Long small RNA76113 targets CYCLIC NUCLEOTIDE-GATED ION CHANNEL 5 to repress disease resistance in rice.

Small RNAs are widely involved in plant immune responses. However, the role of long small RNAs (25 to 40 nt) in monocot plant disease resistance is largely unknown. Here, we identified a long small RNA (lsiR76113) from rice (Oryza sativa) that is downregulated by Magnaporthe oryzae infection and targets a gene encoding CYCLIC NUCLEOTIDE-GATED CHANNEL 5 (CNGC5). The cngc5 mutant lines were more susceptible to M. oryzae than the wild type, while knocking down lsiR76113 in transgenic rice plants promoted pathogen resistance. A protoplast transient expression assay showed that OsCNGC5 promotes Ca2+ influx. These results demonstrate that OsCNGC5 enhances rice resistance to rice blast by increasing the cytosolic Ca2+ concentration. Importantly, exogenous Ca2+ application enhanced rice M. oryzae resistance by affecting reactive oxygen species (ROS) production. Moreover, cngc5 mutants attenuated the PAMP-triggered immunity response, including chitin-induced and flg22-induced ROS bursts and protein phosphorylation in the mitogen-activated protein kinase cascade, indicating that OsCNGC5 is essential for PAMP-induced calcium signaling in rice. Taken together, these results suggest that lsiR76113-mediated regulation of Ca2+ influx is important for PTI responses and disease resistance in rice.

Long-Term Field Application of a Plant Growth-Promoting Rhizobacterial Consortium Suppressed Root-Knot Disease by Shaping the Rhizosphere Microbiota.

Root-knot nematodes (Meloidogyne spp.) are one of the most economically important plant parasitic nematodes, infecting almost all cultivated plants and resulting in severe yield losses every year. Plant growth-promoting rhizobacteria (PGPR) have been extensively used to prevent and control root-knot diseases and increase yield. In this study, the effect of a consortium of three PGPR strains (Bacillus cereus AR156, B. subtilis SM21, and Serratia sp. XY21; hereafter "BBS") on root-knot disease of cucumber was evaluated. The application of BBS significantly reduced the severity of root-knot disease by 56 to 72%, increased yield by 36 to 55%, and improved fruit quality by 14 to 90% and soil properties by 1 to 90% relative to the control in the cucumber fields of the Nanjing suburb, Jiangsu Province, from 2015 to 2018. BBS altered the rhizosphere bacterial community. Compared with the control group, it significantly (false discovery rate, P < 0.05) increased the abundance of 14 bacterial genera that were negatively correlated with disease severity. Additionally, the redundancy analysis suggested that BBS-treated rhizosphere soil samples were dominated by disease-suppressive bacteria, including the genera Iamia, Kutzneria, Salinibacterium, Mycobacterium, Kribbella, Pseudonocardia, Sporichthya, Sphaerisporangium, Actinomadura, Flavisolibacter, Phenylobacterium, Bosea, Hyphomicrobium, Agrobacterium, Sphingomonas, and Nannocystis, which were positively related to total organic carbon, total nitrogen, total organic matter, dissolved organic carbon, [Formula: see text]-N, and available phosphorus contents. This suggests that BBS suppresses root-knot nematodes and improves the soil chemical properties of cucumber by altering the rhizosphere microbial community.

First report of leaf rot on Lonicera japonica caused by Rhizopus arrhizus in China.

