Biosurfactant based formulation of Pseudomonas guariconensis LE3 with multifarious plant growth promoting traits controls charcoal rot disease in Helianthus annus.

Biosurfactants are environment compatible surface-active biomolecules with multifunctional properties which can be utilized in various industries. In this study a biosurfactant producing novel plant growth promoting isolate Pseudomonas guariconensis LE3 from the rhizosphere of Lycopersicon esculentum is presented as biostimulant and biocontrol agent. Biosurfactant extracted from culture was characterized to be mixture of various mono- and di-rhamnolipids with antagonistic activity against Macrophomina phaseolina, causal agent of charcoal rot in diverse crops. Fourier transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance (1H NMR) analysis confirmed the rhamnolipid nature of biosurfactant. PCR analysis established the presence of genes involved in synthesis of antibiotics diacetylphloroglucinol, phenazine 1-carboxylic acid and pyocyanin, and lytic enzymes chitinase and endoglucanase suggesting biocontrol potential of the isolate. Plant growth promoting activities shown by LE3 were phosphate solubilization and production of siderophores, indole acetic acid (IAA), ammonia and 1-aminocyclopropane-1-carboxylate deaminase (ACCD). To assemble all the characteristics of LE3 various bioformuations were developed. Amendment of biosurfactant in bioformulation of LE3 cells improved the shelf life. Biosurfactant amended formulation of LE3 cells was most effective in biocontrol of charcoal rot disease of sunflower and growth promotion in field conditions. The root adhered soil mass of plantlets inoculated with LE3 plus biosurfactant was significantly higher over control. Biosurfactant amended formulation of LE3 cells caused maximum yield enhancement (80.80%) and biocontrol activity (75.45%), indicating that addition of biosurfactant improves the plant-bacterial interaction and soil properties leading to better control of disease and overall improvement of plant health and yield.

Revisiting the plant growth-promoting rhizobacteria: lessons from the past and objectives for the future.

Plant beneficial rhizobacteria (PBR) is a group of naturally occurring rhizospheric microbes that enhance nutrient availability and induce biotic and abiotic stress tolerance through a wide array of mechanisms to enhance agricultural sustainability. Application of PBR has the potential to reduce worldwide requirement of agricultural chemicals and improve agro-ecological sustainability. The PBR exert their beneficial effects in three major ways; (1) fix atmospheric nitrogen and synthesize specific compounds to promote plant growth, (2) solubilize essential mineral nutrients in soils for plant uptake, and (3) produce antimicrobial substances and induce systemic resistance in host plants to protect them from biotic and abiotic stresses. Application of PBR as suitable inoculants appears to be a viable alternative technology to synthetic fertilizers and pesticides. Furthermore, PBR enhance nutrient and water use efficiency, influence dynamics of mineral recycling, and tolerance of plants to other environmental stresses by improving health of soils. This report provides comprehensive reviews and discusses beneficial effects of PBR on plant and soil health. Considering their multitude of functions to improve plant and soil health, we propose to call the plant growth-promoting bacteria (PGPR) as PBR.

Multifaceted Interactions Between Endophytes and Plant: Developments and Prospects.

Microbial endophytes are present in all known plant species. The ability to enter and thrive in the plant tissues makes endophytes unique, showing multidimensional interactions within the host plant. Several vital activities of the host plant are known to be influenced by the presence of endophytes. They can promote plant growth, elicit defense response against pathogen attack, and can act as remediators of abiotic stresses. To date, most of the research has been done assuming that the interaction of endophytes with the host plant is similar to the plant growth-promoting (PGP) microbes present in the rhizosphere. However, a new appreciation of the difference of the rhizosphere environment from that of internal plant tissues is gaining attention. It would be interesting to explore the impact of endosymbionts on the host's gene expression, metabolism, and other physiological aspects essential in conferring resistance against biotic and abiotic stresses. A more intriguing and inexplicable issue with many endophytes that has to be critically evaluated is their ability to produce host metabolites, which can be harnessed on a large scale for potential use in diverse areas. In this review, we discuss the concept of endophytism, looking into the latest insights related to the multifarious interactions beneficial for the host plant and exploring the importance of these associations in agriculture and the environment and in other vital aspects such as human health.

Phenetic, genetic diversity and symbiotic compatibility of rhizobial strains nodulating pigeon pea in Northern India.

