Sustained growth promotion in arabidopsis with long-term exposure to the beneficial soil bacterium Bacillus subtilis (GB03)

Volatile emissions from the commercial growth promoting soil bacterium Bacillus subtilis (GB03) are effective in augmenting short-term growth, photosynthetic capacity and salt tolerance in Petri-dish grown arabidopsis seedlings. In contrast, the impact sustained GB03 volatile exposure on plant growth and development has yet to be examined. here is provided physical and physiological data establishing that bacterial volatiles induce long-term growth promotion, elevated photosynthetic capacity and iron accumulation, as well as delayed albeit higher seed count compared with water-treated control plants. Plants were grown unrestricted in double Magenta boxes containing solid MS media for up to twelve weeks with GB03 volatiles introduced in separate containers within the chamber so that plant bacterial interactions were only by air-borne transmission. These results establish that GB03 volatiles induce sustained beneficial effects on Arabidopsis growth including robust and extended vegetative growth followed by elevated seed set.Key words: Bacillus subtilis (GB03), photosynthetic efficiency, plant growth promoting rhizobacteria (PGPR), seed set, volatile organic compound (VOCs)     

GC-MS SPME profiling of rhizobacterial volatiles reveals prospective inducers of growth promotion and induced systemic resistance in plants

Chemical and plant growth studies of Bacilli strains GB03 and IN937a revealed that the volatile components 2,3-butanediol and acetoin trigger plant growth promotion in Arabidopsis. Differences in growth promotion when cytokinin-signaling mutants are exposed to GB03 versus IN937a volatiles suggest a divergence in chemical signaling for these two bacterial strains. To provide a comprehensive chemical profile of bacterial volatiles emitted from these biologically active strains, headspace solid phase microextraction (SPME) coupled with software extraction of overlapping GC-separated components was employed. Ten volatile metabolites already reported from GB03 and IN937a were identified as well as 28 compounds not previously characterized. Most of the newly identified compounds were branched-chain alcohols released from IN937a, at much higher levels than in GB03. Principal component analysis clearly separated GB03 from IN937a, with GB03 producing higher amounts of 3-methyl-1-butanol, 2-methyl-1-butanol and butane-1-methoxy-3-methyl. The branched-chain alcohols share a similar functional motif to that of 2,3-butanediol and may afford alternative structural patterns for elicitors from bacterial sources.     

One shot-two pathogens blocked: Exposure of Arabidopsis to hexadecane,a long chain volatile organic compound,confers induced resistance against both Pectobacterium carotovorum and Pseudomonas syringae

Bacteria and plant derived volatile organic compounds have been reported as the chemical triggers that elicit induced resistance in plants. Previously, volatile organic compounds (VOCs), including acetoin and 2,3-butanediol, were found to be emitted from plant growth-promoting rhizobacteria (PGPR) Bacillus subtilis GB03, which had been shown to elicit ISR and plant growth promotion. More recently, we reported data that stronger induced resistance could be elicited against Pseudomonas syringae pv maculicola ES4326 in plants exposed to C13 VOC from another PGPR Paenibacillus polymyxa E681 compared with that of strain GB03. Here, we assessed whether another long hydrocarbon C16 hexadecane (HD) conferred protection to Arabidopsis from infection of a biotrophic pathogen, P. syringae pv maculicola and a necrotrophic pathogen, Pectobacterium carotovorum subsp carotovorum. Collectively, long-chain VOCs can be linked to a plant resistance activator for protecting plants against both biotrophic and necrotrophic pathogens at the same time.     

Assessment of root-associated paenibacillus polymyxa groups on growth promotion and induced systemic resistance in pepper

Twenty-nine P. polymyxa strains isolated from rhizospheres of various crops were clustered into five genotypic groups on the basis of BOX-PCR analysis. The characteristics of several plant growth-promoting factors among the isolates revealed the distinct attributes in each allocated group. Under gnotobiotic conditions, inoculation of pepper roots with P. polymyxa isolates significantly increased the biomass in 17 of total 29 treated plants with untreated plants. Experiments on induced systemic resistance (ISR) against bacterial spot pathogen Xanthomonas axonopodis pv. vesicatoria in pepper by P. polymyxa strains were conducted and only one isolate (KNUC265) was selected. Further studies into ISR mediation by the KNUC265 strain against the soft-rot pathogen Erwinia carotovora subsp. carotovora in tobacco demonstrated that the tobacco seedlings exposed to either bacterial volatiles or diffusible metabolites exhibited a reduction in disease severity. In conclusion, ISR and plant growth promotion triggered by P. polymyxa isolates were systemically investigated on pepper for the first time. The P. polymyxa KNUC265 strain, which elicited both ISR and plant growth promotion, could be potentially used in improving the yield of pepper and possibly of other crops.     

