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.     

Pigment Diverse Mutants of Pseudomonas sp.: Inhibition of Fungal Growth and Stimulation of Growth of Cicer arietinum

A Pseudomonas strain MRS16 inhibited growth of different pathogenic fungi (Aspergillus sp., Fusarium oxysporum, Pythium aphanidermatum and Rhizoctonia solani) in vitro. Larger inhibition zones were obtained on nutrient agar and King's B media compared to potato dextrose agar and pigment production media. Mutants altered in production of fluorescent pigment were derived by nitrosoguanidine mutagenesis. The pigment overproducer mutant MRS16M-1 was more inhibitory whereas nonproducer mutant MRS16M-5 was less inhibitory than parent strain on nutrient agar medium. Addition of iron (100 µM FeCl₃) in the medium decreased inhibition of fungal growth, suggesting the involvement of siderophores and other antifungal secondary metabolites. Seed bacterization of two cultivars of chickpea (Cicer arietinum cvs. H8618 and C235) differing in susceptibility to wilt caused initial root and shoot stunting at 5 d of growth followed by proliferation of secondary root growth at 10 d. Coinoculation of chickpea with Pseudomonas strain MRS16 or mutants and Rhizobium sp. Cicer strain Ca181 enhanced nodulation, nitrogen fixation and plant dry mass as compared to single inoculation with Rhizobium strain under sterile conditions.     

Disease suppression and crop improvement in moong beans (<Emphasis Type="Italic">Vigna radiata</Emphasis>) through <Emphasis Type="Italic">Pseudomonas</Emphasis> and <Emphasis Type="Italic">Burkholderia</Emphasis> strains isolated from semi arid region of Rajasthan,India

Pseudomonas fluorescens BAM-4, Burkholderia cepacia BAM-6 and B. cepacia BAM-12 isolated from the rhizosphere of moong bean (Vigna radiata L.) showed significant growth-inhibitory activity against a range of phytopathogenic fungi. Light and scanning electron microscopic (SEM) studies showed morphological abnormalities such as fragmentation, swelling, perforation and lysis of hyphae of pathogens by Pseudomonas and Burkholderia. Two of the strains (BAM-4 and BAM-6) produced siderophore in CAS agar plates, whereas all three strains produced chitinase. Bacterization of seeds of moong bean with pseudomonads has been reported as a potential method for enhancing plant growth and yield, and for providing protection against Macrophomina phaseolina. Seed bacterization with these plant growth-promoting rhizobacteria (PGPR) showed a significant increase in seed germination, shoot length, shoot fresh and dry weight, root length, root fresh and dry weight, leaf area and rhizosphere colonization. Yield parameters such as pods, number of seeds, and grain yield per plant also enhanced significantly in comparison to control. The disease suppression and plant growth enhancement along with the positive rhizosphere colonization by these strains indicate their possible use as PGPR/biocontrol agents against charcoal rot.     

Enhancement of Green Gram Nodulation and Growth by Bacillus Species

Rhizobacteria belonging to Bacillus sp. were isolated from the rhizosphere of green gram (Vigna radiata). Seed inoculation with the rhizobacteria showed stunting effect on root growth whereas four Bacillus strains caused stimulation of shoot growth at both 4 and 7 d of observations. Coinoculation of some Bacillus strains with effective Bradyrhizobium strain S24 resulted in enhanced nodulation and plant growth of green gram. The shoot dry mass (ratio to uninoculated control) varied from 1.32 to 6.33 at day 30 and from 1.28 to 3.55 at day 40 of plant growth. Nodule promoting effect after 40 d of plant growth was observed with majority of Bacillus strains except for MRS9 and MRS26. Maximum gains in nodulation, nitrogenase activity and plant growth were observed with Bacillus strains MRS12, MRS18, MRS22 and MRS27 after 40 d of plant growth, suggesting the usefulness of introduced rhizobacteria in improving crop productivity.     

