Is phosphate solubilizing ability in plant growth-promoting rhizobacteria isolated from chickpea linked to their ability to produce ACC deaminase?

AIMS: Since most phosphate solubilizing bacteria (PSB) also produce 1-aminocyclopropane-1-carboxylate (ACC) deaminase, we investigated if there was an association between these two plant growth-promoting properties under in vitro conditions. METHODS AND RESULTS: A total of 841 bacterial isolates were obtained using selective and enrichment isolation methods. ACC deaminase was investigated using in vitro methods and by sequencing the acdS gene. The effect of ACC deaminase on P solubilization was investigated further using five efficient PSB. ACC deaminase production ability was found amongst a wide range of bacteria belonging to the genera Bacillus, Burkholderia, Pseudomonas and Variovorax. The amount of ACC deaminase produced by PSB was significantly associated with the liberation of Pi from Ca-P when ACC was the sole N source. Ca-P solubilization was associated with the degree of acidification of the medium. Additionally, the P solubilization potential of PSB with (NH4 )2 SO4 was determined by the type of carboxylates produced. An in-planta experiment was conducted using Burkholderia sp. 12F on chickpea cv. Genesis-863 in sand : vermiculite (1 : 1 v/v) amended with rock phosphate and inoculation of this efficient PSB significantly increased growth, nodulation and P uptake of chickpea fertilized with rock phosphate. CONCLUSION: ACC deaminase activity influenced the capacity of PSB to solubilize P from Ca-P when ACC was the sole N source and Burkholderia sp. 12F promoted the chickpea-Mesorhizobium symbiosis. SIGNIFICANCE AND IMPACT OF THE STUDY: ACC deaminase activity could enhance the P solubilizing activity of rhizobacteria that improve plant growth.

Mechanisms in plant growth-promoting rhizobacteria that enhance legume-rhizobial symbioses.

Nitrogen fixation is an important biological process in terrestrial ecosystems and for global crop production. Legume nodulation and N2 fixation have been improved using nodule-enhancing rhizobacteria (NER) under both regular and stressed conditions. The positive effect of NER on legume-rhizobia symbiosis can be facilitated by plant growth-promoting (PGP) mechanisms, some of which remain to be identified. NER that produce aminocyclopropane-1-carboxylic acid deaminase and indole acetic acid enhance the legume-rhizobia symbiosis through (i) enhancing the nodule induction, (ii) improving the competitiveness of rhizobia for nodulation, (iii) prolonging functional nodules by suppressing nodule senescence and (iv) upregulating genes associated with legume-rhizobia symbiosis. The means by which these processes enhance the legume-rhizobia symbiosis is the focus of this review. A better understanding of the mechanisms by which PGP rhizobacteria operate, and how they can be altered, will provide opportunities to enhance legume-rhizobial interactions, to provide new advances in plant growth promotion and N2 fixation.

Impact of seed-applied pesticides on rhizobial survival and legume nodulation.

AIMS: Compatibility of seed-applied pesticides and rhizobial inoculants is an important consideration for farmers when sowing legumes. Some of the seed-applied pesticides may influence rhizobial growth and nodulation, but there is currently little available information on the potential inhibitory effects. Therefore, common seed fungicidal and insecticidal treatments were assessed to determine adverse impacts on rhizobial inoculants both in vitro, on treated seed, and in the field. METHODS AND RESULTS: Initially, the in vitro toxicity of the seed-applied fungicides Thiram 600, P-Pickel T (PPT), their active ingredients (thiram and thiabendazole) and the insecticide Gaucho to rhizobia was measured with filter discs containing varying concentrations of the pesticides. Pea and chickpea seed was then coated with the same pesticides and inoculated with rhizobia in different inoculant substrates to determine bacterial survival and nodulation. Finally, a field trial using the fungicide PPT and commercial inoculants was conducted. Some seed fungicide treatments were found to be inhibitory to rhizobia and reduce nodulation under monoxenic conditions and in the field. SIGNIFICANCE AND IMPACT OF THE STUDY: These data provide more detailed information on the compatibility of specific rhizobial inoculants with common seed-applied pesticides. This research will provide information on the compatibility of rhizobia and seed-applied pesticides, and assist farmers to select sowing practices which reduce the risk of crop nodulation failures.

Feeding of the Nematode Acrobeloides nanus on Bacteria.

