Phylogeny of HCN synthase-encoding hcnBC genes in biocontrol fluorescent pseudomonads and its relationship with host plant species and HCN synthesis ability

Hydrogen cyanide (HCN) is a broad-spectrum antimicrobial compound involved in biological control of root diseases by many plant-associated fluorescent pseudomonads. The HCN synthase is encoded by three biosynthetic genes (hcnA, hcnB, and hcnC), but little is known about the diversity of these genes in fluorescent Pseudomonas spp. and in other bacteria. Here, the partial hcnBC sequence was determined for a worldwide collection of biocontrol fluorescent Pseudomonas spp. Phylogenies based on hcnBC and deduced protein sequences revealed four main bacterial groups, but topological incongruences were found between hcnBC and rrs-based phylogenies, suggesting past lateral transfer of hcnBC among saprophytic root-colonizing pseudomonads. Three of the four groups included isolates from different countries and host plants. Yet, these groups corresponded to distinct, ecologically-adapted populations of HCN-producing biocontrol fluorescent pseudomonads, as indicated by high hcnBC distinctness ratio values and the differences in production levels of HCN in vitro found between groups. This is in accordance with previous results on catabolic properties and biocontrol abilities of these strains. HCN synthase gene diversity may thus reflect the adaptive radiation of HCN+ biocontrol fluorescent pseudomonads. Positive correlations were found between HCN production in vitro and plant protection in the cucumber/Pythium ultimum and tomato/Fusarium oxysporum f. sp. radicis-lycopersici pathosystems.     

Is the ability of biocontrol fluorescent pseudomonads to produce the antifungal metabolite 2,4-diacetylphloroglucinol really synonymous with higher plant protection?

The antifungal compound 2,4-diacetylphloroglucinol (Phl) contributes to biocontrol in pseudomonads, but whether or not Phl(+) biocontrol pseudomonads display higher plant-protecting activity than Phl(-) biocontrol pseudomonads remains to be demonstrated. This issue was addressed by assessing 230 biocontrol fluorescent pseudomonads selected from a collection of 3132 bacterial isolates obtained from 63 soils worldwide. One-third of the biocontrol pseudomonads were Phl(+) and almost all Phl(+) isolates also produced hydrogen cyanide (HCN). The only Phl(+) HCN(-) strain did harbor hcn genes, but with the deletion of a 134 bp hcnC fragment corresponding to an ADP-binding motif. Statistical analysis of biocontrol isolate distributions indicated that Phl production ability was associated with superior disease suppression activity in the Pythium-cucumber and Fusarium-tomato pathosystems, but this was also the case with HCN production ability. However, HCN significance was not as strong, as indicated both by the comparison of Phl(-) HCN(+) and Phl(-) HCN(-) strains and by correlation analyses. This is the first population-level demonstration of the higher plant-protecting activity of Phl(+) biocontrol pseudomonads in comparison with Phl(-) biocontrol pseudomonads.     

Frequency of Antibiotic-Producing Pseudomonas spp. in Natural Environments

The antibiotics phenazine-1-carboxylic acid (PCA) and 2,4-diacetylphloroglucinol (Phl) are major determinants of biological control of soilborne plant pathogens by various strains of fluorescent Pseudomonas spp. In this study, we described primers and probes that enable specific and efficient detection of a wide variety of fluorescent Pseudomonas strains that produce various phenazine antibiotics or Phl. PCR analysis and Southern hybridization demonstrated that specific genes within the biosynthetic loci for Phl and PCA are conserved among various Pseudomonas strains of worldwide origin. The frequency of Phl- and PCA-producing fluorescent pseudomonads was determined on roots of wheat grown in three soils suppressive to take-all disease of wheat and four soils conducive to take-all by colony hybridization followed by PCR. Phenazine-producing strains were not detected on roots from any of the soils. However, Phl-producing fluorescent pseudomonads were isolated from all three take-all-suppressive soils at densities ranging from approximately 5 x 10(sup5) to 2 x 10(sup6) CFU per g of root. In the complementary conducive soils, Phl-producing pseudomonads were not detected or were detected at densities at least 40-fold lower than those in the suppressive soils. We speculate that fluorescent Pseudomonas spp. that produce Phl play an important role in the natural suppressiveness of these soils to take-all disease of wheat.     

