Induction of pre-infection thread structures in the leguminous host plant by mitogenic lipo-oligosaccharides of Rhizobium.

Root nodules of leguminous plants are symbiotic organs in which Rhizobium bacteria fix nitrogen. Their formation requires the induction of a nodule meristem and the formation of a tubular structure, the infection thread, through which the rhizobia reach the nodule primordium. In the Rhizobium host plants pea and vetch, pre-infection thread structures always preceded the formation of infection threads. These structures consisted of cytoplasmic bridges traversing the central vacuole of outer cortical root cells, aligned in radial rows. In vetch, the site of the infection thread was determined by the plant rather than by the invading rhizobia. Like nodule primordia, pre-infection thread structures could be induced in the absence of rhizobia provided that mitogenic lipo-oligosaccharides produced by Rhizobium leguminosarum biovar viciae were added to the plant. In this case, cells in the two outer cortical cell layers containing cytoplasmic bridges may have formed root hairs. A common morphogenetic pathway may be shared in the formation of root hairs and infection threads.

Molecular basis of plant growth promotion and biocontrol by rhizobacteria.

Plant-growth-promoting rhizobacteria (PGPRs) are used as inoculants for biofertilization, phytostimulation and biocontrol. The interactions of PGPRs with their biotic environment, for example with plants and microorganisms, are often complex. Substantial advances in elucidating the genetic basis of the beneficial effects of PGPRs on plants have been made, some from whole-genome sequencing projects. This progress will lead to a more efficient use of these strains and possibly to their improvement by genetic modification.

Molecular determinants of rhizosphere colonization by Pseudomonas.

Rhizosphere colonization is one of the first steps in the pathogenesis of soilborne microorganisms. It can also be crucial for the action of microbial inoculants used as biofertilizers, biopesticides, phytostimulators, and bioremediators. Pseudomonas, one of the best root colonizers, is therefore used as a model root colonizer. This review focuses on (a) the temporal-spatial description of root-colonizing bacteria as visualized by confocal laser scanning microscopal analysis of autofluorescent microorganisms, and (b) bacterial genes and traits involved in root colonization. The results show a strong parallel between traits used for the colonization of roots and of animal tissues, indicating the general importance of such a study. Finally, we identify several noteworthy areas for future research.

Selection of a plant-bacterium pair as a novel tool for rhizostimulation of polycyclic aromatic hydrocarbon-degrading bacteria.

We developed a novel procedure for the selection of a microbe-plant pair for the stable and efficient degradation of naphthalene. Based on the rationale that root exudate is the best nutrient source available in soil, the grass (Lolium multiflorum) cultivar Barmultra was selected because of its abilities to produce a highly branched root system, root deeply, and carry a high population of Pseudomonas spp. bacteria on its roots. Starting with a mixture of total rhizobacteria from grass-like vegetation collected from a heavily polluted site and selecting for stable naphthalene degradation as well as for efficient root colonization, Pseudomonas putida strain PCL1444 was isolated. The strain's ability to degrade naphthalene was shown to be stable in the rhizosphere. Moreover, it had superior root-colonizing properties because, after the inoculation of grass seedlings, it appeared to colonize the root tip up to 100-fold better than the efficient root colonizer Pseudomonas fluorescens WCS365. Strain PCL1444 uses root exudate as the dominant nutrient source because the presence of grass seedlings in soil results in up to a 10-fold increase of PCL1444 cells. Moreover, the root colonized by strain PCL1444 was able to penetrate through an agar layer, resulting in the degradation of naphthalene underneath this layer. In addition, the inoculation of grass seeds or seedlings with PCL1444 protected them against naphthalene phytotoxicity. Finally, this plant-microbe combination appeared able to degrade naphthalene from soil that was heavily polluted with a complex mixture of polycyclic aromatic hydrocarbons. To our knowledge, this is the first time that a naturally occurring bacterium has been selected for the combination of the abilities to degrade a pollutant and colonize plant roots. We suggest that the principle described here, to select a bacterium which combines efficient root colonization with a beneficial activity, also can be used to improve the selection of other more efficient plant-bacterium pairs for beneficial purposes such as biocontrol, biofertilization, and phytostimulation.

