Frequency and biodiversity of 2,4-diacetylphloroglucinol-producing bacteria isolated from the maize rhizosphere at different stages of plant growth

A Pseudomonas 2,4-diacetylphloroglucinol (DAPG)-producing population that occurred naturally on the roots, in rhizosphere soil of Zea mays and in the nonrhizosphere soil was investigated in order to assess the microbial diversity at five stages of plant growth. A total of 1,716 isolates were obtained, and 188 of these isolates were able to produce DAPG. DAPG producers were isolated at each stage of plant growth, indicating that the maize rhizosphere is colonized by natural DAPG producers throughout development. The frequency of DAPG producers was very low in the first stage of plant growth and increased over time. An analysis of the level of biodiversity of the DAPG producers at the species level was performed by comparing the AluI restriction patterns of the 16S ribosomal DNAs (rDNAs) amplified by PCR from 167 isolates. This comparison allowed us to cluster the isolates into four amplified rDNA restriction analysis (ARDRA) groups, and the main group (ARDRA group 1) contained 89.8% of the isolates. The diversity of the 150 isolates belonging to ARDRA group 1 was analyzed by the random amplified polymorphic DNA (RAPD) technique. An analysis of RAPD patterns by a molecular variance method revealed that there was a high level of genetic diversity in this population and that the genetic diversity was related to plant age. Finally, we found that some of the DAPG producers, which originated from all stages of plant growth, had the same genotype. These DAPG producers could be exploited in future screening programs for biocontrol agents.     

Identification and differentiation of Lactobacillus, Streptococcus and Bifidobacterium species in fermented milk products with bifidobacteria

The aim of this study was to identify and discriminate bacteria contained in commercial fermented milks with bifidobacteria by the use of amplified ribosomal DNA restriction analysis (ARDRA) and randomly amplified polymorphic DNA (RAPD) techniques. ARDRA of the 16S rDNA gene and RAPD were performed on 13 Lactobacillus strains, 13 Streptococcus and 13 Bifidobacterium strains isolated from commercial fermented milk. Lactobacillus delbrueckii, Streptococcus thermophilus and Bifidobacterium animalis isolates were identified by genus- and species-PCR and also, they were differentiated at genus and species level by ARDRA using MwoI restriction enzyme. The ARDRA technique allowed for the discrimination among these three related genus with the use of only one restriction enzyme, since distinctive profiles were obtained for each genus. Therefore it can be a simple, rapid and useful method for routine identification. Also, RAPD technique allowed the discrimination of all bacteria contained in dairy products, at genus- and strain-level by the performance of one PCR reaction.     

Frequency and Biodiversity of 2,4-Diacetylphloroglucinol-Producing Bacteria Isolated from the Maize Rhizosphere at Different Stages of Plant Growth

A Pseudomonas 2,4-diacetylphloroglucinol (DAPG)-producing population that occurred naturally on the roots, in rhizosphere soil of Zea mays and in the nonrhizosphere soil was investigated in order to assess the microbial diversity at five stages of plant growth. A total of 1,716 isolates were obtained, and 188 of these isolates were able to produce DAPG. DAPG producers were isolated at each stage of plant growth, indicating that the maize rhizosphere is colonized by natural DAPG producers throughout development. The frequency of DAPG producers was very low in the first stage of plant growth and increased over time. An analysis of the level of biodiversity of the DAPG producers at the species level was performed by comparing the AluI restriction patterns of the 16S ribosomal DNAs (rDNAs) amplified by PCR from 167 isolates. This comparison allowed us to cluster the isolates into four amplified rDNA restriction analysis (ARDRA) groups, and the main group (ARDRA group 1) contained 89.8% of the isolates. The diversity of the 150 isolates belonging to ARDRA group 1 was analyzed by the random amplified polymorphic DNA (RAPD) technique. An analysis of RAPD patterns by a molecular variance method revealed that there was a high level of genetic diversity in this population and that the genetic diversity was related to plant age. Finally, we found that some of the DAPG producers, which originated from all stages of plant growth, had the same genotype. These DAPG producers could be exploited in future screening programs for biocontrol agents.     

