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.

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.

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.

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.

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.

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.

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.

Comparative Transcriptome Analysis Reveals the Biocontrol Mechanism of Bacillus velezensis F21 Against Fusarium Wilt on Watermelon.

The watermelon (Citrullus lanatus) is one of the most important horticultural crops for fruit production worldwide. However, the production of watermelon is seriously restricted by one kind of soilborne disease, Fusarium wilt, which is caused by Fusarium oxysporum f. sp. niveum (Fon). In this study, we identified an efficient PGPR strain B. velezensis F21, which could be used in watermelon production for Fon control. The results of biocontrol mechanisms showed that B. velezensis F21 could suppress the growth and spore germination of Fon in vitro. Moreover, B. velezensis F21 could also enhance plant basal immunity to Fon by increasing the expression of plant defense related genes and activities of some defense enzymes, such as CAT, POD, and SOD. To elucidate the detailed mechanisms regulating B. velezensis F21 biocontrol of Fusarium wilt in watermelon, a comparative transcriptome analysis using watermelon plant roots treated with B. velezensis F21 or sterile water alone and in combination with Fon inoculation was conducted. The transcriptome sequencing results revealed almost one thousand ripening-related differentially expressed genes (DEGs) in the process of B. velezensis F21 triggering ISR (induced systemic resistance) to Fon. In addition, the Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment indicated that numerous of transcription factors (TFs) and plant disease resistance genes were activated and validated by using quantitative real-time PCR (qRT-PCR), which showed significant differences in expression levels in the roots of watermelon with different treatments. In addition, genes involved in the MAPK signaling pathway and phytohormone signaling pathway were analyzed, and the results indicated that B. velezensis F21 could enhance plant disease resistance to Fon through the above related genes and phytohormone signal factors. Taken together, this study substantially expands transcriptome data resources and suggests a molecular framework for B. velezensis F21 inducing systemic resistance to Fon in watermelon. In addition, it also provides an effective strategy for the control of Fusarium wilt in watermelon.

Evaluation of root-knot nematode disease control and plant growth promotion potential of biofertilizer Ning shield on Trichosanthes kirilowii in the field.

Biofertilizer Ning shield was composed of different strains of plant growth promotion bacteria. In this study, the plant growth promotion and root-knot nematode disease control potential on Trichosanthes kirilowii in the field were evaluated. The application of Ning shield significantly reduced the diseases severity caused by Meloidogyne incognita, the biocontrol efficacy could reached up to 51.08%. Ning shield could also promote the growth of T. kirilowii in the field by increasing seedling emergence, height and the root weight. The results showed that the Ning shield could enhance the production yield up to 36.26%. Ning shield could also promote the plant growth by increasing the contents of available nitrogen, phosphorus, potassium and organic matter, and increasing the contents of leaf chlorophyll and carotenoid pigment. Moreover, Ning shield could efficiently enhance the medicinal compositions of Trichosanthes, referring to the polysaccharides and trichosanthin. Therefore, Ning shield is a promising biofertilizer, which can offer beneficial effects to T. kirilowii growers, including the plant growth promotion, the biological control of root-knot disease and enhancement of the yield and the medicinal quality.

Whole-Genome Sequence of Bacillus cereus AR156, a Potential Biocontrol Agent with High Soilborne Disease Biocontrol Efficacy and Plant Growth Promotion.

Bacillus cereus AR156 was originally isolated from the forest soil of Zhenjiang, a city in China. To shed new light on the molecular mechanisms underlying the biological control of soilborne pathogens, the whole genome of this strain was sequenced. Here, we report the draft genome sequence of this strain, consisting of a single circularized contig measuring 5.66 Mb, with an average GC content of 35.5% and 5,367 open reading frames.

An improved strategy for stable biocontrol agents selecting to control rice sheath blight caused by Rhizoctonia solani.

Rice sheath blight caused by Rhizoctonia solani Kühnis increasingly threatening rice production in China. DNA fingerprints of 220 R. solani strains isolated in 11 provinces of China were established by random amplified polymorphic DNA (RAPD)-PCR. Cluster analysis of strains isolated from the same region showed high similarity, indicating that the genetic diversity of R. solani strains is significantly related to geographical origin. We assessed potential bio-control abilities of bio-control agents (BCAs) by values according to inhibition zones against R. solani, extracellular hydrolytic enzymes activity and siderophores production in vitro. Fourteen strains with diverse expected bio-control potential were tested for their bio-control efficacy against rice sheath blight caused by 11 pathogenic exemplars and for growth promoting ability, separately. Bio-control efficacy of single bacterium against various R. solani strains differed significantly (-36.23%∼88.24%), while Pseudomonas fluorescens 4aYN11 achieved a relatively stable control efficacy of 32.26%-78.79% and growth promotion of 18.43%. Pearson correlation coefficient between bio-control efficacy of each BCAs and their assessment is 0.717. In the present study, we established an improved strategy for screening stable bio-control agents based on an assessment system, their growth promotion potential and phylogenetic diversity of pathogen R. solani, and the result provides us not only one promising bio-control strain 4aYN11 with an average bio-control efficacy of 56.50%, but also a practical way for future screen of novel BCAs.

