants, but the controls remained asymptomatic. Colletotrichum karstii was re-isolated from the infected leaves and identified by both morphological characteristics and DNA sequence analysis. The pathogenicity test was repeated thrice, which showed similar results, confirming Koch's postulates. To our knowledge, this is the first report of brown leaf spot on J. mimosifolia caused by C. karstii in China. C. karstii was previously reported as the causal agent of anthracnose on Fatsia japonica (Xu et al. 2020) and Nandina domestica (Li et al. 2017) in China. This finding provides an important basis for further research on the control of this disease.

Brown leaf spot of Prunus serrulata Caused by Colletotrichum fioriniae in Sichuan, China.

Prunus serrulate Lindl is widely cultivated in urban areas of China. It is mainly used for wood cultivation and urban landscaping. In May 2021, new leaf spot disease was observed in Chengdu city (30°42' to 30°45'N, 103°51' to 104°7'E), with 69.3% disease incidence, which could inhibit leaf growth and reduce their biomass. A planting area of more than 1000 square meters was investigated. The diseased leaves were mostly concentrated in the lower position of plants, where the humidity was higher. The disease infected P. serrulata leaves and occurred in the field from March to October, with the highest incidence in early May. The typical symptoms initially appeared as brown necrotic lesions on the margin of the leaves. The lesions then enlarged gradually and developed into reddish brown spots, eventually coalescing into large irregular, necrotic lesions with dark brown margins. Finally, the diseased leaves withered and died. Conidiomata were not formed on the diseased tissue. Ten symptomatic leaves were collected from 5 different trees in the planting area. Infected tissues from ten samples were cut into small pieces of 3 × 3 mm. The infected tissues were surface-sterilized by 3% sodium hypochlorite and 75% ethanol respectively for 30s and 60s, and rinsed three times in sterile water. Then they were blot-dried with autoclaved paper towels and cultured on potato dextrose agar (PDA) amended with streptomycin sulfate (50 µg/mL), and incubated at 25°C for 4 to 8 days. After culturing for 8 days at 25℃ and 12 h/12 h light/dark on PDA, the colony diameter reached 67.5 to 78.6 mm. The colonies were initially white, cottony, then became light pink to misty rose at the center, and the reverse side of the colony turned dark red to red and had pale yellowish borders. The conidia were straight, smooth-walled, colorless, fusiform with acute ends, measuring 8.2 to 16.7 × 3.1 to 5.9 µm in size (n = 100). For molecular identification, the genomic DNA of the representative isolate RBWY202105 was extracted using a fungal genomic DNA extraction kit (Solarbio, Beijing). The internal transcribed spacer (ITS) [ITS1/ITS4 (White et al., 1990)], histone3 (HIS3) [CYLH3F/CYLH3R (Crous et al. 2004)], chitin synthase (CHS-1) [CHS-79F/CHS-345R (Carbone & Kohn, 1999)], actin (ACT) [ACT512F/ACT (Carbone & Kohn, 1999)], ß-tubulin (TUB2) [BT2A/BT2B (O'Donnell et al., 1997)], and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [GDF/GDR (Templeton et al. 1992)] were amplified. Sequences were deposited in GenBank (ITS: ON000436, HIS3: ON014581, CHS-1: ON014579, ACT: ON014583, TUB2: ON014582, and GAPDH: ON014580). BLAST results indicated that the ITS, HIS3, CHS-1, ACT, TUB2 and GAPDH sequences showed >99% identity with Colletotrichum fioriniae (Marcelino & Gouli) R.G. Shivas & Y.P sequences at NCBI (GenBank MW497230 (561/582), MT740312 (415/415), KU736865 (258/258), MK680659 (246/246), MK967342 (757/757), and MW656269 (263/263)). The conidial suspension (1 × 106 conidia/ml) was used for inoculation by spraying leaves of ten 4-year-old P. serrulata plants for pathogenicity test. Fifteen leaves of each plant were inoculated with spore suspensions on the leaves (600 µl per leaf). The same amount of control leaves was sprayed with sterilized distilled water as a control. Finally, all potted plants were placed in a greenhouse at 25°C under a 16 h/8 h photoperiod and 67 to 78% relative humidity. After ten days, the symptoms observed on the inoculated plants were similar to those of the original diseased plants, but the controls remained asymptomatic. Colletotrichum fioriniae was re-isolated from the infected leaves and identified by both morphological characteristics and DNA sequence analysis (The ITS, HIS3, TUB2, CHS-1, GAPDH and ACT genes). The pathogenicity test was repeated thrice, which showed similar results, confirming Koch's postulates. To our knowledge, this is the first report of brown leaf spot on P. serrulata caused by C. fioriniae in China. The identification of C. fioriniae could provide relevant information for taking management strategies and further research on the Prunus serrulata disease.

