Xanthomonas oryzae pv. oryzae (Xoo) is widely prevalent and causes Bacterial Leaf Blight (BLB) in Basmati rice grown in different areas of Pakistan. There is a need to use environmentally safe approaches to overcome the loss of grain yield in rice due to this disease. The present study aimed to develop inocula, based on native antagonistic bacteria for biocontrol of BLB and to increase the yield of Super Basmati rice variety. Out of 512 bacteria isolated from the rice rhizosphere and screened for plant growth promoting determinants, the isolate BRp3 was found to be the best as it solubilized 97 μg/ mL phosphorus, produced 30 μg/mL phytohormone indole acetic acid and 15 mg/ L siderophores in vitro. The isolate BRp3 was found to be a Pseudomonas aeruginosa based on 16S rRNA gene sequencing (accession no. HQ840693). This bacterium showed antagonism in vitro against different phytopathogens including Xoo and Fusarium spp. Strain BRp3 showed consistent pathogen suppression of different strains of BLB pathogen in rice. Mass spectrometric analysis detected the production of siderophores (1-hydroxy-phenazine, pyocyanin, and pyochellin), rhamnolipids and a series of already characterized 4-hydroxy-2-alkylquinolines (HAQs) as well as novel 2,3,4-trihydroxy-2-alkylquinolines and 1,2,3,4-tetrahydroxy-2-alkylquinolines in crude extract of BRp3. These secondary metabolites might be responsible for the profound antibacterial activity of BRp3 against Xoo pathogen. Another contributing factor toward the suppression of the pathogen was the induction of defense related enzymes in the rice plant by the inoculated strain BRp3. When used as an inoculant in a field trial, this strain enhanced the grain and straw yields by 51 and 55%, respectively, over non-inoculated control. Confocal Laser Scanning Microscopy (CLSM) used in combination with immunofluorescence marker confirmed P. aeruginosa BRp3 in the rice rhizosphere under sterilized as well as field conditions. The results provide evidence that novel secondary metabolites produced by BRp3 may contribute to its activity as a biological control agent against Xoo and its potential to promote the growth and yield of Super Basmati rice.

Plant growth promoting rhizobacteria (PGPR) are found to control the plant diseases by adopting various mechanisms. Induced systemic resistance (ISR) is an important defensive strategy manifested by plants against numerous pathogens especially infecting at aerial parts. Rhizobacteria elicit ISR by inducing different pathways in plants through production of various metabolites. In the present study, potential of Bacillus spp. KFP-5, KFP-7, KFP-17 was assessed to induce antioxidant enzymes against Pyricularia oryzae infection in rice. The antagonistic Bacillus spp. significantly induced antioxidant defense enzymes i-e superoxide dismutase (1.7–1.9-fold), peroxidase (3.5–4.1-fold), polyphenol oxidase (3.0–3.8-fold), phenylalanine ammonia-lyase (3.9–4.4-fold), in rice leaves and roots under hydroponic and soil conditions respectively. Furthermore, the antagonistic Bacillus spp significantly colonized the rice plants (2.0E+00–9.1E+08) and secreted multiple biocontrol determinants like protease (1.1–5.5 U/mg of soil or U/mL of hydroponic solution), glucanase, (1.0–1.3 U/mg of soil or U/mL of hydroponic solution), siderophores (6.5–42.8 μg/mL or mg) in the rhizosphere of different rice varieties. The results showed that treatment with Bacillus spp. enhanced the antioxidant defense activities in infected rice, thus alleviating P. oryzae induced oxidative damage and suppressing blast disease incidence.

Plant associated rhizobacteria prevailing in different agro-ecosystems exhibit multiple traits which could be utilized in various aspect of sustainable agriculture. Two hundred thirty four isolates were obtained from the roots of basmati-385 and basmati super rice varieties growing in clay loam and saline soil at different locations of Punjab (Pakistan). Out of 234 isolates, 27 were able to solubilize zinc (Zn) from different Zn ores like zinc phosphate [Zn3 (PO4)2], zinc carbonate (ZnCO3) and zinc oxide (ZnO). The strain SH-10 with maximum Zn solubilization zone of 24 mm on Zn3 (PO4)2ore and strain SH-17 with maximum Zn solubilization zone of 14–15 mm on ZnO and ZnCO3ores were selected for further studies. These two strains solubilized phosphorous (P) and potassium (K) in vitro with a solubilization zone of 38–46 mm and 47–55 mm respectively. The strains also suppressed economically important rice pathogens Pyricularia oryzae and Fusarium moniliforme by 22–29% and produced various biocontrol determinants in vitro. The strains enhanced Zn translocation toward grains and increased yield of basmati-385 and super basmati rice varieties by 22–49% and 18–47% respectively. The Zn solubilizing strains were identified as Bacillus sp. and Bacillus cereus by 16S rRNA gene analysis.