Lonicera japonica is a perennial shrub that has been used since ancient times as a medicine to clear heat and detoxify poisons. Its branches (the vine of L. japonica) and unopened flower buds (honeysuckle) can be used as medicine to treat external wind heat or febrile disease fever (Shang, Pan, Li, Miao, & Ding, 2011). In July 2022, a serious disease was observed in L. japonica individuals planted in an area of experimental base of Nanjing Agricultural University (N 32°02', E 118°86'), Nanjing, Jiangsu Province, China. More than 200 Lonicera plants were surveyed, and the incidence of leaf rot in Lonicera leaves was over 80%. The initial symptoms were of chlorotic spots and gradual development of visible white mycelia and powdery substances (fungal spores) were observed on the leaves. Both the front and back of the leaves gradually appeared as brown diseased spots. Thus, a combination of multiple disease spots causes leaf wilting and the leaves eventually fall off. Leaves with typical symptoms were collected and cut into approximately 5 mm square fragments. The tissues were sterilized in 1% NaOCl for 90 s and 75% ethanol for 15 s and then washed with sterile water three times. The treated leaves were cultured on Potato Dextrose Agar (PDA) medium at 25℃. When mycelia grew around the leaf pieces, fungal plugs were collected along the outer edge of the colony and transferred to fresh PDA plates using a cork borer. Eight fungal strains with the same morphology were obtained after three rounds of subculturing. The colony was initially white with a fast growth rate, and occupied a 9-cm-diameter culture dish within 24 h. The colony turned gray-black in the later stages. After 2 days, small black sporangia spots appeared on top of the hyphae. The sporangia were yellow when immature, and black at maturity. The spores were oval with an average size of 29.6 (22.4-36.9) × 35.3 (25.8-45.2) µm (n = 50) in diameter. To identify the pathogen, fungal hyphae were scraped, and the fungal genome was extracted using a kit (BioTeke, Cat#DP2031). The internal transcribed spacer region (ITS) of the fungal genome was amplified with primers ITS1/ITS4, and the results of ITS sequencing were uploaded to the GenBank database with accession number OP984201. The phylogenetic tree was constructed using the neighbor-joining method with MEGA11 software. Phylogenetic analysis based on ITS showed that the fungus was grouped together with Rhizopus arrhizus (MT590591) and had high bootstrap support. Thus, the pathogen was identified as R. arrhizus. To verify Koch's postulates, 60 ml of a spore suspension (1×104 conidia/ml) was sprayed onto the surface of 12 healthy Lonicera plants, and the other 12 plants were sprayed with sterile water as a control. All plants were kept in the greenhouse at 25°C with 60% relative humidity. After 14 d, the infected plants showed symptoms similar to those of the original diseased plants. The strain was isolated again from the diseased leaves of artificially inoculated plants and verified as the original strain by sequencing. The results showed that R. arrhizus was the pathogen responsible for Lonicera leaf rot. Previous studies have shown that R. arrhizus causes garlic bulb rot (Zhang et al., 2022) and Jerusalem artichoke tuber rot (Yang et al., 2020). To our knowledge, this is the first report of R. arrhizus causing Lonicera leaf rot disease in China. Information regarding the identification of this fungus may be helpful for controlling the leaf rot disease.

Kurstakin Triggers Multicellular Behaviors in Bacillus cereus AR156 and Enhances Disease Control Efficacy Against Rice Sheath Blight.

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.

Bacillus-Secreted Oxalic Acid Induces Tomato Resistance Against Gray Mold Disease Caused by Botrytis cinerea by Activating the JA/ET Pathway.

Bacillus spp. are known for their ability to control plant diseases; however, the mechanism of disease control by Bacillus spp. is still unclear. Previously, bacterial organic acids have been implicated in the process of disease suppression. We extracted the total organic acid from Bacillus cereus AR156 culture filtrate and identified oxalic acid (OA) as the programmed cell death-inducing factor. OA strongly suppressed the lesion caused by Botrytis cinerea without significant antagonism against the fungus. Low concentration of OA produced by Bacillus spp. inhibited cell death caused by high concentrations of OA in a concentration- and time-dependent manner. Pretreatment with a low concentration of OA led to higher accumulation of active oxygen-scavenging enzymes in tomato leaves and provoked the expression of defense-related genes. The activation of gene expression relied on the jasmonic acid (JA) signaling pathway but not the salicylic acid (SA) pathway. The disease suppression capacity of OA was confirmed on wild-type tomato and its SA accumulation-deficient line, while the control effect was diminished in JA synthesis-deficient mutant, suggesting that the OA-triggered resistance relied on JA and ethylene (ET) signaling transduction. OA secretion ability was widely distributed among the tested Bacillus strains and the final environmental OA concentration was under strict regulation by a pH-sensitive degradation mechanism. This study provides the first systematic analysis on the role of low-concentration OA secreted and maintained by Bacillus spp. in suppression of gray mold disease and determines the dependence of OA-mediated resistance on the JA/ET signaling pathway. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2022.