Detection and separation of Rhizobium and Bradyrhizobium Nod metabolites using thin-layer chromatography.

Using radioactive acetate as a precursor, it was shown that the common nodABC genes of Rhizobium and Bradyrhizobium strains are involved in the production of one or more metabolites that are excreted into the growth medium. A rapid thin-layer chromatography (TLC) system has been developed to separate these so-called Nod metabolites that can then be visualized by autoradiography. Different patterns of Nod metabolites were observed in the tested strains of the cross-inoculation groups of R. leguminosarum bv. viceae, R. l. bv. trifolii, R. meliloti, and B. japonicum. Only Nod metabolites of R. meliloti became labeled when radioactive sulphate was present in the medium. The role of the other nodulation genes of R. l. bv. viceae in the production of the detected Nod metabolites was tested in further detail. In addition to the common nodABC genes, the nodFE and nodL genes are involved in the production of Nod metabolites. In contrast, the chromosomal background did not influence the number of detected Nod metabolites or their mobilities on TLC plates. Nod metabolites could also be produced and excreted in Escherichia coli cells in which the appropriate nodulation genes were expressed.

A Rhizobium leguminosarum biovar trifolii locus not localized on the sym plasmid hinders effective nodulation on plants of the pea cross-inoculation group.

Introduction of the Sym plasmid pRL1JI into the cured Rhizobium leguminosarum bv. trifolii strain RCR5 resulted in a strain, designated RBL5523, that was expected to nodulate plants of the pea cross-inoculation group. However, effective nodulation occurred only on Vicia sativa plants, not on V. hirsuta or Pisum sativum. After random Tn5 mutagenesis, a derivative of RBL5523 was isolated that effectively nodulated and fixed nitrogen on P. sativum and V. hirsuta. Characterization of the mutant, RBL5787, indicated the cell surface components, extracellular polysaccharides, lipopolysaccharides, and outer membrane proteins, as well as the pattern of Nod metabolites, were indistinguishable from those of the parental strain. To obtain an indication of the function of the mutated locus, the flanking regions were sequenced and used to perform searches in protein and nonredundant nucleotide data-bases. No significant similarity or homology with any known sequence was detected.

Simultaneous imaging of Pseudomonas fluorescens WCS365 populations expressing three different autofluorescent proteins in the rhizosphere: new perspectives for studying microbial communities.

To visualize simultaneously different populations of pseudomonads in the rhizosphere at the single cell level in a noninvasive way, a set of four rhizosphere-stable plasmids was constructed expressing three different derivatives of the green fluorescent protein (GFP), namely enhanced cyan (ECFP), enhanced green (EGFP), enhanced yellow (EYFP), and the recently published red fluorescent protein (RFP; DsRed). Upon tomato seedling inoculation with Pseudomonas fluorescens WCS365 populations, each expressing a different autofluorescent protein followed by plant growth for 5 days, the rhizosphere was inspected using confocal laser scanning microscopy. We were able to visualize simultaneously and clearly distinguish from each other up to three different bacterial populations. Microcolonies consisting of mixed populations were frequently observed at the base of the root system, whereas microcolonies further toward the root tip predominantly consisted of a single population, suggesting a dynamic behavior of microcolonies over time. Since the cloning vector pME6010 has a broad host range for gram-negative bacteria, the constructed plasmids can be used for many purposes. In particular, they will be of great value for the analysis of microbial communities, for example in processes such as biocontrol, biofertilization, biostimulation, competition for niches, colonization, and biofilm formation.

Introduction of the phzH gene of Pseudomonas chlororaphis PCL1391 extends the range of biocontrol ability of phenazine-1-carboxylic acid-producing Pseudomonas spp. strains.

