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.

Drying without dying: A genome database for desiccation-tolerant plants and evolution of desiccation tolerance.

Desiccation is typically fatal, but a small number of land plants have evolved vegetative desiccation tolerance (VDT), allowing them to dry without dying through a process called anhydrobiosis. Advances in sequencing technologies have enabled the investigation of genomes for desiccation-tolerant plants over the past decade. However, a dedicated and integrated database for these valuable genomic resources has been lacking. Our prolonged interest in VDT plant genomes motivated us to create the "Drying without Dying" database, which contains a total of 16 VDT-related plant genomes (including 10 mosses) and incorporates 10 genomes that are closely related to VDT plants. The database features bioinformatic tools, such as blast and homologous cluster search, sequence retrieval, Gene Ontology term and metabolic pathway enrichment statistics, expression profiling, co-expression network extraction, and JBrowser exploration for each genome. To demonstrate its utility, we conducted tailored PFAM family statistical analyses, and we discovered that the drought-responsive ABA transporter AWPM-19 family is significantly tandemly duplicated in all bryophytes but rarely so in tracheophytes. Transcriptomic investigations also revealed that response patterns following desiccation diverged between bryophytes and angiosperms. Combined, the analyses provided genomic and transcriptomic evidence supporting a possible divergence and lineage-specific evolution of VDT in plants. The database can be accessed at http://desiccation.novogene.com. We expect this initial release of the "Drying without Dying" plant genome database will facilitate future discovery of VDT genetic resources.

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.

The maize ATP-binding cassette (ABC) transporter ZmMRPA6 confers cold and salt stress tolerance in plants.

KEY MESSAGE: ZmMRPA6 was cloned and characterized as the first ATP-binding cassette (ABC) transporter in maize to be proven to participate in cold and salt tolerance. Homologous genes AtABCC4 and AtABCC14 of ZmMRPA6 also responded to salt stress. ATP-binding cassette (ABC) proteins are major transmembrane transporters that play significant roles in plant development against various abiotic stresses. However, available information regarding stress-related ABC genes in maize is minimal. In this study, a maize ABC transporter gene, ZmMRPA6, was identified through genome-wide association analysis (GWAS) for cold tolerance in maize seeds germination and functionally characterized. During germination and seedling stages, the zmmrpa6 mutant exhibited enhanced resistance to cold or salt stress. Mutated of ZmMRPA6 did not affect the expression of downstream response genes related cold or salt response at the transcriptional level. Mass spectrometry analysis revealed that most of the differential proteins between zmmrpa6 and wild-type plants were involved in response to stress process including oxidative reduction, hydrolase activity, small molecule metabolism, and photosynthesis process. Meanwhile, the plants which lack the ZmMRPA6 homologous genes AtABCC4 or AtABCC14 were sensitive to salt stress in Arabidopsis. These results indicated that ZmMRPA6 and its homologous genes play a conserved role in cold and salt stress, and functional differentiation occurs in monocotyledonous and dicotyledonous plants. In summary, these findings dramatically improved our understanding of the function of ABC transporters resistance to abiotic stresses in plants.

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.

Morphological and molecular analyses reveal two new species of Microcera (Nectriaceae, Hypocreales) associated with scale insects on walnut in China.

The fungal genus Microcera consists of species mostly occurring as parasites of scale insects, but are also commonly isolated from soil or lichens. In the present study, we surveyed the diversity and assess the taxonomy of entomopathogenic fungi in Sichuan Province, China. Two new species of Microcera, viz. M.chrysomphaludis and M.pseudaulacaspidis, were isolated from scale insects colonising walnut (Juglansregia). Maximum Likelihood and Bayesian Inference analyses of ITS, LSU, tef1-α, rpb1, rpb2, acl1, act, tub2, cmdA and his3 sequence data provide evidence for the validity of the two species and their placement in Nectriaceae (Hypocreales). Microcerapseudaulacaspidis primarily differs from similar species by having more septate and smaller cylindrical macroconidia, as well as DNA sequence data. Meanwhile, Microcerachrysomphaludis has elliptical, one-septate ascospores with acute ends and cylindrical, slightly curved with 4-6 septate macroconidia up to 78 µm long. Morphological descriptions with illustrations of the novel species and DNA-based phylogeny generated from analyses of multigene dataset are also provided to better understand species relationships.