Phosphate solubilization and growth promotion by <Emphasis Type="Italic">Pseudomonas fragi</Emphasis> CS11RH1 (MTCC 8984), a psychrotolerant bacterium isolated from a high altitude Himalayan rhizosphere

Phosphate solubilization and growth promotion by Pseudomonas fragi CS11RH1 (MTCC 8984), a psychrotolerant bacterium isolated from a high altitude garlic rhizosphere from the Indian Himalayas, are reported here. The identity of the isolate was arrived on the basis of its biochemical features and sequencing of the 16S rRNA gene. The isolate grew and solubilized phosphate at temperatures ranging from 4 to 30°C. Besides solubilizing P it produced indole acetic acid (IAA) and hydrogen cyanide (HCN). Seed bacterization with the isolate significantly increased the percent germination, rate of germination, plant biomass and nutrient uptake of wheat seedlings. While Pseudomonas fragi is normally associated with the spoilage of dairy products stored at cold temperatures, this is an early report on the plant growth promoting ability of the bacterium.     

<Emphasis Type="Italic">Pseudomonas lurida</Emphasis> M2RH3 (MTCC 9245), a psychrotolerant bacterium from the Uttarakhand Himalayas,solubilizes phosphate and promotes wheat seedling growth

Pseudomonas lurida strain M2RH3 (MTCC 9245) is a Gram negative, non spore forming, fluorescent bacterium isolated from a high altitude rhizospheric soil from the Uttarakhand Himalayas. The identity of the bacterium was arrived by sequencing of the 16S rRNA gene and subsequent phylogenetic analysis. It grew and exhibited plant growth promoting traits at 4, 15 and 30°C, under in vitro conditions. The expression of plant growth promoting (PGP) traits by the bacterium was highest at 30°C, with a proportionate reduction in PGP activity at lower temperatures. Determination of phosphate solubilization by the bacterium at three incubation temperatures revealed a steady increase in the soluble P levels across the incubation temperatures, coupled with a concomitant drop in the pH levels of the culture supernatant, till the 14th day of incubation. Seed bacterization with the isolate positively influenced the growth and nutrient uptake parameters of wheat seedlings cv. VL 804 in pot culture conditions at controlled cold growing temperature. This is an early report on the phosphate solubilization and plant growth promotion by Pseudomonas lurida, which is a relatively new species of the genus Pseudomonas and opens up a hitherto unknown facet of this bacterium.     

Two rhizobacterial strains,individually and in interactions with Rhizobium sp., enhance fusarial wilt control,growth, and yield in pigeon pea

A Pseudomonas aeruginosa strain, RRLJ 04, and a Bacillus cereus strain, BS 03, were tested both individually and in combination with a Rhizobium strain, RH 2, for their ability to enhance plant growth and nodulation in pigeon pea (Cajanus cajan L.) under gnotobiotic, greenhouse and field conditions. Both of the rhizobacterial strains exhibited a positive effect on growth in terms of shoot height, root length, fresh and dry weight, nodulation and yield over the non-treated control. Co-inoculation of seeds with these strains and Rhizobium RH 2 also reduced the number of wilted plants, when grown in soil infested with Fusarium udum. Gnotobiotic studies confirmed that the suppression of wilt disease was due to the presence of the respective PGPR strains. Seed bacterization with drug-marked mutants of RRLJ 04 and BS 03 confirmed their ability to colonize and multiply along the roots. The results suggest that co-inoculation of these strains with Rhizobium strain RH 2 can be further exploited for enhanced growth, nodulation and yield in addition to control of fusarial wilt in pigeon pea.     

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.     

Improvement in symbiotic efficiency of chickpea (Cicer arietinum) by coinoculation of Bacillus strains with Mesorhizobium sp. Cicer