A new DGGE protocol targeting 2,4-diacetylphloroglucinol biosynthetic gene phlD from phylogenetically contrasted biocontrol pseudomonads for assessment of disease-suppressive soils

In the rhizosphere, biocontrol pseudomonads producing 2,4-diacetylphloroglucinol (Phl) can protect plants from soil-borne pathogens. DGGE of phlD has been proposed to monitor these bacteria, but two distinct protocols were needed for analysis of both the 'Pseudomonas fluorescens' species complex and the strains from rrs restriction group ARDRA-1. Here, a single DGGE protocol performed on 668-bp GC-clamp-containing phlD amplicons was effective with both types of pseudomonads, and 36 reference biocontrol strains from the 'P. fluorescens' complex or group ARDRA-1 gave a total of 11 distinct DGGE bands. phlD amplicons with at least two to seven nucleotidic differences could be discriminated, and the discrimination level was similar to that of phlD restriction analysis with four enzymes. Multiple phlD-DGGE bands were obtained when studying rhizosphere soil containing indigenous phlD+ pseudomonads, and phlD diversity was higher when DGGE was implemented after incubation of tobacco rhizosphere extracts in semi-selective medium (MPN approach) in comparison with approaches based on direct analysis of rhizosphere DNA extracts or assessment of phlD+colonies. phlD-DGGE profiles differed for a soil suppressive and a soil conducive to black root rot of tobacco, and each soil yielded new phlD sequences. In conclusion, this DGGE protocol was useful for monitoring indigenous rhizosphere consortia of phlD+ pseudomonads.     

Polymorphism of the polyketide synthase gene phID in biocontrol fluorescent pseudomonads producing 2,4-diacetylphloroglucinol and comparison of PhID with plant polyketide synthases

Many biocontrol fluorescent pseudomonads can protect plants from soilborne fungal pathogens through production of the antifungal secondary metabolite 2,4-diacetylphloroglucinol (Phl). One of the phl biosynthetic genes, phlD, encodes a polyketide synthase similar to plant chalcone synthases. Here, restriction analysis of phlD from 39 Phl+ biocontrol fluorescent pseudomonads yielded seven different banding patterns. The gene was sequenced in seven strains, representing the different restriction patterns. Cluster analysis of phlD restriction data or phlD sequences indicated that phlD polymorphism was high, and two main clusters were obtained when predicted PhlD sequences were compared. When the seven PhlD sequences were studied with those of other procaryotic polyketide synthases (gram-positive bacteria) and plant chalcone synthases, however, Phl+ pseudomonads, gram-positive bacteria, and plants clustered separately. Yet, sequence analysis of active site regions for PhlD and plant chalcone synthases revealed that PhlD can be considered a member of the chalcone synthase family, which may be interpreted as convergent evolution of key enzymes involved in secondary metabolism. For the 39 Phl+ pseudomonads, a relationship was found among phlD restriction patterns, phylogenetic groups defined by 16S rDNA restriction analysis (confirmed by 16S rDNA sequencing), and production levels of Phl in vitro.     

Effect of long-term vineyard monoculture on rhizosphere populations of pseudomonads carrying the antimicrobial biosynthetic genes phlD and/or hcnAB