Protecting tobacco plants from O3 injury by Bacillus velezensis with production of acetoin

Plant growth-promoting rhizobacteria (PGPRs) confer benefits to crops by producing volatile organic compounds (VOCs) to trigger induced systemic tolerance (IST). Here we show that Bacillus velezensis GJ11, a kind of PGPRs, produce VOCs such as 2,3-butanediol and acetoin to trigger IST and cause stomatal closure against O₃ injury in tobacco plants. Compared to 2,3-butanediol, acetoin was more effective on triggering IST against O₃ injury. The bdh-knockout strain GJ11Δbdh with a blocked metabolic pathway from acetoin to 2,3-butanediol produced more acetoin triggering stronger IST against O₃ injury than GJ11. Both acetoin and GJ11Δbdh effectively enhance the antioxidant enzymes activity (e.g. superoxide dismutase and catalases) that is favorable for scavenging the reactive oxygen species like H₂O₂ in leaves after exposure to O₃. Consequently, less H₂O₂ accumulation was observed, and reasonably less chlorophylls and proteins were damaged by H₂O₂ in the tobacco leaves treated with acetoin or GJ11Δbdh. The field experiment also showed that both acetoin and GJ11Δbdh could protect tobacco plants from O₃ injury after application by root-drench. This study provides new insights into the role of rhizobacterial B. velezensis and its volatile component of acetoin in triggering defense responses against stresses such as O₃ in plants.     

Induced systemic resistance (ISR) in plants: mechanism of action

Plants possess a range of active defense apparatuses that can be actively expressed in response to biotic stresses (pathogens and parasites) of various scales (ranging from microscopic viruses to phytophagous insect). The timing of this defense response is critical and reflects on the difference between coping and succumbing to such biotic challenge of necrotizing pathogens/parasites. If defense mechanisms are triggered by a stimulus prior to infection by a plant pathogen, disease can be reduced. Induced resistance is a state of enhanced defensive capacity developed by a plant when appropriately stimulated. Systemic acquired resistance (SAR) and induced systemic resistance (ISR) are two forms of induced resistance wherein plant defenses are preconditioned by prior infection or treatment that results in resistance against subsequent challenge by a pathogen or parasite. Selected strains of plant growth-promoting rhizobacteria (PGPR) suppress diseases by antagonism between the bacteria and soil-borne pathogens as well as by inducing a systemic resistance in plant against both root and foliar pathogens. Rhizobacteria mediated ISR resembles that of pathogen induced SAR in that both types of induced resistance render uninfected plant parts more resistant towards a broad spectrum of plant pathogens. Several rhizobacteria trigger the salicylic acid (SA)-dependent SAR pathway by producing SA at the root surface whereas other rhizobacteria trigger different signaling pathway independent of SA. The existence of SA-independent ISR pathway has been studied in Arabidopsis thaliana, which is dependent on jasmonic acid (JA) and ethylene signaling. Specific Pseudomonas strains induce systemic resistance in viz., carnation, cucumber, radish, tobacco, and Arabidopsis, as evidenced by an enhanced defensive capacity upon challenge inoculation. Combination of ISR and SAR can increase protection against pathogens that are resisted through both pathways besides extended protection to a broader spectrum of pathogens than ISR/SAR alone. Beside Pseudomonas strains, ISR is conducted by Bacillus spp. wherein published results show that several specific strains of species B. amyloliquifaciens, B. subtilis, B. pasteurii, B. cereus, B. pumilus, B. mycoides, and B.sphaericus elicit significant reduction in the incidence or severity of various diseases on a diversity of hosts.     