Rhizobacteria belonging to Bacillus sp. were isolated from the rhizosphere of chickpea (Cicer arietinum). Ten Bacillus strains were studied for their antifungal activity, effect on seedling emergence and plant growth promotion. Two Bacillus strains CBS127 and CBS155 inhibited the growth of all the four pathogenic fungi tested on nutrient agar medium plates in vitro. Seed inoculation with different Bacillus strains showed stimulatory effect on root and shoot growth at 10 d of observation in comparison to control whereas four Bacillus strains CBS24, CBS127, CBS129 and CBS155 caused retardation of shoot growth at 10 d. Maximum nodule-promoting effect was observed with Bacillus strains CBS106, CBS127 and CBS155. The symbiotic effectiveness of Mesorhizobium sp. Cicer strain Ca181 was further improved on coinoculation with six Bacillus strains i.e. CBS9, CBS17, CBS20, CBS106, CBS127 and CBS155 at 80 d of plant growth under sterile conditions and shoot dry weight ratios increased 1.62 to 1.74 times those of Mesorhizobium-inoculated treatments, suggesting the usefulness of introduced rhizobacteria in improving crop productivity.     

Impact of two fluorescent pseudomonads and an arbuscular mycorrhizal fungus on tomato plant growth,root architecture and P acquisition

The ability of fluorescent pseudomonads and arbuscular mycorrhizal fungi (AMF) to promote plant growth is well documented but knowledge of the impact of pseudomonad-mycorrhiza mixed inocula on root architecture is scanty. In the present work, growth and root architecture of tomato plants (Lycopersicon esculentum Mill. cv. Guadalete), inoculated or not with Pseudomonas fluorescens 92rk and P190r and/or the AMF Glomus mosseae BEG12, were evaluated by measuring shoot and root fresh weight and by analysing morphometric parameters of the root system. The influence of the microorganisms on phosphorus (P) acquisition was assayed as total P accumulated in leaves of plants inoculated or not with the three microorganisms. The two bacterial strains and the AMF, alone or in combination, promoted plant growth. P. fluorescens 92rk and G. mosseae BEG12 when co-inoculated had a synergistic effect on root fresh weight. Moreover, co-inoculation of the three microorganisms synergistically increased plant growth compared with singly inoculated plants. Both the fluorescent pseudomonads and the myco-symbiont, depending on the inoculum combination, strongly affected root architecture. P. fluorescens 92rk increased mycorrhizal colonization, suggesting that this strain is a mycorrhization helper bacterium. Finally, the bacterial strains and the AMF, alone or in combination, improved plant mineral nutrition by increasing leaf P content. These results support the potential use of fluorescent pseudomonads and AMF as mixed inoculants for tomato and suggest that improved tomato growth could be related to the increase in P acquisition.     

Extracellular Chitinases of Fluorescent Pseudomonads Antifungal to Fusarium oxysporum f. sp. dianthi Causing Carnation Wilt

Vascular wilt of carnation caused by Fusarium oxysporum f. sp. dianthi (Prill. & Delacr.) W. C. Synder & H.N. Hans inflicts substantial yield and quality loss to the crop. Mycolytic enzymes such as chitinases are antifungal and contribute significantly to the antagonistic activity of fluorescent pseudomonads belonging to plant-growth-promoting rhizobacteria. Fluorescent pseudomonads antagonistic to the vascular wilt pathogen were studied for their ability to grow and produce chitinases on different substrates. Bacterial cells grown on chitin-containing media showed enhanced growth and enzyme production with increased anti-fungal activity against the pathogen. Furthermore, the cell-free bacterial culture filtrate from chitin-containing media also significantly inhibited the mycelial growth. Both the strains and their cell-free culture filtrate from chitin-amended media showed the formation of lytic zones on chitin agar, indicating chitinolytic ability. Extracellular proteins of highly antagonistic bacterial strain were isolated from cell-free extracts of media amended with chitin and fungal cell wall. These cell-free conditioned media contained one to seven polypeptides. Western blot analysis revealed two isoforms of chitinase with molecular masses of 43 and 18.5 kDa. Further plate assay for mycelial growth inhibition showed the 43-kDa protein to be antifungal. The foregoing studies clearly established the significance of chitinases in the antagonism of fluorescent pseudomonads, showing avenues for possible exploitation in carnation wilt management.     

Potential plant growth-promoting activity of <Emphasis Type="Italic">Serratia nematodiphila</Emphasis> NII-0928 on black pepper (<Emphasis Type="Italic">Piper nigrum</Emphasis> L.)