Contribution of phlA and some metabolites of fluorescent pseudomonads to antifungal activity

Fluorescent Pseudomonas species are an important group of PGPR that suppress fungal root and seedling disease by production of antifungal metabolites such as 2,4-diacetylphloroglucinol (2,4-DAPG), pyoluteorin, pyrolinitrin, siderophores and HCN. The compound 2,4-DAPG is a major determinant in biocontrol of plant pathogens. A 7.2 kbp chromosomal DNA region, carrying DAPG biosynthetic genes (phlA, phlC, phlB, phlD, phIE and phlF). Detecting the ph1 genes make them an ideal marker gene for 2,4-DAPG-producing fluorescent pseudomonad's. In this study we detected ph1A gene (that convert MAPG to 2,4-DAPG) using PCR assay with primers phlA-1r and phlA- f that enabled amplification of phlA sequences from fluorescent pseudomonad's from ARDRA group 1 and 3. We could detect phlA gene in P. fluorescens strains CHAO, Pf-44, Pf-1, Pf-2, Pf-3, Pf-17, Pf-62 and Pf-64, native isolates of Iran. The efficacy of this method for rapid assay characterizing rhizosphere population of 2,4-DAPG producing bacteria from soil of different area of Iran is in progress. We used a collection of 48 fluorescent pseudomonas strains in vitro, with known biological control activity against some soil born phytopathogenic fungi such as, Macrophomina phaseoli, Rhizoctonia solani Vericillium dahlia, Phytophthora nicotiana, Pythium spp. and Fusarium spp. and the potential to produce known secondary metabolites such as protease. Strains Pf-1, Pf-2, Pf-3, Pf-17, Pf-33 and Pf-44 showed the best antifungal activity against all fungi used in this study. Thirty-eight of 48 strains produced protease. The ability to rapidly characterize populations of 2,4-DAPG producers will greatly enhance our understanding of their role in the suppression of root disease.     

Combination of ARDRA and RAPD genotyping techniques in identification of Acinetobacter spp. genomic species

A total of 10 non-repetitive multi-drug-resist-ant Acinetobacter strains were collected. With reference to A. calcoaceticus (ATCC23055), A. baumannii (ATCC19606), A. lwoffii (ATCC17986), and A. junii (NCTC5866), DNA fingerprint technique, amplified ribo-somal DNA restriction analysis (ARDRA), and random amplified polymorphism DNA (RAPD) were carried out to identify the genomic species of Acinetobacter spp. The distances between them were calculated by the unweighted pair group method with arithmetic (UPGMA). Genotypes ofAcinetobacter spp. were effectively classified and an A. junii together with nine A. baumannii isolates was genomically identified. The combination of ARDRA and RAPD DNA-fingerprint technique shows high com-plementarity, and could be a useful tool in Acinetobacter genomic species identification.     

Molecular and catalytic properties of monoacetylphloroglucinol acetyltransferase from Pseudomonas sp. YGJ3

Monoacetylphloroglucinol (MAPG) acetyltransferase, catalyzing the conversion of MAPG to 2,4-diacetylphloroglucinol (DAPG), was purified from Pseudomonas sp. YGJ3 grown without Cl(-). Cl(-) and pyoluteorin repressed expression of the enzyme. SDS-polyacrylamide gel electrophoresis showed that the purified enzyme (M(r)=330 kDa) was composed of three subunits of 17, 38, and 43 kDa, and protein sequencing identified these as PhlB, PhlA, and PhlC respectively. The enzyme catalyzed the reversible disproportionation of 2 moles of MAPG to phloroglucinol (PG) and DAPG. The equilibrium constant K (=[DAPG][PG]/[MAPG](2)) was estimated to be about 1.0 at 25 °C. A KpnI 20-kb DNA fragment was cloned from the genomic DNA of strain YGJ3, and a 12,598-bp long DNA region containing the phl gene cluster phlACBDEFGHI was sequenced. PCR cloning and expression of the phl genes in Escherichia coli confirmed that expression of phlACB genes produced MAPG ATase.     

