Interactions Between Strains of 2,4-Diacetylphloroglucinol-Producing Pseudomonas fluorescens in the Rhizosphere of Wheat

ABSTRACT Strains of fluorescent Pseudomonas spp. that produce the antibiotic 2,4-diacetylphoroglucinol (2,4-DAPG) are among the most effective rhizobacteria controlling diseases caused by soilborne pathogens. The genotypic diversity that exists among 2,4-DAPG producers can be exploited to improve rhizosphere competence and biocontrol activity. Knowing that D-genotype 2,4-DAPG-producing strains are enriched in some take-all decline soils and that P. fluorescens Q8r1-96, a representative D-genotype strain, as defined by whole-cell repetitive sequence-based polymerase chain reaction (rep-PCR) with the BOXA1R primer, is a superior colonizer of wheat roots, we analyzed whether the exceptional rhizosphere competence of strain Q8r1-96 on wheat is characteristic of other D-genotype isolates. The rhizosphere population densities of four D-genotype strains and a K-genotype strain introduced individually into the soil were significantly greater than the densities of four strains belonging to other genotypes (A, B, and L) and remained above log 6.8 CFU/g of root over a 30-week cycling experiment in which wheat was grown for 10 successive cycles of 3 weeks each. We also explored the competitive interactions between strains of different genotypes inhabiting the same soil or rhizosphere when coinoculated into the soil. Strain Q8r1-96 became dominant in the rhizosphere and in nonrhizosphere soil during a 15-week cycling experiment when mixed in a 1:1 ratio with either strain Pf-5 (A genotype), Q2-87 (B genotype), or 1M1-96 (L genotype). Furthermore, the use of the de Wit replacement series demonstrated a competitive disadvantage for strain Q2-87 or strong antagonism by strain Q8r1-96 against Q2-87 in the wheat rhizosphere. Amplified rDNA restriction analysis and sequence analysis of 16S rDNA showed that species of Arthrobacter, Chryseobacterium, Flavobacterium, Massilia, Microbacterium, and Ralstonia also were enriched in culturable populations from the rhizosphere of wheat at the end of a 30-week cycling experiment in the presence of 2,4-DAPG producers. Identifying the interactions among 2,4-DAPG producers and with other indigenous bacteria in the wheat rhizosphere will help to elucidate the variability in biocontrol efficacy of introduced 2,4-DAPG producers and fluctuations in the robustness of take-all suppressive soils.     

Host Crop Affects Rhizosphere Colonization and Competitiveness of 2,4-Diacetylphloroglucinol-Producing Pseudomonas fluorescens

ABSTRACT Strains of Pseudomonas fluorescens producing the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) are biocontrol agents which play a key role in the suppressiveness of some soils against soilborne pathogens. We evaluated the effect of the host plant genotype on rhizosphere colonization by both indigenous and introduced 2,4-DAPG-producing P. fluorescens. First, population densities of indigenous 2,4-DAPG producers in the rhizospheres of alfalfa, barley, bean, flax, lentil, lupine, oat, pea, and wheat grown in a Fusarium wilt-suppressive Puget silt loam were determined. Population densities differed among the various crops and among pea cultivars, with lentil and oat supporting the highest and lowest densities of 2,4-DAPG producers, respectively. Second, to determine the interactions among 2,4-DAPG producers in the rhizosphere, a Shano sandy loam was inoculated individually and with all possible combinations of P. fluorescens Q8r1-96 (genotype D), F113 (genotype K), and MVP1-4 (genotype P) and sown to wheat or pea, and the rhizosphere population dynamics of each strain was monitored. All three strains were similar in ability to colonize the rhizosphere of wheat and pea when introduced alone into the soil; however, when introduced together in equal densities, the outcome of the interactions differed according to the host crop. In the wheat rhizosphere, the population density of strain F113 was significantly greater than that of Q8r1-96 in the mixed inoculation studies, but no significant differences were observed on pea. The population density of strain Q8r1-96 was greater than that of MVP1-4 in the mixed inoculation on wheat, but the opposite occurred on pea. In the wheat rhizosphere, the population of MVP1-4 dropped below the detection limit (log 3.26 CFU g(-1) of root) in the presence of F113; however, on pea, the population density of MVP1-4 was higher than that of F113. When all three strains were present together, F113 had the greatest density in the wheat rhizosphere, but MVP1-4 was dominant in the pea rhizosphere. Finally, eight pea cultivars were grown in soil inoculated with either MVP1-4 or Q8r1-96. The effect of the pea cultivar on rhizosphere colonization was dependent on the bacterial strain inoculated. Rhizosphere population densities of MVP1-4 did not differ significantly among pea cultivars, whereas population densities of Q8r1-96 did. We conclude from these studies that the host crop plays a key role in modulating both rhizosphere colonization by 2,4-DAPG-producing P. fluorescens and the interactions among different genotypes present in the same rhizosphere.     