First report of Leaf spot Caused by Colletotrichum siamense on Pharbitis purpurea in Sichuan, China.

The Pharbitis purpurea (L.) Voisgt, a member of the Convolvulaceae, is a graceful plant with an air purifying function and ornamental values. It is often cultivated in parks and roadsides. In April 2021, leaf spots (with approximately 67.9% disease incidence) were observed on P. purpurea grown in Xichang city (27°49'N; 102°16'E). More than 1000 square meters of planting area were investigated. Initially, yellowish-brown spots were of different sizes with a yellow irregular border, and slightly sunken necrotic lesions. Gradually, the necrotic lesions expanded and developed into brown spots that often coalesced and expanded to cover the entire leaves. Finally, the leaves wilted, died and fell off. For fungal isolation, infected tissues from ten samples were cut into small pieces of (2.5 × 2.5 mm) sterilized with 3% NaOCl for 30 s and 75% ethanol for 60 s, rinsed three times with sterilized water, blot-dried and cultured on potato dextrose agar (PDA) at 25°C in dark for 8 days. After culturing for 8 days, the colony diameter reached 75.2 to 79.7 mm. The pure colonies were grayish-white with pale yellowish borders and grayish black and pale yellowish borders on the reverse side. The conidia were hyaline, single-celled, cylindrical, smooth-walled, subcylindrical with obtuse to slightly rounded ends, measuring 11.6 to 17.9 × 3.7 to 5.8 µm (n = 100; average=14.7 × 4.9µm). These morphological characteristics were consistent with the description of Colletotricum siamense (Zhang et al. 2021). For molecular identification, the genomic DNA of the representative isolate LBH202104 was extracted using a fungal genomic DNA extraction kit (Solarbio, Beijing). Partial of internal transcribed spacer (ITS) regions, actin (ACT), calmodulin (CAL), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified using the primers ITS1/ITS4, ACT-512F/ACT-783R, CL1C/CL2C, and GDF/GDR, respectively (Weir et al. 2012). BLAST results of obtained sequences (ITS: OM948680, ACT: OM959361, CAL: OM959366, and GAPDH: OM959364), showed >99% identity with C. siamense sequences (MN305712, MZ461478, MK141754, and MK361203) in GenBank. Based on morphology and phylogenetic analysis, the representative isolate was identified as Colletotrichum siamense (Fig. S1&S2). For pathogenicity test, the conidial suspension (1 × 106 conidia/ml) was sprayed on the leaves of 4-year-old eight potted P. purpurea plants. Fifteen leaves of each plant were inoculated. For negative controls, 8 plants were sprayed with sterilized distilled water. Finally, all pots were kept in a greenhouse at 26°C under a 16 h/8 h photoperiod and 68 to 75% relative humidity. The inoculated plants showed symptoms similar to those of the original diseased plants, while controls remained asymptomatic. C. siamense cultures were re-isolated from the infected leaves and identified by both morphological characteristics and DNA sequence analysis. The pathogenicity test was repeated thrice, which showed similar results, confirming Koch's postulates. To our knowledge, this is the first report of leaf spot caused by C. siamense on P. purpurea worldwide. The identification of this pathogen provides a foundation for the management of Leaf spot in P. purpurea.

