Screening of Antagonistic Bacillus against Brown Rot in Dendrocalamus latiflorus and Preparation of Applying Bacterial Suspension.

The aim of this study was to isolate biocontrol bacteria that could antagonize brown rot of Dendrocalamus latiflorus, optimize the culture conditions, and develop an effective biocontrol preparation for brown rot of D. latiflorus. This study isolated a bacterium with an antagonistic effect on bamboo brown rot from healthy D. latiflorus rhizosphere soil. Morphology, molecular biology, and physiological biochemistry methods identified it as Bacillus siamensis. The following culturing media and conditions improved the inhibition effect of B. siamensis: the best culturing media were 2% sucrose, 1.5% yeast extract, and 0.7% potassium chloride; the optimal culturing time, temperature, pH, and inoculation amount were 48 h, 30℃, 6, and 20%. The optimum formula of the applying bacterial suspension was 14% sodium dodecyl benzene sulfonate emulsifier, 4% Na2HPO4·2H2O, 0.3% hydroxypropyl methylcellulose thickener, and 20% B. siamensis. The pot experiment results showed the control effect of applying bacterial suspension, diluted 1,000 times is still better than that of 24% fenbuconazole suspension. The applying bacterial suspension enables reliable control of brown rot in D. latiflorus.

The Targeted Regulation of BDUbc and BDSKL1 Enhances Resistance to Blight in Bambusa pervariabilis × Dendrocalamopsis grandis.

Arthrinium phaeospermum is the major pathogen responsible for the significant stem disease "blight" in B. pervariabilis × D. grandis. The interacting proteins of the key pathogenic factor ApCtf1ß, BDUbc and BDSKL1, have previously been obtained by two-hybrid, BiFC, GST pull-down yeast assays. However, the functions of these interacting proteins remain unknown. This study successfully obtained transgenic plants overexpressing BDUbc, BDSKL1, and BDUbc + BDSKL1 via Agrobacterium-mediated gene overexpression. qRT-PCR analysis revealed significantly increased expression levels of BDUbc and BDSKL1 in the transgenic plants. After infection with the pathogenic spore suspension, the disease incidence and severity index significantly decreased across all three transgenic plants, accompanied by a marked increase in defense enzyme levels. Notably, the co-transformed plant, OE-BDUbc + BDSKL1, demonstrated the lowest disease incidence and severity index among the transgenic variants. These results not only indicate that BDUbc and BDSKL1 are disease-resistant genes, but also that these two genes may exhibit a synergistic enhancement effect, which further improves the resistance to blight in Bambusa pervariabilis × Dendrocalamopsis grandis.

The Identification and Role of the Key Mycotoxin of Pestalotiopsis kenyana Causing Leaf Spot Disease of Zanthoxylum schinifolium.

Leaf spot is a common disease of Zanthoxylum schinifolium (Z. schinifolium), which can seriously harm the plant's ability to grow, flower, and fruit. Therefore, it is important to identify the mechanism of leaf spot caused by Pestalotiopsis kenyana (P. kenyana) for thorough comprehension and disease control. In this study, to verify whether the mycotoxins produced by P. kenyana cause leaf spot disease, the best medium for P. kenyana, namely PDB, was used. The mycotoxins were determined by ammonium sulfate precipitation as non-protein substances. The crude mycotoxin of P. kenyana was prepared, and the optimal eluent was eluted with petroleum either/ethyle acetate (3:1, v/v) and purified by silica gel column chromatography and preparative high-performance liquid chromatography to obtain the pure mycotoxins PK-1, PK-2, and PK-3. The PK-3 had the highest toxicity to Z. schinifolium, which may be the primary mycotoxin, according to the biological activity test using the spray method. The physiological and biochemical indexes of Z. schinifolium plants treated with PK-3 mycotoxin were determined. Within 35 days after mycotoxin treatment, the results showed that the protein content and malondialdehyde content of leaves increased over time. The soluble sugar and chlorophyll content decreased over time. The superoxide dismutase activity and catalase activity of the leaves increased first and then decreased, and the above changes were the same as those of Z. schinifolium inoculated with the spore suspension of the pathogen. Therefore, it is believed that the mycotoxin pestalopyrone could be a virulence factor that helps P. kenyana induce the infection of Z. schinifolium. In this study, the pathogenic mechanism of Z. schinifolium leaf spot was discussed, offering a theoretical foundation for improved disease prevention and control.

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.

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.

Seasonal changes in the abundance Fusarium proliferatium, microbial endophytes and nutrient levels in the roots of hybrid bamboo Bambusa pervariabilis × Dendrocalamopsis grandis.

