Yinggao Liu

Oil tea (Camellia oleifera), as one of the four major woody oilseed crops, possesses considerable economic and medicinal significance. The distinctive geographical and climatic conditions of Sichuan Province provide an ideal environment for the growth of C. oleifera, making it a major hub for the C. oleifera industry. However, in recent years, it has suffered severe fungal disease outbreaks, particularly those caused by Pestalotiopsis and Neopestalotiopsis, which infect a wide range of hosts. From August 2023 to May 2024, a comprehensive survey of 66.67 hectares of Camellia oleifera plantations in Sichuan was conducted to identify fungal pathogens. By integrating morphological characterization and phylogenetic analyses of the internal transcribed spacer (ITS), translation elongation factor 1-α (tef1-α), and beta-tubulin (tub2) genes, three pestalotioid species were identified. Among these, N. folii was identified as a novel pathogenic fungus. Furthermore, two new host records were established for P. trachycarpicola and P. hispanica on C. oleifera. Pathogenicity tests following Koch’s postulates confirmed strain virulence and revealed variations in pathogenic effects. This study formally recognizes N. folii as a novel species, enhancing current knowledge of pestalotioid fungi associated with C. oleifera.

Rhombic-spot disease, caused mainly by Neostagonosporella sichuanensis, significantly impacts the yield and quality of fishscale bamboo (Phyllostachys heteroclada). Xylanases are essential for pathogenic fungi infection, yet their specific functions in the physiology and pathogenicity of N. sichuanensis remain unclear. Here, we characterized three xylanase proteins with glycosyl hydrolase 11 domains from the N. sichuanensis SICAUCC 16–0001 genome and examined the function of Nsxyn1 and Nsxyn2. Purified Nsxyn1 and Nsxyn2 proteins displayed specific xylanase activity in vitro and induced cell death in Nicotiana benthamiana, independent of their enzymatic function. Both proteins possessed signal peptides and were confirmed as secreted proteins using a yeast secretion system. Subcellular localization revealed that Nsxyn1 and Nsxyn2 localized in both the cytoplasm and nucleus and can trigger cell death in N. benthamiana through Agrobacterium tumefaciens-mediated transient transformation. qRT-PCR results showed notable upregulation of Nsxyn1 and Nsxyn2 during infection, with Nsxyn1 exhibiting an 80-fold increase at 15 days post-inoculation. Deletion of Nsxyn1 and Nsxyn2 in N. sichuanensis impaired xylan degradation, adaptation to osmotic and oxidative stress, and pathogenic full virulence. Deletion of Nsxyn1 notably slowed fungal growth and reduced spore production, whereas only a reduction in microconidial production was observed in Nsxyn2 mutants. Complementation of Nsxyn1 and Nsxyn2 only partially restored these phenotypic defects in the ∆Nsxyn1 and ∆Nsxyn2 mutants. These findings suggest that Nsxyn1 and Nsxyn2 contribute to N. sichuanensis virulence and induced plant defense responses, providing new insights into the function of xylanases in the interaction between fishscale bamboo and N. sichuanensis.

Bamboo resources have garnered significant global attention due to their excellent capacity for regeneration and high yield. Rhombic-spot disease, a substantial threat to fishscale bamboo (Phyllostachys heteroclada), is primarily caused by Neostagonosporella sichuanensis. This study first reported the genome assemblies and characteristics of two N. sichuanensis isolates using PacBio and Illumina sequencing platforms. The genomes of N. sichuanensis strain SICAUCC 16–0001 and strain SICAUCC 23–0140, with sizes of 48.0 Mb and 48.4 Mb, respectively, revealed 10,289 and 10,313 protein-coding genes. Additionally, they contained 34.99 and 34.46% repetitive sequences within AT-rich regions, with notable repeat-induced point mutation activity. Comparative genome analysis identified 1,049 contracted and 45 expanded gene families in the genome of N. sichuanensis, including several related to pathogenicity. Several gene families involved in mycotoxin metabolism, secondary metabolism, sterol biosynthesis and transport, and cell wall degradation were contracted. Compared to most analyzed necrotrophic, hemibiotrophic, and phaeosphaeriacous pathogens, the genomes of two N. sichuanensis isolates exhibited fewer secondary metabolite enzymes, carbohydrate-active enzymes, plant cell wall degrading enzymes, secreted proteins, and effectors. Comparative genomics analysis suggested that N. sichuanensis shares more similar characteristics with hemibiotrophic pathogens. Based on single carbon source tests, N. sichuanensis strains demonstrated a higher potential for xylan decomposition than pectin and cellulose. The proportion of cell wall-degrading enzyme effectors occupied a high proportion of the total effectors of the N. sichuanensis genomes. These findings provide valuable insights into uncovering the pathogenesis of N. sichuanensis toward the efficient management of rhombic-spot disease of fishscale bamboo.