A potential bacterial strain designated as NII-0928 isolated from Western ghat forest soil with multiple plant growth promoting attributes, and it has been identified and characterized. Plant growth promoting traits were analyzed by determining the P-solubilization efficiency, Indole acetic acid production, HCN, siderophore production and growth in nitrogen free medium. It was able to solubilize phosphate (76.6 μg ml⁻¹), and produce indole acetic acid (58.9 μg ml⁻¹) at 28 ± 2°C. Qualitative detection of siderophore production and HCN were also observed. At 5°C it was found to express all the plant growth promotion attributes except HCN production. The ability to colonize roots is a sine qua non condition for a rhizobacteria to be considered a true plant growth-promoting rhizobacteria (PGPR). 16S rRNA gene sequencing reveals the identity of the isolate as Serratia nematodiphila with which it shares highest sequence similarity (99.4%). Seed bacterization with black pepper cuttings in greenhouse trials using Sand: Soil: FYM with three individual experimental sets with their respective control showed clearly the growth promoting activity. Hence, Serratia nematodiphila NII-0928 is a promising plant growth promoting isolate showing multiple PGPR attributes that can significantly influence black pepper cuttings. The result of this study provides a strong basis for further development of this strain as a bioinoculants to attain the desired plant growth promoting activity in black pepper growing fields.     

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.     

High abundance and role of antifungal bacteria in compost-treated soils in a wildfire area

Compost has been widely used in order to promote vegetation growth in post-harvested and burned soils. The effects on soil microorganisms were scarcely known, so we performed the microbial analyses in a wildfire area of the Taebaek Mountains, Korea, during field surveys from May to September 2007. Using culture-dependent and -independent methods, we found that compost used in burned soils influenced a greater impact on soil fungi than bacteria. Compost-treated soils contained higher levels of antifungal strains in the genera Bacillus and Burkholderia than non-treated soils. When the antifungal activity of Burkholderia sp. strain O1a_RA002, which had been isolated from a compost-treated soil, was tested for the growth inhibition of bacteria and fungi isolated from burned soils, the membrane-filtered culture supernatant inhibited 19/37 fungal strains including soil fungi, Eupenicillium spp. and Devriesia americana; plant pathogens, Polyschema larviformis and Massaria platani; an animal pathogen, Mortierella verticillata; and an unidentified Ascomycota. However, this organism only inhibited 11/151 bacterial strains tested. These patterns were compatible with the culture-independent DGGE results, suggesting that the compost used in burned soils had a greater impact on soil fungi than bacteria through the promotion of the growth of antifungal bacteria. Our findings indicate that compost used in burned soils is effective in restoring soil conditions to a state closer to those of nearby unburned forest soils at the early stage of secondary succession.     

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.     

GacS-dependent regulation of enzymic and antifungal activities and synthesis of <Emphasis Type="Italic">N</Emphasis>-acylhomoserine lactones in rhizospheric strain <Emphasis Type="Italic">Pseudomonas chlororaphis</Emphasis> 449

Pseudomonas chlororaphis strain 449 isolated from the rhizosphere of maize suppresses numerous plant pathogens in vitro. The strain produces phenazine antibiotics and synthesizes at least three types of quorum sensing signaling molecules, N-acylhomoserine lactones. Here we have shown that the rhizospheric P. chlororaphis strains 449, well known strain 30–84 as well as two other P. chlororaphis strains exhibit polygalacturonase activity. Using mini-Tn5 transposon mutagenesis, four independent mutants of strain P. chlororaphis 449 with insertion of mini-Tn5 Km2 in gene gacS of two-component GacA-GacS system of global regulation were selected. All these mutant strains were deficient in production of extracellular proteinase(s), phenazines, N-acylhomoserine lactones synthesis, and did not inhibit the growth of G⁺ bacteria in comparison with the wild type strain. The P. chlororaphis 449-06 gacS ⁻ mutant studied in greater detail was deficient in polygalacturonase, pectin methylesterase activities, swarming motility and antifungal activity. It is the first time the involvement of GacA-GacS system in the regulation of enzymes of pectin metabolism, polygalacturonase and pectin methylesterase, was demonstrated in fluorescent pseudomonads.     

