Plant growth-promoting activities of fluorescent pseudomonads,isolated from the Iranian soils

Fluorescent pseudomonads are among the most influencing plant growth-promoting rhizobacteria in plants rhizosphere. In this research work the plant growth-promoting activities of 40 different strains of Pseudomonas fluorescens and Pseudomonas putida, previously isolated from the rhizosphere of wheat (Triticum aestivum L.) and canola (Brassica napus L.) and maintained in the microbial collection of Soil and Water Research Institute, Tehran, Iran, were evaluated. The ability of bacteria to produce auxin and siderophores and utilizing P sources with little solubility was determined. Four strains of Wp1 (P. putida), Cfp10 (Pseudomonas sp.), Wp150 (P. putida), and Wp159 (P. putida) were able to grow in the DF medium with ACC. Thirty percent of bacterial isolates from canola rhizosphere and 33% of bacterial isolates from wheat rhizosphere were able to produce HCN. The results indicate that most of the bacteria, tested in the experiment, have plant growth-promoting activities. This is the first time that such PGPR species are isolated from the Iranian soils. With respect to their great biological capacities they can be used for wheat and canola inoculation in different parts of the world, which is of very important agricultural implications.     

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.     

Identification of conserved traits in fluorescent pseudomonads with antifungal activity

A collection of 29 fluorescent pseudomonads, some with known biological control activity against a range of phytopathogenic fungi, were characterized phenotypically and genotypically by comparing carbon source utilization patterns, suppression of Pythium ultimum both in planta and in vitro and the potential to produce known secondary metabolites. Fatty acid profiling and restriction fragment length polymorphism (RFLP) analysis of the ribosomal DNA operon (ribotyping) were used to determine the diversity of isolates. A small group of genetically related Pseudomonas spp. with similar properties was identified; each isolate produced a diffusible bioactive product in vitro and was active against Pythium ultimum in planta . However, other isolates that were able to suppress damping off disease but did not inhibit hyphal extension in vitro clustered outside this group. Phenotypic analyses revealed that the accumulation of C17:0 cyclopropane fatty acid (17CFA) and the production of hydrogen cyanide correlated significantly with biological control activity and with the antagonism of fungal development. The potential of 17CFA as a marker for the selection of fluorescent pseudomonads with biocontrol agent (BCA) potential was demonstrated by the isolation of a novel active strain. This was selected after the screening of 13 clonal groups of fluorescent pseudomonads identified from 500 isolates from the phytosphere of sugar beet. Levels of 17CFA synthesis possibly reflect the efficacy of the rpoS allele in particular strains.     

Disease suppression and crop improvement through fluorescent pseudomonads isolated from cultivated soils

Three fluorescent pseudomonads isolated from rhizosphere/rhizoplane of crop plants showed in vitro antibiosis against seven fungal and two bacterial plant pathogens on iron-deficient KB medium. Seed bacterization of chick- pea (Cicer arientinum L.), egg plant (Solanum melongena L.), soybean (Glycine max Merr.) and tomato (Lycopersicon esculentum Mill.) with these organisms showed an increased seed germination, shoot height, root length, fresh weight, dry weight and yield. Seed bacterization with one of these strains, RB 8, reduced the number of chick-pea wilted plants in wilt-sick (Fusarium oxysporum f.sp. ciceris) soil. Addition of iron into the soil eliminated the disease suppression. The disease suppression and/or growth enhancement along with the positive root colonization by these organisms indicate their possible use as plant growth-promoting rhizobacteria (PGPR)/biocontrol agents against chick-pea wilt.     

Characterization and classification of fluorescent pseudomonads isolated from tap water and surface water

A total of 665 fluorescent pseudomonads, isolated from surface water and from different types of tap water, were classified into 22 groups defined by the results of the following 5 tests: hydrolysis of casein or gelatin, production of N₂ from NO _inf3 ^sup- , and growth on sucrose, ethanol and d-sorbitol, respectively. Differences in colonial morphology and in the degree of proteolysis revealed that these groups were inhomogeneous. A more detailed subdivision was achieved by adding the following characters: growth on l-arabinose, d-mannitol, meso-inositol, and adonitol, respectively, and hydrolysis of Tween-80. Repeating the tests for hydrolyzing enzymes, denitrification, and growth on the carbohydrates and alcohols with strains stored in the laboratory for 1 to 3 years revealed that most characters, except hydrolysis of Tween-80, denitrification, and growth on sucrose, were very stable.Forty-five biotypes of the fluorescent pseudomonads were defined, based on the results of the repeated tests and the results of tests on nine aromatic compounds. The observed changes of some characters in a number of isolates did not diminish the value of this classification, but indicated that close relationships exist between many biotypes. About half of the biotypes described in this paper are similar to those defined by Stanier, Palleroni and Doudoroff (1966) and by Doudoroff and Palleroni (1974a), confirming the wide-spread occurrence of well-definable biotypes of fluorescent pseudomonads.Representatives of some biotypes were most frequently isolated from surface water and from tap water prepared from surface water. Tap water prepared from anaerobic or aerobic ground water contained representatives of biotypes which were typical of these water types. Some pseudomonads were found to grow especially in filters. The observed relationships between origin of the fluorescent pseudomonads and their classification into biotypes as defined in this paper supported the presented classification.     