Plant serine/arginine-rich proteins: versatile players in RNA processing.

MAIN CONCLUSION: Serine/arginine-rich (SR) proteins participate in RNA processing by interacting with precursor mRNAs or other splicing factors to maintain plant growth and stress responses. Alternative splicing is an important mechanism involved in mRNA processing and regulation of gene expression at the posttranscriptional level, which is the main reason for the diversity of genes and proteins. The process of alternative splicing requires the participation of many specific splicing factors. The SR protein family is a splicing factor in eukaryotes. The vast majority of SR proteins' existence is an essential survival factor. Through its RS domain and other unique domains, SR proteins can interact with specific sequences of precursor mRNA or other splicing factors and cooperate to complete the correct selection of splicing sites or promote the formation of spliceosomes. They play essential roles in the composition and alternative splicing of precursor mRNAs, providing pivotal functions to maintain growth and stress responses in animals and plants. Although SR proteins have been identified in plants for three decades, their evolutionary trajectory, molecular function, and regulatory network remain largely unknown compared to their animal counterparts. This article reviews the current understanding of this gene family in eukaryotes and proposes potential key research priorities for future functional studies.

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.

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.

Identification and pathogenicity analysis of leaf brown spot of Juglans regia in China.

English walnut (Juglans regia), has high economic and ecological value. As an important tree species for eliminating poverty, it is planted in many Provinces of China. In 2021, new pathogenic fungi were observed in English walnut in Guangyuan City, Sichuan Province, China. The initial symptom of leaf infection is that the leaves are covered with small black spots, which gradually expand into larger brown spots. Most of the spots appeared at the edges of the leaves, and yellow whorls were observed at the junction between the spots and the healthy leaves. The pathogenic fungi were isoalted form collecting disease samples and purified by single-spore culturing. In vitro and field experiments showed that the pathogen could cause brown spots on walnut leaves. The inoculation experiment showed that the symptoms in the field experiment were the same as those observed on the spot; however, slight differences were observed in the in vitro experiment. Ten isolates were obtained from walnut leaves with brown spot symptoms, and these were further characterized based on morphology and DNA sequencing. ITS (internal transcribed spacer), LSU (large sub-unit rDNA), rpb2 (second largest subunit of RNA polymerase) and tub2 (beta-tubulin) gene regions were used to construct phylogenetic trees and determine the evolutionary relationships among the collected strains. The isolate was identified as Nothophoma quercina by morphological and polygene analyses. As far as we are aware, the brown spots on walnut leaves caused by N. quercina is the first report of its kind.

Trunk rot of Juglans regia caused by Myrmaecium fulvopruinatum in China.

