A Novel Bio-Fertilizer Produced by Prickly Ash Seeds with Biochar Addition Induces Soil Suppressiveness against Black Shank Disease on Tobacco

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.

Whole Genome Sequence of Bacillus velezensis Strain GUMT319: A Potential Biocontrol Agent Against Tobacco Black Shank Disease

Phytophthora nicotianae causes black shank, a serious soil-borne disease, in tobacco. In this study, the Bacillus strain GUMT319 was isolated from the rhizosphere of healthy tobacco plants grown in a field in Guizhou with a high incidence of tobacco black shank. Genome sequencing revealed that GUMT319 contained a single circular chromosome 3,940,023 bp in length, with 4,053 predicted genes and an average GC content of 46.6%. Based on phylogenomic analyses, GUMT319 was designated as Bacillus velezensis. The genome of GUMT319 contained more than 60 genes and 13 gene clusters that have previously been found to be active in antifungal mechanisms, biofilm formation, and chemotaxis motility. Additionally, confocal laser scanning microscopy and scanning electron microscopy showed that GUMT319 formed a spatially organized biofilm in vivo. In addition, lauric acid negatively regulated biofilm formation. This is the first study to report that nicotine in tobacco root exudates was a chemoattractant for biocontrol Bacillus strains. In this study, we identified new interactions between beneficial microorganisms and tobacco roots in the rhizosphere. Moreover, dual culture tests in vitro showed that GUMT319 inhibited the growth of P. nicotianae and also displayed inhibitory effects against eight other plant pathogens, namely, Colletotrichum scovillei, Colletotrichum capsici, Fusarium carminascens, Sclerotinia sclerotiorum, Alternaria alternata, Phomopsis sp., Phyllosticta sorghina, and Exserohilum turcicum. Furthermore, GUMT319 exhibited > 70% control efficiency against tobacco black shank in field experiments conducted in 2018–2020. Thus, GUMT319 was more effective in controlling the incidence of tobacco black shank than other treatments including fungicide application. Overall, these results suggested that GUMT319 (B. velezensis) could be used as a potential biocontrol agent against tobacco black shank.

Prickly Ash Seed Kernel: A New Bio-Fumigation Material Against Tobacco Black Shank

The present study evaluated the fumigant effect and potential mechanisms of Chinese prickly ash seed kernel (PSK) against tobacco black shank caused by Phytophthora nicotianae Breda de Haan. The results showed that PSK fumigant increased the soil organic matter, hydrolysable nitrogen, available potassium, and total phosphorus significantly, while the pH was decreased. Application of 2% PSK promoted plant growth, with higher plant height, root length, and dry weight compared to those in the treatment with no PSK. Both in vitro and in vivo tests indicated that PSK fumigant effectively inhibited mycelial growth of P. nicotianae, and significantly reduced the severity of tobacco black shank. Based on the analysis of volatiles in PSK by headspace solid-phase microextraction (HS-SPME) gas chromatography-mass spectrometry (GC-MS), 28 components were identified, among which monoterpenes were the major components (accounting for 79.46% of peak area). Among the six top components, linalool, piperitone, sabinene, and limonene exhibited strong mycelial inhibition of P. nicotianae. Linalool was the most effective with an IC50 value of 18.03 μL/L. Results of GC-MS revealed that the main components of PSK extract were cis-Vaccenic acid (40.81%), n-Hexadecanoic acid (15.67%), 9,12-Octadecadienoic acid (Z,Z)- (15.25%), Ethanone-1-(2-hydroxy-4,6-dimethoxyphenyl)- (10.32%), cis-9-Hexadecenoic acid (6.21%), and Psilocin (11.75%). Among them, Ethanone-1-(2-hydroxy-4,6-dimethoxyphenyl)- and 9,12-Octadecadienoic acid (Z,Z)- have an obvious inhibitory effect on P. nicotianae. Additionally, changes in soil bacterial and fungal communities were observed. The relative abundance of some microbes, such as Pseudomonas, Azospirillum, Thermomonas, Glutamicibacter, Dyella, and Trichoderma, increased, which have many beneficial microbes in these genera, indicating that microbial community shift could be one of the important outcomes of the PSK fumigant effect. In conclusion, PSK could be a new bio-fumigation material against plant soilborne pathogens, which can function as both a fungicide and fertilizer.