A novel bio-fertilizer, produced from prickly ash seeds (PAS), Bacillus subtilis and biochar, was evaluated for its disease-preventing potential on tobacco black shank caused by Phytophthora nicotianae. The results showed that biochar promoted the growth of Tpb55 in PAS and increased the pH of the organic fertilizer. The final concentration of B. subtilis could reach 1.7 × 10¹⁰ cfu g⁻¹ in the biological organic fertilizer (PBB) under the optimal medium under conditions of solid-state fermentation. PBB exhibited a strong fumigation effect on P. nicotianae, including inhibiting mycelium growth, reducing the disease severity and decreasing the pathogen population in rhizospheric soil. PBB treatment also could significantly increase the pH of acidified soil and improve soil nutrition content such as available K, alkali hydrolysable N and organic carbon. High-throughput pyrosequencing of 16S and 18S rRNA genes revealed that 4% PBB addition in soil had significant effects on the diversity and richness of fungi but not on that of bacteria. The microbial community structure was also shifted after PBB treatment. Some potentially beneficial microbes such as Bacillus, Mucor, Cunninghamella, Chitinophaga and Phenylobacterium were enriched, while potential pathogen Fusarium was significantly decreased. In conclusion, the agricultural waste PAS combined with biochar can replace soybean as a source for the production of biocontrol B. subtilis Tpb55, and the novel bio-fertilizer could effectively control tobacco black shank by pathogen inhibition, soil nutrient improvement and shifting the rhizomicrobial community.

Trichoderma spp., Metarhizium spp., and Bacillus spp. are commonly used as biocontrol microorganisms domestically and internationally. However, microbial pesticides currently prepared from single living microorganisms have problems such as a short shelf life, particularly under stressful environment conditions. Secondary metabolites produced from biocontrol microorganisms are comparatively stable when used under field conditions. This study screened the optimal combination of biocontrol metabolites, referred to as TMB, composed of culture filtrates from certain isolates of Trichoderma asperellum 10264, Bacillus subtilis S4-4-10, and Metarhizium anisopliae 3.11962 (1:4:1 (v/v)). RNA-seq analysis and transmission electron microscope observations were carried out to identify the major functions of the most effective culture filtrates against Magnaporthe oryzae (the pathogen causing rice blast disease) and Chilo suppressalis (an insect pest in rice cultivation). TMB was found to disrupt the midgut subcellular structure of C. suppressalis larvae and inhibit the expression of genes related to immunity, membrane components, protein synthesis, and other functions in C. suppressalis larvae and M. oryzae, thereby interfering with their normal growth, reproduction, and infection potential in rice. In addition, TMB was also able to promote rice growth and trigger host defense responses against infections by the target pests and pathogens. In summary, TMB generated different inhibitory activities against multiple targets in C. suppressalis and M. oryzae and induced plant immunity in rice. Therefore, it can be used as a new environmentally friendly agent or alternative to control rice pests and diseases.

The pathogenic plant fungus Bipolaris maydis is responsible for southern corn leaf blight (SCLB), a widespread agricultural disease that significantly reduces maize yield in various agroecological zones. The present research focuses on characterizing the role of Trichoderma harzianum cellobiohydrolase (CBH) Thph2 in induced maize resistance to SCLB by triggering the production of reactive oxygen species (ROS) in leaves. First of all, we demonstrated the potential activities of Thph2 in triggering ROS burst and PDC in a model plant, Nicotiana benthamiana. Cell death, ROS burst, and programmed cell death (PCD) were observed in N. benthamiana leaves following transient expression of Thph2, indicating its defensive role against Sclerotinia sclerotiorum infection. The removal of the signal peptide from Thph2 resulted in the complete loss of the cell death phenotype and the accumulation of reactive oxygen species (ROS), confirming that Thph2 functions as a microbial elicitor that primes host plant immunity through ROS-mediated signaling, thereby inducing systemic resistance (ISR). Furthermore, the Thph2 protein conferred resistance against B. maydis in maize, significantly increasing reactive oxygen species (ROS) accumulation (1.5-fold compared to the control) at 48 h post-inoculation (hpi),and leading to the reduction in the lesion area of SCLB by 15.9% at 2 days post-inoculation (dpi). Our results demonstrated that the Thph2 protein markedly enhanced the expression of lox5, aos, and hpl in maize leaves, thereby confirming its function in triggering plant defense mechanisms primarily via the jasmonic acid signaling pathway. This research reveals new molecular mechanisms by which T. harzianum enhances plant defense and showcases the biocontrol efficacy of Thph2 against southern corn leaf blight (SCLB).

Fusarium root rot (caused by Fusarium verticillioides) is a destructive soilborne disease in maize, significantly reducing crop yields. The root symbiotic fungi Trichoderma species have been confirmed as effective biocontrol microbes for Fusarium root rot; however, the mechanistic role of Trichoderma-induced endophytes in suppressing Fusarium root rot in maize remains unclear. This study found that Trichoderma harzianum T30 significantly reduced the abundance of pathogens by 48.9% and increased the abundance of potentially antagonistic Bacillus strains (33%) in the root endophytic bacterial community. In addition, the hyd1 gene in T. harzianum T30 induced a 7.5-fold upregulation of ZmOPR7 in maize roots compared to the Δhyd1 mutant treatment, a gene related to the jasmonic acid (JA) pathway. Further, several endophytic Bacillus strains were specifically induced by a hyd1-overexpressing strain, including B. amyloliquefaciens MX66, B. velezensis C9, and B. velezensis GAGAN3. Three endophytes significantly (p < 0.05) reduced Fusarium root rot incidence in maize by 46.6–55.0% and upregulated the expression of jasmonic acid/ethylene (JA/ET) pathway-related genes (ZmOPR7, ZmOPR8 and ZmEIL1) by 5.4-, 1.5-, and 4.6-fold, respectively, compared to untreated controls. Meanwhile, the Bacillus strain also improved maize plant growth. This study examined how overexpression of the T. harzianum elicitor gene hyd1 (in the OE-hyd1 strain) affects the colonization dynamics of beneficial endophytic bacteria in maize roots. Additionally, it further suggested the contribution of selected endophytic Bacillus strains in suppressing Fusarium root rot in maize.