Characterization of the biosynthetic operon for the antibacterial peptide herbicolin in Pantoea vagans biocontrol strain C9-1 and incidence in Pantoea species

Pantoea vagans C9-1 is a biocontrol strain that produces at least two antibiotics inhibiting the growth of Erwinia amylovora, the causal agent of fire blight disease of pear and apple. One antibiotic, herbicolin I, was purified from culture filtrates of P. vagans C9-1 and determined to be 2-amino-3-(oxirane-2,3-dicarboxamido)-propanoyl-valine, also known as N_ß-epoxysuccinamoyl-DAP-valine. A plasposon library was screened for mutants that had lost the ability to produce herbicolin I. It was shown that mutants had reduced biocontrol efficacy in immature pear assays. The biosynthetic gene cluster in P. vagans C9-1 was identified by sequencing the flanking regions of the plasposon insertion sites. The herbicolin I biosynthetic gene cluster consists of 10 coding sequences (CDS) and is located on the 166-kb plasmid pPag2. Sequence comparisons identified orthologous gene clusters in Pantoea agglomerans CU0119 and Serratia proteamaculans 568. A low incidence of detection of the biosynthetic cluster in a collection of 45 Pantoea spp. from biocontrol, environmental, and clinical origins showed that this is a rare trait among the tested strains.     

Fermentation and isolation of herbicolin A,a peptide antibiotic produced byErwinia herbicola strain A 111

Summary Erwinia herbicola (Enterobacter agglomerans), belonging to the Enterobacteriaceae, produces the lipopeptide antibiotics herbicolin A and B, which are active against sterol-containing fungi. Fermentation of these antibiotics was performed in 20-1 stirred glass fermentors in a batch process. Best yields of antibiotic production were found at low cultivation temperatures in a TRIS-buffered chemically defined medium. Under these conditions the amount of impurities aggravating the purification was minimized. Isolation was performed by adsorption, and gel and ion exchange chromatographic techniques. In a final purification step preparative high performance liquid chromatography (PHPLC) yielded pure herbicolin A. Offprint requests to: G. Winkelmann     

Characterization of a versatile rhizospheric organism from cucumber identified as Ochrobactrum haematophilum

Several rhizobacteria play a vital role in promoting plant growth and protecting plants against fungal diseases and degrading pesticides in the environment. In this study, a bacterial strain, designated H10, was isolated from the rhizosphere at Laixi in Shandong Province, China, and was identified as Ochrobactrum haematophilum based on API 20 NE tests and 16S rRNA gene sequence analysis. The plant growth-promoting characteristics of the strain were further characterized, and the results showed that strain H10 produces siderophore, indol-3-acetic (IAA) and solubilized phosphate but lacks 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity. Inoculation with the strain was found to significantly increase (p < 0.05) the growth of cucumber in pot experiments. Strain H10 was assessed in vitro for antagonism against several pathogenic fungi and showed high antifungal activity. The cell-free culture filtrates, which had high extracellular chitinase, β-1,3-glucanase and protease activities, could inhibit the growth of all pathogenic fungi tested, indicating that growth suppression was partly due to extracellular antifungal metabolites present in the culture filtrates. Changes in hyphal morphology were observed in phytopathogenic fungi after treatment with the culture filtrates. Additionally, strain H10 was able to degrade 80%, 85% and 58% of the pesticides chlorpyrifos, β-cypermethrin and imidacloprid, respectively, within 60 h in liquid culture. The inoculation of strain H10 into soil treated with 100 mg kg(-1) of the three pesticides accordingly resulted in a higher degradation rate than in noninoculated soils. These results highlight the potential of this bacterium for use as a biofertilizer and biopesticide and suggest that it may provide an alternative to the use of chemical fertilizers and pesticides in agriculture. Additionally, it may represent a bioremediation agent that can remove contaminating chemical pesticide residues from the environment.     