The impact of repeated culture of perennial plants (i.e. in long-term monoculture) on the ecology of plant-beneficial bacteria is unknown. Here, the influence of extremely long-term monocultures of grapevine (up to 1603 years) on rhizosphere populations of fluorescent pseudomonads carrying the biosynthetic genes phlD for 2,4-diacetylphloroglucinol and/or hcnAB for hydrogen cyanide was determined. Soils from long-term and adjacent short-term monoculture vineyards (or brushland) in four regions of Switzerland were baited with grapevine or tobacco plantlets, and rhizosphere pseudomonads were studied by most probable number (MPN)-PCR. Higher numbers and percentages of phlD ⁺ and of hcnAB ⁺ rhizosphere pseudomonads were detected on using soil from long-term vineyards. On focusing on phlD , restriction fragment length polymorphism profiling of the last phlD -positive MPN wells revealed seven phlD alleles (three exclusively on tobacco, thereof two new ones). Higher numbers of phlD alleles coincided with a lower prevalence of the allele displayed by the well-studied biocontrol strain Pseudomonas fluorescens F113. The prevalence of this allele was 35% for tobacco in long-term monoculture soils vs. >60% in the other three cases. We conclude that soils from long-term grapevine monocultures represent an untapped resource for isolating novel biocontrol Pseudomonas strains when tobacco is used as bait.     

Comparison of ATPase-encoding type III secretion system hrcN genes in biocontrol fluorescent Pseudomonads and in phytopathogenic proteobacteria

Type III protein secretion systems play a key role in the virulence of many pathogenic proteobacteria, but they also occur in nonpathogenic, plant-associated bacteria. Certain type III protein secretion genes (e.g., hrcC) have been found in Pseudomonas sp. strain SBW25 (and other biocontrol pseudomonads), but other type III protein secretion genes, such as the ATPase-encoding gene hrcN, have not been found. Using both colony hybridization and a PCR approach, we show here that hrcN is nevertheless present in many biocontrol fluorescent pseudomonads. The phylogeny of biocontrol Pseudomonas strains based on partial hrcN sequences was largely congruent with the phylogenies derived from analyses of rrs (encoding 16S rRNA) and, to a lesser extent, biocontrol genes, such as phlD (for 2,4-diacetylphloroglucinol production) and hcnBC (for HCN production). Most biocontrol pseudomonads clustered separately from phytopathogenic proteobacteria, including pathogenic pseudomonads, in the hrcN tree. The exception was strain KD, which clustered with phytopathogenic pseudomonads, such as Pseudomonas syringae, suggesting that hrcN was acquired from the latter species. Indeed, strain KD (unlike strain SBW25) displayed the same organization of the hrpJ operon, which contains hrcN, as P. syringae. These results indicate that the occurrence of hrcN in most biocontrol pseudomonads is not the result of recent horizontal gene transfer from phytopathogenic bacteria, although such transfer might have occurred for a minority of biocontrol strains.     

Detection of phlD gene in some fluorescent pseudomonads isolated from Iran and its relative with antifungal activities

Fluorescent pseudomonads that produce antibiotic 2,4-diacetylphloroglocinol (2,4-DAPG) are important group of PGRP that inhibit a broad spectrum of plant pathogenic fungi. Studying on genetic diversity of 2,4-diacetylphloroglucinol-producing fluorescent pseudomonads has been shown with special importance. The first step to investigate the genetic diversity of these bacteria is detecting of the genes required for the biosynthesis of this antibiotic. The objectives of the current study were detection of phlD gene within fluorescent pseudomonads by a PCR-based assay, and comparison of phenotypic and genotypic characteristics of fluorescent pseudomonads with proven biocontrol potential against some soil-borne phytopathogenic fungi. We used a collection of 47 fluorescent Pseudomonas spp. some with known biological control activity against Macrophomina phaseolina, Rhizoctonia solani, Phytophthora nicotianae var. parasitica, Pythium sp. and Fusarium sp. in vitro and the potential to produce known secondary metabolites such as, siderophore, HCN and protease. The results indicated that 66, 40.42, 63.82,48.94 and 27.65% of strains revealed antagonistic activity against R. solani, M. phaseolina, Pythium sp., P. nicotianae and Fusarium sp., respectively. Rhizoctonia solani recognized as the most vulnerable fungus. Among 47 strains, 76.59, 97.87 and 17% of strains produced protease, siderophore and HCN, respectively. We could detect phlD gene in strains P-5, P-32, P-47. Strain CHA0 was used as positive control for the detection this gene. Overall, there was no obvious link between the existence of phlD gene and inhibition of fungal growth or production of the antifungal metabolites in vitro. But in some strains such as CHA0 and P-5, we saw a link between the existence of phlD and antifungal activities. Studying on detection and diversity of phlD provides a fundamental knowledge for developing a rapid genetic screening system to identify a potential biocontrol strains.     

