Assessment of the Role of Chemotaxis and Biofilm Formation as Requirements for Colonization of Roots and Seeds of Soybean Plants by <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> BNM339

This article correlates colonization with parameters, such as chemotaxis, biofilm formation, and bacterial growth, that are believed to be connected. We show here, by using two varieties of soybean plants that seeds axenically produced exudates, induced a chemotactic response in Bacillus amyloliquefaciens, whereas root exudates did not, even when the exudates, also collected under axenic conditions, were concentrated up to 200-fold. Root exudates did not support bacterial cell division, whereas seed exudates contain compounds that support active cell division and high cell biomass at stationary phase. Seed exudates of the two soybean varieties also induced biofilm formation. B. amyloliquefaciens colonized both seeds and roots, and plant variety significantly affected bacterial root colonization, whereas it did not affect seed colonization. Colonization of roots in B. amyloliquefaciens occurred despite the lack of chemotaxis and growth stimulation by root exudates. The data presented in this article suggest that soybean seed colonization, but not root colonization, by B. amyloliquefaciens is influenced by chemotaxis, growth, and biofilm formation and that this may be caused by qualitative changes of the composition of root exudates.     

Recognition of dominant attractants by key chemoreceptors mediates recruitment of plant growth-promoting rhizobacteria

Chemotaxis to plant root exudates is supposed to be a prerequisite for efficient root colonization by rhizobacteria. This is a highly multifactorial process since root exudates are complex compound mixtures of which components are recognized by different chemoreceptors. Little information is available as to the key components in root exudates and their receptors that drive colonization related chemotaxis. We present here the first global assessment of this issue using the plant growth-promoting rhizobacterium (PGPR) Bacillus velezensis SQR9 (formerly B. amyloliquefaciens). This strain efficiently colonizes cucumber roots, and here, we show that chemotaxis to cucumber root exudates was essential in this process. We conducted chemotaxis assays using cucumber root exudates at different concentrations, individual exudate components as well as recomposed exudates, taking into account their concentrations detected in root exudates. Results indicated that two key chemoreceptors, McpA and McpC, were essential for root exudate chemotaxis and root colonization. Both receptors possess a broad ligand range and recognize most of the exudate key components identified (malic, fumaric, gluconic and glyceric acids, Lys, Ser, Ala and mannose). The remaining six chemoreceptors did not contribute to exudate chemotaxis. This study provides novel insight into the evolution of the chemotaxis system in rhizobacteria.     

Impact of soil stockpiling on ericoid mycorrhizal colonization and growth of velvetleaf blueberry (Vaccinium myrtilloides) and Labrador tea (Ledum groenlandicum)

Soil stockpiling is a common practice prior to the reclamation of surface mines. In this study, velvetleaf blueberry and Labrador tea plants were grown from seed in fresh soil, stockpiled soil (1 year), and autoclaved stockpiled soil (1 year) obtained from the Canadian boreal forest. After 7 months of growth, the root colonization intensity with ericoid mycorrhizal (ERM) fungi in both plants growing in stockpiled soil was lower compared to plants growing in the fresh soil. The diversity of ERM fungal species in roots also decreased due to soil stockpiling and Pezoloma ericae was absent from the plants growing in stockpiled soil. Changes in the ERM root colonization in plants growing in stockpiled soil were accompanied by decreases in root and shoot dry weights. Leaf chlorophyll, nitrogen, and phosphorus concentrations of velvetleaf blueberry were higher in fresh soil compared to 1‐year stockpiled soil. Plants grown in the autoclaved stockpiled soil became colonized by the thermotolerant ERM fungus Leohumicola verrucosa and showed higher root and shoot biomass compared to the nonautoclaved stockpiled soil. The results point to the importance of ERM fungi for growth of ericaceous plants, even under favorable environmental conditions and adequate fertilization, and suggest that reduced ERM colonization intensity and ERM fungal diversity in roots likely contributed to the negative effects of soil stockpiling on growth of velvetleaf blueberry and Labrador tea.     