Plant growth-promoting rhizobacteria (PGPR): emergence in agriculture

Plant growth-promoting rhizobacteria (PGPR) are the rhizosphere bacteria that can enhance plant growth by a wide variety of mechanisms like phosphate solubilization, siderophore production, biological nitrogen fixation, rhizosphere engineering, production of 1-Aminocyclopropane-1-carboxylate deaminase (ACC), quorum sensing (QS) signal interference and inhibition of biofilm formation, phytohormone production, exhibiting antifungal activity, production of volatile organic compounds (VOCs), induction of systemic resistance, promoting beneficial plant-microbe symbioses, interference with pathogen toxin production etc. The potentiality of PGPR in agriculture is steadily increased as it offers an attractive way to replace the use of chemical fertilizers, pesticides and other supplements. Growth promoting substances are likely to be produced in large quantities by these rhizosphere microorganisms that influence indirectly on the overall morphology of the plants. Recent progress in our understanding on the diversity of PGPR in the rhizosphere along with their colonization ability and mechanism of action should facilitate their application as a reliable component in the management of sustainable agricultural system. The progress to date in using the rhizosphere bacteria in a variety of applications related to agricultural improvement along with their mechanism of action with special reference to plant growth-promoting traits are summarized and discussed in this review.     

Studies on the microbial populations of the rhizosphere of big sagebrush (<Emphasis Type="Italic">Artemisia tridentata</Emphasis>)

Prolonged use of broad-spectrum antibiotics has led to the emergence of drug-resistant pathogens, both in medicine and in agriculture. New threats such as biological warfare have increased the need for novel and efficacious antimicrobial agents. Natural habitats not previously examined as sources of novel antibiotic-producing microorganisms still exist. One such habitat is the rhizosphere of desert shrubs. Here, we show that one desert shrub habitat, the rhizosphere of desert big sagebrush (Artemisia tridentata) is a source of actinomycetes capable of producing an extensive array of antifungal metabolites. Culturable microbial populations from both the sagebrush rhizosphere and nearby bulk soils from three different sites were enumerated and compared, using traditional plate-count techniques and antibiotic activity bioassays. There were no statistical differences between the relative numbers of culturable non-actinomycete eubacteria, actinomycetes and fungi in the rhizosphere versus bulk soils, but PCR amplification of the 16S rRNA gene sequences of the total soil DNA and denaturing gradient gel electrophoresis showed that the community structure was different between the rhizosphere and the bulk soils. A high percentage of actinomycetes produced antimicrobials; and the percentage of active producers was significantly higher among the rhizosphere isolates, as compared with the bulk soil isolates. Also, the rhizosphere strains were more active in the production of antifungal compounds than antibacterial compounds. 16S rRNA gene sequence analysis showed that sagebrush rhizospheres contained a variety of Streptomyces species possessing broad spectrum antifungal activity. Scanning electron microscopy studies of sagebrush root colonization by one of the novel sagebrush rhizosphere isolates, Streptomyces sp. strain RG, showed that it aggressively colonized young sagebrush roots, whereas another plant rhizosphere-colonizing strain, S. lydicus WYEC108, not originally isolated from sagebrush, was a poor colonizer of the roots of this plant, as were two other Streptomyces isolates from forest soil. These results support the hypothesis that the rhizosphere of desert big sagebrush is a promising source of habitat-adapted actinomycetes, producing antifungal antibiotics.     

Enhanced stability of a rumen-derived xylanase using SpyTag/SpyCatcher cyclization

A phosphate solubilizing bacterium ZB was isolated from the rhizosphere soil of Araucaria, which falls into the species Pantoea agglomerans. Optimization for phosphate solubilization by strain ZB was performed. At optimum culture conditions, the isolate showed great ability of solubilizing different insoluble inorganic phosphate sources viz. Ca₃(PO₄)₂ (TCP), Hydroxyapatite (HP), CaHPO₄, AlPO₄, FePO₄ along with rock phosphates (RPs). Inoculation with planktonic cells was found to enhance dissolved phosphorous as compared to that achieved by symplasma inoculation. Besides inoculation with different status of cells, pre-incubation could also exert a great effect on phosphate solubilization ability of P. agglomerans. When isolate ZB was cultured with glucose as carbon sources, phosphorous was more efficiently dissolved from HP and RP without pre-incubation in comparison to that obtained with pre-cultivation. Pre-cultivation, however, was more suitable for P solubilization than no pre-cultivation when bacteria were grown with xylose. A positive correlation was detected between the production of organic acids and phosphate solubilization. P. agglomerans ZB possessed many plant growth promotion traits such as N₂ fixation and production of indole 3-acetic acid, phytase, alkaline phosphatase. Pot experiment showed inoculation with single isolate ZB or biofertilizer prepared from semi-solid fermentation of isolate ZB with spent mushroom substrate (SMS) compost could enhance plant growth with respect to number of leaves, plant leave area, stem diameter, root length, root dry mass, shoot dry mass and biomass when compared to the abiotic control, revealing strain ZB could be a promising environmental-friendly biofertilizer to apply for agricultural field.