Influence of plant species on population dynamics, genotypic diversity and antibiotic production in the rhizosphere by indigenous Pseudomonas spp

The population dynamics, genotypic diversity and activity of naturally-occurring 2,4-diacetylphloroglucinol (DAPG)-producing Pseudomonas spp. was investigated for four plant species (wheat, sugar beet, potato, lily) grown in two different soils. All four plant species tested, except lily and in some cases wheat, supported relatively high rhizosphere populations (5 x 10(4) to 1 x 10(6) CFU/g root) of indigenous DAPG-producing Pseudomonas spp. during successive cultivation in both a take-all suppressive and a take-all conducive soil. Although lily supported on average the highest population densities of fluorescent Pseudomonas spp., it was the least supportive of DAPG-producing Pseudomonas spp. of all four plant species. The genotypic diversity of 492 DAPG-producing Pseudomonas isolates, assessed by Denaturing Gradient Gel Electrophoresis (DGGE) analysis of the phlD gene, revealed a total of 7 genotypes. Some of the genotypes were found only in the rhizosphere of a specific plant, whereas the predominant genotypes were found at significantly higher frequencies in the rhizosphere of three plant species (wheat, sugar beet and potato). Statistical analysis of the phlD(+) genotype frequencies showed that the diversity of the phlD(+) isolates from lily was significantly lower than the diversity of phlD(+) isolates found on wheat, sugar beet or potato. Additionally, soil type had a significant effect on both the phlD(+) population density and the phlD(+) genotype frequencies, with the take-all suppressive soil being the most supportive. HPLC analysis further showed that the plant species had a significant effect on DAPG-production by the indigenous phlD(+) population: the wheat and potato rhizospheres supported significantly higher amounts of DAPG produced per cell basis than the rhizospheres of sugar beet and lily. Collectively, the results of this study showed that the host plant species has a significant influence on the dynamics, composition and activity of specific indigenous antagonistic Pseudomonas spp.     

PCR amplification of hydrogen cyanide biosynthetic locus hcnAB in Pseudomonas spp

A PCR-based assay targeting hcnAB, essential genes for hydrogen cyanide (HCN) biosynthesis, allowed sensitive detection of HCN(+) pseudomonads between logs 2.9 and 3.5 cells per PCR reaction tube. RFLP analysis revealed 13 allele combinations among selected 2,4-diacetylphloroglucinol-producing (Phl(+))HCN(+), and 13 alleles in Phl(-) HCN(+) strains from a global collection.     

Differential ability of genotypes of 2,4-diacetylphloroglucinol-producing Pseudomonas fluorescens strains to colonize the roots of pea plants

Indigenous populations of 2,4-diacetylphloroglucinol (2,4-DAPG)-producing fluorescent Pseudomonas spp. that occur naturally in suppressive soils are an enormous resource for improving biological control of plant diseases. Over 300 isolates of 2,4-DAPG-producing fluorescent Pseudomonas spp. were isolated from the rhizosphere of pea plants grown in soils that had undergone pea or wheat monoculture and were suppressive to Fusarium wilt or take-all, respectively. Representatives of seven genotypes, A, D, E, L, O, P, and Q, were isolated from both soils and identified by whole-cell repetitive sequence-based PCR (rep-PCR) with the BOXA1R primer, increasing by three (O, P, and Q) the number of genotypes identified previously among a worldwide collection of 2,4-DAPG producers. Fourteen isolates representing eight different genotypes were tested for their ability to colonize the rhizosphere of pea plants. Population densities of strains belonging to genotypes D and P were significantly greater than the densities of other genotypes and remained above log 6.0 CFU (g of root)(-1) over the entire 15-week experiment. Genetic profiles generated by rep-PCR or restriction fragment length polymorphism analysis of the 2,4-DAPG biosynthetic gene phlD were predictive of the rhizosphere competence of the introduced 2,4-DAPG-producing strains.     