Host specificity of Rhizobium strains isolated from nitrogen-fixing trees and nitrogenase activities of strain GRH2 in symbiosis with Prosopis chilensis

Host plant specificity was examined in symbiosis between Rhizobium strains isolated from legume-tree root nodules and herbaceous or woody legumes from which they were isolated. Strain GRH2 isolated from Acacia cyanophylla formed effective nodules on Acacia, Prosopis and Medicago sativa as well. Nitrogenase activity, measured as acetylene reduction, of strain GRH2 in symbiosis with Prosopis chilensis was the highest (P 0.05) among the tropical legumes studied and was similar to those found for other associations involving herbaceous legumes. Relative efficiency of nitrogenase varied from 0.3 to near 1 during the light time of the photoperiod. However no hydrogen uptake activity was detected by the amperometric method used. Rhizobium strains GRH3, GRH5 and GRH9 isolated from A. melanoxylon, P. chilensis and Sophora microphylla, respectively, also showed a very low host-range specificity. All isolates were infective and effective on at least one of the herbaceous legumes tested. These data demonstrate the lack of specificity of Rhizobium strains isolated from nitrogen-fixing tree root nodules and that these strains can form effective nodules on herbaceous legumes.     

Suppression of fusarium wilts by fluorescent pseudomonads: Mechanisms and applications

Fluorescent pseudomonads are involved in the natural suppressiveness of some soils to fusarium wilts. These bacteria have been applied successfully to suppress fusarium wilts of various plant species grown in conducive soils and growing substrates. Suppression of fusarium wilts by fluorescent pseudomonads can be ascribed to direct and indirect effects against pathogenic Fusarium oxysporum. Direct effects are expressed by a reduction of the saprophytic growth of the pathogen leading to delay and reduction of root infections. This antagonism was demonstrated to be related to siderophore‐mediated iron competition. Iron competition was also shown to enhance the antagonistic effect of non‐pathogenic F. oxysporum by making the pathogen more susceptible to fungal competition for carbon. Indirect effects of fluorescent pseudomonads against pathogenic F. oxysporum are mainly related to the induction of host plant resistance which can be associated with the bacterial lipopolysaccharides. Suppression of fusarium wilts by some fluorescent pseudomonads could also be related to their ability to detoxify fungal metabolites such as fusaric acid. Association of different mechanisms of suppression increases the efficacy and consistency of the biological control under various experimental conditions. Increased knowledge of mechanisms of suppression now enables management of the environment, to some extent, to favour expression of the beneficial activities. These activities are only expressed if the bacterial density is sufficiently high.     

Root Exudates of Tomato Plants and Their Effect on the Growth and Antifungal Activity of Pseudomonas Strains

The study of the effect of the root exometabolites of tomato plants on the growth and antifungal activity of plant growth–promoting Pseudomonas strains showed that the antifungal activity of plant growth–promoting rhizobacteria in the plant rhizosphere may depend on the sugar and organic acid composition of root exudates.     

The Growth-promoting Effect of Beijerinckia mobilis and Clostridium sp. Cultures on Some Agricultural Crops

New strains of Beijerinckia mobilis and Clostridium sp. isolated from the pea rhizosphere were studied with respect to their promoting effect on the growth and development of some agricultural crops. Seed soaking in bacterial suspensions followed by the soil application of the suspensions or their application by means of foliar spraying was found to be the most efficient method of bacterization. The application of B. mobilis andClostridium sp. cultures in combination with mineral fertilizers increased the crop production by 1.5–2.5 times. The study of the population dynamics of B. mobilis by the method of genetic marking showed that this bacterium quickly colonized the rhizoplane of plants and, therefore, had characteristics of an r-strategist. At the same time, Clostridiumsp. was closer to K-strategists, since this bacterium slowly colonized the econiches studied. The introduction of the bacteria into soil did not affect the indigenous soil bacterial complex. The presence of Clostridium sp. slowed down the colonization of roots by the fungal mycelium. The possible mechanisms of the plant growth–promoting activity of B. mobilisand Clostridiumsp. are discussed.