     

Metabolites of rhizobacteria antagonistic towards fungal plant pathogens

Biological control of insect, plant pathogens and weeds is the only major alternative to the use of pesticides in agriculture and forestry. A double-layer technique was used for isolation of antagonistic bacteria from rhizosphere against plant pathogenic fungi. Four potential rhizobacteria was selected in dual culture plate method based on their antifungal activity against several soil-borne fungal plant pathogens. The selected rhizobacteria, identified based on their morphological, biochemical and molecular traits, belong to the species of fluorescentPseudomonas (SAB8, GM4) andBacillus (A555, GF23). The active antifungal metabolites produced by these strains in culture filtrates were tested for the growth inhibition ofFusarium semitectum used as test fungus. The active fraction of antifungal metabolite/(s) from fluorescentPseudomonas (SAB8, GM4) and their effects on hyphal growth were observed under microscope. Two kinds of alterations were detected: inhibition of hyphal tip elongation and an extensive branching of hyphae with closer septa.     

Spent growth medium of Pantoea agglomerans primes wheat suspension cells for augmented accumulation of hydrogen peroxide and enhanced peroxidase activity upon elicitation

Induced disease resistance in plants is based on multiple mechanisms, including cell “priming”, i.e. an enhancement of the capacity to mobilize cellular defense responses upon pathogen attack. Potent inducers of priming are, for example, salicylic acid, synthetic compounds such as a benzothiadiazole, and certain rhizosphere bacteria. While priming is well characterized for a number of dicot plants, only few cases of priming are documented in monocots. Here, we report that the spent growth medium of the Gram negative bacterium Pantoea agglomerans is capable of priming wheat cells (Triticum aestivum L. cv Prelude-Sr5) for elicitor-induced defense responses. Pre-incubation of suspension-cultured wheat cells with growth medium of P. agglomerans led to a strong enhancement of an oxidative burst that has been induced by chitin or chitosan and to an increase in extracellular peroxidase activity. Moreover, exopolysaccharides (EPS) were isolated from the spent growth medium and demonstrated to be sufficient for the induction of H₂O₂ priming. The EPS-induced priming was shown to be time- and concentration-dependent. We conclude that EPS are the or one of several priming-active component(s) in the spent growth medium of P. agglomerans. The present work is the first report of priming in a monocot plant by a specific component of bacterial origin. A comparison with known chemical inducers of resistance revealed that a benzothiadiazole was able to enhance the oxidative burst similar to the spent growth medium or the EPS of P. agglomerans, while salicylic acid was not.     

A Genome-Wide Analysis of Antibiotic Producing Genes in Streptomyces globisporus SP6C4

Soil is the major source of plant-associated microbes. Several fungal and bacterial species live within plant tissues. Actinomycetes are well known for producing a variety of antibiotics, and they contribute to improving plant health. In our previous report, Streptomyces globisporus SP6C4 colonized plant tissues and was able to move to other tissues from the initially colonized ones. This strain has excellent antifungal and antibacterial activities and provides a suppressive effect upon various plant diseases. Here, we report the genome-wide analysis of antibiotic producing genes in S. globisporus SP6C4. A total of 15 secondary metabolite biosynthetic gene clusters were predicted using antiSMASH. We used the CRISPR/Cas9 mutagenesis system, and each biosynthetic gene was predicted via protein basic local alignment search tool (BLAST) and rapid annotation using subsystems technology (RAST) server. Three gene clusters were shown to exhibit antifungal or antibacterial activity, viz. cluster 16 (lasso peptide), cluster 17 (thiopeptide-lantipeptide), and cluster 20 (lantipeptide). The results of the current study showed that SP6C4 has a variety of antimicrobial activities, and this strain is beneficial in agriculture.     

