Bacillus subtilis NCD-2 demonstrates exceptional biocontrol potential against cotton Verticillium wilt. While previous studies have established its direct antifungal activity (e.g., inhibiting Verticillium dahliae mycelial growth and spore germination), our work reveals a novel mechanism: NCD-2 primes systemic resistance in cotton by activating plant immune-signaling pathways. Firstly, transcriptional profiling uncovered that NCD-2 triggers a defense response in roots analogous to V. dahliae infection, allowing cotton to maintain a more balanced state when confronted with pathogen attacks. Meanwhile, the mutant strains ∆fen and ∆srf—defective in lipopeptide synthesis—also improved cotton resistance to Verticillium wilt by activating partially identical defense pathways in cotton roots. Furthermore, the application of lipopeptide compounds derived from NCD-2, particularly surfactin and fengycin, could enhance host resistance to V. dahliae. Using an RT-qPCR approach, we found that numerous resistance-related genes were induced by these lipopeptide compounds. The up-regulation of SA/JA pathway markers (e.g., NPR1, ICS1, COI1, and LOX1) revealed NCD-2’s activation of plant immune signaling. Using virus-induced gene silencing (VIGS), we conclusively linked SA and JA signaling to NCD-2-induced defense priming. Silencing either pathway abolished resistance, highlighting their indispensable coordination. By bridging mechanistic insights and agricultural applicability, our work positions NCD-2 as a sustainable alternative to conventional fungicides, addressing both crop productivity and environmental health.

Bacillus subtilis strain NCD-2 is a promising biocontrol agent for soil-borne plant diseases and shows potential for promoting the growth of some crops. The purposes of this study were to analyze the colonization ability of strain NCD-2 in different crops and reveal the plant growth promotion mechanism of strain NCD-2 by rhizosphere microbiome analysis. qRT-PCR was used to determine the populations of strain NCD-2, and microbial communities’ structures were analyzed through amplicon sequencing after application of strain NCD-2. Results demonstrated that strain NCD-2 had a good growth promotion effect on tomato, eggplant and pepper, and it was the most abundant in eggplant rhizosphere soil. There were significantly differences in the types of beneficial microorganisms recruited for different crops after application of strain NCD-2. PICRUSt analysis showed that the relative abundances of functional genes for amino acid transport and metabolism, coenzyme transport and metabolism, lipid transport and metabolism, inorganic ion transport and metabolism, and defense mechanisms were enriched in the rhizospheres of pepper and eggplant more than in the rhizospheres of cotton, tomato and maize after application of strain NCD-2. In summary, the colonization ability of strain NCD-2 for five plants was different. There were differences in microbial communities’ structure in rhizosphere of different plants after application of strain NCD-2. Based on the results obtained in this study, it was concluded that the growth promoting ability of strain NCD-2 were correlated with its colonization quantity and the microbial species it recruited.