Mitogen-Activated Protein Kinases Associated Sites of Tobacco Repression of Shoot Growth Regulates Its Localization in Plant Cells.

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.

AtMC1 Associates With LSM4 to Regulate Plant Immunity Through Modulating Pre-mRNA Splicing.

Alternative splicing of pre-mRNAs is an important gene regulatory mechanism shaping the transcriptome. AtMC1 is an Arabidopsis thaliana type I metacaspase that positively regulates the hypersensitive response. Here, we found that AtMC1 is involved in the regulation of plant immunity to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 and is physically associated with Sm-like4 (LSM4), which is involved in pre-mRNA splicing. AtMC1 and LSM4 protein levels both increased with their coexpression as compared with their separate expression in vivo. Like AtMC1, LSM4 negatively regulates plant immunity to P. syringae pv. tomato DC3000 infection. By RNA sequencing, AtMC1 was shown to modulate the splicing of many pre-mRNAs, including 4CL3, which is a negative regulator of plant immunity. Thus, AtMC1 plays a regulatory role in pre-mRNA splicing, which might contribute to AtMC1-mediated plant immunity.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Insights into the mechanism of the effects of rhizosphere microorganisms on the quality of authentic Angelica sinensis under different soil microenvironments.

BACKGROUND: Angelica sinensis (Oliv.) Diels (A. sinensis) is a Chinese herb grown in different geographical locations. It contains numerous active components with therapeutic value. Rhizosphere microbiomes affect various aspects of plant performance, such as nutrient acquisition, growth and development and plant diseases resistance. So far, few studies have investigated how the microbiome effects level of active components of A. sinensis. This study investigated whether changes in rhizosphere microbial communities and metabolites of A. sinensis vary with the soil microenvironment. Soils from the two main A. sinensis-producing areas, Gansu and Yunnan Province, were used to conduct pot experiments. The soil samples were divided into two parts, one part was sterilized and the other was unsterilized planting with the seedling variety of Gansu danggui 90-01. All seedlings were allowed to grow for 180 days. At the end of the experiment, radix A. sinensis were collected and used to characterize growth targets and chemical compositions. Rhizosphere soils were subjected to microbial analyses. RESULTS: Changes in metabolic profiles and rhizosphere microbial communities of A. sinensis grown under different soil microenvironments were similar. The GN (Gansu non-sterilized), YN (Yunnan non-sterilized), GS (Gansu sterilized), and YS (Yunnan sterilized) groups were significantly separated. Notably, antagonistic bacteria such as Sphingomonas, Pseudomonas, Lysobacter, Pseudoxanthomonas, etc. were significantly (p < 0.05) enriched in Gansu soil compared with Yunnan soil. Moreover, senkyunolide I and ligustilide dimers which were enriched in GS group were strongly positively correlated with Pseudomonas parafulva; organic acids (including chlorogenic acid, dicaffeoylquinic acid and 5-feruloylquinic acid) and their ester coniferyl ferulate which were enriched in YS Group were positively associated with Gemmatimonadetes bacterium WY71 and Mucilaginibater sp., respectively. CONCLUSIONS: The soil microenvironment influences growth and level/type of active components in A. sinensis. Further studies should explore the functional features of quality-related bacteria, identify the key response genes and clarify the interactions between genes and soil environments. This will reveal the mechanisms that determine the quality formation of genuine A. sinensis.

Function of miR825 and miR825* as Negative Regulators in Bacillus cereus AR156-elicited Systemic Resistance to Botrytis cinerea in Arabidopsis thaliana.

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.

Biofilms Positively Contribute to Bacillus amyloliquefaciens 54-induced Drought Tolerance in Tomato Plants.

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.

The spo0A-sinI-sinR Regulatory Circuit Plays an Essential Role in Biofilm Formation, Nematicidal Activities, and Plant Protection in Bacillus cereus AR156.