2-Amino-3-(Oxirane-2,3-Dicarboxamido)-Propanoyl-Valine,an Effective Peptide Antibiotic from the Epiphyte Pantoea agglomerans 48b/90

The epiphyte Pantoea agglomerans 48b/90, which has been isolated from soybean leaves, belongs to the Enterobacteriaceae, as does the plant pathogen Erwinia amylovora, which causes fire blight on rosaceous plants such as apples and leads to severe economic losses. Since P. agglomerans efficiently antagonizes phytopathogenic bacteria, the P. agglomerans strain C9-1 is used as a biocontrol agent (BlightBan C9-1). Here we describe the bioassay-guided isolation of a peptide antibiotic that is highly active against the plant pathogen E. amylovora and pathovars of Pseudomonas syringae, and we elucidate its structure. Bioassay-guided fractionation using anion-exchange chromatography followed by hydrophobic interaction liquid chromatography yielded the bioactive, highly polar antibiotic. The compound was identified as 2-amino-3-(oxirane-2,3-dicarboxamido)-propanoyl-valine by using high-resolution electrospray ionization mass spectrometry and nuclear magnetic resonance techniques. This peptide was found to be produced by three of the nine P. agglomerans strains analyzed. Notably, the biocontrol strain P. agglomerans C9-1 also produces 2-amino-3-(oxirane-2,3-dicarboxamido)-propanoyl-valine. Previously, 2-amino-3-(oxirane-2,3-dicarboxamido)-propanoyl-valine has been characterized only from Serratia plymuthica. 2-Amino-3-(oxirane-2,3-dicarboxamido)-propanoyl-valine has been shown to inhibit the growth of the human pathogen Candida albicans efficiently, but its involvement in the defense of epiphytes against phytopathogenic bacteria has not been investigated so far.Microbial pathogens pose a major threat to many plants and can cause enormous losses in agriculture. Microorganisms that antagonize pathogens can offer a way to fight plant diseases that is more environmentally friendly than chemical treatment. Such diseases include fire blight, which is caused by Erwinia amylovora and affects many rosaceous plants, e.g., apple and pear (18, 25, 29, 38).Suitable strains for biocontrol agents are often plant-associated microorganisms that are forced to defend their ecological niches under natural conditions and are thus adapted to competition with plant pathogens (2, 3). The species Pantoea agglomerans (formerly Erwinia herbicola) comprises many strains that are promising sources for biocontrol agents (8, 15, 30, 32, 43). P. agglomerans strains are ubiquitous in nature, inhabiting plant surfaces, water, soil, animals, and humans (9, 11). Several Pantoea isolates are known to inhibit E. amylovora efficiently in planta (39, 42). In vitro experiments have revealed some antibiotics from P. agglomerans and uncovered how they act against E. amylovora (22, 43). The known antibiotics produced by P. agglomerans strains, which belong to diverse chemical classes and affect different molecular targets, exhibit both narrow- and broad-spectrum activities (21).For example, P. agglomerans Eh318, isolated from apple leaves, produces two peptide antibiotics, pantocin A and pantocin B; both interfere with amino acid biosynthesis. Pantocin A blocks l-histidinol phosphate aminotransferase (20), and pantocin B acts as an N-acetylornithine transaminase inhibitor (5). Consequently, their inhibitory effects can be compensated for by supplementation with l-histidine and l-arginine, respectively (43). Giddens et al. (2002) described a phenazine antibiotic and its precursors, which were produced by P. agglomerans Eh1087 (10). Andrimid, a hybrid nonribosomal peptide polyketide antibiotic from P. agglomerans Eh335, selectively blocks the carboxyl transfer reaction of prokaryotic acetyl coenzyme A carboxylase; this reaction catalyzes the first committed step of fatty acid biosynthesis (19, 26). P. agglomerans E325 sold as Bloomtime Biological (Northwest Agricultural Products, Pasco, WA) acidifies flower stigmata, thus reducing the growth of E. amylovora. Simultaneously, it produces an antibiotic that has high specificity against E. amylovora and is effective under low-phosphate and low-pH conditions (34).P. agglomerans C9-1, which is registered as the biocontrol agent BlightBan C9-1 (Nufarm Agricultural Inc.), produces two antibiotics, herbicolin O and herbicolin I (16). Like pantocin A, herbicolin O loses its activity in the presence of histidine. However, herbicolin I does not become ineffective in the presence of amino acids (17). Although C9-1 is registered as a biocontrol agent, the chemical nature of herbicolins has remained largely unknown (13, 14).P. agglomerans 48b/90 (Pa48b), an epiphyte from soybean leaves (40), attracted our attention because it strongly inhibits the growth of E. amylovora and Pseudomonas syringae pv. glycinea (27), the pathogen that causes the bacterial blight of soybean. Since the mode of action of Pa48b against plant pathogens, in particular E. amylovora, is elusive, we looked for the molecular basis for the biocontrol potential of Pa48b. Here we describe the isolation, structure elucidation, and bioactivity of a potent antibiotic against plant pathogens that is produced by several P. agglomerans strains. The properties of this antibiotic perfectly match those of the chemically unidentified herbicolin I from P. agglomerans C9-1 (BlightBan C9-1).     