Pseudomonas chlororaphis PCL1391 controls tomato foot and root rot caused by Fusarium oxysporum f. sp. radicis-lycopersici. Its biocontrol activity is mediated by the production of phenazine-1-carboxamide (PCN). In contrast, the take-all biocontrol strains P. fluorescens 2-79 and P. aureofaciens 30-84, which produce phenazine-1-carboxylic acid (PCA), do not control this disease. To determine the role of the amide group in biocontrol, the PCN biosynthetic genes of strain PCL1391 were identified and characterized. Downstream of phzA through phzG, the novel phenazine biosynthetic gene phzH was identified and shown to be required for the presence of the 1-carboxamide group of PCN because a phzH mutant of strain PCL1391 accumulated PCA. The deduced PhzH protein shows homology with asparagine synthetases that belong to the class II glutamine amidotransferases, indicating that the conversion of PCA to PCN occurs via a transamidase reaction catalyzed by PhzH. Mutation of phzH caused loss of biocontrol activity, showing that the 1-carboxamide group of PCN is crucial for control of tomato foot and root rot. PCN production and biocontrol activity of the mutant were restored by complementing the phzH gene in trans. Moreover, transfer of phzH under control of the tac promoter to the PCA-producing biocontrol strains P. fluorescens 2-79 and P. aureofaciens 30-84 enabled these strains to produce PCN instead of PCA and suppress tomato foot and root rot. Thus, we have shown, for what we believe is the first time, that the introduction of a single gene can efficiently extend the range of the biocontrol ability of bacterial strains.

Isolation of ropB, a gene encoding a 22-kDa Rhizobium leguminosarum outer membrane protein.

As judged from immunochemical detection, the levels of outer membrane antigen groups II and III of Rhizobium leguminosarum bv. viciae strain 248 decrease during bacteroid differentiation (R. A. de Maagd, R. de Rijk, I. H. M. Mulders, and B. J. J. Lugtenberg, J. Bacteriol. 171:1136-1142, 1989). Using a newly developed colony blot assay, a cosmid clone expressing the Mab8 epitope of the outer membrane antigen group II of R. l. bv. viciae strain 248 was selected in Rhizobium meliloti LPR2120. From this cosmid the gene encoding this epitope was cloned and characterized. An open reading frame of 636 nucleotides was found and predicted to encode a protein with a calculated molecular mass of 22.5 kDa. After subtraction of the predicted 23 amino acid signal peptide, a M(r) of 20.3 kDa was calculated for the mature protein. This gene, designated ropB, was not active in Escherichia coli under the control of its own promoter. The C-terminal amino acid of the protein is a phenylalanine residue which is required for efficient translocation of outer membrane proteins. Membrane spanning amphipathic beta-sheets are predicted to represent a major part of the secondary structure of the protein. A model of the topology of the protein is presented. We determined the start of transcription in order to analyze the promoter region. No homology was found with other known promoter sequences. The ropB gene appeared to be well-conserved in R. leguminosarum and Agrobacterium tumefaciens strains. An attempt was made to mimic the immunochemical decrease of RopB ex planta. Neither the various growth conditions tested nor the addition of nodule or plant extracts resulted in a reduction of the Mab8 epitope to bacteroid levels.

Increased uptake of putrescine in the rhizosphere inhibits competitive root colonization by Pseudomonas fluorescens strain WCS365.

Sequence analysis of the chromosomal Tn5lacZ flanking regions of the Pseudomonas fluorescens WCS365 competitive root colonization mutant PCL1206 showed that the Tn5lacZ is inserted between genes homologous to bioA and potF. The latter gene is the first gene of the potF1F2GHI operon, which codes for a putrescine transport system in Escherichia coli. The position of the Tn5lacZ suggests an effect on the expression of the pot operon. A mutation in the potF1 gene as constructed in PCL1270, however, had no effect on competitive root colonization. The rate of uptake of [1,4-14C]putrescine by cells of mutant PCL1206 appeared to be increased, whereas cells of strain PCL1270 were strongly impaired in the uptake of putrescine. Dansylation of tomato root exudate and subsequent thin-layer chromatography showed the presence of a component with the same Rf value as dansyl-putrescine, which was identified as dansyl-putrescine by mass spectrometric analyses. Other polyamines such as spermine and spermidine were not detected in the root exudate. Growth of mutant strains, either alone or in competition with the wild type, was tested in media containing putrescine, spermine, or spermidine as the sole nitrogen source. The results show that mutant PCL1206 is strongly impaired in growth on putrescine and slightly impaired on spermine and spermidine. The presence of the polyamines had a similar effect on the growth rate of strain PCL1270 in the presence of putrescine but a less severe effect in the presence of spermine and spermidine. We conclude that an increased rate of putrescine uptake has a bacteriostatic effect on Pseudomonas spp. cells. We have shown that putrescine is an important tomato root exudate component and that root-colonizing pseudomonads must carefully regulate their rate of uptake because increased uptake causes a decreased growth rate and, therefore, a decreased competitive colonization ability.