Pigeon pea (Cajanus cajan) is one of the most important legumes grown in the northern province of Uttar Pradesh, India. However, its productively in Uttar Pradesh is lower than the average yield of adjoining states. During the course of the present study, a survey of pigeon pea growing agricultural fields was carried out and it was found that 80% of plants were inadequately nodulated. The study was aimed to evaluate the pigeon pea symbiotic compatibility and nodulation efficiency of root nodulating bacteria isolated from various legumes, and to explore the phenetic and genetic diversity of rhizobial population nodulating pigeon pea growing in fields of Uttar Pradesh. Amongst all the 96 isolates, 40 isolates showed nodulation in pigeon pea. These 40 isolates were further characterized by phenotypic, biochemical and physiological tests. Intrinsic antibiotic resistance pattern was taken to generate similarity matrix revealing 10 phenons. The study shows that most of the isolates nodulating pigeon pea in this region were rapid growers. The dendrogram generated using the NTSYSpc software grouped RAPD patterns into 19 clusters. The high degree of phenetic and genetic diversity encountered is probably because of a history of mixed cropping of legumes. The assessment of diversity is a very important tool and can be used to improve the nodulation and quality of pigeon pea crop. It is also concluded that difference between phenetic and RAPD clustering pattern is an indication that rhizobial diversity of pigeon pea is not as yet completely understood and settled.

Screening for MCL-PHA-producing fluorescent pseudomonads and comparison of MCL-PHA production under iso-osmotic conditions induced by PEG and NaCl.

The medium chain length polyhydroxyalkanoates (MCL-PHA) have attracted much attention from academic and industrial communities for their interesting applications in medical field. The aim of this study was to screen high MCL-PHA-producing fluorescent pseudomonads, and to compare the effect of osmotic stress generated by NaCl (ionic) and polyethylene glycol (PEG, non-ionic inert polymer) on PHA production. A total of 50 fluorescent pseudomonads isolated from rhizospheric soil were screened for PHA production by Sudan Black staining. Out of all the PHA-producing isolates only five were MCL-PHA producers as detected by MCL-PCR. Isolate Bar1 identified as Pseudomonas fluorescens by 16S rRNA gene sequencing was selected for further analysis due to its high MCL-PHA production ability. The iso-osmotic stress generated by NaCl and PEG-6000 showed 5.75- and 3.19-fold enhanced production of PHA at -2 bar osmotic potential, over control (0 bar), respectively. There was 1.8-fold enhanced production of PHA at -2 bar osmotic stress induced by NaCl over PEG. PEG reduces availability of water to microorganisms without reducing exogenously provided nutrients which appear to be responsible for its down performance over NaCl. The FTIR analysis of PHA sample purified from cells showed strong marker bands near 1742, 2870, 1170, 1099, and 2926 cm(-1), corresponding to MCL-PHA. The study reported that supplementation of NaCl (electrolyte) in growth media enhances the production of MCL-PHA which can be very useful for its industrial production.

Practical use of CMC-amended rhizobial inoculant for Mucuna pruriens cultivation to enhance the growth and protection against Macrophomina phaseolina.

In many parts of the world Mucuna pruriens is used as an important medicinal, forage and green manure crop. In the present investigation the effect of the addition of CMC in carrier during development of bioformulation on shelflife, plant growth promotive and biocontrol activity against Macrophomina phaseolina was screened taking M. pruriens as a test crop. Ensifer meliloti RMP6(Ery+Kan+) and Bradyrhizobium sp. BMP7(Tet+Kan+) (kanamycin resistance engineered by Tn5 transposon mutagenesis) used in the study showed production of siderophore, IAA, solubilizing phosphate and biocontrol of M. phaseolina. RMP6(Ery+Kan+) also showed ACC deaminase activity. The survival of both the strains in sawdust-based bioformulation was enhanced with an increase in the concentration of CMC from 0 to 1%. At 0% CMC Bradyrhizobium sp. BMP7(Tet+Kan+) showed more increase in nodule number/plant (500.00%) than E. meliloti RMP6(Ery+Kan+) (52.38%), over the control in M. phaseolina-infested soil. There was 185.94% and 59.52% enhancement in nodule number/plant by RMP6(Ery+Kan+) and BMP7(Tet+Kan+) with an increase in the concentration of CMC from 0% to 1% in the bioformulations. However further increase in concentration of CMC did not result in enhancement in survival of either the strains or nodule number/plant.

Dual activity of pyocyanin from Pseudomonas aeruginosa--antibiotic against phytopathogen and signal molecule for biofilm development by rhizobia.

The purified pyocyanin from Pseudomonas aeruginosa TO3 was investigated for its antagonistic activity against Macrophomina phaseolina and as a signaling molecule for development of biofilm by rhizobial strain Ca2. The antagonistic activity of purified pyocyanin, as determined by a dry mass method, showed inhibition of M. phaseolina. Biofilm formation by strain Ca2 was performed by crystal violet assay. There was an increase in biofilm development by Ca2 with an increase in pyocyanin concentration up to 0.12 nmol·L(-1), followed by a reduction. Using a well-diffusion method, we determined the effect of pyocyanin on disease suppression and biofilm formation by strain Ca2 on radicles of groundnut ( Arachis hypogaea L. ) placed in three concentric whorls on water agar plates. Pyocyanin suppressed disease better at high concentration; however, at lower concentrations increased colony-forming units of Ca2 on radicles of seedlings was observed. A field study in soil infested with M. phaseolina showed that a coinoculant of P. aeruginosa TO3 and rhizobial strain Ca2 enhanced nodule mass and nitrogenase activity by 264.38% and 269.06%, respectively, over that of the control. This study reports that application of pyocyanin-producing pseudomonads together with rhizobia contributes to the enhancement of nodulation ability and better sustains the growth and productivity of groundnut even in the presence of M. phaseolina.