The rhizosphere bacterium Bacillus cereus AR156 is capable of forming biofilms, killing nematodes, and protecting plants. However, the underlying molecular mechanisms of these processes are not well understood. In this study, we found that the isogenic mutants ΔBcspo0A and ΔBcsinI have significantly reduced colonization and nematicidal activity in vitro and biological control efficacy on the tomato plant under greenhouse conditions. We further investigated the role of the spo0A-sinI-sinR regulatory circuit in biofilm formation, killing against nematodes, and biological control in AR156. Results from mutagenesis of those regulatory genes in AR156 and their heterologous expression in B. subtilis suggested that the spo0A-sinI-sinR genetic circuit is not only essential for biofilm formation and cell differentiation in AR156 but also able to functionally replace their counterparts in B. subtilis in a nearly indistinguishable fashion. Genome-wide transcriptional profiling in the wild type and the ΔBcspo0A and ΔBcsinI mutants further revealed hundreds of differentially expressed genes, likely positively regulated by both Spo0A and SinI (via SinR) in AR156. Among them, 29 genes are predicted to be directly controlled by SinR, whose counterpart in B. subtilis is a biofilm master repressor. Collectively, our studies demonstrated the essential role of the spo0A-sinI-sinR regulatory circuit in biofilm formation, cell differentiation, and bacteria-host interactions in B. cereus AR156.

Transcription factors WRKY70 and WRKY11 served as regulators in rhizobacterium Bacillus cereus AR156-induced systemic resistance to Pseudomonas syringae pv. tomato DC3000 in Arabidopsis.

The activation of both the SA and JA/ETsignalling pathways may lead to more efficient general and broad resistance to Pst DC3000 by non-pathogenic rhizobacteria. However, the mechanisms that govern this simultaneous activation are unclear. Using Arabidopsis as a model system, two transcription factors, WRKY11 and WRKY70, were identified as important regulators involved in Induced Systemic Resistance (ISR) triggered by Bacillus cereus AR156. The results revealed that AR156 treatment significantly stimulated the transcription of WRKY70, but suppressed that of WRKY11 in Arabidopsis leaves. Furthermore, they were shown to be required for AR156 enhancing the activation of cellular defence responses and the transcription level of the plant defence response gene. Overexpression of the two transcription factors in Arabidopsis also showed that they were essential for AR156 to elicit ISR. AR156-triggered ISR was completely abolished in the double mutant of the two transcription factors, but still partially retained in the single mutants, indicating that the regulation of the two transcription factors depend on two different pathways. The target genes of the two transcription factors and epistasis analysis suggested that WRKY11 regulated AR156-triggered ISR through activating the JA signalling pathway, and WRKY70 regulated the ISR through activating the SA signalling pathway. In addition, both WRKY11 and WRKY70 modulated AR156-triggered ISR in a NPR1-dependent manner. In conclusion, WRKY11 and WRKY70 played an important role in regulating the signalling transduction pathways involved in AR156-triggered ISR. This study is the first to illustrate the mechanism by which a single rhizobacterium elicits ISR by simultaneously activating both the SA and JA/ET signalling pathways.

Bacillus cereus AR156 primes induced systemic resistance by suppressing miR825/825* and activating defense-related genes in Arabidopsis.

Small RNAs play an important role in plant immune responses. However, their regulatory function in induced systemic resistance (ISR) is nascent. Bacillus cereus AR156 is a plant growth-promoting rhizobacterium that induces ISR in Arabidopsis against bacterial infection. Here, by comparing small RNA profiles of Pseudomonas syringae pv. tomato (Pst) DC3000-infected Arabidopsis with and without AR156 pretreatment, we identified a group of Arabidopsis microRNAs (miRNAs) that are differentially regulated by AR156 pretreatment. miR825 and miR825* are two miRNA generated from a single miRNA gene. Northern blot analysis indicated that they were significantly downregulated in Pst DC3000-infected plants pretreated with AR156, in contrast to the plants without AR156 pretreatment. miR825 targets two ubiquitin-protein ligases, while miR825* targets toll-interleukin-like receptor (TIR)-nucleotide binding site (NBS) and leucine-rich repeat (LRR) type resistance (R) genes. The expression of these target genes negatively correlated with the expression of miR825 and miR825*. Moreover, transgenic plants showing reduced expression of miR825 and miR825* displayed enhanced resistance to Pst DC3000 infection, whereas transgenic plants overexpressing miR825 and miR825* were more susceptible. Taken together, our data indicates that Bacillus cereus AR156 pretreatment primes ISR to Pst infection by suppressing miR825 and miR825* and activating the defense related genes they targeted.

