Biocontrol of Bacterial Leaf Blight of Rice and Profiling of Secondary Metabolites Produced by Rhizospheric Pseudomonas aeruginosa BRp3.

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.

Differential response of kabuli and desi chickpea genotypes toward inoculation with PGPR in different soils.

Pakistan is among top three chickpea producing countries but the crop is usually grown on marginal lands without irrigation and fertilizer application which significantly hampers its yield. Soil fertility and inoculation with beneficial rhizobacteria play a key role in nodulation and yield of legumes. Four kabuli and six desi chickpea genotypes were, therefore, evaluated for inoculation response with IAA-producing Ochrobactrum ciceri Ca-34(T) and nitrogen fixing Mesorhizobium ciceri TAL-1148 in single and co-inoculation in two soils. The soil type 1 was previously unplanted marginal soil having low organic matter, P and N contents compared to soil type 2 which was a fertile routinely legume-cultivated soil. The effect of soil fertility status was pronounced and fertile soil on average, produced 31% more nodules, 62% more biomass and 111% grain yield than marginal soil. Inoculation either with O. ciceri alone or its co-inoculation with M. ciceri produced on average higher nodules (42%), biomass (31%), grains yield (64%) and harvest index (72%) in both chickpea genotypes over non-inoculated controls in both soils. Soil 1 showed maximum relative effectiveness of Ca-34(T) inoculation for kabuli genotypes while soil 2 showed for desi genotypes except B8/02. Desi genotype B8/02 in soil type 1 and Pb-2008 in soil type 2 showed significant yield increase as compared to respective un-inoculated controls. Across bacterial inoculation treatments, grain yield was positively correlated to growth and yield contributing parameters (r = 0.294(*) to 0.838(***) for desi and r = 0.388(*) to 0.857(**) for kabuli). PCA and CAT-PCA analyses clearly showed a site-specific response of genotype x bacterial inoculation. Furthermore, the inoculated bacterial strains were able to persist in the rhizosphere showing colonization on root and within nodules. Present study shows that plant growth promoting rhizobacteria (PGPR) inoculation should be integrated with national chickpea breading program in Pakistan especially for marginal soils. Furthermore, the study shows the potential of phytohormone producing strain Ca-34(T) as promising candidate for development of biofertilizer alongwith nodulating strains to get sustainable yield of kabuli and desi chickpea with minimum inputs at marginal land.

Biological control of red rot in sugarcane by native pyoluteorin-producing Pseudomonas putida strain NH-50 under field conditions and its potential modes of action.

BACKGROUND: Rhizobacteria have a good potential to suppress soilborne diseases, but their efficacy against sugarcane pests is rarely reported. Bacterial strains isolated from the rhizosphere of sugarcane were evaluated for their potential to suppress red rot disease on two susceptible varieties, Co-1148 and SPF-234, under field conditions. The strains were also characterised for the production of secondary metabolites associated with their antagonistic activity. RESULTS: One out of four strains, the Pseudomonas putida strain NH-50 (EU627168), reduced disease severity by 44-60% in different field trials. This potent antagonistic strain produced pyoluteorin antibiotic, as confirmed by high-performance liquid chromatography (HPLC). The PltB gene involved in pyoluteorin synthesis was amplified from the P. putida strain NH-50 and sequenced. The extracellular metabolites and volatile and diffusible antibiotics secreted by the tested strains inhibited mycelial growth of Glomerella tucumensis (Speg.) Arx & E Mull in vitro by 7-55%. CONCLUSION: The pyoluteorin-producing bacteria P. putida strain NH-50 significantly reduced disease severity on both sugarcane varieties, irrespective of fungal inoculation, i.e. either inoculated through stem or through soil. This strain also possesses other plant growth characteristics and can be used as a biopesticide for sugarcane crop.

Biological control of potato black scurf by rhizosphere associated bacteria

The present work was carried out to study the potential of plant rhizosphere associated bacteria for the biocontrol of potato black scurf disease caused by Rhizoctonia solani Khun AG-3. A total of twenty-eight bacteria isolated from diseased and healthy potato plants grown in the soil of Naran and Faisalabad, Pakistan were evaluated for their antagonistic potential. Nine bacterial strains were found to be antagonistic in vitro, reduced the fungal growth and caused the lysis of sclerotia of R. solani in dual culture assay as well as in extracellular metabolite efficacy test. The selected antagonistic strains were further tested for the production and efficacy of volatile and diffusible antibiotics, lytic enzymes and siderophores against R. solani. Selected antagonistic bacteria were also characterized for growth promoting attributes i.e., phosphate solubilization, nitrogen fixation and indole acetic acid production. Biocontrol efficacy and percent yield increase by these antagonists was estimated in greenhouse experiment. Statistical analysis showed that two Pseudomonas spp. StT2 and StS3 were the most effective with 65.1 and 73.9 percent biocontrol efficacy, as well as 87.3 and 98.3 percent yield increase, respectively. Potential antagonistic bacterial strain StS3 showed maximum homology to Pseudomonas sp. as determined by 16S rRNA gene sequencing. These results suggest that bacterial isolates StS3 and StT2 have excellent potential to be used as effective biocontrol agents promoting plant growth with reduced disease incidence.