Induced systemic resistance in arabidopsis thaliana against Pseudomonas syringae pv. tomato by 2,4-diacetylphloroglucinol-producing Pseudomonas fluorescens

Pseudomonas fluorescens strains that produce the polyketide antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) are among the most effective rhizobacteria that suppress root and crown rots, wilts, and damping-off diseases of a variety of crops, and they play a key role in the natural suppressiveness of some soils to certain soilborne pathogens. Root colonization by 2,4-DAPG-producing P. fluorescens strains Pf-5 (genotype A), Q2-87 (genotype B), Q8r1-96 (genotype D), and HT5-1 (genotype N) produced induced systemic resistance (ISR) in Arabidopsis thaliana accession Col-0 against bacterial speck caused by P. syringae pv. tomato. The ISR-eliciting activity of the four bacterial genotypes was similar, and all genotypes were equivalent in activity to the well-characterized strain P. fluorescens WCS417r. The 2,4-DAPG biosynthetic locus consists of the genes phlHGF and phlACBDE. phlD or phlBC mutants of Q2-87 (2,4-DAPG minus) were significantly reduced in ISR activity, and genetic complementation of the mutants restored ISR activity back to wild-type levels. A phlF regulatory mutant (overproducer of 2,4-DAPG) had ISR activity equivalent to the wild-type Q2-87. Introduction of DAPG into soil at concentrations of 10 to 250 μM 4 days before challenge inoculation induced resistance equivalent to or better than the bacteria. Strain Q2-87 induced resistance on transgenic NahG plants but not on npr1-1, jar1, and etr1 Arabidopsis mutants. These results indicate that the antibiotic 2,4-DAPG is a major determinant of ISR in 2,4-DAPG-producing P. fluorescens, that the genotype of the strain does not affect its ISR activity, and that the activity induced by these bacteria operates through the ethylene- and jasmonic acid-dependent signal transduction pathway.     

A rapid polymerase chain reaction-based assay characterizing rhizosphere populations of 2,4-diacetylphloroglucinol-producing bacteria

ABSTRACT Pseudomonas species that produce 2,4-diacetylphloroglucinol (2,4-DAPG) play a significant role in the suppression of fungal root pathogens in the rhizosphere of crop plants. To characterize the abundance and diversity of these functionally important bacterial populations, we developed a rapid polymerase chain reaction (PCR)-based assay targeting phlD, an essential gene in the phloroglucinol biosynthetic pathway. The phlDgene is predicted to encode a polyketide synthase that synthesizes mono-acetylphloroglucinol, the immediate precursor to 2,4-DAPG. A major portion of the phlD open reading frame was cloned and sequenced from five genotypically distinct strains, and the sequences were screened for conserved regions that could be used as gene-specific priming sites for PCR amplification. Several new phlD-specific primers were designed and evaluated. Using the primers B2BF and BPR4, we developed a PCR-based assay that was robust enough to amplify the target gene from a diverse set of 2,4-DAPG producers and sensitive enough to detect as few as log 2.4 cells per sample when combined with enrichment from a selective medium. Restriction fragment length polymorphism analysis of the amplified phlD sequence allows for the direct determination of the genotype of the most abundant 2,4-DAPG producers in a sample. The method described was useful for characterizing both inoculant and indigenous phlD(+) pseudomonads inhabiting the rhizosphere of crop plants. The ability to rapidly characterize populations of 2,4-DAPG-producers will greatly enhance our understanding of their role in the suppression of root diseases.     