The major volatile organic compound emitted from Arabidopsis thaliana flowers, the sesquiterpene (E)-β-caryophyllene, is a defense against a bacterial pathogen

Flowers have a high risk of pathogen attack because of their rich nutrient and moisture content, and high frequency of insect visitors. We investigated the role of (E)-β-caryophyllene in floral defense against a microbial pathogen. This sesquiterpene is a common volatile compound emitted from flowers, and is a major volatile released from the stigma of Arabidopsis thaliana flowers. Arabidopsis thaliana lines lacking a functional (E)-β-caryophyllene synthase or constitutively overexpressing this gene were challenged with Pseudomonas syringae pv. tomato DC3000, which is a bacterial pathogen of brassicaceous plants. Flowers of plant lines lacking (E)-β-caryophyllene emission showed greater bacterial growth on their stigmas than did wild-type flowers, and their seeds were lighter and misshapen. By contrast, plant lines with ectopic (E)-β-caryophyllene emission from vegetative parts were more resistant than wild-type plants to pathogen infection of leaves, and showed reduced cell damage and higher seed production. Based on in vitro experiments, (E)-β-caryophyllene seems to act by direct inhibition of bacterial growth, rather than by triggering defense signaling pathways. (E)-β-Caryophyllene thus appears to serve as a defense against pathogens that invade floral tissues and, like other floral volatiles, may play multiple roles in defense and pollinator attraction.     

Interactions of Bacillus spp. and plants--with special reference to induced systemic resistance (ISR)

Biological control of soil-borne pathogens comprises the decrease of inoculum or of the disease producing activity of a pathogen through one or more mechanisms. Interest in biological control of soil-borne plant pathogens has increased considerably in the last few decades, because it may provide control of diseases that cannot or only partly be managed by other control strategies. Recent advances in microbial and molecular techniques have significantly contributed to new insights in underlying mechanisms by which introduced bacteria function. Colonization of plant roots is an essential step for both soil-borne pathogenic and beneficial rhizobacteria. Colonization patterns showed that rhizobacteria act as biocontrol agents or as growth-promoting bacteria form microcolonies or biofilms at preferred sites of root exudation. Such microcolonies are sites for bacteria to communicate with each other (quorum sensing) and to act in a coordinated manner. Elicitation of induced systemic resistance (ISR) by plant-associated bacteria was initially demonstrated using Pseudomonas spp. and other Gram-negative bacteria. Several strains of the species Bacillus amyloliquefaciens, B. subtilis, B. pasteurii, B. cereus, B. pumilus, B. mycoides, and B. sphaericus elicit significant reductions in the incidence or severity of various diseases on a diversity of hosts. Elicitation of ISR by these strains has been demonstrated in greenhouse or field trials on tomato, bell pepper, muskmelon, watermelon, sugar beet, tobacco, Arabidopsis sp., cucumber, loblolly pine, and two tropical crops (long cayenne pepper and green kuang futsoi). Protection resulting from ISR elicited by Bacillus spp. has been reported against leaf-spotting fungal and bacterial pathogens, systemic viruses, a crown-rotting fungal pathogen, root-knot nematodes, and a stem-blight fungal pathogen as well as damping-off, blue mold, and late blight diseases. This progress will lead to a more efficient use of these strains which is worthwhile approach to explore in context of biocontrol strategies.     

Achieving similar root microbiota composition in neighbouring plants through airborne signalling

The ability to recognize and respond to environmental signals is essential for plants. In response to environmental changes, the status of a plant is transmitted to other plants in the form of signals such as volatiles. Root-associated bacteria trigger the release of plant volatile organic compounds (VOCs). However, the impact of VOCs on the rhizosphere microbial community of neighbouring plants is not well understood. Here, we investigated the effect of VOCs on the rhizosphere microbial community of tomato plants inoculated with a plant growth-promoting rhizobacterium Bacillus amyloliquefaciens strain GB03 and that of their neighbouring plants. Interestingly, high similarity (up to 69%) was detected in the rhizosphere microbial communities of the inoculated and neighbouring plants. Leaves of the tomato plant treated with strain GB03-released β-caryophyllene as a signature VOC, which elicited the release of a large amount of salicylic acid (SA) in the root exudates of a neighbouring tomato seedling. The exposure of tomato leaves to β-caryophyllene resulted in the secretion of SA from the root. Our results demonstrate for the first time that the composition of the rhizosphere microbiota in surrounding plants is synchronized through aerial signals from plants.Subject terms: Microbial ecology, Soil microbiology     

Erwinia carotovora subsp. carotovora and Erwinia-derived elicitors HrpN and PehA trigger distinct but interacting defense responses and cell death in Arabidopsis