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 ApCtf1β, 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.

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.

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 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 the genes and metabolites related to phenylpropanoid metabolism were highly enriched in resistant varieties after pathogen infection, which indicated that phenylpropanoid metabolism might mediate the resistance of Z. armatum. This finding was further confirmed by 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 content in the leaves between resistant and susceptible varieties further supported the conclusion that the flavonoid pathway is one of the main pathways involved in resistance formation in Chinese pepper, and MeJA is involved in the formation of 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.

Zanthoxylum armatum, a significant forest plant in southwestern China, is crucial for preserving soil and water resources. However, the presence of root rot disease has led to plant death, impacting the pepper sector. Effective control measures for this disease are still lacking. Rhizosphere microorganisms play a vital role in plant health by inhibiting plant pathogens and inducing plant resistance. This research aimed to isolate and characterize the pathogen responsible for root rot disease in Z. armatum. Comparative analysis of fungal and bacterial communities in the rhizosphere soil of healthy and diseased plants revealed Fusarium solani as the pathogenic fungus causing root rot disease. Diseased plants had a higher occurrence of Fusarium spp., while disease-free plants had a higher abundance of ecologically beneficial microbial communities that could potentially serve as biocontrol agents. Three bacterial strains (Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus siamensis) were identified as effective biocontrol agents, inhibiting the growth of the pathogenic fungus F. solani both in vivo and in vitro. This study deepens our understanding of the rhizosphere soil microbial community differences between diseased and healthy Z. armatum, providing potential biocontrol bacteria to enhance plant resistance against root rot disease.

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.

As an important nonwood bioresource, fishscale bamboo (Phyllostachys heteroclada Oliver) is widely distributed in the subtropical region of China. Rhombic-spot disease, caused by Neostagonosporella sichuanensis, is one of the most serious diseases that threatens fishscale bamboo health. However, there is limited knowledge about how rhombic-spot disease influences the diversity and structures of phyllosphere fungal communities. In this study, we investigated the phyllosphere fungal communities from stems, branches, and leaves of fishscale bamboo during a rhombic-spot disease outbreak using 18S rRNA sequencing. We found that only the phyllosphere fungal community from stems was significantly affected by pathogen invasion in terms of community richness, diversity, and structure. FUNGuild analysis revealed that the major classifications of phyllosphere fungi based on trophic modes in stems, branches, and leaves changed from symbiotroph-pathotroph, no obvious dominant trophic mode, and symbiotroph to saprotroph, saprotroph–pathotroph–symbiotroph, and saprotroph–symbiotroph, respectively, after pathogen invasion. The fungal community composition of the three tissues displayed significant differences at the genus level between healthy and diseased plants. The associations among fungal species in diseased samples showed more complex co-occurrence network structures than those of healthy samples. Taken together, our results highlight the importance of plant pathological conditions for the assembly of phyllosphere fungal communities in different tissues.

Thaumatin-like proteins (TLPs) are involved in the plant defense response against pathogens, and most of them exhibit antifungal activity. However, the role of TLPs in pathogen-induced defense responses in spruce is not fully understood. In this study, four TLP genes encoding thaumatin-like protein, designated as PlTLP1–4, were isolated and identified from Picea likiangensis needles. Sequence analysis showed that PlTLP1, PlTLP3, and PlTLP4 contained 16 conserved cysteine residues, while PlTLP2 had only 10 conserved cysteine residues. qPCR analysis showed that PlTLPs were expressed in all tissues tested, PlTLP1, PlTLP3, and PlTLP4 had the highest expression levels in young fruits, while PlTLP2 had the highest expression levels in roots. In addition, the expression levels of four PlTLPs were significantly upregulated during infection by Lophodermium piceae. Four recombinant PlTLPs expressed in Escherichia coli exhibited obvious β-1,3-glucanase activity. The antifungal activity assay showed that four recombinant PlTLPs had significant inhibitory effects on the mycelial growth of L. piceae, Fusarium proliferatum, Botrytis cinerea, and Roussoella doimaesalongensis. Microscopic observation revealed that the recombinant PlTLP1–4 induced the morphological changes of the mycelia of L. piceae, and the recombinant PlTLP2 and PlTLP3 induced the morphological changes of the mycelia of F. proliferatum and R. doimaesalongensis, while all the recombinant PlTLPs had no obvious negative effect on the morphology of B. cinerea mycelium. These results suggest that PlTLP genes may play an important role in the defense response of P. likiangensis against L. piceae invasion.