Walnut (Juglans regia) is a deciduous tree of the Juglandaceae family, widely cultivated in China, and provides value in a variety of ways, including the usage of the wood and nuts, and offers substantial economic, social, and environmental advantages (Wang et al, 2017). Nevertheless, a fungal disease of causing walnut trunk rot was observed in approximately 30% of 50 counted ten-year-old J. regia in Chongzhou City (30°33'34″N, 103°38'35″E, 513 m), Sichuan Province, China, and this disease has greatly delete healthy growth of walnut. The infected bark exhibited purple necrotic lesions, and the sick parts were surrounded by water-soaked plaques. From 10 trunks of the 10 diseased trees, 20 isolated fungal colonies were the same. The ascospores placed in 60 mm plates were almost entirely covered with mycelium within 8 days, colonies on the PDA changed from initial pale to white, ad then turned yellowish to light orange or rosy to yellow-brown (25℃, 90% relative humidity, 12-h photoperiod). On the host, Ectostromata were immersed to erumpent, globose to subglobose, purple and brown, and measured 0.6 - 4.5 × 0.3 - 2.8 mm (x̄ = 2.6 × 1.6 mm, n = 40); Ascomata were flask-shaped to subglobose, dark brown, and measured 0.1 - 0.6 × 0.1 - 0.4 mm (x̄ = 0.35 × 0.25 mm, n = 40); Asci were numerous, cylindrical to subclavate, contained 8 uniseriate ascospores, and measured 80 - 150 × 10 - 20 µm (x̄ = 115 × 15 µm, n = 40), and Ascospores were ellipsoid, 2-celled, dark brown to black, plump or attenuated towards, apices with 1 large drop per cell, and measured 14 - 20 × 6.5 - 9 µm (x̄ = 17 × 7.8 µm, n = 40). These morphological characteristics are consistent with the species Myrmaecium fulvopruinatum (Berk.) Jaklitsch & Voglmayr (Jaklitsch et al. 2015). The genomic DNA of a representative isolate SICAUCC 22-0148 was extracted. The ITS, LSU region, tef1-α, rpb2 genes region were amplified using the primer pairs ITS1/ITS4 primers (White et al. 1990), LR0R/LR5 (Moncalvo et al. 1995), EF1-688F/986R (Alves et al. 2008), fRPB2-5f/fRPB2-7cr (Liu et al. 1999), respectively. The sequences were deposited in NCBI with accession numbers ON287043 (ITS), ON287044 (LSU), ON315870 (tef1-α), and ON315871 (rpb2), rspectively, which showed 99.8, 99.8, 98.1, and 98.5% identities with M. fulvopruinatum CBS 139057 holotype (accession numbers KP687858, KP687858, KP688027, and KP687933 respectively). Based on the analyses of phylogenies and morphologies, the isolates were identified as M. fulvopruinatum. The pathogenicity of SICAUCC 22-0148 was tested by inoculating surface-sterilized trunk wounds of four-year-old trees of J. regia with a mycelial plug (Desai et al. 2019). Sterile PDA plugs were used as controls. Wounds were covered with a film, to ensure humidity and prevent contamination. Each inoculation was repeated twice and included two plants, control and inoculated. A month later, the symptoms observed on inoculated trunks were similar to those in the wild, and M. fulvopruinatum was re-isolated from the inoculated trunk, confirming Koch's postulates. Previous research has reported M. fulvopruinatum as an important fungal species that cause canker delete symptoms on Chinese sweet chestnut in China (Jiang et al. 2018). We carried the taxonomy work of the fungi that caused trunk rot on walnut, and this is the first time that M. fulvopruinatum has been linked to walnut trunk rot on J. regia. Trunk rot of walnut will not only cause weakening of trees, but also affect the yield and quality of walnuts, bringing huge economic losses. This study was supported by the Sichuan Science and Technology Program under Grant 2022NSFSC1011. References: Alves, A., et al. 2008. Fungal Diversity 28:1-13. Desai, D.D., et al. 2019. International Journal of Economic Plants 6:147-149. Jaklitsch., W.M., et al. 2015. Fungal Diversity 73(1):159-202. Jiang, N., et al. 2018. Mycosphere 9(6):1268-1289. Liu, Y.L., et al. 1999. Mol Biol Evol 16:1799-1808. Moncalvo, J.M., et al. 1995. Mycologia 87:223-238. Wang, Q.H., et al. 2017. Australasian Plant Pathology 46:585-595. White, T.J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA.

Phylogenetic Analysis of Spliceosome SF3a2 in Different Plant Species.

The formation of mature mRNA requires cutting introns and splicing exons. The occurrence of splicing involves the participation of the spliceosome. Common spliceosomes mainly include five snRNPs: U1, U2, U4/U6, and U5. SF3a2, an essential component of spliceosome U2 snRNP, participates in splicing a series of genes. There is no definition of SF3a2 in plants. The paper elaborated on SF3a2s from a series of plants through protein sequence similarity. We constructed the evolutionary relationship of SF3a2s in plants. Moreover, we analyzed the similarities and differences in gene structure, protein structure, the cis-element of the promoter, and expression pattern; we predicted their interacting proteins and constructed their collinearity. We have preliminarily analyzed SF3a2s in plants and clarified the evolutionary relationship between different species; these studies can better serve for in-depth research on the members of the spliceosome in plants.