Isolation of phosphate solubilising bacterial strains was carried out from rhizosphere, roots and nodules of chickpea, to study the viability for solubilisation of tri-calcium phosphate and the effect on growth of chickpea plants. The potential of isolated bacterial strains to solubilise phosphate was qualitatively evaluated by the measurement of a clear zone around the colonies. The diameter of this zone ranged from 21 to 83 mm. Phosphate solubilisation, by phosphate solubilising bacterial isolates, was quantified by spectrophotometry and was found to range from 65 to 130.5 μg/mL. The drop in pH ranged from 5.6 to 3.6. The plant growth, shoot phosphorus and nitrogen concentrations, nodulation efficiency and nitrogenase activity were significantly enhanced, showing the positive effect of phosphate solubilising bacteria inoculation. Phosphate solubilising bacterial strains CPS-2, CPS-3 and Ca-18 had the maximum positive effect on shoot length, shoot dry weight and nodulation of chickpea plants. Treatments inoculated with non-phosphate solubilising bacterial strains IFA1 and IFA2 showed the minimum values in all the parameters.

The potential of the Bacillus genus to antagonize phytopathogens is associated with the production of cyclic lipopeptides. Depending upon the type of lipopeptide, they may serve as biocontrol agents that are eco-friendly alternatives to chemical fertilizers. This study evaluates the biocontrol activity of surfactin-producing Bacillus (SPB) strains NH-100 and NH-217 and purified surfactin A from these strains against rice bakanae disease. Biologically active surfactin fractions were purified by HPLC, and surfactin A variants with chain lengths from C12 to C16 were confirmed by LCMS-ESI. In hemolytic assays, a positive correlation between surfactin A production and halo zone formation was observed. The purified surfactin A had strong antifungal activity against Fusarium oxysporum, F. moniliforme, F. solani, Trichoderma atroviride and T. reesei. Maximum fungal growth suppression (84%) was recorded at 2000 ppm against F. moniliforme. Surfactin A retained antifungal activity at different pH levels (5-9) and temperatures (20, 50 and 121 °C). Hydroponic and pot experiments were conducted to determine the biocontrol activity of SPB strains and the purified surfactin A from these strains on Super Basmati rice. Surfactin production in the rice rhizosphere was detected by LCMS-ESI at early growth stages in hydroponics experiments inoculated with SPB strains. However, the maximum yield was observed with a consortium of SPB strains (T4) and purified surfactin A (T5) treatments in the pot experiment. The outcomes of the present study revealed that surfactin A significantly reduced rice bakanae disease by up to 80%. These findings suggest that purified surfactin A could be an effective biocontrol agent against bakanae disease in rice and should be incorporated into strategies for disease management.

Field experiment was conducted on fodder maize to explore the potential of integrated use of chemical, organic and biofertilizers for improving maize growth, beneficial microflora in the rhizosphere and the economic returns. The treatments were designed to make comparison of NPK fertilizer with different combinations of half dose of NP with organic and biofertilizers viz. biological potassium fertilizer (BPF), Biopower, effective microorganisms (EM) and green force compost (GFC). Data reflected maximum crop growth in terms of plant height, leaf area and fresh biomass with the treatment of full NPK; and it was followed by BPF+full NP. The highest uptake of NPK nutrients by crop was recorded as: N under half NP+Biopower; P in BPF+full NP; and K from full NPK. The rhizosphere microflora enumeration revealed that Biopower+EM applied along with half dose of GFC soil conditioner (SC) or NP fertilizer gave the highest count of N-fixing bacteria (Azotobacter, Azospirillum, Azoarcus and Zoogloea). Regarding the P-solubilizing bacteria, Bacillus was having maximum population with Biopower+BPF+half NP, and Pseudomonas under Biopower+EM+half NP treatment. It was concluded that integration of half dose of NP fertilizer with Biopower+BPF / EM can give similar crop yield as with full rate of NP fertilizer; and through reduced use of fertilizers the production cost is minimized and the net return maximized. However, the integration of half dose of NP fertilizer with biofertilizers and compost did not give maize fodder growth and yield comparable to that from full dose of NPK fertilizers.

Bacillus spp. produce a broad spectrum of lipopeptide biosurfactants, among which surfactin, iturin and fengycin are widely studied families. The goals of this study were to characterize the biosurfactant activity of Bacillus spp. and to investigate their motility and biofilm formation capabilities. In addition, we extracted lipopeptides from these bacteria to assess their antifungal activities and analyzed these products by mass spectrometry (MS). B. amyloliquefaciens FZB42, Bacillus sp. NH 217 and B. subtilis NH-100 exhibited excellent biosurfactant and surface spreading activities, whereas B. atrophaeus 176s and Paenibacillus polymyxa C1225 showed moderate activity, and B. subtilis 168 showed no activity. Strains FZB42, NH-100, NH-217, 176s and CC125 exhibited excellent biofilm formation capabilities. Lipopeptide extracts displayed good antifungal activity against various phytopathogens and their associated diseases, such as Fusarium moniliforme (rice bakanae disease), Fusarium oxysporum (root rot), Fusarium solani (root rot) and Trichoderma atroviride (ear rot and root rot). Lipopeptide extracts of these strains also showed hemolytic activity, demonstrating their strong potential to produce surfactants. LCMS-ESI analyses identified the presence of surfactin, iturin and fengycin in the extracts of Bacillus strains. Thus, the strains assayed in this study show potential as biocontrol agents against various Fusarium and Trichoderma species.

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