Jian-Hua Guo

Kurstakin is the latest discovered family of lipopeptides secreted by Bacillus spp. In this study, the effects of kurstakin on the direct antagonism, multicellularity, and disease control ability of Bacillus cereus AR156 were explored. An insertion mutation in the nonribosomal peptide synthase responsible for kurstakin synthesis led to a significant reduction of antagonistic ability of AR156 against the plant-pathogenic fungi Rhizoctonia solani, Ascochyta citrullina, Fusarium graminearum, and F. oxysporum f. sp. cubense. The loss of kurstakin synthesis ability significantly impaired the swarming motility of AR156 and reduced biofilm formation and amyloid protein accumulation. Although the loss of kurstakin synthesis ability did not reduce the competitiveness of AR156 under laboratory conditions, the colonization and environmental adaptability of the mutant was significantly weaker than that of wild-type AR156 on rice leaves. The cell surface of wild-type AR156 colonizing the leaf surface was covered by a thick biofilm matrix under a scanning electron microscope, but not the mutant. The colonization ability on rice roots and control efficacy against rice sheath blight disease of the mutant were also impaired. Thus, kurstakin participates in the control of plant diseases by B. cereus AR156 through directly inhibiting the growth of pathogenic fungi and improving long-term environmental adaptability and colonization of AR156 on the host surface by triggering multicellularity. This study explored the multiple functions of kurstakin in plant disease control by B. cereus.

Plant defense and growth rely on multiple transcriptional factors (TFs). Repression of shoot growth (RSG) is a TF belonging to a bZIP family in tobacco, known to be involved in plant gibberellin feedback regulation by inducing the expression of key genes. The tobacco calcium-dependent protein kinase CDPK1 was reported to interact with RSG and manipulate its intracellular localization by phosphorylating Ser-114 of RSG previously. Here, we identified tobacco mitogen-activated protein kinase 3 (NtMPK3) as an RSG-interacting protein kinase. Moreover, the mutation of the predicted MAPK-associated phosphorylation site of RSG (Thr-30, Ser-74, and Thr-135) significantly altered the intracellular localization of the NtMPK3-RSG interaction complex. Nuclear transport of RSG and its amino acid mutants (T30A and S74A) were observed after being treated with plant defense elicitor peptide flg22 within 5 min, and the two mutated RSG swiftly re-localized in tobacco cytoplasm within 30 min. In addition, triple-point mutation of RSG (T30A/S74A/T135A) mimics constant unphosphorylated status, and is predominantly localized in tobacco cytoplasm. RSG (T30A/S74A/T135A) showed no re-localization effect under the treatments of flg22, B. cereus AR156, or GA₃, and over-expression of this mutant in tobacco resulted in lower expression levels of downstream gene GA20ox1. Our results suggest that MAPK-associated phosphorylation sites of RSG regulate its localization in tobacco, and that constant unphosphorylation of RSG in Thr-30, Ser-74, and Thr-135 keeps RSG predominantly localized in cytoplasm.

Plant beneficial microorganisms improve the health and growth of the associated plants. Application of beneficial microbes triggers an enhanced resistance state, also termed as induced systemic resistance (ISR), in the host, against a broad range of pathogens. Upon the activation of ISR, plants employ long-distance systemic signaling to provide protection for distal tissue, inducing rapid and strong immune responses against pathogens invasions. The transmission of ISR signaling was commonly regarded to be a jasmonic acid- and ethylene-dependent, but salicylic acid-independent, transmission. However, in the last decade, the involvement of both salicylic acid and jasmonic acid/ethylene signaling pathways and the regulatory roles of small RNA in ISR has been updated. In this review, the plant early recognition, responsive reactions, and the related signaling transduction during the process of the plant–beneficial microbe interaction was discussed, with reflection on the crucial regulatory role of small RNAs in the beneficial microbe-mediated ISR.

Postharvest strawberry is susceptible to gray mold disease caused by Botrytis cinerea, which seriously damage the storage capacity of fruits. Biological control has been implicated as an effective and safe method to suppress plant disease. The aim of this study is to evaluate the postharvest disease control ability of Bacillus cereus AR156 and explore the response of strawberry fruit to this biocontrol microorganism. Bacillus cereus AR156 treatment significantly suppressed gray mold disease and postponed the strawberry senescence during storage. The bacterium pretreatment remarkably enhanced the reactive oxygen-scavenging and defense-related activities of enzymes. The promotion on the expression of the encoding-genes was confirmed by quantitative real-time PCR (qRT-PCR) that significantly increased the expression of the marker genes of salicylic acid (SA) signaling pathway, such as PR1, PR2, and PR5, instead of that of the jasmonic acid (JA)/ethylene (ET) pathway, which was also shown. Moreover, through transcriptome profiling, about 6,781 differentially expressed genes (DEGS) in strawberry upon AR156 treatment were identified. The gene ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment indicated that AR156 altered the transcription of numerous transcription factors and genes involved in the SA-related plant disease resistance, metabolism, and biosynthesis of benzoxazinoids and flavonoids. This study offered a non-antagonistic Bacillus as a method for postharvest strawberry storage and disease control, and further revealed that the biocontrol effects were arisen from the induction of host responses on the transcription level and subsequent resistance-related substance accumulation.