调控基因gacA在荧光假单胞菌2P24防治土传病害中的作用

 Pseudomonas fluorescens 2P24分离自山东小麦全蚀病自然衰退土壤,该菌株能产生抗生素2,4-二乙酰基藤黄酚(2,4-diacetylphloroglucinol,2,4-DAPG)、氢氰酸,嗜铁素和蛋白酶,且抑菌谱广,可防治多种作物土传病害。本研究应用Tn5转座突变技术,获得1株产嗜铁素过量,同时不产生2,4-DAPG、HCN、蛋白酶、不能形成生物膜(biofilm)的突变菌株PM3390,其表现型与调控基因gacA的突变体表型相似。通过PCR介导的文库筛选方法,从2P24基因组文库中获得2个含有gacA基因的阳性克隆,进一步亚克隆,得到只含有完整gacA开放阅读框的1.2 kb片段,互补实验表明其能恢复突变菌株的多种缺失表型。生测结果表明,gacA-突变菌株与野生型2P24相比,对不同土传病害的生防效果均显著降低。以上结果证实gacA在2P24中具有整体水平的调控功能,并在2P24防治土传病害中起到重要的作用。     

荧光假单胞菌2P24中phlF基因对抗生素2,4-二乙酰基间苯三酚产生的影响

Pseudomonas fluorescens2P24是分离自山东小麦全蚀病自然衰退土的1株生物防治菌株,产生抗生素2,4-二乙酰基间苯三酚(2,4-diacetylphloroglucinol;2,4-DAPG)是其主要防病机制。2,4-DAPG是由phlACBD基因簇合成,受多种调控因子调控。本研究用Tn5转座子插入技术,获得1株phlA基因转录增强的突变体,其突变基因为抗生素合成的负调控基因phlF。与野生菌相比,phlF基因的缺失突变体中phlA的转录增强约100倍,抗生素产量提高492倍。同时,菌株2P24的phlF缺失突变体对病原真菌的拮抗作用明显增强。但2,4-DAPG过量表达菌株对多种作物种子根生长有抑制作用。     

Frequency, Diversity, and Activity of 2,4-Diacetylphloroglucinol-Producing Fluorescent Pseudomonas spp. in Dutch Take-all Decline Soils

ABSTRACT Natural suppressiveness of soils to take-all disease of wheat, referred to as take-all decline (TAD), occurs worldwide. It has been postulated that different microbial genera and mechanisms are responsible for TAD in soils from different geographical regions. In growth chamber experiments, we demonstrated that fluorescent Pseudomonas spp. that produce the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) play a key role in the natural suppressiveness of two Dutch TAD soils. First, 2,4-DAPG-producing fluorescent Pseudomonas spp. were present on roots of wheat grown in both of the TAD soils at densities at or above the threshold density required to control take-all of wheat; in a complementary take-all conducive soil, population densities of 2,4-DAPG-producing Pseudomonas spp. were below this threshold level. Second, introduction of 2,4-DAPG-producing strain SSB17, a representative of the dominant geno-typic group found in the Dutch TAD soils, into the take-all conducive soil at population densities similar to the densities of indigenous 2,4-DAPG producers found in TAD soils provided control of take-all similar to that observed in the TAD soil. Third, a mutant of strain SSB17 deficient in 2,4-DAPG production was not able to control take-all of wheat, indicating that 2,4-DAPG is a key determinant in take-all suppression. These results show that in addition to the physicochemically different TAD soils from Washington State, 2,4-DAPG-producing fluorescent Pseudomonas spp. are also a key component of the natural suppressiveness found in Dutch TAD soils. Furthermore, it is the first time since the initial studies of Gerlagh (1968) that at least part of the mechanisms and microorganisms that operate in Dutch TAD soils are identified. Although quantitatively similar, the genotypic composition of 2,4-DAPG-producing Pseudomonas spp. varied between the Dutch TAD soils and the TAD soils from Washington State.     