Biological control of potato black scurf by rhizosphere associated bacteria.

The present work was carried out to study the potential of plant rhizosphere associated bacteria for the biocontrol of potato black scurf disease caused by Rhizoctonia solani Khun AG-3. A total of twenty-eight bacteria isolated from diseased and healthy potato plants grown in the soil of Naran and Faisalabad, Pakistan were evaluated for their antagonistic potential. Nine bacterial strains were found to be antagonistic in vitro, reduced the fungal growth and caused the lysis of sclerotia of R. solani in dual culture assay as well as in extracellular metabolite efficacy test. The selected antagonistic strains were further tested for the production and efficacy of volatile and diffusible antibiotics, lytic enzymes and siderophores against R. solani. Selected antagonistic bacteria were also characterized for growth promoting attributes i.e., phosphate solubilization, nitrogen fixation and indole acetic acid production. Biocontrol efficacy and percent yield increase by these antagonists was estimated in greenhouse experiment. Statistical analysis showed that two Pseudomonas spp. StT2 and StS3 were the most effective with 65.1 and 73.9 percent biocontrol efficacy, as well as 87.3 and 98.3 percent yield increase, respectively. Potential antagonistic bacterial strain StS3 showed maximum homology to Pseudomonas sp. as determined by 16S rRNA gene sequencing. These results suggest that bacterial isolates StS3 and StT2 have excellent potential to be used as effective biocontrol agents promoting plant growth with reduced disease incidence.

Characterization and screening of bacteria from rhizosphere of maize grown in Indonesian and Pakistani soils.

Thirty rhizobacteria isolated from maize grown in Pakistani and Indonesian soils were evaluated for their morphological characteristics, nitrogen fixation, P-solubilization, indole acetic acid (IAA) and siderophores production. Nitrogenase activity was detected in nineteen isolates ranging from 21.8-3624 n moles C2H4 produced/h/mg protein. Most of the isolates produced IAA, ten were capable of siderophore production while four were P-solubilizers. Ultrastructural studies of Pseudomonas sp. F14 indicated characteristic rhizospheric colonization within 48 h that was observed to change considerably with the passage of time from few bacteria to micro colonies. Random amplified polymorphic DNA (RAPD) analysis of 30 bacterial strains using 30 oligonucleotide primers resulted in considerable level of genetic diversity, with genetic distance ranging from 2-16%. Indonesian isolates were found to be more diverse as compared to Pakistani isolates. The characterization and screening of rhizobacteria of maize rhizosphere has helped in selection of isolates F7, LS-1, 3.1.1.C, F2, F3 and F13 as superior strains for use as bioinoculant. Moreover isolate F14 identified, as Pseudomonas fulgida by partial 16S rRNA sequence analysis is a novel strain regarding its tremendous potentials for inoculum production to enhance the yield of maize.

Isolation, characterization and beneficial effects of rice associated plant growth promoting bacteria from Zanzibar soils.

This study was undertaken to isolate and characterize plant growth promoting bacteria (PGPB) occurring in four soils of Zanzibar, Tanzania as well as to evaluate their potential use as biofertilizers for rice. A total of 12 PGPB strains were isolated from rice and studied for growth characteristics, carbon/nitrogen source utilization patterns using QTS-24 kits, phosphate solubilization, indole acetic acid (IAA) production, antibiotic resistance patterns and growth at different pH, temperature and salt concentrations. All the isolates were motile and gram negative except Z3-4. Acetylene reduction activity was detected in all isolates ranging from 5.9-76.4 nmole C2H2 reduced/h x mg protein while 9 isolates produced IAA ranged from 20-90.8 mg/l. Most of the isolates showed resistance against different environmental stresses like 10-40 degrees C temperature, 0.2-1 M salt concentration and 4-8.5 pH range. Only one isolate Z2-7 formed clear zones on Pikovskaia's medium showing its ability to solubilize phosphates. Z3-2 was used to develop fluorescent antibodies to check the cross reactivity of the isolates. Inoculation of these bacterial isolates resulted in higher plant biomass, root area, and total N and P contents on Tanzanian rice variety BKN PRAT3036B under controlled conditions. Bacillus sp. Z3-4 and Azospirillum sp. Z3-1 are effective strains and, after further testing under field conditions, can be used for inoculum production of rice in Tanzania. The plant growth promoting effects of these PGPRs suggest that these can be exploited to improve crop productivity of rice in Tanzania.