Antifungal activities of plant-beneficial Bacillus have been widely studied in recent years. Numerous studies have studied the antifungal mechanisms of soluble non-volatile bioactive compounds such as lipopeptides and proteins produced by Bacillus against soil-borne diseases. However, the antagonistic mechanisms of volatile organic compounds (VOCs) from Bacillus against airborne phytopathogens are still largely unknown, and the function of Alternaria solani pathogenic genes has not been well identified. Here, we first isolated a Bacillus strain with strong antifungal activity and finally identified it as B. subtilis ZD01. Then, the antagonistic mechanisms of VOCs produced by strain ZD01, against A. solani, an airborne fungal pathogen that can cause early blight diseases of potato, were studied. We showed that VOCs produced by strain ZD01 can reduce the colony size and mycelial penetration and can cause serious morphological changes of A. solani. Scanning electron microscope (SEM) observation showed that VOCs released by ZD01 could cause more flaccid and gapped hyphae of A. solani. Also, we found that VOCs produced by ZD01 can inhibit the conidia germination and reduce the lesion areas and number of A. solani in vivo significantly. Meanwhile, based on gas chromatography/mass spectrometry (GC/MS) analysis, 29 volatile compounds produced by strain ZD01 were identified. Out of 29 identified VOCs, 9 VOCs showed complete growth inhibition activities against A. solani. Moreover, we identified two virulence-associated genes (slt2 and sod) in A. solani. slt2 is a key gene that regulates the mycelial growth, penetration, sporulation, and virulence in vivo in A. solani. In addition, sod plays a significant role in the SOD synthetic pathway in A. solani. Results from qRT-PCR showed that the transcriptional expression of these two genes was down-regulated after being treated by VOCs produced by ZD01. These results are useful for a better understanding of the biocontrol mechanism of Bacillus and offer a potential method for potato early blight disease control.

Alternaria solani is an airborne fungus and the primary causal agent of potato early blight worldwide. No available fungicides that are both effective and environmentally friendly are usable to control this fungus. Therefore, biological control is a potential approach for its suppression. In this study, Bacillus subtilis strain ZD01’s fermentation broth strongly reduced A. solani pathogenicity under greenhouse conditions. The effects of strain ZD01’s secondary metabolites on A. solani were investigated. The exposure of A. solani hyphae to the supernatant resulted in swelling and swollen sacs, and the ZD01 supernatant reduced A. solani conidial germination significantly. Matrix-assisted laser desorption/ionization time of flight mass spectrometry and pure product tests revealed that fengycins were the main antifungal lipopeptide substances. To elucidate the molecular mechanism of the fengycins’ biological control, RNA sequencing analyses were performed. A transcriptome analysis revealed that 304 and 522 genes in A. solani were differentially expressed after 2-h and 6-h fengycin treatments, respectively. These genes were respectively mapped to 53 and 57 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. In addition, the most enriched KEGG pathway analysis indicated that the inhibitory mechanisms of fengycins against A. solani regulated the expression of genes related to cell wall, cell membrane, transport, energy process, protein synthesis and genetic information. In particular, cell wall and cell membrane metabolism were the main processes affected by fengycin stress. Scanning and transmission electron microscope results revealed hyphal enlargement and a wide range of abnormalities in A. solani cells after exposure to fengycins. Furthermore, fengycins induced chitin synthesis in treated cells, and also caused the capture of cellular fluorescent green labeling and the release of adenosine triphosphate (ATP) from outer membranes of A. solani cells, which may enhance the fengycins ability to alter cell membrane permeability. Thus, this study increases the transcriptome data resources available and supplies a molecular framework for B. subtilis ZD01 inhibition of A. solani HWC-168 through various mechanisms, especially damaging A. solani cell walls and membranes. The transcriptomic insights may lead to an effective control strategy for potato early blight.

Continuous cropping of the same crop leads to soil degradation and a decline in crop production, and these impacts could be mitigated through rotation cropping. Although crop rotation enhances soil fertility, microbial community diversity, and potato yield, its effects on the soil ecosystem multifunctionality (EMF) remain unclear. In the present research, we comparatively examined the effects of potato continuous cropping (PP) and rotation cropping [potato–oat rotation (PO) and potato–forage maize rotation (PFM)] on the soil EMF as well as the roles of keystone taxa, microbes abundance, and chemical properties in EMF improvement. It was demonstrated that soil EMF is increased in rotation cropping (PO and PFM) than PP. Soil pH was higher in rotation cropping (PO and PFM) than in PP, while total phosphorus (TP) and available phosphorus (AP) were significantly decreased than that in PP. Rotation cropping (PO and PFM) markedly changed the bacterial and fungal community compositions, and improved the potential plant-beneficial fungi, e.g., Schizothecium and Chaetomium, while reducing the abundances of the potentially phytopathogenic fungi, e.g., Alternaria, Fusarium, Verticillium dahiae, Gibberella, Plectosphaerella, Colletotrichum, Phoma, and Lectera in comparison with PP. Also, co-occurrence patterns for bacteria and fungi were impacted by crop rotation, and keystone taxa, e.g., Nitrospira.1, Lysinibacillus, Microlunatus.1, Sphingomonas.3, Bryobacter.1, Micromonospora, and Schizothecium, were enriched in PO and PFM than PP. The structural equation model (SEM) further demonstrated that cropping systems increased soil ecosystem multifunctionality through regulating SOM and keystone taxa (Schizothecium1), and keystone taxa were mediated by soil pH. This study suggested that rotation cropping might contribute to the improvement of soil ecosystem multifunctionality as well as the development of disease-suppressive soils in comparison with potato continuous cropping.