No role for bacterially produced salicylic Acid in rhizobacterial induction of systemic resistance in Arabidopsis

ABSTRACT The role of bacterially produced salicylic acid (SA) in the induction of systemic resistance in plants by rhizobacteria is far from clear. The strong SA producer Pseudomonas fluorescens WCS374r induces resistance in radish but not in Arabidopsis thaliana, whereas application of SA leads to induction of resistance in both plant species. In this study, we compared P. fluorescens WCS374r with three other SA-producing fluorescent Pseudomonas strains, P. fluorescens WCS417r and CHA0r, and P. aeruginosa 7NSK2 for their abilities to produce SA under different growth conditions and to induce systemic resistance in A. thaliana against bacterial speck, caused by P. syringae pv. tomato. All strains produced SA in vitro, varying from 5 fg cell(-1) for WCS417r to >25 fg cell(-1) for WCS374r. Addition of 200 muM FeCl(3) to standard succinate medium abolished SA production in all strains. Whereas the incubation temperature did not affect SA production by WCS417r and 7NSK2, strains WCS374r and CHA0r produced more SA when grown at 33 instead of 28 degrees C. WCS417r, CHA0r, and 7NSK2 induced systemic resistance apparently associated with their ability to produce SA, but WCS374r did not. Conversely, a mutant of 7NSK2 unable to produce SA still triggered induced systemic resistance (ISR). The possible involvement of SA in the induction of resistance was evaluated using SA-nonaccumulating transgenic NahG plants. Strains WCS417r, CHA0r, and 7NSK2 induced resistance in NahG Arabidopsis. Also, WCS374r, when grown at 33 or 36 degrees C, triggered ISR in these plants, but not in ethylene-insensitive ein2 or in non-plant pathogenesis- related protein-expressing npr1 mutant plants, irrespective of the growth temperature of the bacteria. These results demonstrate that, whereas WCS374r can be manipulated to trigger ISR in Arabidopsis, SA is not the primary determinant for the induction of systemic resistance against bacterial speck disease by this bacterium. Also, for the other SAproducing strains used in this study, bacterial determinants other than SA must be responsible for inducing resistance.     

Involvement of gacA Gene in the Suppression of Tomato Bacterial Wilt by Pseudomonas fluorescens FPT9601

Pseudomonas fluorescens FPT9601, a plant growth-promoting rhizobacterium (PGPR) isolated from tomato rhizosphere, can protect tomato (Lycopersicon esculentum Mill) from bacterial wilt disease caused by Ralstonia solanacearum. This strain produces antibiotics 2,4-diacetylphloroglucinol (2,4-DAPG) and hydrogen cyanide (HCN). It also produces proteases and uncharacterized siderophores (Sid). A mutant strain SM2214, obtained by Tn5 insertion, did not produce 2,4-DAPG, HCN or proteases, but overproduced Sid. Marker-exchange mutagenesis confirmed that a single transposon insertion caused the multiple phenotypic changes of this mutant. Complementation of the mutant with a 1.3-kb DNA fragment that was amplified from genomic DNA of the wild-type P. fluorescens strain by PCR could restore the lost functions of the mutant strain. Nucleotide sequencing revealed that the fragment contained a 642-bp open reading frame (ORF) highly homologous to the regulator responser gene gacA. The in vitro anti-bacterium test and plant protection experiment under greenhouse conditions indicated that the gacA gene played an important role in the suppression of tomato bacterial wilt disease. Received 20 November 2000/ Accepted in revised form 19 January 2001     

