Genome assembly, comparative genomics, and identification of genes/pathways underlying plant growth-promoting traits of an actinobacterial strain, Amycolatopsis sp. (BCA-696).

The draft genome sequence of an agriculturally important actinobacterial species Amycolatopsis sp. BCA-696 was developed and characterized in this study. Amycolatopsis BCA-696 is known for its biocontrol properties against charcoal rot and also for plant growth-promotion (PGP) in several crop species. The next-generation sequencing (NGS)-based draft genome of Amycolatopsis sp. BCA-696 comprised of ~ 9.05 Mb linear chromosome with 68.75% GC content. In total, 8716 protein-coding sequences and 61 RNA-coding sequences were predicted in the genome. This newly developed genome sequence has been also characterized for biosynthetic gene clusters (BGCs) and biosynthetic pathways. Furthermore, we have also reported that the Amycolatopsis sp. BCA-696 produces the glycopeptide antibiotic vancomycin that inhibits the growth of pathogenic gram-positive bacteria. A comparative analysis of the BCA-696 genome with publicly available closely related genomes of 14 strains of Amycolatopsis has also been conducted. The comparative analysis has identified a total of 4733 core and 466 unique orthologous genes present in the BCA-696 genome The unique genes present in BCA-696 was enriched with antibiotic biosynthesis and resistance functions. Genome assembly of the BCA-696 has also provided genes involved in key pathways related to PGP and biocontrol traits such as siderophores, chitinase, and cellulase production.

Streptomyces consortia-mediated plant growth-promotion and yield performance in chickpea.

Fourteen Streptomyces strains reported earlier as plant growth promoters (PGP) in chickpea were characterized for production of ammonia and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase and solubilization of silica and zinc. The results showed that nine (CAI-17, CAI-78, KAI-26, CAI-21, CAI-26, MMA-32, CAI-140, CAI-155 and KAI-180) and six (CAI-17, CAI-21, CAI-26, CAI-13, CAI-93 and KAI-180) strains were found to produce ammonia and ACC deaminase, respectively, while one (KAI-180) and eight (CAI-17, CAI-21, CAI-26, MMA-32, CAI-13, CAI-85, CAI-93 and KAI-180) strains solubilized silica and zinc, respectively. The selected 14 Streptomyces strains were categorized into three consortia groups, consortium-1 (CAI-17, CAI-68, CAI-78, KAI-26 and KAI-27), consortium-2 (CAI-21, CAI-26 and MMA-32) and consortium-3 (CAI-13, CAI-85, CAI-93, CAI-140, CAI-155 and KAI-180), based on their compatibility, and evaluated for their PGP traits in chickpea. The experiment was conducted under field conditions with two chickpea varieties over two years. The consortia-treated plots enhanced nodule number up to 23%, nodule weight up to 36%, root weight up to 27% and shoot weight up to 26% at 30 days after sowing and pod weight up to 35%, pod number up to 34% and grain yield up to 24% at harvest over the un-inoculated control plots. The harvested grains of consortia treatments were found to enhance crude protein up to 14%, crude fibre up to 17% and crude fat up to 16% over the grains from un-inoculated control. The rhizosphere soils of the consortia-treated plots enhanced total nitrogen up to 21%, organic carbon up to 8% and available phosphorous up to 16% over the un-inoculated control plots. This investigation demonstrated the potential use of the selected consortium of Streptomyces strains in the farmers' fields to improve the chickpea yields and soil fertility.

Streptomyces consortia-mediated plant defense against Fusarium wilt and plant growth-promotion in chickpea.

Three strains of Streptomyces griseus (CAI-24, CAI-121 and CAI-127) and one strain each of Streptomyces africanus (KAI-32) and Streptomyces coelicolor (KAI-90) were reported by us as biocontrol agents against Fusarium wilt, caused by Fusarium oxysporum f. sp. ciceri (FOC), and as plant growth-promoters (PGP) in chickpea. In the present study, the combined effect of these Streptomyces strains as a consortium were assessed for their biocontrol potential against Fusarium wilt and PGP in chickpea. Based on their compatibility, biocontrol ability and PGP performance, two consortia were assembled, consortium-1 having all the five strains of Streptomyces sp. and consortium-2 having the two promising strains (CAI-127 and KAI-32). Under greenhouse conditions, consortium-1 and consortium-2 were found to reduce the Fusarium wilt disease incidence by 55% and 74%, while under field conditions, these were by 86% and 96% in year-1 and by 54% and 69% in year-2, respectively, when compared to the positive control (only FOC treated). Shoot samples treated with consortia + FOC contained significantly enhanced antioxidant enzymes such as superoxide dismutase, catalase, ascorbate peroxidase, guaiacol peroxidase, glutathione reductase and phenylalanine ammonia-lyase, when compared to the positive control (only FOC treated) or the negative control samples (neither FOC nor consortia treated). When the consortia were evaluated for their PGP traits under field conditions in two chickpea cultivars, JG11 and ICCV2, and in two consecutive years, nodule number was found to enhance up to 25%, nodule weight up to 49%, leaf area up to 37%, leaf weight up to 43%, root weight up to 23%, shoot weight up to 35%, seed weight up to 30%, seed number up to 29%, total dry matter up to 22% and grain yield up to 22% over the un-inoculated control plants. This study had demonstrated that the selected consortium of Streptomyces spp. has a greater potential for biological control of Fusarium wilt disease and PGP in chickpea.