Global Analysis of Dark- and Heat-Regulated Alternative Splicing in Arabidopsis.

Alternative splicing (AS) is one of the major post-transcriptional regulation mechanisms that contributes to plant responses to various environmental perturbations. Darkness and heat are two common abiotic factors affecting plant growth, yet the involvement and regulation of AS in the plant responses to these signals remain insufficiently examined. In this study, we subjected Arabidopsis seedlings to 6 h of darkness or heat stress and analyzed their transcriptome through short-read RNA sequencing. We revealed that both treatments altered the transcription and AS of a subset of genes yet with different mechanisms. Dark-regulated AS events were found enriched in photosynthesis and light signaling pathways, while heat-regulated AS events were enriched in responses to abiotic stresses but not in heat-responsive genes, which responded primarily through transcriptional regulation. The AS of splicing-related genes (SRGs) was susceptible to both treatments; while dark treatment mostly regulated the AS of these genes, heat had a strong effect on both their transcription and AS. PCR analysis showed that the AS of the Serine/Arginine-rich family gene SR30 was reversely regulated by dark and heat, and heat induced the upregulation of multiple minor SR30 isoforms with intron retention. Our results suggest that AS participates in plant responses to these two abiotic signals and reveal the regulation of splicing regulators during these processes.

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.

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.

Glutathione and neodiosmin feedback sustain plant immunity.

Plants have evolved a two-layer immune system comprising pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) that is activated in response to pathogen invasion. Microbial patterns and pathogen effectors can be recognized by surface-localized pattern-recognition receptors (PRRs) and intracellularly localized nucleotide-binding leucine-rich repeat receptors (NLRs) to trigger PTI and ETI responses, respectively. At present, the metabolites activated by PTI and ETI and their roles and signalling pathways in plant immunity are not well understood. In this study, metabolomic analysis showed that ETI and PTI induced various flavonoids and amino acids and their derivatives in plants. Interestingly, both glutathione and neodiosmin content were specifically up-regulated by ETI and PTI, respectively, which significantly enhanced plant immunity. Further studies showed that glutathione and neodiosmin failed to induce a plant immune response in which PRRs/co-receptors were mutated. In addition, glutathione-reduced mutant gsh1 analysis showed that GSH1 is also required for PTI and ETI. Finally, we propose a model in which glutathione and neodiosmin are considered signature metabolites induced in the process of ETI and PTI activation in plants and further continuous enhancement of plant immunity in which PRRs/co-receptors are needed. This model is beneficial for an in-depth understanding of the closed-loop mode of the positive feedback regulation of PTI and ETI signals at the metabolic level.

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.

Morphology and phylogeny of ascomycetes associated with walnut trees (Juglans regia) in Sichuan province, China.

In Sichuan province, walnuts, consisting of Juglans regia, Juglans sigillata, and the hybrid J. regia × J. sigillata, are commercially important edible nuts, and J. regia is the most widespread plant. To date, the diversity and distribution of fungi inhabiting on Juglans have not received enough attention, although there have been studies focusing on pathogens from fruit and stem. In order to update the checklist of fungi associated with Sichuan walnuts, a survey on fungi associated with the three Juglans species from 15 representative regions in Sichuan was conducted. In this article, ten fungi distributed in two classes of Ascomycota (Dothideomycetes and Sordariomycetes) were described based on morpho-molecular analyses, and two novel species, Neofusicoccum sichuanense and Sphaerulina juglandina, a known species of Ophiognomonia leptostyla, and seven new hosts or geographical records of Cladosporium tenuissimum, Diatrypella vulgaris, Helminthosporium juglandinum, Helminthosporium velutinum, Loculosulcatispora hongheensis, Periconia byssoides, and Rhytidhysteron subrufulum were included. Morphological descriptions and illustrations of these fungi are provided.

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.