产抗菌素2,4-二乙酰基藤黄酚假单胞杆菌的分离及其防治沙打旺根腐病的室内测定

从山东、内蒙古、北京等地土壤中分离筛选出荧光菌 50 0 0余株 ,其中 1 2 0 0余株为抑制性荧光菌。经PCR检测 ,获得 73株 2 ,4 二乙酰基藤黄酚 (2 ,4 DAPG)产生菌。平板筛选结果表明 ,2 ,4 DAPG产生菌CPF 1 0和 2P8对沙打旺根腐病菌Sad1和Sad2均有较好的抑制效果 ,其中CPF 1 0的抑菌带宽分别为 5.0和 1 2 .0mm ;2P8抑菌带宽分别为 3 .5和 7.0mm。温室试验两次调查表明 ,CPF 1 0对沙打旺根腐病防治效果最好 ,达 63 .5%和 67.8% ;2P8防效也在 40 %左右 ,且均达到极显著水平。 2 ,4 DAPG产生菌可以显著促进沙打旺植株根系发育 ,CPF 1 0处理后地上部株高差异不显著 ,但鲜重和干重与对照相比有极显著的增加 ,说明两菌株菌剂处理可以促进植株生长。根部定殖结果表明 ,两菌株在沙打旺根部都有一定的定殖能力 ,在根表种群数量比较稳定 ,根内细菌数量在调查时间内呈逐渐上升的趋势     

Effect of 2,4-diacetylphloroglucinol on pythium: cellular responses and variation in sensitivity among propagules and species

ABSTRACT The antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) plays an important role in the suppression of plant pathogens by several strains of Pseudomonas spp. Based on the results of this study, there is variation within and among Pythium spp. to 2,4-DAPG. Also, various propagules of Pythium ultimum var. sporangiiferum, that are part of the asexual stage of the life cycle, differ considerably in their sensitivity to 2,4-DAPG. Mycelium was the most resistant structure, followed by zoosporangia, zoospore cysts, and zoospores. Additionally, we report for the first time that pH has a significant effect on the activity of 2,4-DAPG, with a higher activity at low pH. Furthermore, the level of acetylation of phloroglucinols is also a major determinant of their activity. Transmission electron microscopy studies revealed that 2,4-DAPG causes different stages of disorganization in hyphal tips of Pythium ultimum var. sporangiiferum, including alteration (proliferation, retraction, and disruption) of the plasma membrane, vacuolization, and cell content disintegration. The implications of these results for the efficacy and consistency of biological control of plant-pathogenic Pythium spp. by 2,4-DAPG-producing Pseudomonas spp. are discussed.     

Genetic Diversity of phlD from 2,4-Diacetylphloroglucinol-Producing Fluorescent Pseudomonas spp

ABSTRACT Fluorescent Pseudomonas spp. that produce 2,4-diacetylphloroglucinol (2,4-DAPG) have biocontrol activity against damping-off, root rot, and wilt diseases caused by soilborne fungal pathogens, and play a key role in the natural suppression of Gaeumannomyces graminis var. tritici, known as take-all decline. Diversity within phlD, an essential gene in the biosynthesis of 2,4-DAPG, was studied by restriction fragment length polymorphism (RFLP) analysis of 123 2,4-DAPG-producing isolates from six states in the United States and six other locations worldwide. Clusters defined by RFLP analysis of phlD correlated closely with clusters defined previously by BOX-polymerase chain reaction (PCR) genomic fingerprinting, indicating the usefulness of phlD as a marker of genetic diversity and population structure among 2,4-DAPG producers. Genotypes defined by RFLP analysis of phlD were conserved among isolates from the same site and cropping history. Random amplified polymorphic DNA analyses of genomic DNA revealed a higher degree of polymorphism than RFLP and BOX-PCR analyses. Genotypic diversity in a subset of 30 strains representing all the phlD RFLP groups did not correlate with production in vitro of monoacetylphloroglucinol, 2,4-DAPG, or total phloroglucinol compounds. Twenty-seven of the 30 representative strains lacked pyrrolnitrin and pyoluteorin biosynthetic genes as determined by the use of specific primers and probes.     

Combining proteolytic and phloroglucinol-producing bacteria for improved biocontrol of Pythium-mediated damping-off of sugar beet

