Characterization of biosurfactant-producing strains of fluorescent pseudomonads in a soilless cultivation system

The use of biosurfactants is a promising alternative in biological control of zoospore-producing plant pathogens. In the present study, biosurfactant production by the indigenous population of fluorescent pseudomonads in a soilless plant cultivation system was studied during the growing season. A total of 600 strains was screened and of these 18.5% were observed to produce biosurfactants. Production of both antibiotics and biosurfactant was uncommon among the isolated strains. A selective effect of the cultivation system filter was observed on the biosurfactant-producing strains and these strains were only occasionally observed after the filter, despite having a significantly higher motility than the nonbiosurfactant-producing strains. The majority of biosurfactant-producing strains were isolated from the filter skin, which suggests that this is a suitable surface for inoculation with biocontrol strains.     

Suppression of disease in tomato infected by <Emphasis Type="Italic">Pythium ultimum</Emphasis> with a biosurfactant produced by <Emphasis Type="Italic">Pseudomonas koreensis</Emphasis>

The use of biosurfactants is a promising alternative in biological control of zoospore-producing oomycetes, which are a major plant pathogen world-wide in a wide variety of crops. Oomycetes are of particular concern in closed hydroponic cultivation systems. The present study investigated the efficacy of a biosurfactant produced by Pseudomonas koreensis and added as a crude extract against the oomycete Pythium ultimum in hydroponic tomato cultivation. A significant reduction in disease was observed. Biosurfactant addition did not affect the indigenous root microflora when evaluated as sole carbon source utilisation. Chemical analysis, using electrospray hybrid mass spectrometry (ESI-MSMS), of the biosurfactant indicated it to be lokisin, a cyclic lipopeptide. These results confirm that biosurfactants are important in developing sustainable biological control strategies for oomycetes.     

Interaction of nutrients and four antifungal antibiotics in their effects on Pythium species in vitro and in soil

Summary Griseofulvin, streptomycin, aureomycin, and actidione, with and without nutrients, were tested againstPythium ultimum Trow (two strains),P. irregulare Buis., andP. rostratum Butler. Streptomycin was tolerated in the highest concentration, and actidione and aureomycin in the lowest.P. rostratum, a saprophytic species, was more resistant to the antibiotics than the pathogenic species, which were inhibited at low concentrations. The two strains ofP. ultimum behaved differently, the oogonial strain being more tolerant than the sporangial strain. Yeast extract was usually superior to sucrose or asparagine in counteracting the toxicity of antibiotics.Germination of sporangia ofPythium irregulare was studiedin vitro and in a natural soil in the presence of antibiotic and antibiotic-nutrient solutions. Very meager germination of sporangia occurred in soil amended with antibiotics. Actidione severely restricted germination in soil. Nutrients nullified the toxicity to varying degrees. Usually higher germination of sporangia occurredin vitro than in soil. Sub-lethal concentrations of antibiotics induced morphological abnormalities in germ tubes. Aureomycin caused a dendroid type of branching; actidione caused distortion, vacuolation, and vesiculation; griseofulvin caused lysis and septation; and streptomycin caused distortion.Germ tubes were lysed in a natural soil and in soils amended with antibiotics, but nutrients, in general, prevented early lysis at low concentrations of antibiotics. No resistant bodies were formed prior to the initiation of lysis of germ tubes.     

Use of Trichoderma harzianum and Gliocladium vitens for the Biological Control of Post-emergence Damping-off and Root Rot of Cucumbers Caused by Pythium ultimum

The antagonist strains Gliocladium virens G2 and Trichoderma harzianum T3 originally isolated from Pythium suppressive peat, and two benomyl-resistant strains of T. harzianum, T12B and T95, were evaluated as biological control agents of damping-off and root rot of cucumbers in sphagnum peat caused by Pythium ultimum. All strains were equally effective when applied as 1 % peat-bran preparations, whereas the effectiveness of disease control was reduced at higher concentrations of the antagonists. The two wild-type strains were also found to be effective when applied as conidial suspensions, and in this case no reduction in disease control was seen at higher concentrations. G. virens G2 and T. harzianum T12B showed antibiotic activity against P. ultimum in in vitro tests; however there were no signs of mycoparasitism of P. ultimum by any of the antagonist strains.     