Physiologically stressed cells of fluorescent pseudomonas EKi as better option for bioformulation development for management of charcoal rot caused by Macrophomina phaseolina in field conditions.

Bioformulation that supports the inoculant under storage condition and on application to field is of prime importance for agroindustry. Pseudomonas strain EKi having biocontrol activity against Macrophomina phaseolina was used in the study. EKi cells were pretreated by carbon starvation, osmotic stress (NaCl), and freeze drying conditions, and talc-based bioformulation was developed. Combined pretreatment with carbon starvation and osmotic stress was given to Pseudomonas cells. Bioformulation of untreated, freeze dried (FD), carbon starved, osmotic stressed, and combined pre-treated cells showed 50.36, 44.76, 45.95, 34.82, and 27.27% reduction in CFU counts after 6 months of storage. The osmotic stressed cells showed one over-expressed protein (11.5 kDa) in common with carbon starved cells responsible for its better shelf life. The plant growth promotory activity of bioformulations was determined taking Cicer arietinum as a test crop in M. phaseolina infested field. Carbon starved + osmotic stressed cells showed maximum enhancement of dry weight (272.56%) followed by osmotic stressed (230.74%), untreated (155.70%), FD (88.93%), and carbon starved (59.34%) cells over uninoculated control. Carbon starved + osmotic stressed, osmotic stressed, untreated, FD, and carbon starved cells showed 156.60, 100, 75, 40, and 16.67% reduction of charcoal rot disease over uninoculated control. The results clearly showed that combined pretreatment by carbon starvation and osmotic stress provides the bacteria potential of rapid adaptation to different environment conditions.

Suppression of charcoal rot of chickpea by fluorescent Pseudomonas under saline stress condition.

The ability of fluorescent Pseudomonas strain EKi, in production of biocontrol and plant growth promotory (PGP) metabolites under saline stress was evaluated. Strain EKi could tolerate NaCl up to 1,550 mM and showed biocontrol of Macrophomina phaseolina (76.19%) in the presence of up to 400 mM NaCl. Strain EKi was able to produce IAA, siderophore and pyocyanin with gradual reduction of up to 76.31, 45.46, and 48.99%, respectively, as NaCl concentration increased from 0 to 500 mM. Reduced growth rate resulted in delayed induction of IAA, siderophore and pyocyanin by the PGPR. Thin layer chromatography of chloroform extract from non-stressed and salt stressed EKi, and inhibition of M. phaseolina by purified pyocyanin clearly indicated its role in biocontrol. In vitro and in vivo results showed the growth promotion and charcoal rot disease suppression of chickpea by strain EKi under both non-stressed and saline stress. There was 76.75 and 65.25% reduction of disease incidence in non-saline and saline conditions, respectively, in vitro conditions. In presence of M. phaseolina strain EKi brought about 67.65 and 58.45% reduction of disease incidence in non-saline and saline soil, respectively.

Effect of indole-3-acetic acid (IAA) produced by Pseudomonas aeruginosa in suppression of charcoal rot disease of chickpea.

The production of indole-3-acetic acid (IAA), by rhizobacteria, has been associated with plant growth promotion, especially root initiation and elongation. Isolate TO3 selected from 103 fluorescent pseudomonads, identified as Pseudomonas aeruginosa, showed maximum production of IAA. Isolate TO3 having biocontrol activity against Macrophomina phaseolina also showed production of siderophore and HCN was used to screen the role of bacterial IAA in reducing the level of charcoal rot disease occurrence in chickpea. Four IAA defective stable mutants of isolate TO3 having biocontrol activity against M. phaseolina were developed through 5-bromouracil mutagenesis. Mutant TO(52) showed 76.47% reduction in production of IAA. Standard IAA was used in similar concentration as present in cell-free culture supernatant of wild isolate TO3 and its mutant TO(52). The in vitro and in vivo study showed that IAA-defective mutant TO(52) caused reduced biocontrol and plant growth promotory activity than wild isolate TO3. Standard IAA showed comparable biocontrol activity to the culture supernatant. To some extent better biocontrol and growth promotory activity in supernatant than standard IAA indicates the synergistic role of siderophore and HCN. The study clearly reports the role of bacterial IAA in suppression of charcoal rot disease of chickpea.