Effects of Pseudomonas putida modified to produce phenazine-1-carboxylic acid and 2,4-diacetylphloroglucinol on the microflora of field grown wheat

Pseudomonas putida WCS358r, genetically modified to have improved activity against soil-borne pathogens, was released into the rhizosphere of wheat. Two genetically modified derivatives carried the phzor the phl biosynthetic gene loci and constitutively produced either the antifungal compound phenazine-1-carboxylic acid (PCA) or the antifungal and antibacterial compound 2,4-diacetylphloroglucinol (DAPG). In 1997 and 1998, effects of single introductions of PCA producing derivatives on the indigenous microflora were studied. A transient shift in the composition of the total fungal microflora, determined by amplified ribosomal DNA restiction analysis (ARDRA), was detected. Starting in 1999, effects of repeated introduction of genetically modified microorganisms (GMMs) were studied. Wheat seeds coated with the PCA producer, the DAPG producer, a mixture of the PCA and DAPG producers, or WCS358r, were sown and the densities, composition and activities of the rhizosphere microbial populations were measured. All introduced strains decreased from 10⁷CFU per gram of rhizosphere sample to below the detection limit after harvest of the wheat plants. The phz genes were stably maintained in the PCA producers, and PCA was detected in rhizosphere extracts of plants treated with this strain or with the mixture of the PCA and DAPG producers. The phl genes were also stably maintained in the DAPG producing derivative of WCS358r. Effects of the genetically modified bacteria on the rhizosphere fungi and bacteria were analyzed by using amplified ribosomal DNA restriction analysis. Introduction of the genetically modified bacterial strains caused a transient change in the composition of the rhizosphere microflora. However, introduction of the GMMs did not affect the several soil microbial activities that were investigated in this study. This revised version was published online in August 2006 with corrections to the Cover Date.     

Phloroglucinol mediates cross-talk between the pyoluteorin and 2,4-diacetylphloroglucinol biosynthetic pathways in Pseudomonas fluorescens Pf-5

The antibiotics pyoluteorin and 2,4-diacetylphloroglucinol (DAPG) contribute to the biological control of soilborne plant diseases by some strains of Pseudomonas fluorescens, including Pf-5. These secondary metabolites also have signalling functions with each compound reported to induce its own production and repress the other's production. The first step in DAPG biosynthesis is production of phloroglucinol (PG) by PhlD. In this study, we show that PG is required at nanomolar concentrations for pyoluteorin production in Pf-5. At higher concentrations, PG is responsible for the inhibition of pyoluteorin production previously attributed to DAPG. DAPG had no effect on pyoluteorin production, and monoacetylphloroglucinol showed both stimulatory and inhibitory activities but at concentrations 100-fold greater than the levels of PG required for similar effects. We also demonstrate that PG regulates pyoluteorin production in P. aeruginosa and that a phlD gene adjacent to the pyoluteorin biosynthetic gene cluster in P. aeruginosa strain LESB58 can restore pyoluteorin biosynthesis to a ΔphlD mutant of Pf-5. Bioinformatic analyses show that the dual role of PhlD in the biosynthesis of DAPG and the regulation of pyoluteorin production could have arisen within the pseudomonads during the assembly of these biosynthetic gene clusters from genes and gene subclusters of diverse origins.     