Deciphering the antagonistic effect of Streptomyces spp. and host-plant resistance induction against charcoal rot of sorghum.

MAIN CONCLUSION: The findings of this study suggest that the selected five strains of Streptomyces spp. could be used for biological control of charcoal rot disease in sorghum. Two strains each of Streptomyces albus (CAI-17 and KAI-27) and Streptomyces griseus (KAI-26 and MMA-32) and one strain of Streptomyces cavourensis (SAI-13) previously reported to have plant growth-promotion activity in chickpea, rice and sorghum were evaluated for their antagonistic potential against Macrophomina phaseolina, which causes charcoal rot in sorghum. The antagonistic potential of these strains against M. phaseolina was assessed through dual culture assay, metabolite production assay, blotter paper assay in greenhouse and field disease screens. In both dual culture and metabolite production assays, the selected strains significantly inhibited the growth of M. phaseolina (63-74%). In the blotter paper assay, all the five strains of Streptomyces spp. inhibited the pathogen (80-90%). When these five strains were tested for their antagonistic potential under the greenhouse (two times) and field (two seasons) conditions by toothpick method of inoculation, significant differences were observed for charcoal rot severity. Principal component analysis capturing 91.3% phenotypic variations, revealed that the shoot samples treated with both Streptomyces and the pathogen exhibited significantly enhanced antioxidant parameters including superoxide dismutase, catalase, ascorbate peroxidase, guaiacol peroxidase, glutathione reductase, phenylalanine ammonia-lyase, polyphenol oxidase, and total phenolic contents when compared to shoot samples treated with only M. phaseolina. Scanning electron microscope analysis revealed that the phloem and xylem tissues of the Streptomyces treated stem samples were intact compared to that of pathogen inoculated plants. This study indicated that the selected strains of Streptomyces spp. have the potential for biological control of charcoal rot disease in sorghum.

Identification and Characterization of a Streptomyces albus Strain and Its Secondary Metabolite Organophosphate against Charcoal Rot of Sorghum.

Streptomycesalbus strain CAI-21 has been previously reported to have plant growth-promotion abilities in chickpea, pigeonpea, rice, and sorghum. The strain CAI-21 and its secondary metabolite were evaluated for their biocontrol potential against charcoal rot disease in sorghum caused by Macrophomina phaseolina. Results exhibited that CAI-21 significantly inhibited the growth of the pathogen, M. phaseolina, in dual-culture (15 mm; zone of inhibition), metabolite production (74% inhibition), and blotter paper (90% inhibition) assays. When CAI-21 was tested for its biocontrol potential under greenhouse and field conditions following inoculation of M. phaseolina by toothpick method, it significantly reduced the number of internodes infected (75% and 45% less, respectively) and length of infection (75% and 51% less, respectively) over the positive control (only M. phaseolina inoculated) plants. Under greenhouse conditions, scanning electron microscopic analysis showed that the phloem and xylem tissues of the CAI-21-treated shoot samples were intact compared to those of the diseased stem samples. The culture filtrate of the CAI-21 was purified by various chromatographic techniques, and the active compound was identified as "organophosphate" by NMR and MS. The efficacy of organophosphate was found to inhibit the growth of M. phaseolina in the poisoned food technique. This study indicates that S.albus CAI-21 and its active metabolite organophosphate have the potential to control charcoal rot in sorghum.

Deciphering the tri-dimensional effect of endophytic Streptomyces sp. on chickpea for plant growth promotion, helper effect with Mesorhizobium ciceri and host-plant resistance induction against Botrytis cinerea.