Pseudomonas fluorescens F113 and Stenotrophomonas maltophilia W81 protect sugar beet from Pythium -mediated damping-off through production of the antifungal secondary metabolite 2,4-diacetylphloroglucinol and extracellular proteolytic activity, respectively. In this study, the two biocontrol strains were combined in a consortium, with the objective of improving upon the level of protection achieved when using each strain singly. Growth and in vitro production of 2,4-diacetylphloroglucinol by F113 and extracellular lytic enzymes by W81 were not affected when inoculated in combination. The abilities of W81 and F113 to colonize the rhizosphere of sugar beet were essentially similar when the two strains were applied singly or coinoculated onto seeds in a 1 : 1 ratio, both in natural soil microcosms and under field conditions. Concomitantly, single inoculation with W81 or F113 effectively prevented colonization of sugar beet seeds by Pythium spp. in soil microcosms, without the necessity for combining both strains. However, this parity was not reflected in seed emergence where the combination of W81 and F113 significantly enhanced final sugar beet stands (to the level achieved with chemical pesticides) under microcosm conditions at 28 days after sowing. In a field experiment, the only inoculation treatment capable of conferring effective protection of sugar beet was that in which W81 and F113 were coinoculated, and this treatment proved equivalent to the use of chemical fungicides. In conclusion, when compared with single inoculations of either biocontrol strain, the combined use of a phloroglucinol-producing P. fluorescens and a proteolytic S. maltophilia improved protection of sugar beet against Pythium -mediated damping-off.     

Salicylic Acid Biosynthetic Genes Expressed in Pseudomonas fluorescens Strain P3 Improve the Induction of Systemic Resistance in Tobacco Against Tobacco Necrosis Virus

ABSTRACT Application of salicylic acid induces systemic acquired resistance in tobacco. pchA and pchB, which encode for the biosynthesis of salicylic acid in Pseudomonas aeruginosa, were cloned into two expression vectors, and these constructs were introduced into two root-colonizing strains of P. fluorescens. Introduction of pchBA into strain P3, which does not produce salicylic acid, rendered this strain capable of salicylic acid production in vitro and significantly improved its ability to induce systemic resistance in tobacco against tobacco necrosis virus. Strain CHA0 is a well-described biocontrol agent that naturally produces salicylic acid under conditions of iron limitation. Introduction of pchBA into CHA0 increased the production of salicylic acid in vitro and in the rhizosphere of tobacco, but did not improve the ability of CHA0 to induce systemic resistance in tobacco. In addition, these genes did not improve significantly the capacity of strains P3 and CHA0 to suppress black root rot of tobacco in a gnotobiotic system.     

Effect of Population Density of Pseudomonas fluorescens on Production of 2,4-Diacetylphloroglucinol in the Rhizosphere of Wheat

ABSTRACT The role of antibiotics in biological control of soilborne pathogens, and more generally in microbial antagonism in natural disease-suppressive soils, often has been questioned because of the indirect nature of the supporting evidence. In this study, a protocol for high pressure liquid chromatography/mass spectrometry is described that allowed specific identification and quantitation of the antibiotic 2,4-diacetylphloroglucinol (Phl) produced by naturally occurring fluorescent Pseudomonas spp. on roots of wheat grown in a soil suppressive to take-all of wheat. These results provide, for the first time, biochemical support for the conclusion of previous work that Phl-producing fluorescent Pseudomonas spp. are key components of the natural biological control that operates in take-all-suppressive soils in Washington State. This study also demonstrates that the total amount of Phl produced on roots of wheat by P. fluorescens strain Q2-87, at densities ranging from approximately 10(5) to 10(7) CFU/g of root, is proportional to its rhizosphere population density and that Phl production per population unit is a constant (0.62 ng/10(5) CFU). Thus, Phl production in the rhizosphere of wheat is strongly related to the ability of the introduced strain to colonize the roots.     

The global regulator GacA of Pseudomonas fluorescens CHA0 is required for suppression of root diseases in dicotyledons but not in Gramineae