Plant rhizosphere-associated bacterial communities play key roles in affecting host health in response to diverse biotic stresses. Currently, the effect of continuous cropping of potato on soil bacterial communities and physiochemical parameters has not been well documented. Herein, we compared bacterial composition and diversity in rotationally and continuously (5, 10, and 30 years) cropped soils, and clarified the correlations between soil properties and the bacterial communities revealed by Illumina MiSeq sequencing. Our results demonstrated that Proteobacteria, Actinobacteria and Firmicutes were the predominant phyla in all the tested soil samples. While the abundance of Proteobacteria showed an increase, the abundance of Actinobacteria and Firmicutes displayed a reduction with the increase of continuous cropping years. At the genus level, as continuous cropping years increasing, the abundance of Pseudarthrobacter, Bacillus and Pseudomonas decreased, but the abundance of Rhodanobacte, Sphingobium, Mizugakiibacter and Devosia increased. Our results also demonstrated that the abundance of plant growth-promoting rhizobacteria in the rotationally cropped soil was significantly higher than that of continuously cropped soil. Furthermore, our results showed that soil organic matter, available nitrogen, available phosphorus and available potassium were significantly correlated with bacterial community distribution. Overall, our work provides a comprehensive view of altered structure and composition of bacterial communities between the continuously cropped soil and rotationally cropped soil.

The antagonistic mechanisms of soluble non-volatile bioactive compounds, such as proteins and lipopeptides emitted from Bacillus have been widely studied. However, there are limited studies on the antifungal mechanisms of volatile organic compounds (VOCs) produced by Bacillus against plant fungal diseases. In this study, the antagonistic mechanisms of one specific VOC, 6-methyl-2-heptanone, against Alternaria solani were investigated. To optimize the extraction conditions of headspace solid-phase microextraction, a 50/30-μm divinylbenzene/carboxen/polydimethylsiloxane fiber at 50°C for 40 min was used. For gas chromatography-mass spectrometry using a free fatty acid phase capillary column, 6-methyl-2-heptanone accounted for the highest content, at 22.27%, of the total VOCs from Bacillus subtilis ZD01, which inhibited A. solani mycelial growth strongly in vitro. Therefore, 6-methyl-2-heptanone was selected as the main active chemical to elucidate the action mechanisms against A. solani. Scanning and transmission electron microscopy analyses revealed that after exposure to an EC₅₀ dose of 6-methyl-2-heptanone, A. solani hyphal cells had a wide range of abnormalities. 6-Methyl-2-heptanone also caused the capture of cellular fluorescent green label and the release of adenosine triphosphate (ATP) from outer membranes A. solani cells, which may enhance 6-methyl-2-heptanone ability to reach the cytoplasmic membrane. In addition, 6-methyl-2-heptanone showed strong inhibitory effect on A. solani conidial germination. It also damaged conidial internal structures, with the treated group having collapsed shrunken small vesicles as observed by transmission electron microscopy. Because 6-methyl-2-heptanone showed strong effects on mycelial integrity and conidial structure, the expression levels of related pathogenic genes in A. solani treated with 6-methyl-2-heptanone were investigated. The qRT-PCR results showed that transcriptional expression levels of slt2 and wetA genes were strongly down-regulated after exposure to 6-methyl-2-heptanone. Finally, because identifying the functions of pathogenic genes will be important for the biological control of A. solani, the wetA gene was identified as a conidia-associated gene that plays roles in regulating sporulation yield and conidial maturation. These findings provide further insights into the mechanisms of VOCs secreted by Bacillus against A. solani.

Bacillus velezensis HN-Q-8, isolated in our previous study, has an antagonistic effect on Alternaria solani. After being pretreated with a fermentation liquid with HN-Q-8 bacterial cell suspensions, the potato leaves inoculated with A. solani displayed smaller lesion areas and less yellowing than the controls. Interestingly, the activity levels of superoxide dismutase, peroxidase, and catalase in potato seedlings were enhanced by the addition of the fermentation liquid with bacterial cells. Additionally, the overexpression of key genes related to induced resistance in the Jasmonate/Ethylene pathway was activated by the addition of the fermentation liquid, suggesting that the HN-Q-8 strain induced resistance to potato early blight. In addition, our laboratory and field experiments showed that the HN-Q-8 strain can promote potato seedling growth and significantly increase tuber yield. The root activity and chlorophyll content of potato seedlings were significantly increased along with the levels of indole acetic acid, gibberellic acid 3, and abscisic acid upon addition of the HN-Q-8 strain. The fermentation liquid with bacterial cells was more efficient in inducing disease resistance and promoting growth than bacterial cell suspensions alone or the fermentation liquid without bacterial cells. Thus, the B. velezensis HN-Q-8 strain is an effective bacterial biocontrol agent, augmenting the options available for potato cultivation.