影响引人微生物根部定殖的因素

从外界引入的各类有益微生物如生防菌(BCA)和根际促生菌或增产菌(PGPR,YIB)到种子表面随其生根发芽而蔓延或直接到根表沿根分布定殖．外来微生物在根际定殖的过程为与根尖接触,沿根分布,最后在根际建立自己的种群．定殖的位点以PGPR为例,是表皮细胞间隙,或侧根、根毛基部．外来微生物在根际定殖动态变化的原因,由于根际生物的和非生物的因素引起的．生物因子除去外来微生物本身的生理特性,还有根际土著微生物与外来微生物的相互作用,更重要的是植物基因型对微生物定殖的影响．非生物因子包括土壤环境、土壤结构和含水量,土壤温度和土壤pH值均能影响外来微生物在根部的定殖．     

Flagella-driven chemotaxis towards exudate components is an important trait for tomato root colonization by Pseudomonas fluorescens

Motility is a major trait for competitive tomato root-tip colonization by Pseudomonas fluorescens. To test the hypothesis that this role of motility is based on chemotaxis toward exudate components, cheA mutants that were defective in flagella-driven chemotaxis but retained motility were constructed in four P. fluorescens strains. After inoculation of seedlings with a 1:1 mixture of wild-type and nonmotile mutants all mutants had a strongly reduced competitive root colonizing ability after 7 days of plant growth, both in a gnotobiotic sand system as well as in nonsterile potting soil. The differences were significant on all root parts and increased from root base to root tip. Significant differences at the root tip could already be detected after 2 to 3 days. These experiments show that chemotaxis is an important competitive colonization trait. The best competitive root-tip colonizer, strain WCS365, was tested for chemotaxis toward tomato root exudate and its major identified components. A chemotactic response was detected toward root exudate, some organic acids, and some amino acids from this exudate but not toward its sugars. Comparison of the minimal concentrations required for a chemotactic response with concentrations estimated for exudates suggested that malic acid and citric acid are among major chemo-attractants for P. fluorescens WCS365 cells in the tomato rhizosphere.     

Effect of Botanical Aromatic Compounds and Seed-surface pH on Growth and Colonization of Cotton Plant Growth-promoting Rhizobacteria

Citral (3 , 7 - dimethyl - 2 , 6 - octadienal) , furfural (2 - furaldehyde) and benzaldehyde (benzoic adel hyde) previously demonstrated control activity against Meloidogyne incognita and fungal diseases on cotton . Plant growth - promoting rhizobacteria (PGPR) applied to cotton were previously found to promote plant growth and reduce seedling disease . Studies were under taken to determine if these compounds were compatible with PGPR . In tests with 12 PGPR strains , vapor of citral inhibited in vitro growth of most strains , and vapor of furfural and benzaldehyde , with one exception , killed all but the Bacillus spp . tested . When 0 . 35 ml kg 1 soil of each compound were applied to the soil 9 - 10 days prior to planting the cotton cultivar Deltapine 51 , only furfural significantly reduced rhizosphere colonization across all strains from 4 . 70 colony - forming units (CFUs) / g of root to 4 . 42 CFUs / g root . In greenhouse studies , the low seed - surface pH (2 . 3) of commercial seed did not reduce root colonization , compared with colonization on roots from seed at pH 5 . 4 . There were no synergistic interactions between seed - surface pH and any of the compounds . Although previous research indicated that application of both furfural and benzaldehyde increased the proportion of Burkholderia spp . in the soil , there is no indication that they increased cotton root colonization by the B. cepacia strain tested . These results indicate PGPR can be combined with citral and benzaldehyde in integrated management systems and that the low seed - surface pH of acid - delinted cotton will not limit their application .     