Diversity and activity of biosurfactant-producing Pseudomonas in the rhizosphere of black pepper in Vietnam

Aims: Phytophthora capsici is a major pathogen of black pepper and zoospores play an important role in the infection process. Fluorescent pseudomonads that produce biosurfactants with zoosporicidal activities were isolated from the black pepper rhizosphere in Vietnam, and their genotypic diversity and potential to control Phy. capsici root rot was determined. Methods: Biosurfactant‐producing pseudomonads were genotypically and biochemically characterized by BOX‐polymerase chain reaction (PCR), 16S‐rDNA sequencing, reverse‐phase‐high‐performance liquid chromatography and liquid chromatography‐masss spectrometry analyses. Results: Biosurfactant‐producing fluorescent pseudomonads make up c. 1.3% of the culturable Pseudomonas population in the rhizosphere of black pepper. Although BOX‐PCR revealed substantial genotypic diversity, the isolates were shown to produce the same biosurfactants and were all identified as Pseudomonas putida. When applied to black pepper stem cuttings, several of the biosurfactant‐producing strains provided significant disease control. In absence of the disease, several of the bacterial strains promoted shoot and root growth of black pepper stem cuttings. Conclusions: Biosurfactant‐producing pseudomonads indigenous to the rhizosphere of black pepper plants are genotypically diverse and provide a novel resource for the control of Phy. capsici root rot and growth promotion of black pepper stem cuttings. Significance and Impact of the Study: The results of this study provide a strong basis for further development of supplementary strategies with antagonistic bacteria to control foot and root rot of black pepper and to promote plant growth.     

Antagonistic actions of Pythium oligandrum and Trichoderma harzianum against phytopathogenic fungi (Fusarium oxysporum and Pythium ultimum var. ultimum)

Abstract

The possible biological control of damping-off fungi, Fusarium oxysporum and Pythium ultimum by Pythium oligandrum or Trichoderma harzianum was in vitro investigated. Results of comparing the antagonistic activity of P. oligandrum and T. harzianum in dual plates against the tested phytopathogens indicated different degrees of antagonism. After 12 days of incubation colony of the phytopathogenic fungus was completely overgrown by the antagonist, except for the interaction between T. harzianum and F. oxysporum which showed no overgrowth or any hyphal penetration by the antagonist. However, growth and proliferation of F. oxysporum colony was repressed. T. harzianum and P. oligandrum produced chitinase and β-1,3-glucanase when they were grown on liquid culture medium supplemented with chitin or fungal dried mycelium as a sole carbon source, and enzyme production was higher by T. harzianum comparing with P. oligandrum under the same condition. Fungal dried mycelium of F. oxysporum was the most selective carbon source for enzyme production, on the other hand, chitinase production was significant locked when P. ultimum dried mycelium was used as a carbon source. Production of volatile compounds by P. oligandrum or T. harzianum against F. oxysporum and P. ultimum was examined using the inverted plates method. F. oxysporum was inhibited by the antagonist volatile compounds and it is inhibited 100% by increasing the amount of inoculum size. Production of potential biocontrol agents provided with economically features and working under field conditions are recommended.     

Assessment of genetic and functional relationship of antagonistic fluorescent pseudomonads of rice rhizosphere by repetitive sequence,protein coding sequence and functional gene analyses

Antagonistic fluorescent pseudomonads isolated from rice rhizospheric soil were characterized using biochemical, taxonomical and molecular tools. Production of cyclopropane fatty acid (CFA) was correlated with their antagonistic potential. Strains were grouped into 18 different genotypes on the basis of amplified ribosomal DNA restriction analysis (ARDRA) and repetitive (rep)-PCR based genotypic fingerprinting analyses. High phylogenetic resolution among antagonistic fluorescent pseudomonad strains was obtained based on the DNA gyrase B subunit (gyrB) and RNA polymerase sigma factor 70 (rpoD) gene sequence analyses. Combined gyrB and rpoD sequence analysis resulted in the accurate estimation of molecular phylogeny and provided a significant correlation between the genetic distances among strains. Present study demonstrated the genetic and functional relationship of fluorescent pseudomonads. The knowledge on genetic and functional potential of fluorescent pseudomonads associated with rice rhizosphere is useful to understand their ecological role and for their utilization in sustainable agriculture.     