Genetic diversity and antifungal activity of native Pseudomonas isolated from maize plants grown in a central region of Argentina

Pseudomonas strains producing antimicrobial secondary metabolites play an important role in the biocontrol of phytopathogenic fungi. In this study, native Pseudomonas spp. isolates were obtained from the rhizosphere, endorhizosphere and bulk soil of maize fields in Córdoba (Argentina) during both the vegetative and reproductive stages of plant growth. However, the diversity based on repetitive-element PCR (rep-PCR) and amplified ribosomal DNA restriction analysis (ARDRA) fingerprinting was not associated with the stage of plant growth. Moreover, the antagonistic activity of the native isolates against phytopathogenic fungi was evaluated in vitro. Several strains inhibited members of the genera Fusarium, Sclerotinia or Sclerotium and this antagonism was related to their ability to produce secondary metabolites. A phylogenetic analysis based on rpoB or 16S rRNA gene sequences confirmed that the isolates DGR22, MGR4 and MGR39 with high biocontrol potential belonged to the genus Pseudomonas. Some native strains of Pseudomonas were also able to synthesise indole acetic acid and to solubilise phosphate, thus possessing potential plant growth-promoting (PGPR) traits, in addition to their antifungal activity. It was possible to establish a relationship between PGPR or biocontrol activity and the phylogeny of the strains. The study allowed the creation of a local collection of indigenous Pseudomonas which could be applied in agriculture to minimise the utilisation of chemical pesticides and fertilisers.     

The purB gene controls rhizosphere colonization by Pantoea agglomerans

Aims: To isolate the rhizosphere competence‐defective transposon Tn5 mutant of Pantoea agglomerans NBRISRM (SRM) and to identify the gene causing defect in its root colonization ability. Methods and Results: From over 5000 clones containing Tn5, one mutant P. agglomerans NBRISRMT (SRMT) showing 6 log units less colonization when compared with SRM, after 30 days in sand‐nonsterilized soil assay system was selected for further work to determine the effects of the mutation on rhizosphere competence. Southern hybridization analysis of restricted genomic DNA of SRMT demonstrated that the mutant had a single Tn5 insert. SRM increased in titre to about 2 × 10⁸ CFU g^?1 root, compared with the indigenous bacterial population of heterotrophs of about 5 × 10⁷ CFU g^?1 root. In contrast, 30 days later, the titre value of SRMT was almost undetectable at 1 × 10² CFU g^?1 root, demonstrating its inability to survive and colonize the rhizosphere. Sequencing of the flanking region of the Tn5 mutant revealed that Tn5 disrupted the purB gene. Conclusions: A defect in the colonization phenotype of the SRMT was attributed to the disruption in adenylosuccinate lyase (EC 4.3.2.2) which is encoded by the pur B gene and is required for rhizosphere colonization in P. agglomerans. Significantly less exopolysaccharide and biofilm was formed by SRMT when compared to SRM, because of the disruption of the purB gene. Significance and Impact of the Study: This work provides the first evidence for a functional role of purB gene in rhizosphere competence and root colonization by any rhizobacteria.     

pMH2, a small plasmid bearing the nif gene cluster of Enterobacter agglomerans 333 as an excisable cassette

A small plasmid containing the entire nif gene cluster of Enterobacter agglomerans 333 as an excisable cassette has been constructed, using pACYC177 as a vector. Two cosmid clones taken from a gene library of E. agglomerans plasmid pEA3 were used as a source of nif genes. A SmaI fragment of peaMS2-2, containing the H,D,K,Y,E,N,X,U,S,V,W,Z,M,L,A and B genes and an ApaI fragment of peaMS2-16 containing nifA,B,Q,F and J were selected to construct pMH2. The resulting plasmid of 33 kb carries the complete nif gene cluster as a nif cassette on a single XbaI fragment. The nif construct pMH2 in Escherichia coli strains has significant nitrogenase activity compared to wild-type E. agglomerans 333. The nif gene cluster construct was found to be very stable.     