Bacillus cereus NJ01 induces SA- and ABA-mediated immunity against bacterial pathogens through the EDS1-WRKY18 module.

Emerging evidence suggests a beneficial role of rhizobacteria in ameliorating plant disease resistance in an environment-friendly way. In this study, we characterize a rhizobacterium, Bacillus cereus NJ01, that enhances bacterial pathogen resistance in rice and Arabidopsis. Transcriptome analyses show that root inoculation of NJ01 induces the expression of salicylic acid (SA)- and abscisic acid (ABA)-related genes in Arabidopsis leaves. Genetic evidence showed that EDS1, PAD4, and WRKY18 are required for B. cereus NJ01-induced bacterial resistance. An EDS1-PAD4 complex interacts with WRKY18 and enhances its DNA binding activity. WRKY18 directly binds to the W box in the promoter region of the SA biosynthesis gene ICS1 and ABA biosynthesis genes NCED3 and NCED5 and contributes to the NJ01-induced bacterial resistance. Taken together, our findings indicate a role of the EDS1/PAD4-WRKY18 complex in rhizobacteria-induced disease resistance.

Small RNAs: Efficient and miraculous effectors that play key roles in plant-microbe interactions.

Plants' response to pathogens is highly complex and involves changes at different levels, such as activation or repression of a vast array of genes. Recently, many studies have demonstrated that many RNAs, especially small RNAs (sRNAs), are involved in genetic expression and reprogramming affecting plant-pathogen interactions. The sRNAs, including short interfering RNAs and microRNAs, are noncoding RNA with 18-30 nucleotides, and are recognized as key genetic and epigenetic regulators. In this review, we summarize the new findings about defence-related sRNAs in the response to pathogens and our current understanding of their effects on plant-pathogen interactions. The main content of this review article includes the roles of sRNAs in plant-pathogen interactions, cross-kingdom sRNA trafficking between host and pathogen, and the application of RNA-based fungicides for plant disease control.

Plant Disease Resistance-Related Pathways Recruit Beneficial Bacteria by Remodeling Root Exudates upon Bacillus cereus AR156 Treatment.

The environmentally friendly biological control strategy that relies on beneficial bacterial inoculants to improve plant disease resistance is a promising strategy. Previously, it has been demonstrated that biocontrol bacteria treatments can change the plant rhizosphere microbiota but whether plant signaling pathways, especially those related to disease resistance, mediate the changes in rhizosphere microbiota has not been explored. Here, we investigated the complex interplay among biocontrol strains, plant disease resistance-related pathways, root exudates, rhizosphere microorganisms, and pathogens to further clarify the biocontrol mechanism of biocontrol bacteria by using plant signaling pathway mutants. Bacillus cereus AR156, which was previously isolated from forest soil by our laboratory, can significantly control tomato bacterial wilt disease in greenhouse and field experiments. Moreover, compared with the control treatment, the B. cereus AR156 treatment had a significant effect on the soil microbiome and recruited 35 genera of bacteria to enrich the rhizosphere of tomato. Among them, the relative rhizosphere abundance of nine genera, including Ammoniphilus, Bacillus, Bosea, Candidimonas, Flexivirga, Brevundimonas, Bordetella, Dyella, and Candidatus_Berkiella, was regulated by plant disease resistance-related signaling pathways and B. cereus AR156. Linear correlation analysis showed that the relative abundances of six genera in the rhizosphere were significantly negatively correlated with pathogen colonization in roots. These rhizosphere bacteria were affected by plant root exudates that are regulated by signaling pathways. IMPORTANCE Our data suggest that B. cereus AR156 can promote the enrichment of beneficial microorganisms in the plant rhizosphere by regulating salicylic acid (SA) and jasmonic acid (JA)/ethylene (ET) signaling pathways in plants, thereby playing a role in controlling bacterial wilt disease. Meanwhile, Spearman correlation analysis showed that the relative abundances of these beneficial bacteria were correlated with the secretion of root exudates. Our study reveals a new mechanism for SA and JA/ET signals to participate in the adjustment of plant resistance whereby the signaling pathways adjust the rhizosphere microecology by changing the root exudates and thus change plant resistance. On the other hand, biocontrol strains can utilize this mechanism to recruit beneficial bacteria by activating disease resistance-related signaling pathways to confine the infection and spread of pathogens. Finally, our data also provide a new idea for the in-depth study of biocontrol mechanisms.

