Anthracnose, caused by Colletotrichum spp., is a significant disease affecting oil tea (Camellia oleifera Abel.). Extensive molecular studies have demonstrated that Colletotrichum fructicola is the dominant pathogen of oil tea anthracnose in China. This study aims to investigate differences in molecular processes and regulatory genes at a late stage of infection of C. fructicola, to aid in understanding differences in pathogenic mechanisms of C. fructicola of different geographic populations. We compared the pathogenicity of C. fructicola from different populations (Wuzhishan, Hainan province, and Shaoyang, Hunan province) and gene expression of representative strains of the two populations before and after inoculation in oil tea using RNA sequencing. The results revealed that C. fructicola from Wuzhishan has a more vital ability to impact oil tea leaf tissue. Following infection with oil tea leaves, up-regulated genes in the strains from two geographic populations were associated with galactosidase activity, glutamine family amino acid metabolism, arginine, and proline metabolism. Additionally, up-regulated gene lists associated with infection by Wuzhishan strains were significantly enriched in purine metabolism pathways, while Shaoyang strains were not. These results indicate that more transcriptional and translational activity and the greater regulation of the purine metabolism pathway in the C. fructicola of the Wuzhishan strain might contribute to its stronger pathogenicity.

To mitigate soil degradation and decrease dependency on chemical inputs in agriculture, this study examined the joint effects of coconut shell biochar and Bacillus strain Ya-1 on soil fertility, rhizosphere bacterial communities, and the growth of chili (Capsicum annuum L.). A controlled pot experiment with four treatments was conducted: control (CK), biochar only (C), Bacillus strain Ya-1 only (B), and a combination of both (BC). The BC treatment significantly enhanced the soil carbon and available phosphorus contents by approximately 20% and the soil nitrogen content and pH by 18% and 0.3 units, respectively, compared to the control. It also increased microbial biomass carbon and nitrogen by 25% and 30%, respectively, indicating improved soil microbial diversity as shown by the highest Pielou evenness index and Shannon index values. The combined application of biochar and the Ya-1 strain resulted in a 15% increase in chili plant height and a 40% improvement in root dehydrogenase activity, suggesting enhanced nutrient uptake and metabolism. Metabolic profiling showed shifts in stress response and nutrient assimilation under different treatments. Collectively, these results indicate the potential of biochar and microbial inoculants to significantly promote soil and plant health, providing a sustainable strategy to improve agricultural productivity and reduce reliance on chemical inputs.

Cowpea (Vigna unguiculata L.), a significant vegetable crop in China, holds particular prominence in the tropical island of Hainan. This region serves as the primary production area for the winter cultivation of cowpea. Phytoplasmas are an idiopathic parasitic pathogen and cannot be cultured in vitro. It is mainly transmitted by the insect vectors with the piercing and sucking mouthparts, such as leafhoppers, plant hoppers, and psyllids. (Kumari et al. 2019). On September 11, 2023, typical characteristics of phytoplasma diseases on cowpeas were observed in the experimental base of Hainan Academy of Agricultural Sciences (20°0'38.6964″N, 110°21'35.4024″E, Haikou City, Hainan Province, China), including reduced leaf size, chlorosis, and the development of broom-like branch deformities reminiscent, as depicted in Figure 1. At the same time, we found a large number of leafhoppers near the diseased plants, and we speculated that leafhoppers are the insect carriers that spread the disease. Following an on-site investigation, it was determined that the disease incidence ranges from 10% to 15%, leading to a consequential decrease of about 10% in yield, which is a potential disease that seriously threatens the cowpea industry in Hainan. Ten disease and healthy samples were meticulously collected and subsequently preserved at -80°C within the laboratory refrigerator. Three disease samples denoted as HNNKY-1, HNNKY-2, and HNNKY-3, were randomly chosen, and total DNA extraction was carried out employing the NuClean Plant Genomic DNA Kit (CWBIO, Taizhou, China), while three healthy samples were randomly selected as control. The 16S rRNA gene was amplified by PCR using the primer pairs P1/P7 (Schneider et al. 1995) and R16F2n / R16R2 (Lee et al. 1993) and the secA gene was amplified by PCR using the primer pairs secAfor1/secArev3 (Hodgetts et al. 2008). After agarose gel electrophoresis analysis, no DNA fragments were observed in the healthy leaf samples, whereas all three disease samples yielded amplification products. The PCR products were subsequently sequenced by Hainan Nanshan Biotech Co., Ltd., Haikou, China. After sequence analysis, it was found that the 16S rRNA gene and secA gene sequences HNNKY-1, HNNKY-2, and HNNKY-3 were identical to each other. We selected two gene sequences of strain HNNKY-3 to submission to the GenBank database, The length of the 16S rRNA gene sequence is 1193 base pairs, identified by the accession number OR666421, while the secA gene sequence is 825 base pairs in length, associated with the accession number OR661282. The phytoplasma strain HNNKY-3 was named 'Vigna unguiculata' witches'-broom phytoplasma. A BLAST analysis of the 16S rRNA gene revealed that strain HNNKY-3 displayed a 100% sequence match with 'Emilia sonchifolia' witches'-broom phytoplasma (MT420682), Peanut witches'-broom phytoplasma (OR239773), and 'Raphanus sativus' witches'-broom phytoplasma (OK491387). All of these phytoplasmas were classified within the 16SrII group. Based on the BLAST analysis of partial secA gene sequences, it was discerned that sequence homogeneity ranged from 99.27% to 99.74% among the studied sequences. These sequences were collectively classified as members of the 16SrII group. In addition, a phylogenetic tree was constructed by MEGA 11 (version 11.0.13) based on the 16Sr RNA gene and secA gene by the neighbor-joining method (Tamura et al. 2004). The results demonstrated the clustering of HNNKY-3 phytoplasma strains within the 16SrII group, as illustrated in Figures 2 and 3. A virtual RFLP analysis based on the 16S rRNA gene fragment of HNNKY-3 was conducted using the interactive online phytoplasma classification tool, iPhyClassifier (Zhao et al. 2009). The results indicated that the phytoplasma strain was the same as the reference pattern of the onion yellows phytoplasma of 16SrII-A (GenBank accession: L33765), and the similarity coefficient was 1.00. To best of our knowledge, this is the inaugural documentation of 16SrII Group-related phytoplasma infecting cowpea in Hainan, China, and lays the groundwork for further research on the dissemination of cowpea phytoplasma disease within China.