Information on the effect of bacteria-feeding nematodes on bacterial populations in the soil is sparse. We have isolated, cultured, and microscopically examined bacteria and nematodes coexisting within an agricultural soil and have studied their feeding relationship. The bacterium Pseudomonas corrugata isolate 2140R is a biocontrol agent against the pathogenic fungus Gaeumannomyces graminis var. tritici. The nematode Acrobeloides nanus is a cosmopolitan, bacteria-feeding organism widespread in agricultural and arid soils throughout Australia. Using light and electron microscopy, we observed the ingestion and breakdown of P. corrugata in the pharynx of A. nanus and bacterial passage through the nematode intestine as well as the accumulation of fluorescent compounds from ingested and broken P. fluorescens in the lumen of the nematode's intestine. We also observed A. nanus feeding, growing, and reproducing on the Gram-positive bacterium Clavibacter toxicus, the causative agent of the disease annual ryegrass toxicity, and detected crushed bacteria in the nematode's intestine.

[Introduction of the chromogenic gene to the plant growth-promoting rhizobacteria of cucumber].

Using a bicomponent transposition system with the E. coli lacZY gene cloned between Tn7 termini, a sensitive, selectable marker based on expression of the E. coli lac operon genes encoding beta-galactosidase and lactose permease was transformed into the rifampicin resistant mutant of plant growth-promoting rhizobacteria of cucumber, Pseudomonas aeruginosa CN116 and Pseudomonas corrugata CN31, respectively. Transformants were conferred the ability to utilize lactose as a sole carbon source and the ability to cleave the chromogenis substrate X-Gal to show a specific blue color. Southern blotting analysis showed that lacZY gene was inserted into the genome DNA of target strains. Compared with the wild type strains, the cultural characters, morphological features, growth promoting and disease control effects of transformants were almost unchanged, except the new marked phenotype. This marker system enabled the detection of lac+ transformants at sensitivity of 10 CFU/g soil, which makes the further studies on PGPR more easily.

The effect of Penicillium fungi on plant growth and phosphorus mobilization in neutral to alkaline soils from southern Australia.

The phosphate solubilizing fungi Penicillium radicum, Penicillium bilaiae (strain RS7B-SD1), and an unidentified Penicillium sp. designated strain KC6-W2 were tested for their ability to increase the growth and phosphorus (P) nutrition of wheat, medic, and lentil in three soils of neutral to alkaline pH reaction. The strongest plant growth promoting (PGP) strain was Penicillium sp. KC6-W2, which stimulated significant increases in shoot growth and dry mass in seven of the nine experiments conducted. Levels of PGP by Penicillium sp. KC6-W2 ranged from 6.6% to 19% and were associated with increased uptake of P to the shoot. The PGP properties of Penicillium sp. KC6-W2 were evident on each of the three different plant species and soil types, a level of reliability not observed in other strains tested. Inoculation of seed with P. radicum increased lentil growth by 5.5% (P < 0.05) in soil from Tarlee but did not affect plant growth in the eight other experiments. Inoculation of plant seed with P. bilaiae RS7B-SD1 resulted in significant PGP in two of the nine experiments conducted. However, when significant, stimulation of PGP by P. bilaiae RS7B-SD1 was strong and resulted in increases in medic dry matter (19%) and lentil shoot dry matter (15%). A soil microcosm experiment investigated the effect of Penicillium fungi on cycling of soil P. Penicillium bilaiae RS7B-SD1 was the only fungus to significantly increase HCO3-extractable P (23% increase; P < 0.05). Production of phosphatase enzymes was not associated with increased HCO3-extractable P. Addition of carbon in the form of ryegrass seed significantly increased microbial respiration and movement of P to the microbial biomass (P < 0.05), but these parameters were irrespective of Penicillium treatment. This work has established the potential for use of Penicillium inoculants to increase plant growth on alkaline soils in Australia. The role of Penicillium fungi in plant P uptake and soil P cycling requires further exploration.

Early production of rhizopine in nodules induced by Sinorhizobium meliloti strain L5-30.

The rhizopine L-3-O-methyl-scyllo-inosamine (3-O-MSI) is metabolized by approximately 10% of the strains of Rhizobium leguminosarum by. viciae and Sinorhizobium meliloti. Rhizopine strains enjoy a substantial competitive advantage in nodulation, which is manifest before 14 days post-inoculation, implying that rhizopine is produced before this time. We were able to detect this compound in the roots of alfalfa (Medicago sativum L. cv. Hunter River) four days after germination (six days post-infection) with S. meliloti strain L5-30 by gas chromatography-mass spectrometry (GC-MS). At four days, nodules were not visible, and the concentration of rhizopine was extremely low, estimated at 67 pg/gfw (picograms/gram fresh weight). The amount increased gradually but remained low until 16 days, when there was a 50-fold increase from day four, by which time nodules were well established. This pattern of synthesis is consistent with previous studies indicating that rhizopine synthesis is regulated by nifA/ntrA regulatory genes, which are maximally expressed in bacteroids at the onset of nitrogen fixation. However, the low level of rhizopine synthesis must be responsible for the early effects on competition for nodulation. Production of rhizopine at this time most likely results from micro-aerobic induction of mos genes in free-living bacteria, either in the infection threads or in the rhizosphere.