Rhizosphere competent <Emphasis Type="Italic">Pantoea agglomerans</Emphasis> enhances maize (<Emphasis Type="Italic">Zea mays</Emphasis>) and chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.) growth,without altering the rhizosphere functional diversity

Plant growth promoting Pantoea agglomerans NBRISRM (NBRISRM) was able to produce 60.4 μg/ml indole acetic acid and solubilize 77.5 μg/ml tri-calcium phosphate under in vitro conditions. Addition of 2% NaCl (w/v) in the media induced the IAA production and phosphate solubilization by 11% and 7%, respectively. For evaluating the plant growth promotory effect of NBRISRM inoculation a micro plot trial was conducted using maize and chickpea as host plants. The results revealed significant increase in all growth parameters tested in NBRISRM inoculated maize and chickpea plants, which were further confirmed by higher macronutrients (N, P and K) accumulation as compared to un-inoculated controls. Throughout the growing season of maize and chickpea, rhizosphere population of NBRISRM were in the range 10⁷–10⁸ CFU/g soil and competing with 10⁷–10⁹ CFU/g soil with heterogeneous bacterial population. Functional richness, diversity, and evenness were found significantly higher in maize rhizosphere as compared to chickpea, whereas NBRISRM inoculation were not able to change it, in both crops as compared to their un-inoculated control. To the best of our knowledge this is first report where we demonstrated the effect of P. agglomerans strain for improving maize and chickpea growth without altering the functional diversity.     

Biological control of phytopathogenic bacteria Pantoea agglomerans and Erwinia chrysanthemi using 100 essential oils

Plants interact with a wide variety of pathogenic organisms by virtue of their sessile lifestyle. The Pantoea agglomerans and Erwinia chrysanthemi are major plant pathogen amongst them. They are known to cause significant losses in many crop plants. In the present study, bacteria isolated from infected Cajanus cajan and Arachis hypogaea seed are identified by 16S rDNA sequencing as P. agglomerans and E. chrysanthemi, respectively. In vitro antimicrobial properties of 100 essential oils (EOs) were evaluated against P. agglomerans and E. chrysanthemi. The chemical composition of most active EOs was investigated by gas chromatography–mass spectral analysis. The potential properties of these EOs as ecofriendly and economical biocontrol in agriculture is discussed.     

Improved germination efficiency of Salicornia ramosissima seeds inoculated with Bacillus aryabhattai SP1016‐20

Saline agriculture and the crop cultivation of halophytes represent an alternative for the reclamation of salinized soils and for the management of irrigation water. Halotolerant plant growth promoting bacteria with biocontrol effect, as an alternative to commercial fungicides, may contribute to improve crop productivity while mitigating saline stress effects. The objective of this work was to isolate autochthonous rhizobacteria with biocontrol features, to be used as germination enhancers and plant‐growth promoting agents in the crop cultivation of Salicornia ramosissima. A set of isolates obtained from the rhizosphere of S. ramosissima was characterised in terms of Gram, motility, salt tolerance and biocontrol traits (hydrogen cyanide production, antifungal activity and production of extracellular lipases and proteases). One Gram‐positive motile isolate that tested positive for all biocontrol traits was identified by 16S rRNA gene sequencing as Bacillus aryabhattai. The inoculation of S. ramosissima seeds with B. aryabhattai SP1016‐20 reduced the negative effect of salinity on the germination efficiency. At the highest tested salinity (30 g/L NaCl) the final germination efficiency of inoculated seeds doubled in relation to non‐inoculated controls. Although the mechanisms involved in the biocontrol effect were not defined in the current work, the results highlight the potential of Bacillus aryabhattai SP1016‐20 as a plant‐growth promoting agent for the crop cultivation of Salicornia and contribute to the strengthening of the scientific basis of biosaline agriculture and plant growth promoting rhizobacteria‐assisted crop cultivation of halophytes in saline soils and estuarine sediments.     

The Beta vulgaris-derived resistance gene Rz2 confers broad-spectrum resistance against soilborne sugar beet-infecting viruses from different families by recognizing triple gene block protein 1

Sugar beet cultivation is dependent on an effective control of beet necrotic yellow vein virus (BNYVV, family Benyviridae), which causes tremendous economic losses in sugar production. As the virus is transmitted by a soilborne protist, the use of resistant cultivars is currently the only way to control the disease. The Rz2 gene product belongs to a family of proteins conferring resistance towards diverse pathogens in plants. These proteins contain coiled-coil and leucine-rich repeat domains. After artificial inoculation of homozygous Rz2 resistant sugar beet lines, BNYVV and beet soilborne mosaic virus (BSBMV, family Benyviridae) were not detected. Analysis of the expression of Rz2 in naturally infected plants indicated constitutive expression in the root system. In a transient assay, coexpression of Rz2 and the individual BNYVV-encoded proteins revealed that only the combination of Rz2 and triple gene block protein 1 (TGB1) resulted in a hypersensitive reaction (HR)-like response. Furthermore, HR was also triggered by the TGB1 homologues from BSBMV as well as from the more distantly related beet soilborne virus (family Virgaviridae). This is the first report of an R gene providing resistance across different plant virus families.     