Blueberry is an important agricultural crop with high nutritional, health, and economic value. Despite the well‐studied blueberry cultivation methods and soil requirements, little is known about how beneficial bacteria function in organic blueberry cultivation systems and their effects on acidic soils. In this study, a single bacteria Bacillus amyloliquefaciens JC65 and three biocontrol bacteria consortiums containing JC65 were applied to organic system. The effect of bacteria to blueberry growth, yield, fruit quality, and soil quality was investigated. A consortium of three mixed Bacillus (B. amyloliquefaciens JC65, B. licheniforims HS10 and B. subtilis 7ze3) showed the highest growth improvement efficiency. The bacterial inoculation increased blueberry leaf chlorophyll content, net photosynthetic rate by 21.50%, 13.21% at 30 days, and increased average plant height by 2.72% at 69 days. Compared with the control, the inoculated plants showed an increased yield of 14.56%. Interestingly, blueberry fruit quality was also improved with supplement of the bacterial consortium. Fruit anthocyanin, soluble sugar, vitamin C, soluble solids, and soluble protein content were increased by 5.99%, 4.21%, 17.31%, 2.41%, and 21.65%, respectively. Besides, beneficial bacterial consortium also enables sustainable agriculture by improving soil ammonium nitrogen and organic matter by 3.77% and 2.96% after blueberry planting. In conclusion, the combination of beneficial bacteria showed a synergistic activity in organic system to promote the blueberry yield, fruit quality, and soil nutrient preservation.

Small RNAs play an important role in plant innate immunity. However, their regulatory function in induced systemic resistance (ISR) triggered by plant growth‐promoting rhizobacteria remains unclear. Here, using Arabidopsis as a model system, one plant endogenous small RNA, miR472, was identified as an important regulator involved in the process of Bacillus cereus AR156 ISR against Pseudomonas syringae pv. tomato (Pst) DC3000. The results revealed that miR472 was down‐regulated with B. cereus AR156 treatment by comparing small RNA profiles and northern blot analysis of Arabidopsis with or without B. cereus AR156 treatment. Plants overexpressing miR472 showed higher susceptibility to Pst DC3000; by contrast, plant lines with miR472 knocked down/out showed the opposite. The transcriptome sequencing revealed thousands of differentially expressed genes in the transgenic plants. Target prediction showed that miR472 targets lots of coiled coil nucleotide‐binding site (NBS) and leucine‐rich repeat (LRR) type resistance genes and the expression of these targets was negatively correlated with the expression of miR472. In addition, transgenic plants with knocked‐out target genes exhibited decreased resistance to Pst DC3000 invasion. Quantitative reverse transcription PCR results indicated that target genes of miR472 were expressed during the process of B. cereus AR156‐triggered ISR. Taken together, our results demonstrate that the miR472‐mediated silencing pathway is an important regulatory checkpoint occurring via post‐transcriptional control of NBS‐LRR genes during B. cereus AR156‐triggered ISR in Arabidopsis.

Drought stress is a major obstacle to agriculture. Although many studies have reported on plant drought tolerance achieved via genetic modification, application of plant growth-promoting rhizobacteria (PGPR) to achieve tolerance has rarely been studied. In this study, the ability of three isolates, including Bacillus amyloliquefaciens 54, from 30 potential PGPR to induce drought tolerance in tomato plants was examined via greenhouse screening. The results indicated that B. amyloliquefaciens 54 significantly enhanced drought tolerance by increasing survival rate, relative water content and root vigor. Coordinated changes were also observed in cellular defense responses, including decreased concentration of malondialdehyde and elevated concentration of antioxidant enzyme activities. Moreover, expression levels of stress-responsive genes, such as lea, tdi65, and ltpg2, increased in B. amyloliquefaciens 54-treated plants. In addition, B. amyloliquefaciens 54 induced stomatal closure through an abscisic acid-regulated pathway. Furthermore, we constructed biofilm formation mutants and determined the role of biofilm formation in B. amyloliquefaciens 54-induced drought tolerance. The results showed that biofilm-forming ability was positively correlated with plant root colonization. Moreover, plants inoculated with hyper-robust biofilm (ΔabrB and ΔywcC) mutants were better able to resist drought stress, while defective biofilm (ΔepsA-O and ΔtasA) mutants were more vulnerable to drought stress. Taken altogether, these results suggest that biofilm formation is crucial to B. amyloliquefaciens 54 root colonization and drought tolerance in tomato plants.