Multilocus sequence analysis of biocontrol fluorescent Pseudomonas spp. producing the antifungal compound 2,4-diacetylphloroglucinol

The genetic and evolutionary relationship among 2,4-diacetylphloroglucinol (Phl)-producing pseudomonads that protect plants from soil-borne pathogens were investigated by multilocus sequence typing. A total of 65 pseudomonads consisting of 58 Phl-positive biocontrol strains of worldwide origin and seven Phl-negative representatives of characterized Pseudomonas species were compared using 10 housekeeping genes (i.e. rrs, dsbA, gyrB, rpoD, fdxA, recA, rpoB, fusA, rpsL and rpsG). Multilocus sequence typing differentiated 51 strains among 58 Phl-positive pseudomonads and proved to be as discriminative as enterobacterial repetitive intergenic consensus polymerase chain reaction profiling. As phylogenetic trees inferred from each locus were rather incongruent with one another, we derived the topology from all concatenated loci, which led to the identification of six main groups of Phl-producing Pseudomonas spp. Taxonomically, these groups could correspond to at least six different species. Linkage disequilibrium analysis pointed to a rather clonal structure, even when the analysis was restricted to Phl-producing pseudomonads from a same geographic location or a same phylogenetic group. Intragenic recombination was evidenced for gyrB, rpoD and fdxA, but was shown to be a weaker force than mutation in the origin of intragenetic diversity. This is the first multilocus assessment of the phylogeny and population structure of an ecologically important bacterial group involved in plant disease suppression.     

Evaluation of fluorescent Pseudomonads and <Emphasis Type="Italic">Bacillus</Emphasis> isolates for the biocontrol of a wilt disease complex of pigeonpea

Summary Twenty isolates of fluorescent pseudomonads and Bacillus spp. were obtained from pathogen suppressive soil of a pigeonpea (Cajanus cajan) field showing wilt disease complex. These isolates were evaluated in the laboratory and screen-house for the biocontrol of wilt disease complex. Six isolates were considered to have potential for the biocontrol of the disease on the basis of antibiotic sensitivity, antifungal activity, fluorescence produced by Pseudomonas, inhibitory effect on the hatching and penetration of nematodes and colonization of pigeonpea roots by these isolates. These isolates will be further tested for their biocontrol of wilt disease complex of pigeonpea under field conditions.     

Effect of sorghum seedlings, and previous crop, on soil fluorescent Pseudomonas spp.

Hypotheses in which sorghum seedlings [Sorghum bicolor (L.) Moench] of different genotypes will differentially modify soil microorganisms and will affect subsequent planting of wheat (Triticum aestivum L.) seedlings, were tested. Wheat cultivar Lewjain, and sorghum genotypes Redlan and RTx433, were planted into soils previously planted with wheat or sorghum in growth chamber experiments. Total culturable fungi and oomycetes, and fluorescent Pseudomonas spp. numbers (cfu) were determined. Pseudomonads were screened for hydrogen cyanide (HCN) production, for the presence of the phlD gene for 2,4-diacetylphloroglucinol production (Phl) and for a region of the operon involved in phenazine-1-carboxylic acid (PCA) production. Pasteurized soils were inoculated with rifampicin-marked strains of Pseudomonas fluorescens then planted with Lewjain, Redlan and RTx433 to assess rhizosphere and soil colonization. Effects of plant species, sorghum genotype and previous crop on culturable fungi and oomycetes, and pseudomonad numbers (cfu g^?1 soil) were statistically significant. Soils planted with RTx433 or Lewjain had greater numbers of fungal cfu than soils planted with Redlan. When Lewjain seedlings were grown in soil previously planted with RTx433, there were greater numbers of fungal cfu than when Lewjain was planted into Redlan soil. Wheat planted into wheat soil resulted in statistically significantly fewer numbers of pseudomonads than when planted into sorghum soil. Overall, percentages of HCN-producing pseudomonads increased, especially when wheat seedlings were planted in wheat soil. For most treatments, percent of isolates with Phl declined, except when Redlan was planted into Redlan soil, which resulted in increased Phl isolates. When rifampicin-marked P. fluorescens isolates were applied to pasteurized soil, sorghum seedlings sustained rhizosphere and soil populations similar to those on wheat. Sorghum genotypes may differ in associations with soil microorganisms, suggesting that they may differentially affect numbers of fluorescent pseudomonads in cropping systems.     