We have used an hrp-positive strain of the soft rot pathogen Erwinia carotovora subsp. carotovora to elucidate plant responses to this bacterial necrotroph. Purified virulence determinants, harpin (HrpN) and polygalacturonase (PehA), were used as tools to facilitate this analysis. We show that HrpN elicits lesion formation in Arabidopsis and tobacco and triggers systemic resistance in Arabidopsis. Establishment of resistance is accompanied by the expression of salicylic acid (SA)-dependent, but also jasmonate/ethylene (JA/ET)-dependent, marker genes PR1 and PDF1.2, respectively, suggesting that both SA-dependent and JA/ET-dependent defense pathways are activated. Use of pathway-specific mutants and transgenic NahG plants show that both pathways are required for the induction of resistance. Arabidopsis plants treated simultaneously with both elictors PehA, known to trigger only JA/ET-dependent defense signaling, and HrpN react with accelerated and enhanced induction of the marker genes PR1 and PDF1.2 both locally and systemically. This mutual amplification of defense gene expression involves both SA-dependent and JA/ET-dependent defense signaling. The two elicitors produced by E. carotovora subsp. carotovora also cooperate in triggering increased production of superoxide and lesion formation.     

Jasmonate-dependent induction of indole glucosinolates in Arabidopsis by culture filtrates of the nonspecific pathogen Erwinia carotovora

Elicitors from the plant pathogen Erwinia carotovora trigger coordinate induction of the tryptophan (Trp) biosynthesis pathway and Trp oxidizing genes in Arabidopsis. To elucidate the biological role of such pathogen-induced activation we characterized the production of secondary defense metabolites such as camalexin and indole glucosinolates derived from precursors of this pathway. Elicitor induction was followed by a specific increase in 3-indolylmethylglucosinolate (IGS) content, but only a barely detectable accumulation of the indole-derived phytoalexin camalexin. The response is mediated by jasmonic acid as shown by lack of IGS induction in the jasmonate-insensitive mutant coi1-1. In accordance with this, methyl jasmonate was able to trigger IGS accumulation in Arabidopsis. In contrast, ethylene and salicylic acid seem to play a minor role in the response. They did not trigger alterations in IGS levels, and methyl jasmonate- or elicitor-induced IGS accumulation in NahG and ethylene-insensitive ein2-1 mutant plants was similar as in the wild type. The breakdown products of IGS and other glucosinolates were able to inhibit growth of E. carotovora. The results suggest that IGS is of importance in the defense against bacterial pathogens.     

Photoperiod regulates elicitation of growth promotion but not induced resistance by plant growth-promoting rhizobacteria

For several years, we have noticed that plant growth-promoting rhizobacteria (PGPR), which consistently promote plant growth in greenhouse tests during spring, summer, and fall, fail to elicit plant growth promotion during the midwinter under ambient light conditions. This report tests the hypothesis that photoperiod regulates elicitation of growth promotion and induced systemic resistance (ISR) by PGPR. A commercially available formulation of PGPR strains Bacillus subtilis GB03 and Bacillus amyloliquefaciens IN937a (BioYield) was used to grow tomato and pepper transplants under short-day (8 h of light) (SD) and long-day (12 h of light) (LD) conditions. Results of many experiments indicated that under LD conditions, BioYield consistently elicited significant increases in root and shoot mass as well as in several parameters of root architecture. However, under SD conditions, such increases were not elicited. Differential root colonization of plants grown under LD and SD conditions and changes in leachate quality partially account for these results. BioYield elicited ISR in tomato and pepper under both LD and SD conditions, indicating that although growth promotion was not elicited under SD conditions, induced resistance was. Overall, the results indicate that PGPR-mediated growth promotion is regulated by photoperiod, while ISR is not.     

Root-secreted malic acid recruits beneficial soil bacteria

Beneficial soil bacteria confer immunity against a wide range of foliar diseases by activating plant defenses, thereby reducing a plant's susceptibility to pathogen attack. Although bacterial signals have been identified that activate these plant defenses, plant metabolites that elicit rhizobacterial responses have not been demonstrated. Here, we provide biochemical evidence that the tricarboxylic acid cycle intermediate L-malic acid (MA) secreted from roots of Arabidopsis (Arabidopsis thaliana) selectively signals and recruits the beneficial rhizobacterium Bacillus subtilis FB17 in a dose-dependent manner. Root secretions of L-MA are induced by the foliar pathogen Pseudomonas syringae pv tomato (Pst DC3000) and elevated levels of L-MA promote binding and biofilm formation of FB17 on Arabidopsis roots. The demonstration that roots selectively secrete L-MA and effectively signal beneficial rhizobacteria establishes a regulatory role of root metabolites in recruitment of beneficial microbes, as well as underscores the breadth and sophistication of plant-microbial interactions.     