Plant root pathogens invade the soil around plant roots, disturbing the systemic balance, reducing plant defenses, and causing severe disease. At present, there are few studies on the severity of plant diseases caused by pathogen invasion in different seasons and how pathogens affect root microecology. In this study, we compared the levels of nutrients in the root tissues of the two groups of plants. We used 16S and ITS amplicon sequencing with Illumina NovaSeq 6000 to compare seasonal changes in the composition and structure of microbial communities from healthy roots of bamboo Bambusa pervariabilis × Dendrocalamopsis grandis and roots infected by the soilborne pathogen Fusarium proliferatum. We have found that the invasion of the pathogen led to a substantial decrease in nutrient elements in bamboo roots, except for nitrogen. The pathogen presence correlated with seasonal changes in the bamboo root microbiome and decreased bacterial richness in diseased plants. The root microbial community structure of healthy plants was more stable than that of their diseased counterparts. Furthermore, we identified the lesion area and relative abundance of F. proliferatum were significant predictors of disease progression. The potassium tissue content and the disease lesion area were identified as factors linked with the observed changes in the bamboo root microbiome. This study provides a theoretical foundation for understanding the seasonal dynamics F. proliferatum, an economically important soilborne pathogen of hybrid bamboo grown in Sichuan Province, China.

Using A Protoplast Transformation System to Enable Functional Studies in Mangifera indica L.

Mangoes (Mangifera indica L.) are an important kind of perennial fruit tree, but their biochemical testing method and transformation technology were insufficient and had not been rigorously explored. The protoplast technology is an excellent method for creating a rapid and effective tool for transient expression and transformation assays, particularly in plants that lack an Agrobacterium-mediated plant transformation system. This study optimized the conditions of the protoplast isolation and transformation system, which can provide a lot of help in the gene expression regulation study of mango. The most beneficial protoplast isolation conditions were 150 mg/mL of cellulase R-10 and 180 mg/mL of macerozyme R-10 in the digestion solution at pH 5.6 and 12 h of digestion time. The 0.16 M and 0.08 M mannitol in wash solution (WI) and suspension for counting (MMG), respectively, were optimal for the protoplast isolation yield. The isolated leaf protoplasts (~5.4 × 105 cells/10 mL) were transfected for 30 min mediated by 40% calcium-chloride-based polyethylene glycol (PEG)-4000-CaCl2, from which 84.38% of the protoplasts were transformed. About 0.08 M and 0.12 M of mannitol concentration in MMG and transfection solutions, respectively, were optimal for protoplast viability. Under the florescence signal, GFP was seen in the transformed protoplasts. This showed that the target gene was successfully induced into the protoplast and that it can be transcribed and translated. Experimental results in this paper show that our high-efficiency protoplast isolation and PEG-mediated transformation protocols can provide excellent new methods for creating a rapid and effective tool for the molecular mechanism study of mangoes.

The influence of national policies on the evolution of industrial design education in China.

This study systematically describes research trends of "industrial design education in China" using bibliometrics mapping from 1992 to 2021. This study aims to sort out industrial design education's historical flow in China and analyze its intrinsic links with Chinese national policies. A combination of quantitative and qualitative methods is used to describe and analyze this study. A technique that combines policy historical analysis with a bibliometric review based on Citespace's knowledge mapping is used in this research. The study was conducted on 492 ″Industrial Design Education" papers included in the core collection database of the China National Knowledge Infrastructure between 1992 and 2021. The results obtained from this study are 1) Research on industrial design education in China has grown steadily over the past three decades and remained high-quality level; 2) The three main research themes are "Chinese culture," "interdisciplinary cooperation" and "government, industry, academia and research" cooperation; 3) Innovation and entrepreneurship, evaluation system, Interdisciplinary, new engineering, and new liberal arts are the research hot spots of Chinese core journals; 4) Interdisciplinary construction in the context of new engineering, assessment system research in the context of high-quality development, and innovation and entrepreneurship education in the context of creative industry development are the future research directions.

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.

Verification of the Interaction Target Protein of the Effector ApCE22 of Arthrinium phaeospermum in Bambusa pervariabilis × Dendrocalamopsis grandis.