Occurrence of Pythium spp. and Phytophthora spp. in Norwegian Greenhouses and their Pathogenicity on Cucumber Seedlings

Samples of tomato, lettuce and cucumber submitted for diagnosis to the Plant Protection Centre at the Norwegian Crop Research Institute and samples of soil, water and cucumber collected from greenhouses employing hydroponic cultures were examined for the occurrence of Pythium spp. and Phytophthora spp. Two species of Phytophthora and 16 species of Pythium were identified. Phytophthora cryptogea was found on tomato and lettuce. Phytophthora nicotianae was found on tomato fruit. Phytophthora was not found on cucumbers. Pythium irregulare and Pythium group F were the two most commonly found Pythium species in hydroponically cultivated cucumbers. A pathogenicity test with 56 isolates was performed on cucumber seedlings. The most aggressive species were Pythium aphanidermatum, P. irregulare, Pythium paroecandrum and Pythium ultimum.     

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.     

Role of antibiotics and siderophores in biocontrol of take-all disease of wheat

Both antibiotics and siderophores have been implicated in the control of soilborne plant pathogens by fluorescent pseudomonads. In Pseudomonas fluorescens 2–79, which suppresses take-all of wheat, the importance of the antibiotic phenazine-1-carboxylic acid was established with mutants deficient or complemented for antiobiotic production and by isolation of the antibiotic from the roots of wheat colonized by the bacteria. Genetic and biochemical studies of phenazine synthesis have focused on two loci; the first is involved in production of both anthranilic acid and phenazine-1-carboxylic acid, and the second encodes genes involved directly in phenazine synthesis. Because the antibiotic does not account fully for the suppressiveness of strain 2-79, additional mutants were analyzed to evaluate the role of the fluorescent siderophore and of an antifungal factor (Aff, identified as anthranilic acid) that accumulates when iron is limiting. Whereas strains producing only the siderophore conferred little protection against take-all, Aff⁺ strains were suppressive, but much less so than phenazine-producing strains. Iron-regulated nonsiderophore antibiotics may be produced by fluorescent pseudomonads more frequently than previously recognized, and could be partly responsible for beneficial effects that were attributed in the past to fluorescent siderophores.     

Growth response of marigold (Tagetes erecta L.) to inoculation withGlomus mosseae,Trichoderma aureoviride andPythium ultimum in a peat-perlite mixture

The interactions between the mycorrhizal fungusGlomus mosseae, the plant pathogenPythium ultimum, and a pathogen-antagonistTrichoderma aureoviride in the rhizosphere ofTagetes erecta (marigold) were studied for their effects on plant growth in a peat-perlite substrate. Mycorrhizal fungus inoculation protected the plant againstP. ultimum, since both phytomass production and foliar development were higher in mycorrhizal plants.T. aureoviride had no effect on nonmycorrhizal plants in the presence or absence ofP. ultimum. However, more biomass was produced by mycorrhizal plants whenT. aureoviride was present, whether or not soil was infested withP. ultimum. ei]R Rodriguez-Kabana     

Bioassay of metalaxyl in plant tissue

A method for determining the concentration of metalaxyl in methanolic extracts of plant tissue is described. Extracts were applied to filter papers in Petri dishes and covered with small volumes of lima bean agar. The growth of an isolate of Pythium ultimum sensitive to metalaxyl was related to the quantity of fungicide applied. The bioassay could detect quantities as low as 0·025 μg in the filter paper, a sensitivity which was confirmed by using ¹⁴C-metalaxyl. Whilst untreated extracts of lettuce and brassicas were not fungitoxic to the isolate of P. ultimum employed, this was not the case with tobacco and grape-vine. Hence alternative test organisms may be required before the method can be generally applied to all species.     

Involvement of G-alpha protein GNA3 in production of cell wall-degrading enzymes by Trichoderma reesei (Hypocrea jecorina) during mycoparasitism against Pythium ultimum

The involvement of the G-alpha protein GNA3 in the production of cell wall-degrading enzymes (CWDEs) by Trichoderma reesei during antagonism against Pythium ultimum was investigated. cAMP content was 2.8-fold higher in the T. reesei mutant gna3QL than in the parental TU-6. The gna3QL, like TU-6, inhibited the growth of P. ultimum in dual culture assays. Scanning electron microscopy showed that the gna3QL promoted more morphological alterations of P. ultimum cell wall than TU-6. In general, gna3QL produced higher activities of CWDEs than TU-6. We therefore suggest that CWDEs production during mycoparasitism by T. reesei against P. ultimum may be associated with the level of GNA3 activity.     