Pseudomonas fluorescens strain CHA0 suppresses various plant diseases caused by soil-borne fungi. The pseudomonad produces the antimicrobial metabolites 2,4-diacetylphloroglucinol (Phl), pyoluteorin (Plt) and hydrogen cyanide, which are important for disease suppression, as well as the siderophores pyoverdine (Pvd), salicylic acid (Sal) and pyochelin (Pch). In the current work, a derivative of CHA0 with a mutation in the global regulator gene gacA (GacA⁻), which is unable to produce Phl, Plt and HCN, failed to protect the dicotyledonous plants cress and cucumber against damping-off caused by Pythium ultimum . In contrast, the GacA⁻ mutant could still protect the Gramineae wheat and maize against damping-off mediated by the same strain of P. ultimum , and wheat against take-all caused by Gaeumannomyces graminis . However, the GacA⁻ mutant overproduced Pch and Pvd. To gain more insight into disease protection afforded by the GacA⁻ mutant, a GacA⁻ Pvd⁻ double mutant (strain CHA496) was constructed by gene replacement. Strain CHA496 overproduced Pch and Sal compared with CHA0 and protected wheat against P. ultimum and G. graminis , whereas cress and cucumber were not protected. Addition of FeCl₃ repressed Pch and Sal production by strain CHA496 in vitro and impaired the protection of wheat in soil microcosms. In conclusion, a functional gacA gene was necessary for the protection of dicotyledons against root diseases, but not for that of Gramineae. Results indicated also that Pch and/or Sal were involved in the ability of the GacA⁻ Pvd⁻ mutant of CHA0 to suppress root diseases in Gramineae.     

Characterization of fluorescent Pseudomonas spp. associated with roots and soil of two sorghum genotypes

Sorghum is used as bioenergy feedstock, animal feed, and food. Economical methods for disease prevention and control are valuable for producers. Fluorescent Pseudomonas spp. were isolated from sorghum roots and surrounding soil with the goal of finding isolates that significantly inhibited sorghum fungal pathogens. Fluorescent pseudomonads were collected from seedlings of sorghum cultivars RTx433 and Redlan and wheat cultivar Lewjain, grown in two soils. Lewjain is known to support growth of producers of the antibiotic, 2,4-diacetylphloroglucinol (2,4-DAPG). Isolates from all three plants were assessed for hydrogen cyanide (HCN) and extracellular protease production, and for a 2,4-DAPG gene, phlD. Both soil type and plant type affected HCN- and protease-production, but phlD was not affected. Subsets of phlD ⁺ isolates were chosen to determine phlD genotypes and to conduct in vitro inhibition assays against sorghum pathogens. Most isolates from sorghum and wheat were genotype D, previously associated with superior root colonization. phlD ⁺ sorghum isolates were co-cultured with five sorghum pathogens. One isolate from each sorghum line exhibited inhibition to all five pathogens but more Redlan isolates were inhibitory to the virulent pathogen, Fusarium thapsinum, than RTx433 isolates. Nearly all inhibitory isolates from either sorghum cultivar were from one soil type. This is consistent with what had been previously observed in field studies: that soil type played a significant role in determining characteristics of fluorescent Pseudomonas spp. isolated from roots or soil, but sorghum genotype also had a considerable effect.     

Influence of plant species on disease suppression by Pseudomonas fluorescens strain CHAO with enhanced antibiotic production

Introduction of the recombinant cosmid pME3090 into Pseudomonas fluorescens strain CHAO, a good biocontrol agent of various diseases caused by soilborne pathogens, increased three- to five-fold the production of the antibiotic metabolites pyoluteorin (Pit) and 2,4-diacetylphlorogIucinol (Phi) in vitro. Strain CHAO/pME3090 also overproduced Pit and Phi in the rhizosphere of wheat infected or not infected with Pythium ultimum. The biocontrol activity of the wild-type and recombinant Straitis was compared using various plant pathogen-host combinations in a gnotobiotic system. Antibiotic overproduction affected neither the protection of wheat against P. ultimum and Gaeumannomyces graminis var. tritici nor the growth of wheat plants. In contrast, strain CHA0/pME3090 showed an increased capacity to protect cucumber against Fusarium oxysporum f. sp. cucumerinum and Phomopsis sclerotioides , compared with the wild-type strain CHAO, The antibiotic overproducing strain protected tobacco roots significantly better against Thielaviopsis basicola than the wild-type strain but drastically reduced the growth of tobacco plants and was also toxic to the growth of sweet com. On King's B agar and on malt agar, the recombinant strain CHA0/pME3090 inhibited all pathogens more than did the parental strain CHAO. Synthetic Pit and Phi were toxic to all fungi tested. Tobacco and sweet com were more sensitive to synthetic Pit and Phi than were cucumber and wheat. There was no correlation between the sensitivity of the pathogens to the synthetic antibiotics and the degree of disease suppression by strain CHAO pME3090. However, there was a correlation between the sensitivity of the plants and the toxicity of the recombinant strain. We conclude that the plant species rather than the pathogen determines whether cosmid pME3090 in P. fluorescens strain CHAO leads to improved disease suppression.     