Plant growth-promoting rhizobacteria modulate root-system architecture in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> through volatile organic compound emission

Extensive communication occurs between plants and microorganisms during different stages of plant development in which signaling molecules from the two partners play an important role. Volatile organic compounds (VOCs) emission by certain plant-growth promoting rhizobacteria (PGPR) has been found to be involved in plant growth. However, little is known about the role of bacterial VOCs in plant developmental processes. In this work, we investigated the effects of inoculation with twelve bacterial strains isolated from the rhizosphere of lemon plants (Citrus aurantifolia) on growth and development of Arabidopsis thaliana seedlings. Several bacterial strains showed a plant growth promoting effect stimulating biomass production, which was related to differential modulation of root-system architecture. The isolates L263, L266, and L272a stimulated primary root growth and lateral root development, while L254, L265a and L265b did not significantly alter primary root growth but strongly promoted lateral root formation. VOC emission analysis by SPME-GC-MS identified aldehydes, ketones and alcohols as the most abundant compounds common to most rhizobacteria. Other VOCs, including 1-octen-3-ol and butyrolactone were strain specific. Characterization of L254, L266 and L272a bacterial isolates by 16S rDNA analysis revealed the identity of these strains as Bacillus cereus, Bacillus simplex and Bacillus sp, respectively. Taken together, our data suggest that rhizospheric bacterial strains can modulate both plant growth promotion and root-system architecture by differential VOC emission.     

A two-strain mixture of rhizobacteria elicits induction of systemic resistance against Pseudomonas syringae and Cucumber mosaic virus coupled to promotion of plant growth on Arabidopsis thaliana

We evaluated a commercial biopreparation of plant growth-promoting rhizobacteria (PGPR) strains Bacillus subtilis GB03 and B. amyloliquefaciens IN937a formulated with the carrier chitosan (BioYield) for its capacity to elicit growth promotion and induced systemic resistance against infection by Cucumber Mosaic Virus (CMV) and Pseudomonas syringae pv. tomato DC3000 in Arabidopsis thaliana. The biopreparation promoted plant growth of Arabidopsis hormonal mutants, which included auxin, gibberellic acid, ethylene, jasmonate, salicylic acid, and brassinosteroid insensitive lines as well as each wild-type. The biopreparation protected plants against CMV based on disease severity in wild-type plants. However, virus titre was not lower in control plants and those treated with biopreparation, suggesting that the biopreparation induced tolerance rather than resistance against CMV. Interestingly, the biopreparation induced resistance against CMV in NahG plants, as evidenced by both reduced disease severity and virus titer. The biopreparation also elicited induced resistance against P. syringae pv. tomato in the wild-type but not in NahG transgenic plants, which degrade endogenous salicylic acid, indicating the involvement of salicylic acid signaling. Our results indicate that some PGPR strains can elicit plant growth promotion by mechanisms that are different from known hormonal signaling pathways. In addition, the mechanism for elicitation of induced resistance by PGPR may be pathogen-dependent. Collectively, the two-Bacilli strain mixture can be utilized as a biological inoculant for both protection of plant against bacterial and viral pathogens and enhancement of plant growth.     

植物根际促生枯草芽孢杆菌JC01筛选

【目的】筛选高效植物根际促生细菌,阐明产挥发性有机化合物(VOC)菌株JC01的促生机制。【方法】选取从植物根际中分离得到的838株细菌,以固氮、解(溶)磷以及分泌嗜铁素、吲哚乙酸(IAA)活性为指标,对其促生能力进行赋值评估,将赋值在3分以上的107株细菌进行指纹图谱分析,挑选其中不同簇的20株促生潜力细菌进行温室实验,以评价赋值体系与温室促生效果之间的关系,进一步探究具有较好促生效果菌株JC01的作用机理。【结果】共筛选出了来源于指纹图谱中不同簇的4株具有较好促生效果的菌株,细菌的平板活性赋值与促生效果之间的相关系数r大于0.6。其中,菌株JC01分泌的具有促生作用的VOC能够增强番茄植株IAA信号通路关键基因的表达,减弱脱落酸(ABA)、乙烯(ETH)信号通路关键基因的表达。JC01经16S r RNA基因鉴定为枯草芽孢杆菌。【结论】细菌的平板活性赋值与促生效果之间存在较高的正相关性,枯草芽孢杆菌JC01可能通过产生VOC对番茄生长进行调控。