Agrocinopine A, a tumor-inducing plasmid-coded enzyme product, is a phosphodiester of sucrose and L-arabinose.

Opines are unusual compounds found specifically in plant crown gall tumors. Genes for their synthesis and catabolism reside in agrobacteria as tumor-inducing (Ti) plasmid DNA. Only a small Ti-plasmid segment (24 kilobase pairs), the T-DNA, is transferred to the plant cell where it commonly codes for enzymes involved in the biosynthesis of nitrogenous opines such as nopaline (N2-(1,3-D-dicarboxypropyl)-L-arginine) as well as the tumor phenotype. Ellis and Murphy, (Ellis, J.G., and Murphy, P.J. (1981) Mol. Gen. Genet. 181, 36-43) reported the existence of the phosphorylated opines, agrocinopines A and B in tumors containing nopaline. Pure agrocinopine A has now been isolated in a yield of 0.05-0.06 g/100 g, fresh weight, from such tumors. Physical, chemical, and biological data establish the structure of agrocinopine A as an unusual non-nitrogenous opine of sucrose and L-arabinose with a phosphodiester linkage from the 2-hydroxyl of the arabinose to the 4-hydroxyl of the fructose moiety in sucrose. Agrocinopine B is the corresponding phosphodiester, in which the glucose has been hydrolyzed from the sucrose portion of agrocinopine A. Borohydride reduction of the free L-arabinose anomeric carbon of agrocinopine A, to the corresponding arabinitol derivative eliminates the characteristic inhibition zone enhancement produced by both agrocinopines A and B in the agrocin 84 (a fraudulent adenine nucleotide) bioassay. Because of the limited number of genes in the T-DNA, a generalization is proposed, whereby all opines will be found to comprise two common plant cell constituents linked in an uncommon manner by the minimum number of enzymes.

Virulence properties of strains of agrobacterium on the apical and Basal surfaces of carrot root discs.

Most pathogenic strains of Agrobacterium are able to induce crown gall or hairy root on both the apical surface (facing the root tip) and the basal surface (facing the shoot) of carrot (Daucus carota L.) root discs. Tumorigenic strains carrying mutations in the shoot inhibition region of the T-DNA (TL-DNA genes 1 and 2) are markedly attenuated on the basal surface but remain virulent on the apical surface. Coinoculation of two attenuated tumorigenic strains, with mutations in gene 1 and gene 2, respectively, resulted in restoration of virulence on the basal surface. Wild type hairy root-inducing strains can be divided into two groups: those that are virulent on both apical and basal surfaces and those that are virulent only on the apical surface. alpha-Naphthalene acetic acid stimulated virulence of hairy root strain TR7, belonging to the latter group, on the basal surface. Attenuated virulence on the basal surface can be explained in terms of an auxin deficiency in the basal tissues and unidirectional auxin transport to the apical surface.

Genetic analysis of agrocin 84 production and immunity in Agrobacterium spp.

Mutations affecting agrocin production on the 48-kilobase (kb) plasmid, pAgK84, can be complemented in trans with cloned portions of the plasmid. Five complementation groups ranging in minimum size from 1.2 to 5.6 kb were identified within a 14-kb segment. Plasmid pAgK84-encoded immunity to agrocin 84 was located to two separate regions of the plasmid. Either region alone was sufficient to protect sensitive strains, and both loci mapped to the agrocin 84 biosynthesis region. One region is located within complementation group I, while the other forms a part of complementation group IV. Production of agrocin 84 was unaffected by nopaline, agrocinopine A, acetosyringone, or low or high levels of ferric iron. Agrocin 84 production was greatly suppressed when the strain also contained a Ti plasmid nutritionally or mutationally derepressed for agrocinopine A catabolism. RNA dot-blot analysis indicated that decreased agrocin 84 production by such strains was not due to transcriptional repression of agrocin 84 biosynthetic loci. In strains also harboring pAtK84b, the opine catabolic plasmid of Agrobacterium radiobacter K84, induction of the agrocinopine A catabolic locus of this plasmid had no such effect on agrocin 84 production.

An Experimental Test of the Rhizopine Concept in Rhizobium meliloti.

In some Rhizobium-legume symbioses, compounds known as rhizopines are synthesized by bacteroids and subsequently catabolized by free-living cells of the producing strain. It has been suggested than rhizopines act as proprietary growth substrates and enhance the competitive ability of the producing strain in its interactions with the diverse microbial community found within the rhizosphere. Wild-type, rhizopine-producing Rhizobium meliloti L5-30 and mutant L5-30 strains deficient for either rhizopine synthesis or catabolism were inoculated onto lucerne host plants in competition experiments. These experiments demonstrated that no apparent advantage resulted from the ability to synthesize a rhizopine, whereas the ability to catabolize rhizopine provided a clear advantage when an organism was in competition with a strain without this ability. The results suggest that when an organism is in competition with a catabolism-deficient mutant, the ability to catabolize rhizopine results in enhanced rates of nodulation. The results of the experiments were not consistent with the hypothesis that the sole role of rhizopines is to act as proprietary growth substrates for the free-living population of the producing strain.