Survival of native <Emphasis Type="Italic">Pseudomonas</Emphasis> in soil and wheat rhizosphere and antagonist activity against plant pathogenic fungi

Survival of Pseudomonas sp. SF4c and Pseudomonas sp. SF10b (two plant-growth-promoting bacteria isolated from wheat rhizosphere) was investigated in microcosms. Spontaneous rifampicin-resistant mutants derived from these strains (showing both growth rate and viability comparable to the wild-strains) were used to monitor the strains in bulk soil and wheat rhizosphere. Studies were carried out for 60 days in pots containing non-sterile fertilized or non-fertilized soil. The number of viable cells of both mutant strains declined during the first days but then became established in the wheat rhizosphere at an appropriate cell density in both kinds of soil. Survival of the strains was better in the rhizosphere than in the bulk soil. Finally, the antagonism of Pseudomonas spp. against phytopatogenic fungi was evaluated in vitro. Both strains inhibited the mycelial growth (or the resistance structures) of some of the phytopathogenic fungi tested, though variation in this antagonism was observed in different media. This inhibition could be due to the production of extracellular enzymes, hydrogen cyanide or siderophores, signifying that these microorganisms might be applied in agriculture to minimize the utilization of chemical pesticides and fertilizers.     

茎瘤芥根际一株产紫色杆菌素杜擀氏菌的分离鉴定及其基因组分析

【目的】对茎瘤芥根际微生物进行分离鉴定,分析微生物菌群构成,选择具有优良特性的菌株,评估其次级代谢产物合成能力,为茎瘤芥根际微生物多样性和菌种资源的挖掘利用奠定基础。【方法】采集重庆市涪陵区二渡村和邓家村的茎瘤芥根,分离培养根际微生物菌株,通过菌株形态观察和看家基因的序列分析,对菌株进行初步鉴定、归类和保存。选择具有优良性状的菌株,利用Pacbio RS II和Illumina HiSeq平台完成全基因组测序,通过antiSMASH分析评估其次级代谢产物合成潜力,克隆目的基因簇并进行异源表达和产物鉴定。【结果】分离得到256株微生物,初步鉴定120株;从中鉴定了一株产紫色杆菌素的杜擀氏菌BjR8,完成了基因组测序及分析,发现该菌基因组为一条环状染色体,全长7 205 593 bp,GC含量为64.67%,含有6 241个编码基因。生物信息学分析发现基因组含有9个次级代谢产物生物合成基因簇,其中7个基因簇与已知化合物编码基因簇同源性较低,说明该菌具有产生多种新型次级代谢产物的潜力;克隆得到紫色杆菌素生物合成基因簇,并在变铅青链霉菌TK23中完成了异源表达。【结论】从茎瘤芥根际分离得到25...     

Effect of a phosphate solubilizing bacteria on maize growth and root exudation over four levels of labile phosphorus

The influence of inoculation with phosphate-solubilizing bacteria (Enterobacter agglomerans) on maize growth, P uptake and root exudation was studied. Plants were grown in an axenic culture device where P was supplied as soluble phosphate at different contents (0, 5, 15 or 25 ppm) in the nutrient solution and as insoluble rock phosphate added to the culture sand. Experimental device was successfully used to obtain axenic root systems or good establishment of the inoculated strain in the rhizosphere of maize (10⁹ bact. g⁻¹ dry rhizospheric material). Plant growth was promoted by inoculation only for 5 or 15 ppm of soluble P in the nutrient medium without any significant effect on P uptake by the plant, suggesting that the quantities of P released by bacterial rhizospheric activity were very small. Amounts of organic compounds (total C and water-soluble C) exuded were relatively low (3.0 to 6.4% of the total plant biomass) and were reduced by bacterial inoculation when plant growth was largely promoted. Carbon balance modification and plant growth hormone production by the inoculated strain were suspected and discussed.