NodFE-dependent fatty acids that lack an alpha-beta unsaturation are subject to differential transfer, leading to novel phospholipids.

In Rhizobium leguminosarum, the nodABC and nodFEL operons are involved in the production of lipo-chitin oligosaccharide signals that mediate host specificity. A nodFE-determined, highly unsaturated C18:4 fatty acid (trans-2, trans-4, trans-6, cis-11-octadecatetraenoic acid) is essential for the ability of the signals to induce nodule meristems and pre-infection thread structures on the host plant Vicia sativa. Of the nod genes, induction of only nodFE is sufficient to modify fatty acid biosynthesis to yield trans-2, trans-4, trans-6, cis-11-octadecatetraenoic acid, with an absorbance maximum of 303 nm. This unusual C18:4 fatty acid is not only found in the lipo-chitin oligosaccharides but is also associated with the phospholipids (O. Geiger, J. E. Thomas-Oates, J. Glushka, H. P. Spaink, and B. J. J. Lugtenberg, 1994, J. Biol. Chem. 269:11090-11097). Here we report that the phospholipids can contain other nodFE-derived fatty acids, a C18:3 trans-4, trans-6, cis-11-octadecatrienoic acid that has a characteristic absorption maximum at 225 nm, and a C18:2 octadecadienoic acid. Neither this C18:3 nor this C18:2 fatty acid has to date been observed attached to lipo-chitin oligosaccharides, suggesting that an as yet unknown acyl transferase (presumably NodA), responsible for the transfer of the fatty acyl chain to the glycan backbone of the lipo-chitin oligosaccharides, does not transfer all fatty acids synthesized by the action of NodFE to the lipo-chitin oligosaccharides. Rather, it must have a preference for alpha-beta unsaturated fatty acids during transfer.

Role of the O-antigen of lipopolysaccharide, and possible roles of growth rate and of NADH:ubiquinone oxidoreductase (nuo) in competitive tomato root-tip colonization by Pseudomonas fluorescens WCS365.

Colonization-defective, transposon-induced mutants of the efficient root colonizer Pseudomonas fluorescens WCS365 were identified with a gnotobiotic system. Most mutants were impaired in known colonization traits, i.e., prototrophy for amino acids, motility, and synthesis of the O-antigen of LPS (lipopolysaccharide). Mutants lacking the O-antigen of LPS were impaired in both colonization and competitive growth whereas one mutant (PCL1205) with a shorter O-antigen chain was defective only in colonization ability, suggesting a role for the intact O-antigen of LPS in colonization. Eight competitive colonization mutants that were not defective in the above-mentioned traits colonized the tomato root tip well when inoculated alone, but were defective in competitive root colonization of tomato, radish, and wheat, indicating they contained mutations affecting host range. One of these eight mutants (PCL1201) was further characterized and contains a mutation in a gene that shows homology to the Escherichia coli nuo4 gene, which encodes a subunit of one of two known NADH:ubiquinone oxidoreductases. Competition experiments in an oxygen-poor medium between mutant PCL1201 and its parental strain showed a decreased growth rate of mutant PCL1201. The requirement of the nuo4 gene homolog for optimal growth under conditions of oxygen limitation suggests that the root-tip environment is micro-aerobic. A mutant characterized by a slow growth rate (PCL1216) was analyzed further and contained a mutation in a gene with similarity to the E. coli HtrB protein, a lauroyl transferase that functions in lipid A biosynthesis.