The study of interaction proteins of the pathogen A. phaeospermum effector protein is an important means to analyze the disease-resistance mechanism of Bambusa pervariabilis × Dendrocalamopsis grandis shoot blight. To obtain the proteins interacting with the effector ApCE22 of A. phaeospermum, 27 proteins interacting with the effector ApCE22 were initially identified via a yeast two-hybrid assay, of which four interaction proteins were obtained after one-to-one validation. The B2 protein and the chaperone protein DnaJ chloroplast protein were then verified to interact with the ApCE22 effector protein by bimolecular fluorescence complementation and GST pull-down methods. Advanced structure prediction showed that the B2 protein contained the DCD functional domain related to plant development and cell death, and the DnaJ protein contained the DnaJ domain related to stress resistance. The results showed that both the B2 protein and DnaJ protein in B. pervariabilis × D. grandis were the target interaction proteins of the ApCE22 effector of A. phaeospermum and related to the stress resistance of the host B. pervariabilis × D. grandis. The successful identification of the pathogen effector interaction target protein in B. pervariabilis × D. grandis plays an important role in the mechanism of pathogen-host interaction, thus providing a theoretical basis for the control of B. pervariabilis × D. grandis shoot blight.

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.

Function of ALA Content in Porphyrin Metabolism Regulation of Ananas comosus var. bracteatus.

Chlorophyll and heme are essential molecules for photosynthesis and respiration, which are competing branches of the porphyrin metabolism pathway. Chlorophyll and heme balance regulation is very important for the growth and development of plants. The chimeric leaves of Ananas comosus var. bracteatus were composed of central photosynthetic tissue (PT) and marginal albino tissue (AT), which were ideal materials for the study of porphyrin metabolism mechanisms. In this study, the regulatory function of ALA content on porphyrin metabolism (chlorophyll and heme balance) was revealed by comparing PT and AT, 5-Aminolevulinic Acid (ALA) exogenous supply, and interference of hemA expression. The AT remained similar in porphyrin metabolism flow level to the PT by keeping an equal ALA content in both tissues, which was very important for the normal growth of the chimeric leaves. As the chlorophyll biosynthesis in AT was significantly inhibited, the porphyrin metabolism flow was directed more toward the heme branch. Both tissues had similar Mg2+ contents; however, Fe2+ content was significantly increased in the AT. The chlorophyll biosynthesis inhibition in the white tissue was not due to a lack of Mg2+ and ALA. A 1.5-fold increase in ALA content inhibited chlorophyll biosynthesis while promoting heme biosynthesis and hemA expression. The doubling of ALA content boosted chlorophyll biosynthesis while decreasing hemA expression and heme content. HemA expression interference resulted in a higher ALA content and a lower chlorophyll content, while the heme content remained at a relatively low and stable level. Conclusively, a certain amount of ALA was important for the stability of porphyrin metabolism and the normal growth of plants. The ALA content appears to be able to regulate chlorophyll and heme content by bidirectionally regulating porphyrin metabolism branch direction.

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.

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.

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.

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.

Cloning and Characterization of Two Novel PR4 Genes from Picea asperata.

Pathogenesis-related (PR) proteins are important in plant pathogenic resistance and comprise 17 families, including the PR4 family, with antifungal and anti-pathogenic functions. PR4 proteins contain a C-terminal Barwin domain and are divided into Classes I and II based on the presence of an N-terminal chitin-binding domain (CBD). This study is the first to isolate two PR4 genes, PaPR4-a and PaPR4-b, from Picea asperata, encoding PaPR4-a and PaPR4-b, respectively. Sequence analyses suggested that they were Class II proteins, owing to the presence of an N-terminal signal peptide and a C-terminal Barwin domain, but no CBD. Tertiary structure analyses using the Barwin-like protein of papaya as a template revealed structural similarity, and therefore, functional similarity between the proteins. Predictive results revealed an N-terminal transmembrane domain, and subcellular localization studies confirmed its location on cell membrane and nuclei. Real-time quantitative PCR (RT-qPCR) demonstrated that PaPR4-a and PaPR4-b expression levels were upregulated following infection with Lophodermium piceae. Additionally, PaPR4-a and PaPR4-b were induced in Escherichia coli, where the recombinant proteins existed in inclusion bodies. The renatured purified proteins showed antifungal activity. Furthermore, transgenic tobacco overexpressing PaPR4-a and PaPR4-b exhibited improved resistance to fungal infection. The study can provide a basis for further molecular mechanistic insights into PR4-induced defense responses.

Differential Metabolomics Reveals Pathogenesis of Pestalotiopsis kenyana Causing Leaf Spot Disease of Zanthoxylum schinifolium.