Bacillus cereus AR156 Extracellular Polysaccharides Served as a Novel Micro-associated Molecular Pattern to Induced Systemic Immunity to Pst DC3000 in Arabidopsis.

Non-host resistance (NHR) is a broad-spectrum plant defense. Upon colonizing on the surface on the root or leaves of non-host species, pathogens initial encounter preform and induce defense response in plant, such as induced hypersensitive response, PAMPs triggered immunity (PTI), and effector triggered immunity (ETI). The ability of plants to develop an induced systemic response (ISR) in reaction to the colonization by non-pathogenic rhizobacterium depends on interactions between host plants and the colonizing rhizobacterium, and the ISR also can be defined as a NHR. However, how the colonization signal is and how systemic resistance to pathogens is developed is still unclear. In this study, we demonstrated that the extracellular polysaccharides (EPSs) of Bacillus cereus AR156 could act as novel microbe-associated molecular patterns (MAMPs) and function in the early perception status of the ISR of B. cereus AR156. The results revealed that B. cereus AR156 EPS could induce systemic resistance to Pst DC3000 in Arabidopsis. Cellular defense response markers such as hydrogen peroxide accumulation, callose deposition, and defense-associated enzyme were induced upon challenge inoculation in the leaves primed by EPS. Moreover, the defense-related genes PR1, PR2, and PR5 and mitogen-activated kinases (MAPK) cascade marker gene MPK6 were concurrently expressed in the leaves of EPS-treated plants and induced higher resistance to Pst DC3000 in Col-0 than that in the jar1 or etr1 mutants. The protection was absent in the NahG transgenic plants and npr1 mutant, suggesting an activation of the salicylic acid (SA)- and the MAPK-dependent signaling pathways with NPR1-dependent by B. cereus AR156 EPS. In conclusion, B. cereus AR156 EPS play an important role in MAMP perception during the process of rhizobacteria-triggered NHR. This study is the first to illustrate how AR156 induces systemic resistance to Pst DC3000 in Arabidopsis. It also provides the first explanation of how plants perceive colonization of non-pathogenic bacteria and how rhizobacteria trigger ISR to plant pathogens.

Study on screening and antagonistic mechanisms of Bacillus amyloliquefaciens 54 against bacterial fruit blotch (BFB) caused by Acidovorax avenae subsp. citrulli.

Bacterial fruit blotch (BFB) was a serious threat to cucurbitaceae crops. It was caused by the gram-negative bacterium Acidovorax avenae subsp. citrulli. Two hundred strains, which have the potential in controlling plant diseases in our laboratory's biocontrol strain library, were employed to this research to screen some antagonistic bacteria, which can efficiently control bacterial fruit blotch disease. Based on the results of antagonistic activity experiments, greenhouse tests and field trials, 5 of the test strains have high abilities to control BFB. One of the 5 bacteria strains has the highest potential to control BFB named 54. The biocontrol efficacy of 54 was up to 60%. To characterize the strain, we used series of methods to evaluate the bacterium, including morphology analysis, physiological biochemical test and biomolecular assay. We found that the bacterium 54 belongs to the species Bacillus amyloliquefaciens. The colonization test results showed that 54 had the highest colonization levels, and the density of the strain on leaves was up 10(5)colony forming units (CFU) per gram of leaf tissue. Our recent results show that B. amyloliquefaciens 54 can promote the plant growth due to raised the contents of available N, P, K and the leaf chlorophyll. The antagonistic bacterium 54 can significantly control the BF B by increasing the expression level of defense-related gene PR1 and the accumulation the hydrogen peroxide in the plant. The results of trail experiment was also verified this efficient results of bacterium. This is also the first report of B. amyloliquefaciens strain that is able to control BFB.

Evaluation of root-knot nematode disease control and plant growth promotion potential of biofertilizer Ning shield on Trichosanthes kirilowii in the field

Abstract Biofertilizer Ning shield was composed of different strains of plant growth promotion bacteria. In this study, the plant growth promotion and root-knot nematode disease control potential on Trichosanthes kirilowii in the field were evaluated. The application of Ning shield significantly reduced the diseases severity caused by Meloidogyne incognita, the biocontrol efficacy could reached up to 51.08%. Ning shield could also promote the growth of T. kirilowii in the field by increasing seedling emergence, height and the root weight. The results showed that the Ning shield could enhance the production yield up to 36.26%. Ning shield could also promote the plant growth by increasing the contents of available nitrogen, phosphorus, potassium and organic matter, and increasing the contents of leaf chlorophyll and carotenoid pigment. Moreover, Ning shield could efficiently enhance the medicinal compositions of Trichosanthes, referring to the polysaccharides and trichosanthin. Therefore, Ning shield is a promising biofertilizer, which can offer beneficial effects to T. kirilowii growers, including the plant growth promotion, the biological control of root-knot disease and enhancement of the yield and the medicinal quality.