The effects of traits of Bacillus megaterium onseed and root colonization and their correlation withthe suppression of Rhizoctonia root rot of soybean

Bacillus megaterium strainB153-2-2 is a potential bacterial biocontrol agentagainst Rhizoctonia solani isolate 2B12(ISG-2B). To study the role of antagonism (Ant),chemotaxis (Che), motility (Mot), and sporulation(Spo) of the biocontrol agent during seed and rootcolonization and the correlation between rootcolonization and the suppression of soybean (Glycine max) root rot caused by R. solani,strain B153-2-2(Che⁺Mot⁺Ant⁺⁺Spo⁺⁺) and the sevenderived mutants with altered antagonism, chemotaxis,motility, and/or sporulation were used. The bacterialcells were introduced into soil separately either asa soybean seed coating or soil application. Two soilmixtures defined as coarse and fine soil were used. The bacterial cell chemotactic response to soybeanroot and seed exudates and antagonism to R.solani were significantly (p = 0.05) correlatedwith root and seed colonization in some but not alltreatments. The sporulation-defective mutants had lowcell populations immediately after application and,therefore, reduced root colonization. The differencesin root colonization diminished among the mutants andstrain B153-2-2 when R. solani was present inthe soil or, as seedlings grew older. Soybean seedlingroots grown in coarse soil had significantly greatercolonization by B153-2-2 or its mutants and a lowerdisease index than that in fine soil. There was asignificant positive correlation (r ² = 0.78)between root colonization by strain B153-2-2 or itsmutants and suppression of Rhizoctonia root rot.     

嫁接黄瓜地上部的南瓜根系分泌物对种子萌发的影响

经嫁接黄瓜接穗的南瓜根系分泌物对黄瓜和南瓜的发芽率和胚根、胚轴的伸长均具有明显的抑制作用.分析表明:嫁接黄瓜根系分泌物可以促进黄瓜和南瓜体内吲哚乙酸氧化酶的活性,抑制淀粉酶的活性,从而降低其吲哚乙酸(IAA)水平,影响子叶中贮藏物质的转化和利用,抑制其萌发和生长.     

Beneficial bacteria of agricultural importance

The rhizosphere is the soil–plant root interphase and in practice consists of the soil adhering to the root besides the loose soil surrounding it. Plant growth-promoting rhizobacteria (PGPR) are potential agents for the biological control of plant pathogens. A biocontrol strain should be able to protect the host plant from pathogens and fulfill the requirement for strong colonization. Numerous compounds that are toxic to pathogens, such as HCN, phenazines, pyrrolnitrin, and pyoluteorin as well as, other enzymes, antibiotics, metabolites and phytohormones are the means by which PGPR act, just as quorum sensing and chemotaxis which are vital for rhizosphere competence and colonization. The presence of root exudates has a pronounced effect on the rhizosphere where they serve as an energy source, promoting growth and influencing the root system for the rhizobacteria. In certain instances they have products that inhibit the growth of soil-borne pathogens to the advantage of the plant root. A major source of concern is reproducibility in the field due to the complex interaction between the plant (plant species), microbe and the environment (soil fertility and moisture, day length, light intensity, length of growing season, and temperature). This review listed most of the documented PGPR genera and discussed their exploitation.     

Plant protection and rhizosphere colonization of barley by seed inoculated herbicide degrading Burkholderia (Pseudomonas) cepacia DBO1(pRO101) in 2,4-D contaminated soil

The 2,4-dichlorophenoxyacetic acid (2,4-D) degrading bacterium, Burkholderia cepacia (formerly Pseudomonas cepacia) DBO1(pRO101) was coated on non-sterile barley (Hordeum vulgare) seeds, which were planted in two non-sterile soils amended with varying amounts of 2,4-D herbicide. In the presence of 10 or 100 mg 2,4-D per kg soil B. cepacia DBO1(pRO101) readily colonized the root at densities up to 10⁷ CFU per cm root. In soil without 2,4-D the bacterium showed weak root colonization. The seeds coated with B. cepacia DBO1(pRO101) were able to germinate and grow in soils containing 10 or 100 mg kg^–1 2,4-D, while non-coated seeds either did not germinate or quickly withered after germination. The results suggest that colonization of the plant roots by the herbicide-degrading B. cepacia DBO1(pRO101) can protect the plant by degradation of the herbicide in the rhizosphere soil. The study shows that the ability to degrade certain pesticides should be considered, when searching for potential plant growth-promoting rhizobacteria. The role of root colonization by xenobiotic degrading bacteria is further discussed in relation to bioremediation of contaminated soils.     