Pyoluteorin production byPseudomonas fluorescens strain CHA0 is involved in the suppression ofPythium damping-off of cress but not of cucumber

Pseudomonas fluorescens strain CHA0 is an effective biocontrol agent of various soilborne pathogens. It controls damping-off or root rot caused byPythium ultimum on cucumber, wheat and cress. Strain CHA0 synthesizes several antibiotic metabolites such as hydrogen cyanide, 2,4-diacetylphloroglucinol, and pyoluteorin. The role of pyoluteorin in the suppression of damping-off was investigated. Two Tn5 mutants (CHA660 and CHA661) of strain CHA0 were isolated which had lost the capacity to produce pyoluteorin but still produced 2,4-diacteylphloroglucinol and HCN. These mutants still inhibitedP. ultimum on malt agar (which favours the production of 2,4-diacetylphloroglucinol) but had partially lost the ability to inhibit this pathogen on King's B agar (which favours the production of pyoluteorin). The two pyoluteorin-negative mutants showed a reduced capacity to suppress damping-off of cress caused byP. ultimum but were as effective in the protection of cucumber against this pathogen as the wild-type strain.These results indicate that, depending on the plant, pyoluteorin production plays a role in the suppression of damping-off by strain CHA0 without being a major mechanism in disease suppression. We suggest that the contribution of pyoluteorin to the biocontrol activity of strain CHA0 is determined by the quantity of this antibiotic produced in the rhizosphere, which might depend on the root exudates of the host plant.     

Zinc Improves Biocontrol of Fusarium Crown and Root Rot of Tomato by Pseudomonas fluorescens and Represses the Production of Pathogen Metabolites Inhibitory to Bacterial Antibiotic Biosynthesis

ABSTRACT Crown and root rot of tomato caused by Fusarium oxysporum f. sp. radicis-lycopersici is an increasing problem in Europe, Israel, Japan, and North America. The biocontrol agent Pseudomonas fluorescens strain CHA0 provides only moderate control of this disease. A one-time amendment of zinc EDTA at 33 mug of Zn(2+)/ml to hydroponic nutrient solution in soilless rockwool culture did not reduce disease when used alone, but did reduce disease by 25% in the presence of CHA0. In in vitro studies with the pathogen, zinc at concentrations as low as 10 mug/ml abolished production of the phytotoxin fusaric acid, a Fusarium pathogenicity factor, and increased production of microconidia over 100-fold, but reduced total biomass. Copper EDTA at 33 mug of Cu(2+)/ml had a similar effect as zinc on the pathogen in vitro; it reduced disease when used alone, and increased the biocontrol activity of CHA0 in soilless culture. Ammonium-molybdate neither improved the biocontrol activity of CHA0 nor affected production of fusaric acid or microconidia. Strain CHA0 did not degrade fusaric acid. Fusaric acid at concentrations as low as 0.12 mug/ml repressed production by CHA0 of the antibiotic 2,4-diacetylphloroglucinol, a key factor in the biocontrol activity of this strain. Production of pyoluteorin by CHA0 was also reduced, but production of hydrogen cyanide and protease was not affected, suggesting that fusaric acid affects biosynthesis at a regulatory level downstream of gacA and apdA genes. Fusaric acid did not affect the recovery of preformed antibiotics nor did it affect bacterial growth even at concentrations as high as 200 mug/ml. When microbial meta-bolite production was measured in the rockwool bioassay, zinc amendments reduced fusaric acid production and enhanced 2,4-diacetylphloro-glucinol production. We suggest that zinc, which did not alleviate the repression of antibiotic biosynthesis by fusaric acid, improved biocontrol activity by reducing fusaric acid production by the pathogen, which resulted in increased antibiotic production by the biocontrol agent. This demonstrates that pathogens can have a direct negative impact on the mechanism(s) of biocontrol agents.