Watermelon (Citrullus lanatus T.) is one of the most important economic crops in China. Soil-borne diseases are becoming more and more serious with longer growing seasons and continuous cropping of watermelon in greenhouses. In May 2020, symptoms were observed on plants in greenhouses located at Xingtai, Hebei province of China and included wilted leaves, chlorosis and plant death. Among the 26 greenhouses examined, symptomatic plants were observed in 17 greenhouses. The incidences of infected plants ranged from 1% to 35%, and caused an average 10% yield loss. Symptoms began on lower part of the plants and progressed upward to the vines and leaves. At the early stage of infection, the edge of watermelon leaves changed from green to yellow, and became soft. As the disease progressed, infected leaves wilted and desicated. The vascular tissue of the stem exhibited a uniform brown discoloration that often extended throughout the vine. To identify the causal agent, small pieces approximate 3.0×3.0 mm size of infected stem tissues were collected and sterilized with 0.5% sodium hypochlorite solution for 1 min, rinsed three times with sterile water and transferred onto potato dextrose agar (PDA) medium amended with 100 μg·mL-1 of chloramphenicol. The plates were incubated at 25°C for 3 days in the dark and fungal isolates were purified using the single-spore isolation method. A total of 22 fungal isolates with identical colony morphology were collected from diseased plants. The color of the fungal colonies on PDA medium was creamy-white with an abundance of mycelia that darken after 5 days growth due to the formation of microsclerotia. Fungal colonies consisted of fine, hyaline hyphae with verticillate conidiophores producing hyaline, ellipsoidal to oval conidia with an average size of 5.12×3.41 μm (n=50). The morphological characters of the fungal isolates were identical to those of Verticillium dahliae Kleb. described by Hawksworth and Talboys (Hawksworth, D. and Talboys, P, 1970). Pathogenicity tests were performed by soaking 30 watermelon seedlings with wounded root tips in the fungal conidial suspension (1x107 conidium/mL) for 30 min (Ma, et al, 2004). The same number of non-inoculated watermelon seedlings was used as a control. All plants were kept in a greenhouse at 25°C and 90%-95% relative humidity. Seven days post-inoculation (dpi), leaves of treated plants began to show symptoms of wilt. At 10-dpi, lower leaves wilted and dry and by 15-dpi, whole plants were dead. Pathogenicity tests were repeated three times with consistent results. The pathogen was re-isolated from the diseased plants and displayed identical morphological characteristics to the original isolates. To further identity the pathogens, the ribosomal DNA Internal Transcribed Spacer (rDNA-ITS) region was amplified by PCR (White et al., 1990; Liu et al., 1999; Bellemain et al.. 2010). The amplicon was sequenced and showed 99%-100% identity to the ITS region of the V. dahliae reference strains deposited in the NCBI database (MK093977.1, MK287620.1, MT348570.1 and LC549667.1, respectively). Based on morphological and ITS sequence information, the fungal pathogen was identified as V. dahliae. V. dahliae is an economically important pathogen with a wide host range worldwide. The discovery of Verticillium wilt on watermelons indicates that there might be a risk of Verticillium wilt when watermelons are planted in subsequent crops of the host plants of the disease, such as cotton or eggplant. To our knowledge, this is the first report of V. dahliae causing Verticillium wilt of watermelon in China. Financed: the Special Fund for Agro-scientific Research in the Public Interest, China (201503109) References: Hawksworth, D. and Talboys, P. 1970. Description of Pathogenic Fungi and Bacteria, CMI, Surrey. Ma, P., et al. 2004. A New Inoculation Method for Verticillium Wilt on Cotton and Its Application in Evaluating Pathogenesis and Host Resistance. Acta Phytopathologica Sinica, 34(6): 536-541. White, T. J., et al. 1990. Amplification and Direct Sequencing of Fungal Ribosomal RNA Genes for Phylogenetics. PCR protocols: a guide to methods and applications, 18(1), 315-322. Bellemain, E., et al. 2010. ITS as an Environmental DNA Barcode for Fungi: an in Silico Approach Reveals Potential PCR Biases. BMC microbiology, 10(1), 1-9. Liu, Y. J., et al. 1999. Phylogenetic Relationships Among Ascomycetes: Evidence from an RNA Polymerse II SubunitMol. Biol. Evol. 16:1799-1808.

Plant Growth Promoting Rhizobacteria, PGPR, can protect plants against soil-borne diseases and abiotic stress conditions. The primary objective of this study was to evaluate the effects of different PGPRs (TF1, TF2, TF3, and TF4) on the rhizosphere microbial community of silage maize in a saline–alkaline field via Illumina MiSeq high-throughput sequencing technology. Results demonstrated that different PGPRs significantly increased the harvest density (by 21.31–45.16%), plant height (by 9.12–19.98%), stem diameter (by 30.07–45.78%), and biomass (by 33.20–65.36%) of silage maize, TF3 treatment significantly increased the fresh weight (by 32.50%), while the other treatments could increase the fresh weight but not significantly. Four microbial agents significantly reduced the contents of soil available phosphorus (AP), electrical conductivity (EC), and neutral phosphatase activity (NPA), while significantly increasing the contents of available potassium (AK), ammonium nitrogen (NH₄⁺-N), nitrate nitrogen (NO₃⁻-N), chitinase activity (ChtA), and urease activity (UA). Specifically, TF2 and TF3 treatments significantly decreased the soil pH value, while not for TF1 and TF4. Microbiome analysis showed that four microbial agents significantly increased the relative abundances of beneficial microorganisms, such as Arthrobacter, Blastococcus, MNDI, Chaetomidium, Alternaria, Sarocladium, Acremonium, and Clonostachys, and significantly decreased the relative abundances of Gibberella and Fusarium. Mental analysis showed that the soil bacterial community structure did not significantly correlate with soil biochemical properties, while the soil fungal community structure significantly and positively correlated with pH. Maize yield significantly and positively correlated with NH₄⁺-N, OM, AP, EC, UA, ChtA, and NPA.