Genetic diversity and biological control activity of novel species of closely related pseudomonads isolated from wheat field soils in South Australia.

Rhizobacteria closely related to two recently described species of pseudomonads, Pseudomonas brassicacearum and Pseudomonas thivervalensis, were isolated from two geographically distinct wheat field soils in South Australia. Isolation was undertaken by either selective plating or immunotrapping utilizing a polyclonal antibody raised against P. brassicacearum. A subset of 42 isolates were characterized by amplified 16S ribosomal DNA restriction analysis (ARDRA), BIOLOG analysis, and gas chromatography-fatty acid methyl ester (GC-FAME) analysis and separated into closely related phenetic groups. More than 75% of isolates tested by ARDRA were found to have >95% similarity to either Pseudomonas corrugata or P. brassicacearum-P. thivervalensis type strains, and all isolates had >90% similarity to either type strain. BIOLOG and GC-FAME clustering showed a >70% match to ARDRA profiles. Strains representing different ARDRA groups were tested in two soil types for biological control activity against the soilborne plant pathogen Gaeumannomyces graminis var. tritici, the causative agent of take-all of wheat and barley. Three isolates out of 11 significantly reduced take-all-induced root lesions on wheat plants grown in a red-brown earth soil. Only one strain, K208, was consistent in reducing disease symptoms in both the acidic red-brown earth and a calcareous sandy loam. Results from this study indicate that P. brassicacearum and P. thivervalensis are present in Australian soils and that a level of genetic diversity exists within these two novel species but that this diversity does not appear to be related to geographic distribution. The result of the glasshouse pot trial suggests that some isolates of these species may have potential as biological control agents for plant disease.

Diversification of Pseudomonas corrugata 2140 produces new phenotypes altered in GC-FAME, BIOLOG, and in vitro inhibition profiles and taxonomic identification.

Bacteria are known to rapidly produce new phenotypes, but it is unclear how phenotype "plasticity" relates to studies on the population ecology of bacteria in complex environments. We characterised a collection of 14 spontaneous phenotype variants, derived from in vitro and in vivo cultures (wheat roots) of Pseudomonas corrugata 2140, using fatty acid methyl ester profiles (GC-FAME), carbon substrate utilisation (BIOLOG), and in vitro inhibition against seven soil microorganisms. All three phenotype profiles indicated marked differences between some variants and the parent isolate. Some variant types were classified taxonomically by GC-FAME as different species to their wild-type parent, and up to a Euclidian distance of 11 from their parent. Taxonomic identification by the BIOLOG assay was more consistent; however, use of 22 carbon sources were altered (lost or gained) in one or more variants. All variant types had a reduced ability to inhibit one or more test organisms, depending on the variant and test organism. Hierarchical cluster analysis of variants using GC-FAME, BIOLOG, and inhibition profiles produced different groupings. The ability of variants to cross taxonomic boundaries specified by the GC-FAME and BIOLOG libraries at the species level has implications for both taxonomy and the ecological study of bacterial communities.

Survival of a lacZY-marked strain of Pseudomonas corrugata following a field release.

Abstract Pseudomonas corrugata, strain 2140, a biological control agent of take-all disease of wheat, was originally isolated from an acidic red-brown earth soil in New South Wales, Australia. A spontaneous rifampicin-resistant mutant of this bacterium was marked with the disarmed transposon, Tn7::lacZY. This marked strain (2140RlacZY) was introduced into a calcareous sandy loam soil (pH 8) in South Australia. Up to 4 years after its release, P. corrugata 2140RlacZY cells were re-isolated, single colony purified and stored at -80 degrees C. Re-isolated bacteria, including re-isolates obtained 3 (22 re-isolates) and 4 (3 re-isolates) years after release, were examined for stability of the lacZY insert site and for gross chromosomal changes. Hybridization of a cloned lacZY fragment to DNA extracted from the soil re-isolates did not reveal any major changes to the lacZY insert site. Gross chromosomal changes were further examined by restriction endonuclease fingerprinting and PCR based on repetitive sequences (repetitive extragenic palindromic-, enterobacterial repetitive intergeneric consensus- and BOX-PCR). MspI digests distinguished the lacZY-marked strain from the parental strain. None of the genetic techniques used revealed any polymorphisms between the original 2140RlacZY-marked strain and the soil re-isolates. The results demonstrated that the chromosomal landscape within and around the insertion site of the lacZY construct had not altered in the re-isolated bacteria during the 4 years the organism had been in the field.