Potent endogenous allelopathic compounds in Lepidium sativum seed exudate: effects on epidermal cell growth in Amaranthus caudatus seedlings

Many plants exude allelochemicals--compounds that affect the growth of neighbouring plants. This study reports further studies of the reported effect of cress (Lepidium sativum) seed(ling) exudates on seedling growth in Amaranthus caudatus and Lactuca sativa. In the presence of live cress seedlings, both species grew longer hypocotyls and shorter roots than cress-free controls. The effects of cress seedlings were allelopathic and not due to competition for resources. Amaranthus seedlings grown in the presence of cress allelochemical(s) had longer, thinner hypocotyls and shorter, thicker roots--effects previously attributed to lepidimoide. The active principle was more abundant in cress seed exudate than in seedling (root) exudates. It was present in non-imbibed seeds and releasable from heat-killed seeds. Release from live seeds was biphasic, starting rapidly but then continuing gradually for 24 h. The active principle was generated by aseptic cress tissue and was not a microbial digestion product or seed-treatment chemical. Crude seed exudate affected hypocotyl and root growth at ~25 and ~450 μg ml(-1) respectively. The exudate slightly (28%) increased epidermal cell number along the length of the Amaranthus hypocotyl but increased total hypocotyl elongation by 129%; it resulted in a 26% smaller hypocotyl circumference but a 55% greater epidermal cell number counted round the circumference. Therefore, the effect of the allelochemical(s) on organ morphology was imposed primarily by regulation of cell expansion, not cell division. It is concluded that cress seeds exude endogenous substances, probably including lepidimoide, that principally regulate cell expansion in receiver plants.     

Effects of different plant root exudates and their organic acid components on chemotaxis, biofilm formation and colonization by beneficial rhizosphere-associated bacterial strains

Aim

It is necessary to understand the roles of root exudates involved in plant-microbe interactions to inform practical application of beneficial rhizosphere microbial strains.

Methods

Colonization of Bacillus amyloliquefaciens SQR9 (isolated from cucumber rhizosphere) and Bacillus subtilis N11 (isolated from banana rhizosphere) of their original host was found to be more effective as compared to the colonization of the non-host plant. Organic acids in the root exudates of the two plants were identified by High performance liquid chromatography (HPLC). The chemotactic response and effects on biofilm formation were assessed for SQR9 and N11 in response to cucumber and banana root exudates, as well as their organic acids components.

Results

Citric acid detected exclusively in cucumber exudates could both attract SQR9 and induce its biofilm formation, whereas only chemotactic response but not biofilm formation was induced in N11. Fumaric acid that was only detected in banana root exudates revealed both significant roles on chemotaxis and biofilm formation of N11, while showing only effects on biofilm formation but not chemotaxis of SQR9.

Conclusion

The relationship between PGPR strain and root exudates components of its original host might contribute to preferential colonization. This study advances a clearer understanding of the mechanisms relevant to application of PGPR strains in agricultural production.     

Identification of aliphatic and aromatic acids in root and seed exudates of peas,cotton, and barley

Summary We detected aromatic and aliphatic acids in root and seed exudates of aseptic cultures of pea, cotton and barley plants by thin-layer and gas-liquid chromatography. There were traces of p-hydroxybenzoic acid in the root and seed exudates of all three plant species. Acid hydrolysis of pea and barley seed exudates yielded p-hydroxybenzoic, and of cotton seed exudates yielded p-coumaric acid, as the predominant aromatic acid constituents of materials exuded by the germinating seeds. Lactic was the predominant aliphatic acid detected in pea and barley root exudates whereas malic acid was predominant in cotton exudates. With the exception of citric acid in peas, malic acid was the predominant acid found in pea, cotton and barley seed exudates. The germinating seed was responsible for a large portion of the total aliphatic and aromatic acid exudation of the seedling plant grown aseptically for 14 days. Trade names are used in this publication only to provide scientific information. Their use does not constitute a guarantee of the products named and does not signify that they are approved by the U.S. Department of Agriculture to the exclusion of others of suitable composition.     