Characterization of two morphological groups of isolates ofPythium ultimum var.ultimum in a vegetable field

Comparisons were made between two morphological groups ofPythium ultimum var.ultimum strains isolated in a vegetable field in Japan. The groups were distinguished as having smaller or larger sexual organs by the sizes of their antheridia and oogonia. Morphological study indicated that the two groups comprised a single taxon,P. ultimum var.ultimum, by the current taxonomical keys. The smaller group grew faster in the lower temperature range of 4–15°C, whereas the larger group grew faster in the higher temperature range of 25–37°C. Random amplified polymorphic DNA (RAPD) and isozyme analyses revealed genetic dissimilarity between the two groups. Cluster analysis of the isozyme banding patterns with four otherPythium spp. demonstrated that the genetic dissimilarity between the two groups was equivalent to species level. In the field survey, the smaller group was frequently detected in February, May and September but not in July, while the larger group was detected mainly in July and September. The two groups were not distinguishable by their pathogenicity to spinach seedlings.     

Secondary Metabolite- and Endochitinase-Dependent Antagonism toward Plant-Pathogenic Microfungi of Pseudomonas fluorescens Isolates from Sugar Beet Rhizosphere

Forty-seven isolates representing all biovars of Pseudomonas fluorescens (biovars I to VI) were collected from the rhizosphere of field-grown sugar beet plants to select candidate strains for biological control of preemergence damping-off disease. The isolates were tested for in vitro antagonism toward the plant-pathogenic microfungi Pythium ultimum and Rhizoctonia solani in three different plate test media. Mechanisms of fungal inhibition were elucidated by tracing secondary-metabolite production and cell wall-degrading enzyme activity in the same media. Most biovars expressed a specific mechanism of antagonism, as represented by a unique antibiotic or enzyme production in the media. A lipopeptide antibiotic, viscosinamide, was produced independently of medium composition by P. fluorescens bv. I, whereas the antibiotic 2,4-diacetylphloroglucinol was observed only in glucose-rich medium and only in P. fluorescens bv. II/IV. Both pathogens were inhibited by the two antibiotics. Finally, in low-glucose medium, a cell wall-degrading endochitinase activity in P. fluorescens bv. I, III, and VI was the apparent mechanism of antagonism toward R. solani. The viscosinamide-producing DR54 isolate (bv. I) was shown to be an effective candidate for biological control, as tested in a pot experiment with sugar beet seedlings infested with Pythium ultimum. The assignment of different patterns of fungal antagonism to the biovars of P. fluorescens is discussed in relation to an improved selection protocol for candidate strains to be used in biological control.     

Production of Inter- and Intra-strain Hybrids of Trichoderma spp. by Protoplast Fusion and Evaluation of Their Biocontrol Activity Against Soil-borne and Foliar Pathogens

Protoplast fusion techniques were used for the production of new antagonistic strains of Trichoderma spp. Two British and two Italian strains of Trichoderma with different biocontrol potential against Botrytis cinerea, Sclerotinia sclerotiorum and Pythium ultimum, were treated with u.v. radiation and mutants resistant to hygromycin B or propiconzole were selected. Protoplasts were obtained from these mutants after digestion of young hyphae with Novozym 234 and used in inter- and intra-strain protoplast fusion experiments. Hybrids were obtained in 13 crosses isolated on fungicide-amended media by the inheritance of resistance markers from both parental strains. Selected, fast-growing, stable mutants were tested in biocontrol trials against P. ultimum on lettuce seedlings and B. cinera on grape bunches in comparison with their parental strains. Intra-strain hybrids derived from cross XIII were tested in vitro for their mycoparasitic ability on sclerotia of S. sclerotiorum. A high degree of variability in the biocontrol and the mycoparasitic ability of the fusants was observed but no significant increase in the activity was accomplished after fusion, the hybrids being generally less activewas accomplished after fusion, the hybrids being generally less active than their parental strains.     

Potential for improving pea production by co-inoculation with fluorescent Pseudomonas and Rhizobium

Seed bacterization with five plant growth promoting fluorescent Pseudomonas strains isolated from Indian and Swedish soils and three Rhizobium leguminosarumbiovar viceae strains isolated from Swedish soils were shown to promote plant growth in Pisum sativum L. cv. Capella. Co-inoculation of the fluorescent pseudomonads and Rhizobium improved plant growth in terms of shoot height, root length and dry weight. Both the fluorescent pseudomonads and Rhizobium were shown to exhibit a wide range of antifungal activity against pathogens specific to pea. Seed bacterization with plant growth promoting strains alone and together with a rhizobial isolate, R 361-27 reduced the number of infected peas grown in Fusarium oxysporum infested soils. We found that the introduced organisms were able to colonize the roots, which was confirmed using immunofluorescence staining and drug resistant mutant strains. In a synthetic culture medium, all the plant growth promoting fluorescent pseudomonads strains produced siderophores, which shown to express antifungal and antibacterial activity. Our results suggest the potential use of these bacteria to induce plant growth and disease suppression in sustainable agriculture production systems.     