Evaluation of production of some antifungal metabolites by fluorescent pseudomonads in vitro and their inhibitory effect on Penicillium digitatum in semi-commercial condition

Epiphytic fluorescent Pseudomonads isolated from the surfaces of citrus leaves and fruits collected from the citrus orchards located in the north of Iran were screened for antagonistic activity against Penicillium digitatum caused agent of green mold of citrus fruit. 9 isolates revealed antagonistic effect in dual culture and then evaluated for production of antimicrobial metabolites such as production hydrogen cyanide on King's B medium containing glycine, production of siderophore on blue CAS-agar medium and production protease enzyme on Skim Milk Agar (SMA) medium. About 28% strains produced hydrogen cyanide, 83% strains produced siderophore and 70% of them produced protease. Antagonistic effects of the screened 9 isolates were studied by inoculating of the infected orange fruits with the bacterium at the concentration of 10(8) cells/ml under sterilized condition at 20 degrees C. The three final screened antagonist isolates were more studied in semi commercial trails in cold storage with dipping of the orange fruits in the bacterial suspension at the concentration of 10(8) cells/ml and then 5 weeks storage at 4 degrees C. The isolate P39 showed to be the most effective for controlling of this disease and decreased the damage by 77.28% respectively.     

Influence of soil reaction on diversity and antifungal activity of fluorescent pseudomonads in crop rhizospheres

The diversity and antifungal activity of fluorescent pseudomonads isolated from rhizospheres of tea, gladiolus, carnation and black gram grown in acidic soils with similar texture and climatic conditions were studied. Biochemical characterisation including antibiotic resistance assay, RAPD and PCR-RFLP studies revealed a largely homogenous population. At soil pH (5.2), the isolates exhibited growth with varying levels of siderophore production, irrespective of crop rhizospheres. Two isolates with maximum chitinase production showed antagonism. The bacterial populations in general lacked the ability to produce deleterious traits such as cellulase, pectinase and hydrogen cyanide. However, increased pH levels beyond 5.2 caused reduction in metabolite production with reduced antifungal activity. The homogeneity of the bacterial population irrespective of crop rhizospheres together with decreased secondary metabolite production at higher pH levels reinstated the importance of soil over host plant in influencing rhizosphere populations. The studies also yielded acid tolerant chitinase producing antagonistic fluorescent pseudomonads.     

Assessment of genetic and functional diversity of phosphate solubilizing fluorescent pseudomonads isolated from rhizospheric soil

Background  

Phosphorus is an essential macronutrient for the growth of plants. However, in most soils a large portion of phosphorus becomes insoluble and therefore, unavailable to plants. Knowledge on biodiversity of phosphate-solubilizing fluorescent pseudomonads is essential to understand their ecological role and their utilization in sustainable agriculture.     

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.     

Production of biosurfactants and antibiotics by fluorescent pseudomonads isolated from a closed hydroponic system equipped with a slow filter

The presence of antibiotic- and biosurfactant-producing strains of fluorescent pseudomonads in a closed hydroponic system equipped with a slow filter was investigated. A total of 271 strains of pseudomonads were isolated before the filter, from the filter skin and from the effluent. Production of biosurfactants was determined using the drop-collapse method. The ability of the strains to inhibit the growth of the plant pathogens Pythium ultimum, Phytophthora cryptogea and Fusarium oxysporum was determined using dual culture plating. The influence of carbon sources on production was determined for selected strains, which also were identified to species level. Production of antibiotics or biosurfactants was observed to be a common trait among the fluorescent pseudomonads within the closed hydroponic system and it was affected by the filter. Pythium ultimum was the pathogen that was most sensitive to antibiotics produced by the fluorescent pseudomonads. The results indicated a strong influence of nutritional resources on antibiotic and biosurfactant production.     