Gnotobiotic system for studying rhizosphere colonization by plant growth-promoting Pseudomonas bacteria.

A gnotobiotic system for studying tomato rhizosphere colonization by Pseudomonas bacteria was developed. The system is based on sterile seedlings that are inoculated with one or two strains and subsequently grown in a sterile glass tube containing quartz sand. After 7 days of growth in a climate-controlled growth chamber, the number of bacteria present on the root tip was analyzed. The system was optimized with respect to root morphology, inoculation of the seedling, and isolation of root tip bacteria. With this system, rhizosphere colonization on tomato, radish, wheat, and potato was analyzed. For detailed analysis of tomato rhizosphere colonization by some representative plant growth-promoting rhizo-bacteria, the colonization of known poor, moderate, and good potato root-colonizing Pseudomonas strains and of four Rhizobium strains was determined. All strains colonized the root tips when inoculated as single strains. When inoculated in competition with the efficient root colonizer P. fluorescens strain WCS365, many strains were outcompeted. Mutants of Pseudomonas biocontrol bacteria lacking flagella or the O-antigen of lipopolysaccharide (LPS), which were isolated in previous studies and shown to be impaired in potato rhizosphere colonization in field soil systems, showed a reduced colonization ability in the gnotobiotic system also. The gnotobiotic system was used to screen a collection of 300 random P. fluorescens WCS365::Tn5 mutants for colonization-impaired mutants. Three novel mutants were found that were outcompeted by the wild-type strain in tomato root tip colonization but were not impaired in known colonization traits such as motility, amino acid auxotrophy, and presence of the O-antigenic side chain of LPS. One strain appeared to be a thiamine auxotroph, suggesting that the root does not secrete a sufficient amount of thiamine to support growth of this strain. The other two mutants had a reduced growth rate in laboratory media, suggesting that growth rate is an important colonization factor. As the system is gnotobiotic and devoid of field-soil variables, it can also be used to study the effects of selected biotic and abiotic factors on colonization.

Root colonization by phenazine-1-carboxamide-producing bacterium Pseudomonas chlororaphis PCL1391 is essential for biocontrol of tomato foot and root rot.

The phenazine-1-carboxamide-producing bacterium Pseudomonas chlororaphis PCL1391 controls tomato foot and root rot caused by Fusarium oxysporum f. sp. radicislycopersici. To test whether root colonization is required for biocontrol, mutants impaired in the known colonization traits motility, prototrophy for amino acids, or production of the site-specific recombinase, Sss/XerC were tested for their root tip colonization and biocontrol abilities. Upon tomato seedling inoculation, colonization mutants of strain PCL1391 were impaired in root tip colonization in a gnotobiotic sand system and in potting soil. In addition, all mutants were impaired in their ability to control tomato foot and root rot, despite the fact that they produce wild-type levels of phenazine-1-carboxamide, the antifungal metabolite previously shown to be required for biocontrol. These results show, for what we believe to be the first time, that root colonization plays a crucial role in biocontrol, presumably by providing a delivery system for antifungal metabolites. The ability to colonize and produce phenazine-1-carboxamide is essential for control of F. oxysporum f. sp. radicis-lycopersici. Furthermore, there is a notable overlap of traits identified as being important for colonization of the rhizosphere and animal tissues.

The sss colonization gene of the tomato-Fusarium oxysporum f. sp. radicis-lycopersici biocontrol strain Pseudomonas fluorescens WCS365 can improve root colonization of other wild-type pseudomonas spp.bacteria.