Root colonization of maize and lettuce by bioluminescent Rhizobium leguminosarum biovar phaseoli.

Two strains of Rhizobium leguminosarum bv. phaseoli and three other plant growth-promoting rhizobacteria (PGPR) were examined for the potential of maize and lettuce root colonization. All of these strains were selected in vitro for their phosphate-solubilizing abilities. Maize and lettuce seeds were treated with derivatives of all strains marked with lux genes for bioluminescence and resistance to kanamycin and rifampin prior to planting in nonsterile Promix and natural soil. The introduced bacterial strains were quantified on roots by dilution plating on antibiotic media together with observation of bioluminescence. Rhizobia were superior colonizers compared with other tested bacteria; rhizobial root populations averaged log 4.1 CFU/g (fresh weight) on maize roots 4 weeks after seeding and log 3.7 CFU/g (fresh weight) on lettuce roots 5 weeks after seeding. The average populations of the recovered PGPR strains were log 3.5 and log 3.0 CFU/g (fresh weight) on maize and lettuce roots, respectively. One of the three PGPR was not recovered later than the first week after seeding in Promix. Bioluminescence also permitted visualization of in situ root colonization in rhizoboxes and demonstrated the efficiency of rhizobial strains to colonize and survive on maize and lettuce roots.     

ALLELOPATHIC AND HERBICIDAL EFFECTS OF EXTRACTS FROM TREE OF HEAVEN (AILANTHUS ALTISSIMA)

Mature trees of Ailanthus altissima produce one or more potent inhibitors of seed germination and seedling growth. Inhibitor activity is highest in bark, especially of roots, intermediate in leaflets, and low in wood. Crude extracts of Ailanthus root bark and leaflets corresponding to 34 and 119 mg water extractable material/L, respectively, caused 50% inhibition of cress radicle growth. Ailanthus seeds also contain one or more inhibitors. These are bound within the seed by the pericarp but diffuse into water agar when the pericarp is removed. The inhibitor(s) could readily be extracted from Ailanthus tissues with methanol, but not dichloromethane, indicating polar characteristics. Ailanthus leaflets had highest inhibitory activity during expansion in spring, whereas activity of trunk bark peaked just before emergence of leaves. This pattern suggests transport of allelochemicals from bark into new leaves. A comparison of seven plant species for sensitivity to the inhibitor(s) from Ailanthus root bark showed little selectivity, although velvetleaf was somewhat more resistant. The inhibitor(s) from Ailanthus root bark exhibited strong herbicidal effects when sprayed pre- and postemergence on plants in soil in the greenhouse. Postemergence effects were striking, with nearly complete mortality of all species, except velvetleaf, at even the lowest doses tested. The results suggest the allelochemical(s) from Ailanthus may have potential for development as natural-product herbicides.     

Root cap influences root colonisation by Pseudomonas fluorescens SBW25 on maize

We investigated the influence of root border cells on the colonisation of seedling Zea mays roots by Pseudomonas fluorescens SBW25 in sandy loam soil packed at two dry bulk densities. Numbers of colony forming units (CFU) were counted on sequential sections of root for intact and decapped inoculated roots grown in loose (1.0 mg m(-3)) and compacted (1.3 mg m(-3)) soil. After two days of root growth, the numbers of P. fluorescens (CFU cm(-1)) were highest on the section of root just below the seed with progressively fewer bacteria near the tip, irrespective of density. The decapped roots had significantly more colonies of P. fluorescens at the tip compared with the intact roots: approximately 100-fold more in the loose and 30-fold more in the compact soil. In addition, confocal images of the root tips grown in agar showed that P. fluorescens could only be detected on the tips of the decapped roots. These results indicated that border cells, and their associated mucilage, prevented complete colonization of the root tip by the biocontrol agent P. fluorescens, possibly by acting as a disposable surface or sheath around the cap.     