Biological activities of rishitin, an antifungal compound isolated from diseased potato tubers, and its derivatives

Rishitin, a norsesquiterpene alcohol, found in infected, resistantpotato-tuber tissue completely inhibited zoospore germinationand germtube elongation of Phytophthora infestans (MONT.) DEBARY at 10^–3M. There was little difference in sensitivityto rishitin among races of Phytophthora infestans. IAA-inducedelongation of Avcna coleoptile sections and GA₃-induced elongationof wheat leaf sections were also inhibited by rishitin. Theinhibition of IAA-induced elongation of Avena coleoptiles wasrelieved to some extent by increasing IAA concentration. However,little relief of the inhibition of GA₃-induced elongation ofwheat leaf sections was obtained by increasing GA₃ concentration.No plant injury was observed at this concentration of rishitin(10^–3M). Examination of a series of rishitin derivatives indicated thatthe hydroxyl group at C-3 is indispensable for antifungal activity.This activity was intensified by saturating the double bondbetween the rings of rishitin and/or that of the isopropenylgroup at C-7, though activity decreased when oxygenated functionalgroups were introduced into the side chain. Aromatization of the A ring did not lower biological activities.The antifungal activities of most rishitin derivatives almostparalleled their activities as plant growth retardants. However,some compounds without antifungal activity were active as growthretardants. ¹Studies on the phytoalexins (5). (Received August 14, 1968; )     

Phylogenetic diversity of fluorescent pseudomonads in agricultural soils from Korea

AIMS: To identify and compare the relative diversity and distribution of genotypes of culturable fluorescent pseudomonads from soils. METHODS AND RESULTS: Analysis of 160 isolates from seven soil samples using randomly amplified polymorphism DNA methods revealed 53 genotypes, which were subsequently identified by their 16S ribosomal DNA sequences. Phylogenetic analyses of the 53 genotypes along with 43 fluorescent pseudomonad type strains separated the genotypes into 10 distinct clusters that included two phylogenetic groups that were not represented by previously described type strains. CONCLUSIONS: The diversity of genotypes that was obtained from the soil samples was highly variable among the different soils and appeared to be associated with different soil management practices that also influence plant yields. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification and phylogenetic analysis of these genotypes offers opportunities for study of phenotypic traits that may be associated within taxonomically related groups of fluorescent pseudomonad species and how these groups vary in relation to soil management practices.     

Characterization of antifungal metabolite phenazine from rice rhizosphere fluorescent pseudomonads (FPs) and their effect on sheath blight of rice

We have shown, the outcome of antifungal activity of phenazine derivatives which is produced by fluorescent pseudomonads (FPs) for the control of sheath blight of rice. A total of 50 fluorescent pseudomonads (FPs) were isolated from rice rhizosphere. Off which, 36 FPs exhibited antagonistic activity against Rhizoctonia solani, Macrophomina phaseolina, Fusarium oxysporum, Alternaria alternata and Sclerotium rolfsii up to 70–80% compared to control by dual culture method. BOX-PCR analyses of antagonistic isolates indicated that two phylogenetic group, where group I consisted of 28 isolates and eight isolates belongs to group II. Among 36 FPs, a total of 10 FPs revealed that the presence of phenazine derivatives on thin layer chromatography (TLC), which is coincided with that of authentic phenazine with R_f value 0.57. Similar to TLC analysis, antibiotic encoding gene phenazine-1-carboxamide (PCN) was detected in 10 FPs by PCR analysis with respective primer. Among, PCN detected isolates of FPs, a significant biocontrol potential possessing isolate designated as VSMKU1 and it was showed prominent antifungal activity against R. solani and other tested fungal pathogens. Hence, the isolate VSMKU1 was selected for further studies. The selected isolate VSMKU1 was identified as Pseudomonas aeruginosa by 16S rDNA sequence analysis. The antifungal metabolite phenazine like compound produced by VSMKU1 was confirmed by UV, FT-IR and HPLC analysis. The phenazine compound from VSMKU1 significantly arrest the growth of R. solani compared to carbendazim by well diffusion method. The detached leaf assay showed remarkable inhibition of lesion height 80 to 85% by the treatments of culture (VSMKU1), cell free culure filtrate and phenazine like compound compared to control and other treatments was observed in detached leaves of rice. These results emphasized that VSMKU1 isolate can be used as an alternative potential biocontrol agent against sheath blight of rice, instead of using commercial fungicide such as validamycin and carbendazim which cause environmental pollution and health hazards.     