We show that the disease tomato foot and root rot caused by the pathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici can be controlled by inoculation of seeds with cells of the efficient root colonizer Pseudomonas fluorescens WCS365, indicating that strain WCS365 is a biocontrol strain. The mechanism for disease suppression most likely is induced systemic resistance. P. fluorescens strain WCS365 and P. chlororaphis strain PCL1391, which acts through the production of the antibiotic phenazine-1-carboxamide, were differentially labeled using genes encoding autofluorescent proteins. Inoculation of seeds with a 1:1 mixture of these strains showed that, at the upper part of the root, the two cell types were present as microcolonies of either one or both cell types. Microcolonies at the lower root part were predominantly of one cell type. Mixed inoculation tended to improve biocontrol in comparison with single inoculations. In contrast to what was observed previously for strain PCL1391, mutations in various colonization genes, including sss, did not consistently decrease the biocontrol ability of strain WCS365. Multiple copies of the sss colonization gene in WCS365 improved neither colonization nor biocontrol by this strain. However, introduction of the sss-containing DNA fragment into the poor colonizer P. fluorescens WCS307 and into the good colonizer P. fluorescens F113 increased the competitive tomato root tip colonization ability of the latter strains 16- to 40-fold and 8- to 16-fold, respectively. These results show that improvement of the colonization ability of wild-type Pseudomonas strains by genetic engineering is a realistic goal.

A two-component system plays an important role in the root-colonizing ability of Pseudomonas fluorescens strain WCS365.

We describe the characterization of a novel Tn5lacZ colonization mutant of the efficiently colonizing Pseudomonas fluorescens strain WCS365, mutant strain PCL1210, which is at least 300- to 1,000-fold impaired in colonization of the potato root tip after co-inoculation of potato stem cuttings with a 1:1 mixture of mutant and parental cells. Similarly, the mutant is also impaired in colonization of tomato, wheat, and radish, indicating that the gene involved plays a role in the ability of P. fluorescens WCS365 to colonize a wide range of plant species. A 3.1-kb DNA fragment was found to be able to complement the observed mutation. The nucleotide sequence of the region around the Tn5lacZ insertion showed three open reading frames (ORFs). The transcriptional start site was determined. The operon is preceded by an integration host factor (IHF) binding site consensus sequence whereas no clear -10 and -35 sequences are present. The deduced amino acid sequences of the first two genes of the operon, designated as colR and colS, show strong similarity with known members of two-component regulatory systems. ColR has homology with the response regulators of the OmpR-PhoB subclass whereas ColS, the product of the gene in which the mutation resides, shows similarity to the sensor kinase members of these two-component systems. Hydrophobicity plots show that this hypothetical sensor kinase has two transmembrane domains, as is also known for other sensor kinases. The product of the third ORF, Orf222, shows no homology with known proteins. Only part of the orf222 gene is present in the colonization-complementing, 3.1-kb region, and it therefore does not play a role in complementation. No experimental evidence for a role of the ColR/ColS two-component system in the suspected colonization traits chemotaxis and transport of exudate compounds could be obtained. The function of this novel two-component system therefore remains to be elucidated. We conclude that colonization is an active process in which an environmental stimulus, through this two-component system, activates a so far unknown trait that is crucial for colonization.

Comparison of characteristics of the nodX genes from various Rhizobium leguminosarum strains.

We have analyzed the nucleotide sequences of the nodX genes from two strains of Rhizobium leguminosarum bv. viciae able to nodulate Afghan peas (strains A1 and Himalaya) and from two strains of R. leguminosarum bv. trifolii (ANU843 and CSF). The nodX genes of strains A1 and ANU843 were shown to be functional for the induction of nodules on Afghan peas. To analyze the cause of phenotypic differences of strain A1 and strain TOM we have studied the composition of the lipochitin-oligosaccharides (LCOs) produced by strain A1 after induction by the flavonoid naringenin or various pea root exudates. The structural analysis of the LCOs by mass spectrometry revealed that strain A1 synthesizes a family of at least 23 different LCOs. The use of exudates instead of naringenin resulted only in quantitative differences in the ratios of various LCOs produced.

Phenazine-1-carboxamide production in the biocontrol strain Pseudomonas chlororaphis PCL1391 is regulated by multiple factors secreted into the growth medium.

