Cabbage Fusarium Wilt (CFW) is a serious disease caused by Fusarium oxysporum f. sp. conglutinans in many parts of the world. The use of chemical fungicides has placed a heavy burden on the environment and is prone to drug resistance in plant pathogens. As a method with great potential, biological control has attracted the attention of many academics both at home and abroad. In this study, we have found that strains B5 and B6 had a strong inhibitory effect on various pathogens and significantly inhibited mycelium growth. They were both identified as Bacillus velezensis by morphological features, biochemical determinations, 16S rRNA gene and gyrA gene sequence analysis. When different concentrations of bacterial suspension were applied to cabbage seeds, hypocotyl and taproot length increased to varying degrees. The in vivo results showed that B5 and B6 decreased the incidence of cabbage seedling wilt disease, with B6 performing significantly better. Furthermore, B. velezensis B6 had the ability to colonize cabbage plants and rhizosphere soil. Thus, strain B6 has great potential for biocontrol development and this research could lead to the development of a promising biological agent for CFW.

Colletotrichum capsici is one of the most important pathogens on chili peppers. Unreasonable application of chemical fungicides will lead to threats to human and animal health, environmental damage, and increased fungicide resistance to pathogens. As an alternative strategy, biological control has been paid more and more attention by academics. In this study, the Yb-1 strain was isolated from healthy cucumber leaves, which could significantly restrict the mycelium growth of C. capsici and Botrytis cinerea, with inhibition rates of 93.49% and 74.03%, respectively. Strain Yb-1 was identified as Bacillus velezensis by morphological features and 16S rRNA gene, gyrA gene, and gyrB gene sequence analysis. When pepper seeds were treated with different concentrations of bacterial suspension, we found that a medium-concentration treatment (10⁴ CFU/mL and 10⁵ CFU/mL) could promote seed germination and growth, while high-concentration treatments (10⁸ CFU/mL) limited seed germination and growth. In addition, the root-irrigation method, acupuncture-leaf method, and leaf-cutting method were used to evaluate the safety of strain Yb-1 on peppers. The results indicated that Yb-1 did not affect the normal growth of peppers. The results of further field trials showed that the control effect of strain Yb-1 on pepper anthracnose was 59.45%. Thus, the strain B. velezensis Yb-1 has great biocontrol potential for pepper anthracnose and promotes plant growth.

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.

Alpinia oxyphylla Miq. is mainly distributed in Hainan, Guangdong and Guangxi provinces of China. Between July and August 2021, a leaf spot disease was observed in Ledong, Hainan Province, China (18°70′20.50″ N, 109°25′25.47″E) on A.oxyphylla. The incidence of infected leaves ranged from 8% to 10%, and the incidence rate of infected plants was about 50%. Symptoms appeared as primary yellow-brown withered spots on the diseased leaves, which further developed into irregular red-brown spots. The center of the lesions was gray-black, and the tissue was irregularly necrotic, ruptured or perforated, and there were yellow chlorotic halos around the edges of the lesions (Figure 1A). Tissues 5 mm in diameter were taken from the junction of diseased and healthy tissue for pathogen isolation, Successively, a total of 8 isolates were obtained from the affected leaves. Three single spore isolates (YZ-HN-001, YZ-HN-043 and YZ-HN-051) were obtained and confirmed to be identical based on morphological characteristics. Therefore, the representative isolate YZ-HN-001 was selected for morphological and molecular identification. On Potato Dextrose Agar(PDA), the colony was gray-white at first and gradually turned dark green to dark brown with lead gray on the back, growth was slow, and mycelium was short and dense (Figure 1B and Figure 1C). Pycnidia were epiphyllous, globose, brown (about 120-140 µm in diameter), and conidia were elliptical, colorless, single celled and smooth (8-12×4-7 µm) (Figure 1D). Molecular identification was performed by partially sequencing the internal transcribed spacer gene (ITS), 18S rRNA gene and the actin gene (ACT) by using the primers ITS1/ITS4 (White et al. 1990), EF4/Fungi5 (Khodaparase et al. 2005) and ACT-512F/ACT-783R (Carbone and Kohn. 1999). The sequences of the amplified fragments were deposited in GenBank, the ITS sequence (ON005130, 616 bp) showed 100% identity with Phyllosticta capitalensis strain CGMCC3.14345 (JN791605.1), the 18S rRNA sequence (ON005129, 541 bp) showed 99% identity with P. capitalensis isolate MUCC0029 (AB454185.1) and the ACT sequence (ON049348, 251 bp) showed 100% identity with P. capitalensis strain DZSN202005-2 (MW533248.1). A phylogenetic analysis was conducted in MEGA X using the neighbor-joining method and showed that isolate YZ-HN-001 clustered together with P. capitalensis (Figure 2). Based on the above morphological and molecular characteristics, the isolate was determined to be P. capitalensis. Pathogenicity tests were conducted in three replicates by inoculating surface-sterilized leaves of A. oxyphylla. The leaves were wounded and inoculated with colonized PDA plugs (5×5 mm) from 15-day-old cultures. Control leaves wounded in the same way and were inoculated with sterile PDA plugs (5×5 mm). Leaves were moisturized by spraying with sterile water every three days. After 20 days at room temperature (23 to 28℃), similar symptoms were observed in the inoculated leaves as in the field (Figure 1E), but no symptoms were observed on the control leaves (Figure 1F). The same P. capitalensis was reisolated in the inoculated leaves, confirming Koch’s postulates. Phyllosticta capitalensis has been reported to cause leaf spots or black spots on various host plants around the world (Wikee et al. 2013), including on oil palm (Nasehi et al. 2020), tea plant (Cheng et al. 2019 ), and castor (Tang et al. 2020). Nevertheless, to our knowledge, this is the first report of leaf spot caused by P. capitalensis on A. oxyphylla worldwide.