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.

Branch blight of Juglans sigillata caused by Juglanconis appendiculata in China.

Juglans sigillata Dode, an endemic walnut species native in southwest China, is mainly used as nuts in Sichuan Province (Jin et al., 2019). In May 2021, symptoms of branches blight were observed in an orchard measuring 10 hectares located in Mianyang City, Sichuan Province (31°5' 25″N, 105°27'36″E, 365 m above sea level). About 40% of plants were diseased in the quadrat consisting of twenty walnut trees, and 20% of branches were dead on each affected tree. Initially, light brown spots appeared; then, the spots expanded to surround the whole branches; finally, the branches changed from brown to reddish-brown and died. Four symptomatic branches were sampled randomly from different trees. Next, four fungal isolates were obtained from the acervuli of each branch using the single-conidium isolation (Chomnunti et al. 2014) and cultured on potato dextrose agar (PDA). The Petri dishes were placed in an incubator and cultured at 25 °C under a 12-h photoperiod. Colonies were initially white with thin aerial mycelia and gradually turned dark grey with irregular margins. Conidiomata were acervular, black and scattered, with a diameter of 0.3 - 0.7 mm. Conidiophores were narrowly cylindrical, simple or branched at the base, 30 - 43 × 3 - 8 µm (x = 36.5 × 5.5 µm, n = 40). Conidiogenous cells were annellidic with distinct annellations. Conidia were unicellular, brown when mature, narrowly ellipsoid with gelatinous sheaths and truncate scars at the base, 17 - 32 × 7 - 12 µm (x = 27 × 9 µm, n = 40). The genomic DNA of a representative isolate SICAUCC 22-0064 was extracted, and the internal transcribed spacer (ITS) region, guanine nucleotide-binding protein subunit beta gene (ms204), translation elongation factor 1-alpha (tef1-α), and partial sequences of ß-tubulin (tub2) were amplified by polymerase chain reaction and sequenced with primers V9G/LR5 (de Hoog & van den Ende 1998), MS-E1F1/MS-E5R1 (Walker et al. 2012), EF1-728F (Carbone & Kohn 1999)/TEF1LLErev (Jaklitsch et al. 2005), and T1/BtHV2r (Voglmayr et al. 2017), respectively. The sequences of ITS, ms204, tef1-α, and tub2 were deposited in NCBI with accession numbers ON000068, ON112376, ON112374, and ON112375, respectively. With the consideration of the sequence lack of ms204 and tub2 in the ex-type strain (D96) of Juglanconis appendiculata Voglmayr & Jaklitsch, the isolate D140 was used for nucleotide blast. The results showed 99.68%, 100%, 100%, and 100% identities of ITS, ms204, tef1-α, and tub2 with D140 (accession numbers KY427138, KY427157, KY427207, KY427226). Phylogenetic analysis based on a combined dataset showed 100% bootstrap with J. appendiculata, and the morphology was consistent with the asexual stage of J. appendiculata (Voglmayr et al., 2017). To verify Koch's postulates, five branches wounded by pin-prick were sprayed with conidial suspension (1 × 105 conidia/mL) in each plant, and three repetitions were performed on healthy 2-year-old potted plants. The same number of branches were sprayed with sterile distilled water as controls. The plants were placed in a greenhouse at 25 ℃ under 90% relative humidity and a 12-h fluorescent light/dark regime. After five weeks, all the inoculated branches showed brown necrosis similar to that observed in the field, and no symptoms occurred on the controls. The pathogens were re-isolated from the necrotic lesions and identified by morphology and phylogeny. J. appendiculata has been reported on Juglans nigra and J. regia in Austria, France, Spain and Greece (Farr & Rossman 2022). This paper is the first report of branch blight on Juglans sigillata caused by J. appendiculata in China. This result may develop the understanding of walnut diseases and lay a foundation for further management.

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.