Genotypic and phenotypic diversity of phlD-containing Pseudomonas strains isolated from the rhizosphere of wheat

Production of 2,4-diacetylphloroglucinol (2,4-DAPG) in the rhizosphere by strains of fluorescent Pseudomonas spp. results in the suppression of root diseases caused by certain fungal plant pathogens. In this study, fluorescent Pseudomonas strains containing phlD, which is directly involved in the biosynthesis of 2,4-DAPG, were isolated from the rhizosphere of wheat grown in soils from wheat-growing regions of the United States and The Netherlands. To assess the genotypic and phenotypic diversity present in this collection, 138 isolates were compared to 4 previously described 2, 4-DAPG producers. Thirteen distinct genotypes, one of which represented over 30% of the isolates, were differentiated by whole-cell BOX-PCR. Representatives of this group were isolated from eight different soils taken from four different geographic locations. ERIC-PCR gave similar results overall, differentiating 15 distinct genotypes among all of the isolates. In most cases, a single genotype predominated among isolates obtained from each soil. Thirty isolates, representing all of the distinct genotypes and geographic locations, were further characterized. Restriction analysis of amplified 16S rRNA gene sequences revealed only three distinct phylogenetic groups, one of which accounted for 87% of the isolates. Phenotypic analyses based on carbon source utilization profiles revealed that all of the strains utilized 49 substrates and were unable to grow on 12 others. Individually, strains could utilize about two-thirds of the 95 substrates present in Biolog SF-N plates. Multivariate analyses of utilization profiles revealed phenotypic groupings consistent with those defined by the genotypic analyses.     

ARDRA and RAPD-fingerprinting reject the sibling species concept for the ciliate Paramecium caudatum (Ciliophora, Protoctista)

Morphologically indistinguishable sibling species also known as syngens are a characteristic taxonomic feature of the ciliate genus Paramecium . This has been convincingly demonstrated for the P. aurelia species complex. For a long time this feature has also been assumed for P. caudatum . Classical morphology based techniques of taxonomic analysis are often inefficient to study sibling specie. We therefore investigated 14 P. caudatum strains of seven supposedly different syngens using random amplified polymorphic DNA (RAPD)-fingerprinting and amplified ribosomal DNA restriction analyses (ARDRA, Riboprinting). The RAPD patterns revealed by five different random primers were similar between the different strains of the same syngen (similarity index ranging from 73 to 91%) and also between strains of supposedly different syngens (similarity index ranging from 67 to 91%). The amplified 18S rRNA-fragments of supposedly different syngens, as well as the restriction patterns of these fragments digested by five different endonucleases, were identical for all investigated P. caudatum stains. Consequently we reject the sibling species hypothesis for P. caudatum . According to our molecular analysis, P. caudatum is not a species complex, but just one single species.     

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.     

Analysis of genomic diversity among photosynthetic stem-nodulating rhizobial strains from northeast Argentina

The genomic diversity among photosynthetic rhizobia from northeast Argentina was assessed. Forty six isolates obtained from naturally occurring stem and root nodules of Aeschynomene rudis plants were analyzed by three molecular typing methods with different levels of taxonomic resolution: repetitive sequence-based PCR (rep-PCR) genomic fingerprinting with BOX and REP primers, amplified 16S rDNA restriction analysis (ARDRA), and 16S-23S rDNA intergenic spacer-restriction fragment length polymorphism (IGS-RFLP) analysis. The in vivo absorption spectra of membranes of strains were similar in the near infrared region with peaks at 870 and 800 nm revealing the presence of light harvesting complex I, bacteriochlorophyll-binding polypeptides (LHI-Bchl complex). After extraction with acetone-methanol the spectra differed in the visible part displaying peaks belonging to canthaxanthin or spirilloxanthin as the main carotenoid complement. The genotypic characterization by rep-PCR revealed a high level of genomic diversity among the isolates and almost all the photosynthetic ones have identical ARDRA patterns and fell into one cluster different from Bradyrhizobium japonicum and Bradyrhizobium elkanii. In the combined analysis of ARDRA and rep-PCR fingerprints, 7 clusters were found including most of the isolates. Five of those contained only photosynthetic isolates; all canthaxanthin-containing strains grouped in one cluster, most of the other photosynthetic isolates were grouped in a second large cluster, while the remaining three clusters contained a few strains. The other two clusters comprising reference strains of B. japonicum and B. elkanii, respectively. The IGS-RFLP analysis produced similar clustering for almost all the strains. The 16S rRNA gene sequence of one representative isolate was determined and the DNA sequence analysis confirmed the position of photosynthetic rhizobia in a distinct phylogenetic group within the Bradyrhizobium rDNA cluster.     