Small RNAs function to regulate plant defense responses to pathogens. We previously showed that miR825 and miR825* downregulate Bacillus cereus AR156 (AR156)-triggered systemic resistance to Pseudomonassyringae pv. tomato DC3000 in Arabidopsis thaliana (Arabidopsis). Here, Northern blotting revealed that miR825 and miR825* were more strongly downregulated in wild type Arabidopsis Col-0 (Col-0) plants pretreated with AR156 than in nontreated plants upon Botrytis cinerea (B. cinerea) B1301 infection. Furthermore, compared with Col-0, transgenic plants with attenuated miR825 and miR825* expression were more resistant to B. cinerea B1301, yet miR825- and miR825*-overexpressing (OE) plants were more susceptible to the pathogen. With AR156 pretreatment, the transcription of four defense-related genes (PR1, PR2, PR5, and PDF1.2) and cellular defense responses (hydrogen peroxide production and callose deposition) were faster and stronger in miR825 and miR825* knockdown lines but weaker in their OE plants than in Col-0 plants upon pathogen attack. Also, AR156 pretreatment caused stronger phosphorylation of MPK3 and MPK6 and expression of FRK1 and WRKY53 genes upon B. cinerea B1301 inoculation in miR825 and miR825* knockdown plants than in Col-0 plants. Additionally, the assay of agrobacterium-mediated transient co-expression in Nicotiana benthamiana confirmed that AT5G40910, AT5G38850, AT3G04220, and AT5G44940 are target genes of miR825 or miR825*. Compared with Col-0, the target mutant lines showed higher susceptibility to B. cinerea B1301, while still expressing AR156-triggered induced systemic resistance (ISR). The two-way analysis of variance (ANOVA) revealed a significant (P < 0.01) interactive effect of treatment and genotype on the defense responses. Hence, miR825 and miR825*act as negative regulators of AR156-mediated systemic resistance to B. cinerea B1301 in Arabidopsis.

Beneficial microorganisms have been extensively used to make plants more resistant to abiotic and biotic stress. We previously identified a consortium of three plant growth-promoting rhizobacteria (PGPR) strains (Bacillus cereus AR156, Bacillus subtilis SM21, and Serratia sp. XY21; hereafter “BBS”) as a promising and environmentally friendly biocontrol agent. In this study, the effect of BBS on a soil-borne disease of sweet pepper was evaluated. Application of BBS significantly reduced the prevalence of phytophthora blight and improved fruit quality and soil properties relative to the control. BBS was able to alter the soil bacterial community: it significantly increased the abundances of Burkholderia, Comamonas, and Ramlibacter, which were negatively associated with disease severity, relative to the control. A redundancy analysis suggested that BBS-treated soil samples were dominated by Burkholderia, Comamonas, Ramlibacter, Sporichthya, Achromobacter, and Pontibacter; abundance of these genera was related to total organic carbon (TOC), total nitrogen (TN), ammonium nitrogen (AN), total potassium (TP), and available phosphorus (AP) contents. This suggests that BBS treatment shifted the microbe community to one that suppressed soil-borne disease and improved the soil chemical properties.

Application of plant growth‐promoting rhizobacteria (PGPR) has been considered as an environmentally friendly method for crop yield promotion as well as plant disease management. Efforts have been devoted to unraveling mechanisms involved in bacteria–plant and bacteria–pathogen interactions. However, little is known on the effect of the interaction among PGPR, soil, and plant. We compared growth and yield promotion capacity of biofertilizer Ning Shield, a consortium of bacterial preparation used as a biofertilizer (BF), and its mixture with compost of agricultural waste including spent substrate of Pleurotus ostreatus (SSP)/Volvariella volvacea (SSV), chicken manure (CM), and inorganic fertilizer (IOF) in a pepper field, respectively. The disease control efficacy, pepper fruit preservation time, and nutrients were also determined. Soil nutrient parameters including organic matter and available NPK of treatments were assayed before and after one growth season. All of the mixture of BF+organic compost treatment significantly enhanced the yield and quality of pepper fruit. Moreover, disease control capacity was promoted by the mixture of BF+organic compost, with BF+SSV reaching the highest control efficacy of 81% on 60th day after transplanting, and remaining 76% at the 105^th day. The BF+SSV treatment showed soil fertility retention ability with higher soil nutrient contents after one growth season of pepper. This study provides evidence that, when combined with organic fertilizers such as spent mushroom substrate compost, beneficial microbes have the ability to promote plant growth and yield as well as suppress plant disease by sustaining soil fertility through complex bacteria–soil–plant interaction.