小麦内生细菌及其对根茎部主要病原真菌的抑制作用

对小麦植株不同生育期、不同器官的内生细菌进行了分离和数量变化分析.结果表明,根、茎、叶及未成熟籽粒等器官中存在大量的内生细菌,鲜组织中平均约含内生细菌5.0×10⁵ CFU·g^-1,其中根系中内生细菌数量达7.8×10⁵ CFU·g^-1,而茎秆、叶片和未成熟籽粒中内生细菌数量分别为4.8×10⁵、3.2×10⁵和2.8×10⁵ CFU·g^-1.内生细菌数量在不同生育期也存在差异,幼苗期平均约为3.1×10⁵ CFU·g^-1、拔节期和灌浆期分别为5.7×10⁵和7.0×10⁵ CFU·g^-1.不同小麦田块之间存在明显差异,长武县一田块植物鲜组织中内生细菌的数量为6.1×10⁵ CFU·g^-1,而大荔县一田块约为3.9×10⁵ CFU·g^-1.试验结果发现,对小麦全蚀病菌具有拮抗作用的内生细菌有51株、对小麦纹枯病菌具有抑制作用的内生细菌有45株.用平板对峙法测定,有71株对两种病原真菌均有拮抗作用,对小麦全蚀病菌抑菌圈直径大于10 mm的有23株,其中来源于根系、茎秆、叶片和籽粒的分别为6株、7株、9株和1株;对小麦纹枯病菌抑菌圈超过10 mm的有20株,其中来源于根系、茎秆、叶片和籽粒的分别为7株、5株、7株和1株,说明从小麦叶片诱捕分离的内生细菌中,对小麦全蚀病菌和纹枯病菌抑菌作用较强的分离株比率最高,其次为茎秆,而根部和未成熟籽粒中比例明显较低.     

Genotypic and phenotypic diversity of PGPR fluorescent pseudomonads isolated from the rhizosphere of sugarcane (Saccharum officinarum L.)

The genetic diversity of plant growth-promoting rhizobacterial (PGPR) fluorescent pseudomonads associated with the sugarcane (Saccharum officinarum L.) rhizosphere was analyzed. Selected isolates were screened for plant growthpromoting properties including production of indole acetic acid, phosphate solubilization, denitrification ability, and production of antifungal metabolites. Furthermore, 16S rDNA sequence analysis was performed to identify and differentiate these isolates. Based on 16S rDNA sequence similarity, the isolates were designated as Pseudomonas plecoglossicida, P. fluorescens, P. libaniensis, and P. aeruginosa. Differentiation of isolates belonging to the same group was achieved through different genomic DNA fingerprinting techniques, including randomly amplified polymorphic DNA (RAPD), amplified ribosomal DNA restriction analysis (ARDRA), repetitive extragenic palindromic (REP), enterobacterial repetitive intergenic consensus (ERIC), and bacterial repetitive BOX elements (BOX) analyses. The genetic diversity observed among the isolates and rep-PCR-generated fingerprinting patterns revealed that PGPR fluorescent pseudomonads are associated with the rhizosphere of sugarcane and that P. plecoglossicida is a dominant species. The knowledge obtained herein regarding the genetic and functional diversity of fluorescent pseudomonads associated with the sugarcane rhizosphere is useful for understanding their ecological role and potential utilization in sustainable agriculture.     

Comparsion ofin vitro activities of antifungal drugs and propolis against yeasts isolated from patients with superficial mycoses

Thein vitro susceptibilities of propolis and antifungal drugs were determined against some yeasts isolated from patients with superficial mycoses. The agents tested included fluconazole, itraconazole, ketoconazole, terbinafine and propolis. MICs were determined by the broth microdilution technique following National Committee for Clinical Laboratory Standards document M27-P. For allCandida albicans isolates from the patients with superficial mycoses, ketoconazole presented higher (P<0.05) efficiency than that of the other antifungal agents tested. The geometric mean MIC values of antifungal drugs and propolis against the yeasts tested ranged from 0.087 to 12.69 μg/mL and 0.4–0.6 μg/mL, respectively. Propolis also showed an important antifungal activity against the yeasts tested, MIC ranges of the propolis were between 0.01–1.65 μg/mL. Based on these results, propolis requires further investigation as a potential agent for the treatment of superficial mycoses.     

Synthesis,antibacterial and antifungal activities of some carbazole derivatives

A series of N-substituted carbazole derivatives were synthesized and evaluated for antibacterial and antifungal activities against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Bacillus proteus, Candida albicans and Aspergillus fumigatus by two fold serial dilution technique. Some of the synthesized compounds displayed comparable or even better antibacterial and antifungal activities than reference drugs fluconazole, chloramphenicol and norfloxacin against tested strains.