Surface colonization of lodgepole pine (Pinus contorta var. latifolia [Dougl. Engelm.]) roots by Pseudomonas fluorescens and Paenibacillus polymyxa under gnotobiotic conditions

Indirect immunofluorescence techniques and confocal scanning laser microscopy were used to identify rhizobacterial strains on the root surfaces of pine seedlings, which were grown from seeds under gnotobiotic conditions. Conifer plant growth promoting rhizobacterial strains Paenibacillus polymyxa L6 and Pw-2, and the forest soil isolate Pseudomonas fluorescens M20, were inoculated onto surface-disinfested pine seeds, singly, or in dual combinations: strains L6 + M20, or strains Pw-2 + M20. Segments containing particular root microsites (root tip, root hair zone, or areas of lateral root emergence) were sampled randomly from roots 7 or 13 weeks after inoculation, and the colonization of roots by each bacterium was observed. Root segments were also sampled from individual roots at six different points along the length of the root, and the qualitative colonization of younger areas, closer to the root tip, contrasted with that of older areas, closer to the root base. The ability of strain M20 to colonize root areas adjacent to sites of lateral root emergence improves in the presence of either P. polymyxa strain, while the ability of the P. polymyxa strains to colonize these areas was not affected. More rhizobacteria were also generally observed on younger root tissues than on areas closer to the root base.     

Chemotaxis of Bradyrhizobium japonicum to soybean exudates.

The chemotactic response of Bradyrhizobium japonicum toward soybean seed and root exudates was examined. Assays using various isoflavones and fractionated exudate indicated that isoflavones are not the principal attractants in exudates. Likewise, induction of nod genes with isoflavones or seed exudate before assay did not enhance chemotaxis. Screening of numerous compounds revealed that only dicarboxylic acids and the amino acids glutamate and aspartate were strong attractants. The presence of glutamate, aspartate, and dicarboxylic acids in appreciable concentrations in soybean seed and root exudates indicates that these compounds likely represent natural chemoattractants for B. japonicum.     

Chemotaxis of Bradyrhizobium japonicum to soybean exudates.

The chemotactic response of Bradyrhizobium japonicum toward soybean seed and root exudates was examined. Assays using various isoflavones and fractionated exudate indicated that isoflavones are not the principal attractants in exudates. Likewise, induction of nod genes with isoflavones or seed exudate before assay did not enhance chemotaxis. Screening of numerous compounds revealed that only dicarboxylic acids and the amino acids glutamate and aspartate were strong attractants. The presence of glutamate, aspartate, and dicarboxylic acids in appreciable concentrations in soybean seed and root exudates indicates that these compounds likely represent natural chemoattractants for B. japonicum.     

Expression of chitinase in Adenanthera pavonina seedlings

Chitinases (EC 3.2.1.14) are hydrolytic enzymes found in different organisms. In plants, they have been described in different tissues and organs, including seeds. This study was triggered by the isolation of a 30-kDa thermostable chitinase from Adenanthera pavonina L. seeds. The enzyme was submitted to N-terminal amino acid sequencing, and the analysis revealed a high degree of homology with class III chitinases. Bidimensional electrophoresis of the 30-kDa band showed the presence of three isoforms with pIs of 5.2, 5.5 and 5.8. A chitinase was also found in exudates released from the same seeds, which was seen to be immunorelated to the above 30-kDa protein. It was also submitted to N-terminal amino acid sequencing and seen as highly homologous to class III chitinases. In addition, the expression of chitinases during A. pavonina L. seed germination and seedling development was investigated. Seeds were allowed to germinate in the absence of light for approximately 5 days and were grown, for different times, in the absence or presence of light. After each seedling developmental time, samples of exudates, roots and cotyledonary leaves were collected and submitted to protein extraction. The presence of proteins immunorelated to the 30-kDa chitinase was detected in all analyzed samples. Further analyses showed that light significantly interfered with the chitinase expression in some organs. The tissue and subcellular chitinase location in seedling roots was also investigated, and it was majorly localized in the cell wall and in the intercellular spaces of the root hair zone.