ARDRA联合RAPD对不动杆菌基因型鉴定的研究

收集多重耐药的不动杆菌10株,以标准参照株作对照,采用扩增核糖体DNA限制性酶切(ARDRA)DNA指纹技术联合随机扩增多态性DNA(RAPD)技术对其基因亚型进行分析;以非加权组间平均法(UPG-MA)进行聚类分析。该法可以有效地鉴定不动杆菌基因亚型;并从10株不动杆菌中鉴定出1株琼氏不动杆菌及9株鲍曼不动杆菌。ARDRA联合RAPD基因指纹分型技术有良好的互补性,可准确鉴定不动杆菌基因型。     

Biodiversity of a Burkholderia cepacia population isolated from the maize rhizosphere at different plant growth stages.

A Burkholderia cepacia population naturally occurring in the rhizosphere of Zea mays was investigated in order to assess the degree of root association and microbial biodiversity at five stages of plant growth. The bacterial strains isolated on semiselective PCAT medium were mostly assigned to the species B. cepacia by an analysis of the restriction patterns produced by amplified DNA coding for 16S rRNA (16S rDNA) (ARDRA) with the enzyme AluI. Partial 16S rDNA nucleotide sequences of some randomly chosen isolates confirmed the ARDRA results. Throughout the study, B. cepacia was strictly associated with maize roots, ranging from 0.6 to 3.6% of the total cultivable microflora. Biodiversity among 83 B. cepacia isolates was analyzed by the random amplified polymorphic DNA (RAPD) technique with two 10-mer primers. An analysis of RAPD patterns by the analysis of molecular variance method revealed a high level of intraspecific genetic diversity in this B. cepacia population. Moreover, the genetic diversity was related to divergences among maize root samplings, with microbial genetic variability markedly higher in the first stages of plant growth; in other words, the biodiversity of this rhizosphere bacterial population decreased over time.     

Genetic characterization of morphologically variant strains of Paracoccidioides brasiliensis

Molecular characterization of Paracoccidioides brasiliensis variant strains that had been preserved under mineral oil for decades was carried out by random amplified polymorphic DNA analysis (RAPD). On P. brasiliensis variants in the transitional phase and strains with typical morphology, RAPD produced reproducible polymorphic amplification products that differentiated them. A dendrogram based on the generated RAPD patterns placed the 14 P. brasiliensis strains into five groups with similarity coefficients of 72%. A high correlation between the genotypic and phenotypic characteristics of the strains was observed. A 750 bp-RAPD fragment found only in the wild-type phenotype strains was cloned and sequenced. Genetic similarity analysis using BLASTx suggested that this RAPD marker represents a putative domain of a hypothetical flavin-binding monooxygenase (FMO)-like protein of Neurospora crassa.     

Biodiversity of cultivable psychrotrophic marine bacteria isolated from Terra Nova Bay (Ross Sea, Antarctica)

A set of 146 Antarctic marine isolates from the Ross Sea was characterized by a combination of molecular techniques in order to determine the degree of inter- and intraspecific variability. Isolates were analyzed by amplified rDNA restriction analysis (ARDRA) using the tetrameric enzyme AluI, resulting in 52 different groups, corresponding to at least 52 different bacterial species, indicating a high degree of interspecific variability. The phylogenetic position of bacteria belonging to some ARDRA groups was obtained by sequencing of 16S rDNA. Random amplified polymorphic DNA (RAPD) analysis, carried out on the largest ARDRA groups, revealed a high intraspecific genetic variability, too. The analysis of plasmid content revealed the existence of horizontal gene transfer between strains belonging to the same and to different species. A comparison of the whole body of morphological, physiological and biochemical data was finally carried out.     