2,4-diacetylphloroglucinol alters plant root development

Pseudomonas fluorescens isolates containing the phlD gene can protect crops from root pathogens, at least in part through production of the antibiotic 2,4-diacetylphloroglucinol (DAPG). However, the action mechanisms of DAPG are not fully understood, and effects of this antibiotic on host root systems have not been characterized in detail. DAPG inhibited primary root growth and stimulated lateral root production in tomato seedlings. Roots of the auxin-resistant diageotropica mutant of tomato demonstrated reduced DAPG sensitivity with regards to inhibition of primary root growth and induction of root branching. Additionally, applications of exogenous DAPG, at concentrations previously found in the rhizosphere of plants inoculated with DAPG-producing pseudomonads, inhibited the activation of an auxin-inducible GH3 promoter::luciferase reporter gene construct in transgenic tobacco hypocotyls. In this model system, supernatants of 17 phlD+ P. fluorescens isolates had inhibitory effects on luciferase activity similar to synthetic DAPG. In addition, a phlD() mutant strain, unable to produce DAPG, demonstrated delayed inhibitory effects compared with the parent wild-type strain. These results indicate that DAPG can alter crop root architecture by interacting with an auxin-dependent signaling pathway.     

Characterization of CMR5c and CMR12a, novel fluorescent Pseudomonas strains from the cocoyam rhizosphere with biocontrol activity

AIM: To screen for novel antagonistic Pseudomonas strains producing both phenazines and biosurfactants that are as effective as Pseudomonas aeruginosa PNA1 in the biocontrol of cocoyam root rot caused by Pythium myriotylum. MATERIAL AND RESULTS: Forty pseudomonads were isolated from the rhizosphere of healthy white and red cocoyam plants appearing in natural, heavily infested fields in Cameroon. In vitro tests demonstrated that Py. myriotylum antagonists could be retrieved from the red cocoyam rhizosphere. Except for one isolate, all antagonistic isolates produced phenazines. Results from whole-cell protein profiling showed that the antagonistic isolates are different from other isolated pseudomonads, while BOX-PCR revealed high genomic similarity among them. 16S rDNA sequencing of two representative strains within this group of antagonists confirmed their relatively low similarity with validly described Pseudomonas species. These antagonists are thus provisionally labelled as unidentified Pseudomonas strains. Among the antagonists, Pseudomonas CMR5c and CMR12a were selected because of their combined production of phenazines and biosurfactants. For strain CMR5c also, production of pyrrolnitrin and pyoluteorin was demonstrated. Both CMR5c and CMR12a showed excellent in vivo biocontrol activity against Py. myriotylum to a similar level as Ps. aeruginosa PNA1. CONCLUSION: Pseudomonas CMR5c and CMR12a were identified as novel and promising biocontrol agents of Py. myriotylum on cocoyam, producing an arsenal of antagonistic metabolites. SIGNIFICANCE AND IMPACT OF THE STUDY: Present study reports the identification of two newly isolated fluorescent Pseudomonas strains that can replace the opportunistic human pathogen Ps. aeruginosa PNA1 in the biocontrol of cocoyam root rot and could be taken into account for the suppression of many plant pathogens.     