Pseudomonas chlororaphis PCL1391 controls tomato foot and root rot caused by Fusarium oxysporum f. sp. radicis-lycopersici. The production of phenazine-1-carboxamide (PCN) is crucial for this biocontrol activity. In vitro production of PCN is observed only at high-population densities, suggesting that production is under the regulation of quorum sensing. The main autoinducer molecule produced by PCL1391 was identified structurally as N-hexanoyl-L-homoserine lactone (C6-HSL). The two other autoinducers that were produced comigrate with N-butanoyl-L-homoserine lactone (C4-HSL) and N-octanoyl-L-homoserine lactone (C8-HSL). Two PCL1391 mutants lacking production of PCN were defective in the genes phzI and phzR, respectively, the nucleotide sequences of which were determined completely. Production of PCN by the phzI mutant could be complemented by the addition of exogenous synthetic C6-HSL, but not by C4-HSL, C8-HSL, or any other HSL tested. Expression analyses of Tn5luxAB reporter strains of phzI, phzR, and the phz biosynthetic operon clearly showed that phzI expression and PCN production is regulated by C6-HSL in a population density-dependent manner. The introduction of multiple copies of the regulatory genes phzI and phzR on various plasmids resulted in an increase of the production of HSLs, expression of the PCN biosynthetic operon, and consequently, PCN production, up to a sixfold increase in a copy-dependent manner. Surprisingly, our expression studies show that an additional, yet unidentified factor(s), which are neither PCN nor C4-HSL or C8-HSL, secreted into the growth medium of the overnight cultures, is involved in the positive regulation of phzI, and is able to induce PCN biosynthesis at low cell densities in a growing culture, resulting in an increase of PCN production.

Unusual structure of the exopolysaccharide of Rhizobium leguminosarum bv. viciae strain 248.

The exopolysaccharide from R. leguminosarum bv. viciae strain 248 differs from those of other Rhizobium strains with similar symbiotic behavior. 13C-N.m.r. spectroscopy of fragments generated by partial hydrolysis, together with methylation analysis and 13C-n.m.r. spectroscopy of the enzymically depolymerised exopolysaccharide, indicated the following nonasaccharide repeating-unit: [formula: see text] The locations of the acetyl and 3-hydroxybutanoyl substituents in the exopolysaccharide are assigned provisionally. R. leguminosarum bv. viciae strain 248, cured of its Sym plasmid pRL1JI, synthesised an exopolysaccharide in which the sites and degree of substitution were unchanged. A Tn5 mutant, derived from strain 248 and unable to induce nodules, synthesised small amounts of EPS that lacked galactose.

Isolation and characterization of antagonistic Bacillus subtilis strains from the avocado rhizoplane displaying biocontrol activity.

AIM: This study was undertaken to isolate Bacillus subtilis strains with biological activity against soil-borne phytopathogenic fungi from the avocado rhizoplane. METHODS AND RESULTS: A collection of 905 bacterial isolates obtained from the rhizoplane of healthy avocado trees, contains 277 gram-positive isolates. From these gram-positive isolates, four strains, PCL1605, PCL1608, PCL1610 and PCL1612, identified as B. subtilis, were selected on the basis of their antifungal activity against diverse soil-borne phytopathogenic fungi. Analysis of the antifungal compounds involved in their antagonistic activity showed that these strains produced hydrolytic enzymes such as glucanases or proteases and the antibiotic lipopeptides surfactin, fengycin, and/or iturin A. In biocontrol trials using the pathosystems tomato/Fusarium oxysporum f.sp. radicis-lycopersici and avocado/Rosellinia necatrix, two B. subtilis strains, PCL1608 and PCL1612, both producing iturin A, exhibited the highest biocontrol and colonization capabilities. CONCLUSIONS: Diverse antagonistic B. subtilis strains isolated from healthy avocado rhizoplanes have shown promising biocontrol abilities, which are closely linked with the production of antifungal lipopeptides and good colonization aptitudes. SIGNIFICANCE AND IMPACT OF THE STUDY: This is one of the few reports dealing with isolation and characterization of B. subtilis strains with biocontrol activity against the common soil-borne phytopathogenic fungi F. oxysporum f.sp. radicis-lycopersici and R. necatrix.