Survival and Ecological Fitness of Pseudomonas fluorescens Genetically Engineered with Dual Biocontrol Mechanisms

The antibiotic 2,4-diacetylphloroglucinol (Phl) is produced by a range of naturally occurring fluorescent pseudomonads. One isolate, Pseudomonas fluorescens F113, protects pea plants from the pathogenic fungus Pythium ultimum by reducing the number of pathogenic lesions on plant roots, but with a concurrent reduction in the emergence of plants such as pea. The genes responsible for Phl production have been shown to be functionally conserved between the wild-type (wt) P. fluorescens strains F113 and Q2-87. In this study the genes from F113 were isolated using an optimized long PCR method and a 6.7-kb gene cluster inserted into the chromosome of the non-Phl-producing P. fluorescens strain SBW25 EeZY6KX. This strain is a lacZY, km^R marked derivative of the wt SBW25 which effects biological control against the plant pathogen Pythium ultimum by competitive exclusion as a result of its strong rhizosphere-colonizing ability. We describe here the integration of the Phl antifungal and competitive exclusion mechanisms into a single strain, and the impact this has on survival and plant emergence in microcosms. The insertion of the Phl biosynthetic genes from the F113 into the SBW25 chromosome gave a Phl-producing transformant (strain Pa21) able to suppress P. ultimum through antibiotic production. The growth of Pa21 was not reduced in flask culture at 20°C compared with its parent strain. When inoculated on pea seedlings, the strain containing the Phl operon behaved similarly to the SBW25 EeZY6KX parent but did not show the tendency of the wt Phl producer F113 to cause lower pea seed emergence. Pea roots inoculated with SBW25 EeZY6KX have significantly lower indigenous populations than with F113 and the control. This is indicative of this strains strong colonising presence. Pa21, the Phl-modified strain, is able to exclude the resident population from roots to the same degree as the SBW25 EeZY6KX from which it is derived. This suggests that it has maintained its competitiveness around the root systems of plants even with the introduction of the Phl locus. Thus, strain Pa21 possesses the qualities necessary to provide effective integrated biocontrol, through maintaining both its wt trait of competitive exclusion on the plant roots, while also expressing the genes from the F113 biocontrol strain for Phl production. Interestingly, however, an additional beneficial trait appears to emerge with the strain Pa21s lowered survival competence compared with SBW25 EeZY6KX in the rhizosphere soil. With fears of the spread of genetically modified organisms and persistence in the soil, this trait may be of some ecological and commercial benefit and becomes a candidate for further investigation and possible exploitation.     

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.     

Effect of osmolarity and dehydration on alginate production by fluorescent pseudomonads

Alginate is produced as an exopolysaccharide by many fluorescent pseudomonads. However, pseudomonads often have a nonmucoid phenotype in standard laboratory media. Growth in the presence of 0.3M sodium chloride or 3–5% ethanol reportedly can lead to the generation of mucoid variants of nonmucoid strains ofPseudomonas aeruginosa. We wished to determine whether alginate production by other fluorescent pseudomonads is affected by sodium chloride and ethanol. Eight alginate-producing strains of saprophytic and phytopathogenic pseudomonads were grown as broth cultures containing 0–0.7M sodium chloride or 0–5% ethanol for 24–30 h at 28° or 35°C. Culture supernatant fluids were subjected to ethanol precipitation, and the amount of alginate present was estimated by measuring the uronic acid content. The presence of sodium chloride and ethanol caused significant stimulation of alginate production by all strains tested exceptP. viridiflava ATCC 13223 andP. fluorescens W4F1080. The optimal concentration of sodium chloride ranged from 0.2 to 0.5M; that for ethanol ranged from 1 to 3%. Moreover, inclusion of the nonmetabolizable, nonionic solute sorbitol showed a similar stimulation of alginate production. The stimulation of alginate production by high medium osmolarity and dehydration appears to be a trait shared by fluorescent pseudomonads.Reference to brand or firm name does not constitute endorsement by the U.S. Department of Agriculture overothers of a similar nature not mentioned.