Diversity of culturable heterotrophic aerobic bacteria in pristine stream bed sediments

More than 900 culturable, heterotrophic aerobic isolates were obtained from the sediments of a forested, pristine stream and analyzed using three classical microbiological tests: API 20E, amplified ribosomal DNA restriction analysis (ARDRA), and fatty acid analysis. Gram-negative bacteria comprised most of the heterotrophic aerobic isolates (66.7%), similar to other oligotrophic environments. The isolates were assigned to the genus level as Pseudomonas, Flavobacterium, Micrococcus, Bacillus, Chromobacterium, Acinetobacter, Alcaligenes, Aeromonas, Methylobacterium, Enterobacter, Corynebacterium, and Sporolactobacillus. Genotypic analysis by ARDRA facilitated the comparison among strains within Pseudomonas, Bacillus, and Enterobacter groups. Temperature and predation may influence the survival of bacteria during seasons, as shown previously by others. Our results showed that the number of heterotrophic aerobic bacteria, especially Enterobacter, Alcaligenes, and Aeromonas, and Gram-positive bacteria, decreased in winter compared to summer conditions.     

Application of ITS sequence analysis, RAPD and AFLP fingerprinting in characterising the yeast genus Fellomyces

Three molecular techniques, ITS sequence analysis, random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) were used to study phylogenetic and genotypic relationships among strains of the genus Fellomyces. In the analyses were included strains isolated predominantly from epiphytic lichens collected in Indonesia, China and Mexico. The polyphasic approach indicated that the Fellomyces isolates are genotypically heterogeneous and that lichens represent a specific environment for selection of large number of the sterigmatoconidia producing species. The phylogenetic and genotypic analysis confirmed the existence of 11 currently accepted Fellomyces species and indicated that several species may be the new representatives of the genus. The RAPD and AFLP analyses demonstrated a higher potential in distinguishing the Fellomyces strains than the ITS regions. Since the sequence analysis showed low or no divergence among several strains, both RAPD and AFLP fingerprinting indicated that the strains may be discriminated at the species level.     

Application of PCR, rep-PCR and RAPD techniques for typing ofLactococcus lactis strains

A collection of 34 lactococcal strains were characterized using the polymerase chain reaction (PCR) for the acmA gene, and for the 16S rDNA gene, and DNA fingerprinting methods for randomly amplified polymorphic DNA (RAPD) and repetitive extragenic palindrome-PCR (rep-PCR). PCR experiments corroborated the genotypic identification of Lactococcus lactis strains by RAPD; rep-PCR did not distinguish between L. lactis subspecies. In some cases, phenotypic classification of L. lactis subspecies did not correlate with genotypic characterization.     

Intraspecies discrimination of Lactobacillus paraplantarum by PCR

Lactobacillus paraplantarum is a species phenotypically close to Lactobacillus plantarum. Several PCR methods were evaluated to discriminate L. paraplantarum strains and among them, a PCR using an enterobacterial repetitive intergenic consensus (ERIC) sequence differentiated L. paraplantarum from other Lactobacillus species. In addition, a combination of ERIC and random amplified polymorphic DNA (RAPD) analysis distinguished among seven strains of L. paraplantarum tested. ERIC-PCR profiles showed several strain-specific DNA fragments in L. paraplantarum, among them, a 2.2-kb ERIC marker, termed LpF1, found to be specific to strain FBA1, which improved the skin integrity in an animal model. The LpF1 encodes three proteins similar to Lactobacillus fermentum AroA, TyrA, and AroK, which are involved in the shikimate pathway. A primer pair specific to FBA1 based on the internal sequence of LpF1 amplified a 950-bp FBA1-specific fragment LpF2. Southern blot analysis of Dra I-digested genomic DNA of L. paraplantarum strains using LpF2 as a probe showed that LpF2 is distinctive of strain FBA1 among 16 L. paraplantarum strains. Because both ERIC- and RAPD-PCR are fast and technically simple methods, they are useful for the rapid discrimination of L. paraplantarum strains and for the development of new strain-specific DNA markers for identifying industrially important strains.