Pseudomonas community structure and antagonistic potential in the rhizosphere: insights gained by combining phylogenetic and functional gene-based analyses

The Pseudomonas community structure and antagonistic potential in the rhizospheres of strawberry and oilseed rape (host plants of the fungal phytopathogen Verticillium dahliae) were assessed. The use of a new PCR-DGGE system, designed to target Pseudomonas-specific gacA gene fragments in environmental DNA, circumvented common biases of 16S rRNA gene-based DGGE analyses and proved to be a reliable tool to unravel the diversity of uncultured Pseudomonas in bulk and rhizosphere soils. Pseudomonas-specific gacA fingerprints of total-community (TC) rhizosphere DNA were surprisingly diverse, plant-specific and differed markedly from those of the corresponding bulk soils. By combining multiple culture-dependent and independent surveys, a group of Pseudomonas isolates antagonistic towards V. dahliae was shown to be genotypically conserved, to carry the phlD biosynthetic locus (involved in the biosynthesis of 2,4-diacetylphloroglucinol - 2,4-DAPG), and to correspond to a dominant and highly frequent Pseudomonas population in the rhizosphere of field-grown strawberries planted at three sites in Germany which have different land use histories. This population belongs to the Pseudomonas fluorescens phylogenetic lineage and showed closest relatedness to P. fluorescens strain F113 (97% gacA gene sequence identity in 492-bp sequences), a biocontrol agent and 2,4-DAPG producer. Partial gacA gene sequences derived from isolates, clones of the strawberry rhizosphere and DGGE bands retrieved in this study represent previously undescribed Pseudomonas gacA gene clusters as revealed by phylogenetic analysis.     

Characteristics of Inhibition of Suppressive Soil Created hy Monoculture with Radish in the Presence of Rhizoctonia solani

Soils collected from five districts of Hawaii county were infested with Rhtzoctonia solani in small inoculum particles and successfully planted with radish to induce suppression, Suppressiveness was induced in some, but not all, replicates of all. soils. When fresh inoculum was added, suppressiveness was demonstrated in some, but not all, replicates of two soils, but not in the other three soils. Acidity of soil was not important in successful induction of suppression. Characteristics of induced suppression in soil from one site (S. Kohala) were further investigated. Reduction of microbial population by heat treatment of suppressive soil completely nullified its inhibitory effect. The populations of actinomycetes, fungi in general and Trichoderma spp. in suppressive and conducive soil were not significantly different. However, the population of bacteria in suppressive soil was almost four times higher than that in conducive soil. The survival time of R. solani in suppressive soil was shorter than that in conducive soil. Hyphae of R. solani also lysed faster in suppressive soil than in conducive soil. It is suggested that suppressiveness of the South Kohala soil created by monoculture is due to enhanced competitive pressure generated by an increased bacterial population, which in turn causes the rapid autolysis of R. solani hyphae.     

Quantification of 2,4-diacetylphloroglucinol-producing Pseudomonas fluorescens strains in the plant rhizosphere by real-time PCR

A real-time PCR SYBR green assay was developed to quantify populations of 2,4-diacetylphloroglucinol (2,4-DAPG)-producing (phlD+) strains of Pseudomonas fluorescens in soil and the rhizosphere. Primers were designed and PCR conditions were optimized to specifically amplify the phlD gene from four different genotypes of phlD+ P. fluorescens. Using purified genomic DNA and genomic DNA extracted from washes of wheat roots spiked with bacteria, standard curves relating the threshold cycles (C(T)s) and copies of the phlD gene were generated for P. fluorescens strains belonging to genotypes A (Pf-5), B (Q2-87), D (Q8r1-96 and FTAD1R34), and I (FTAD1R36). The detection limits of the optimized real-time PCR assay were 60 to 600 fg (8 to 80 CFU) for genomic DNA isolated from pure cultures of P. fluorescens and 600 fg to 6.0 pg (80 to 800 CFU, corresponding to log 4 to 5 phlD+ strain CFU/rhizosphere) for bacterial DNA extracted from plant root washes. The real-time PCR assay was utilized to quantify phlD+ pseudomonads in the wheat rhizosphere. Regression analysis of population densities detected by real-time PCR and by a previously described phlD-specific PCR-based dilution endpoint assay indicated a significant linear relationship (P = 0.0016, r2 = 0.2). Validation of real-time PCR assays with environmental samples was performed with two different soils and demonstrated the detection of more than one genotype in Quincy take-all decline soil. The greatest advantage of the developed real-time PCR is culture independence, which allows determination of population densities and the genotype composition of 2,4-DAPG producers directly from the plant rhizospheres and soil.