Induced Systemic Resistance for Improving Plant Immunity by Beneficial Microbes.

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.

Comparative transcriptomics reveal different genetic adaptations of biofilm formation in Bacillus subtilis isolate 1JN2 in response to Cd2+ treatment.

Biofilm plays important roles in the life cycle of Bacillus species, such as promoting host and object surface colonization and resisting heavy metal stress. This study utilized transcriptomics to evaluate the impacts of cadmium on the components, morphology, and function of biofilms of Bacillus subtilis strain 1JN2. Under cadmium ion stress, the morphology of the B. subtilis 1JN2 biofilm was flattened, and its mobility increased. Moreover, differential gene expression analysis showed that the main regulator of biofilm formation, Spo0A, decreased in expression under cadmium ion stress, thereby inhibiting extracellular polysaccharide synthesis through the SinI/SinR two-component regulatory system and the AbrB pathway. Cadmium ion treatment also increased the SigD content significantly, thereby increasing the expression of the flagella encoding and assembly genes in the strain. This promoted poly-γ-glutamic acid production via the DegS/DegU two-component regulatory system and the conversion of biofilm extracellular polysaccharide to poly-γ-glutamic acid. This conferred cadmium stress tolerance in the strain. Additionally, the cadmium ion-mediated changes in the biofilm composition affected the colonization of the strain on the host plant root surface. Cadmium ions also induced surfactin synthesis. These findings illustrate the potential of Bacillus species as biocontrol strains that can mitigate plant pathogenic infections and heavy metal stress. The results also provide a basis for the screening of multifunctional biocontrol strains.

The plant growth-promoting rhizobacterium Bacillus cereus AR156 induces systemic resistance in Arabidopsis thaliana by simultaneously activating salicylate- and jasmonate/ethylene-dependent signaling pathways.

Bacillus cereus AR156 is a plant growth-promoting rhizobacterium that induces resistance against a broad spectrum of pathogens including Pseudomonas syringae pv. tomato DC3000. This study analyzed AR156-induced systemic resistance (ISR) to DC3000 in Arabidopsis ecotype Col-0 plants. Compared with mock-treated plants, AR156-treated ones showed an increase in biomass and reductions in disease severity and pathogen density in the leaves. The defense-related genes PR1, PR2, PR5, and PDF1.2 were concurrently expressed in the leaves of AR156-treated plants, suggesting simultaneous activation of the salicylic acid (SA)- and the jasmonic acid (JA)- and ethylene (ET)-dependent signaling pathways by AR156. The above gene expression was faster and stronger in plants treated with AR156 and inoculated with DC3000 than that in plants only inoculated with DC3000. Moreover, the cellular defense responses hydrogen peroxide accumulation and callose deposition were induced upon challenge inoculation in the leaves of Col-0 plants primed by AR156. Also, pretreatment with AR156 led to a higher level of induced protection against DC3000 in Col-0 than that in the transgenic NahG, the mutant jar1 or etr1, but the protection was absent in the mutant npr1. Therefore, AR156 triggers ISR in Arabidopsis by simultaneously activating the SA- and JA/ET-signaling pathways in an NPR1-dependent manner that leads to an additive effect on the level of induced protection.

Transcriptome and Biochemical Analysis Jointly Reveal the Effects of Bacillus cereus AR156 on Postharvest Strawberry Gray Mold and Fruit Quality.

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.