Pepper leaf spot is a common disease of Zanthoxylum schinifolium. When it is serious, it directly affects the growth of Z. schinifolium, making the plant unable to blossom and bear fruit, which seriously restricts the development of the Z. schinifolium industry. Therefore, the pathogenic mechanism of leaf spots should be explored to provide a basis for a comprehensive understanding of the disease. Using liquid chromatography-mass spectrometry (LC-MS) technology combined with the data-dependent acquisition, the full spectrum analysis of pathogen mycelium samples was carried out. Partial least squares discriminant analysis (PLS-DA) was used to reveal the differences in metabolic patterns among different groups. Hierarchical clustering analysis (HCA) and PLS-DA were used to reveal the relationship between samples and metabolites, which reflected the metabolomics changes of Pestalotiopsis kenyana in the logarithmic growth phase of mycelia, the stable growth phase of mycelia, the massive spore stage, the induction culture conditions of PDA and Z. schinifolium leaves, and the possible pathogenic substances were selected for pathogenicity detection. PLS-DA had a strong predictive ability, indicating a clear analysis trend between different groups. The results of the metabolomics analysis showed that the differential metabolites of pathogenic bacteria were abundant at different stages and under different medium conditions, and the content of metabolites changed significantly. There were 3922 differential metabolites in nine groups under positive and negative ion modes, including lipids and lipid molecules, organic acids and their derivatives, organic heterocyclic compounds, organic oxygen compounds, carbohydrate polyketides, nucleosides, nucleotides, and analogs. The results of the pathogenicity test showed that the leaves treated with 3,5-dimethoxy benzoic acid, S-(5-adenosy)-l-homocysteine, 2-(1H-indol-3-yl) acetic acid, l-glutamic acid, and 2-(2-acetyl-3,5-dihydroxy phenyl) acetic acid showed different degrees of yellowish-brown lesions. This indicated that these substances may be related to the pathogenicity of P. kenyana, and the incidence was more serious when treated with 3,5-dimethoxybenzoic acid and S-(5-adenosy)- l -homocysteine. This study provides a basis for further analysis of differential metabolites and provides a theoretical reference for the prevention and treatment of Z. schinifolium leaf spot.

Structural and Dynamic Analysis of Leaf-Associated Fungal Community of Walnut Leaves Infected by Leaf Spot Disease Based Illumina High-Throughput Sequencing Technology.

Leaf-associated microbiota is vital in plant-environment interactions and is the basis for micro-ecological regulation. However, there are no studies on the direct differences in microbial community composition between disease-susceptible and healthy walnut leaves. This study collected five samples of healthy and infected leaves (all leaves with abnormal spots were considered diseased leaves) from May to October 2018. Differences in fungal diversity (Chao1 index, Shannon index, and Simpson index) and community structure were observed by sequencing and analyzing diseased and healthy leaf microbial communities by Illumina HiSeq sequencing technology. The main fungal phyla of walnut leaf-associated were Ascomycota, Basidiomycota, and Glomeromycota. Diversity indices (Shannon and Chao1 index values) of healthy leaves differed significantly in the late stages of disease onset. The results showed that the fungal species that differed considerably between the healthy and infected groups differed, and the fungal species that differed significantly between the healthy and infected groups changed with the development of the leaf disease. Critical control time points were determined by analyzing the population dynamics of pathogenic fungi. Leaf-associated microorganisms are abundant and diverse, and fungal identification and diversity studies are helpful for developing more appropriate walnut management strategies.

Identification of the interacting proteins of Bambusa pervariabilis × Dendrocalamopsis grandis in response to the transcription factor ApCtf1ß in Arthrinium phaeospermum.

Arthrinium phaeospermum is the main pathogen that causes Bambusa pervariabilis × Dendrocalamopsis grandis blight. It secretes the cutinase transcription factor ApCtf1ß, which has been shown to play an important role in B. pervariabilis × D. grandis virulence. However, knowledge about the interaction target genes of ApCtf1ß in B. pervariabilis × D. grandis remains limited. A cDNA library for the yeast two-hybrid system was constructed from B. pervariabilis × D. grandis shoots after 168 h treatment with A. phaeospermum. The library was identified as 1.20 × 107 cfu, with an average insert >1,000 bp in size and a 100% positive rate, providing a database for the subsequent molecular study of the interaction between A. phaeospermum and B. pervariabilis × D. grandis. The yeast two-hybrid (Y2H), bimolecular fluorescence complementation (BiFC), and glutathione-S-transferase (GST) pull-down assays were used to screen for and identify two ApCtf1ß interacting target proteins, BDUbc and BDSKL1, providing a reliable theoretical basis to study the molecular mechanism underlying B. pervariabilis × D. grandis resistance in response to A. phaeospermum, which would, in turn, establish a platform to develop new strategies for the sustainable and effective control of the blight diseases of forest trees.