The climate-driven acceleration of forest disease outbreaks has intensified the demand for sustainable biocontrol strategies. In this study, we evaluated the effects of the endophytic bacterium Bacillus amyloliquefaciens csuftcsp75 on soil properties, microbial communities, and functional metabolism in soils affected by Pinus massoniana shoot blight. Soil physicochemical analysis, carbon substrate utilization profiling (AWCD), and diversity indices (the Shannon, Simpson, and McIntosh indices) were integrated to assess the microbial responses under different inoculation treatments. The csuftcsp75 treatment significantly improved soil nutrient availability—especially available phosphorus and potassium—and was associated with enhanced microbial metabolic activity and sustained functional diversity. Principal component analysis and correlation mapping revealed strong associations between labile nutrients and microbial responses. Comparative analysis showed that csuftcsp75 promoted a balanced and metabolically rich microbial community, while less compatible strains exhibited transient or unstable effects. These findings support a dual-pathway model wherein nutrient-driven metabolic activation and ecological integration jointly determine biocontrol efficacy. This study highlights the importance of matching microbial inoculants with local soil environments to optimize functional outcomes. This work provides a theoretical basis for applying endophytic Bacillus in forest disease management and contributes to the development of ecologically coherent biocontrol strategies.

Heliconia subulata is a common ornamental plant, it has been widely planted in southern China for greening parks, roads, and residential areas. H. subulata plants with spots on their leaves were observed in East Coast Wetland Park (18°16'53.37″N, 109°30'19.36″E), Sanya City, Hainan Province, China on Aug. 31, 2023. The symptoms of the leaves are irregular gray-white, spots, that develop into brown and black, with yellow halos at the disease-health junction. Following an on-the-spot investigation, it was found that the incidence of the disease was 40 to 50%. The leaves were disinfected with 70% ethanol for 1 min, rinsed with sterile water 3 times, disinfected for 1 min with 0.1% HgCl2, rinsed with sterile water 3 times, dried, put on potato dextrose agar (PDA) and incubated at 28℃ for 7 days. The red conidia pile was selected from the culture, dispersed in sterile water and diluted to 20 μL containing 1 to 2 conidia. After absorbing 20 μL spore suspension for many times and inoculating it on the new PDA plate, five pure cultures of single spore, J-1-1 to J-1-5, were obtained. After 7 days of growth, the colonies were grayish aerial mycelium on the front and light orange conidia on the reverse. The white aerial mycelia, conidia, acervulus, and appressorium were observed (Supplementary Fig. S1). The morphological characteristics showed that the isolate had the same characteristics as the previously described Colletotrichum spp. (Wang et al. 2021). The genomic DNA of isolates J-1-1 and J-1-5 were extracted by Fungal DNA Kit (OMEGA bio-tek, Guangzhou, China). The internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GADPH), and β-tubulin 2 genes (TUB2) were amplified by primers ITS1/ITS4, GDF/GDR, and Bt2a/Bt2b, respectively (Weir et al. 2012). Based on sequencing and gene sequence alignment analysis, it was found that the consistency between the ITS sequences of isolates J-1-1 and J-1-5 was 99.82%. The consistency between GADPH and TUB2 sequences was 100%. The gene sequences of isolates J-1-1 and J-1-5 were submitted to GenBank with accession numbers PP455510/PP455511 (ITS), PP510210/PP510211 (GADPH) and PP510212/PP510213 (TUB2) respectively. Based on the BLAST analysis, the three sequences were more than 99% identical to those of the C. tropicale strain FC1 (ITS: MT192648, GAPDH: MT155819, TUB2: MT199874; Duan et al. 2022). A phylogenetic tree was constructed by MEGA 11 based on the ITS, GADPH, and TUB2 gene sequence by the maximum-likelihood method. The results showed that the isolates J-1-1 and J-1-5 were clustered with C. tropicale CBS:124949 (Supplementary Fig. S2). Based on morphological and molecular biological analysis, two isolates were identified as C. tropicale. To further test the pathogenicity of isolates J-1-1 and J-1-5, spore suspensions (1×106 conidia/mL) were prepared and 20 μL spore suspensions were inoculated on the leaves of healthy H. subulata potted plants stabbed with sterile toothpicks. Three leaves were inoculated in each treatment, and sterile water was inoculated as a control. The treated plants were placed in an incubator with a temperature of 28℃, relative humidity of 90%, and light/dark (12h/12h). After 15 days, the spore suspension treatment showed the same symptoms as the naturally diseased H. subulata plants in the field, but the leaves treated with sterile water were not infected (Supplementary Fig. S1). The morphology of the isolates obtained from diseased leaves was the same as that of isolates J-1-1 and J-1-5 on the PDA plate. To our knowledge, this is the first report of H. subulata, a new host of C. tropicale causing anthracnose in China.