A total of 219 endophytic actinobacteria, isolated from roots, stems and leaves of chickpea, were characterized for antagonistic potential against Botrytis cinerea, causal organism of Botrytis grey mold (BGM) disease, in chickpea. Among them, three most potential endophytes, AUR2, AUR4 and ARR4 were further characterized for their plant growth-promoting (PGP) and nodulating potentials and host-plant resistance against B. cinerea, in chickpea. The sequences of 16 S rDNA gene of the three endophytes were matched with Streptomyces but different species. In planta, the isolate AUR4 alone was able to significantly enhance PGP traits including seed numbers (11.8 vs. 9.8/Plant), seed weight (8 vs. 6.8 g/Plant), pod numbers (13.6 vs. 11.5/Plant), pod weight (9.3 vs. 7.5 g/Plant) and biomass (10.9 vs. 8 g/Plant) over the un-inoculated control in chickpea genotype JG11. Interestingly, consortium of the selected endophytes, AUR2, AUR4 and ARR4 were found less effective than single inoculation. Co-inoculation of the selected endophytes with Mesorhizobium ciceri significantly enhanced nodulation and nitrogenase activity in five chickpea genotypes including ICCV2, ICCV10, ICC4958, Annigeri and JG11 over the un-inoculated control. The selected endophytes showed antagonistic potential in planta by significant reduction of disease incidence (28─52%) in both single inoculation and consortium treatments over the un-inoculated control across the genotypes ICC4954 (susceptible), ICCV05530 (moderately resistant) and JG11 (unknown resistance). Further, antioxidant enzymes such as superoxide dismutase, catalase, ascorbate peroxidase, guaiacol peroxidase, glutathione reductase, phenylalanine ammonia-lyase and polyphenol oxidase and phenolics were found induced in the leaves of chickpea inoculated with selected endophytes over un-inoculated control. Principal component analysis revealed that, the antioxidant enzymes and phenolics were found in the magnitude of ICC4954 < JG11 < ICCV05530 which correlates with their resistance level. The selected endophytes enhanced the plant growth and also host plant resistance against BGM in chickpea.

Plant growth-promoting actinobacteria on chickpea seed mineral density: an upcoming complementary tool for sustainable biofortification strategy.

The present study was evaluated to test the potential of plant growth-promoting actinobacteria in increasing seed mineral density of chickpea under field conditions. Among the 19 isolates of actinobacteria tested, significant (p < 0.05) increase of minerals over the uninoculated control treatments was noticed on all the isolates for Fe (10-38 %), 17 for Zn (13-30 %), 16 for Ca (14-26 %), 9 for Cu (11-54 %) and 10 for Mn (18-35 %) and Mg (14-21 %). The increase might be due to the production of siderophore-producing capacity of the tested actinobacteria, which was confirmed in our previous studies by q-RT PCR on siderophore genes expressing up to 1.4- to 25-fold increased relative transcription levels. The chickpea seeds were subjected to processing to increase the mineral availability during consumption. The processed seeds were found to meet the recommended daily intake of FDA by 24-28 % for Fe, 25-28 % for Zn, 28-35 % for Cu, 12-14 % for Ca, 160-167 % for Mn and 34-37 % for Mg. It is suggested that the microbial inoculum can serve as a complementary sustainable tool for the existing biofortification strategies and substantially reduce the chemical fertilizer inputs.

The extent of grain yield and plant growth enhancement by plant growth-promoting broad-spectrum Streptomyces sp. in chickpea.

The physiological and molecular responses of five strains of Streptomyces sp. (CAI-17, CAI-68, CAI-78, KAI-26 and KAI-27), with their proven potential for charcoal rot disease control in sorghum and plant growth-promotion (PGP) in sorghum and rice, were studied to understand the mechanisms causing the beneficial effects. In this investigation, those five strains were evaluated for their PGP capabilities in chickpea in the 2012-13 and 2013-14 post-rainy seasons. All of the Streptomyces sp. strains exhibited enhanced nodule number, nodule weight, root weight and shoot weight at 30 days after sowing (DAS) and pod number, pod weight, leaf area, leaf weight and stem weight at 60 DAS in both seasons over the un-inoculated control. At crop maturity, the Streptomyces strains had enhanced stover yield, grain yield, total dry matter and seed number plant(-1) in both seasons over the un-inoculated control. In the rhizosphere, the Streptomyces sp. also significantly enhanced microbial biomass carbon, dehydrogenase activity, total nitrogen, available phosphorous and organic carbon in both seasons over the un-inoculated control. Of the five strains of Streptomyces sp., CAI-17, CAI-68 and CAI-78 were superior to KAI-26 and KAI-27 in terms of their effects on root and shoot development, nodule formation and crop productivity. Scanning electron microscopy (SEM) micrographs had revealed the success in colonization of the chickpea roots by all five strains. Quantitative real-time PCR (qRT-PCR) analysis of selected PGP genes of actinomycetes revealed the selective up-regulation of indole-3-acetic acid (IAA)-related and siderophore-related genes by CAI-68 and of ß-1,3-glucanase genes by KAI-26.

Plant growth-promoting traits of Pseudomonas geniculata isolated from chickpea nodules.

A bacterium, isolated from nodules of chickpea grown in alluvial soils of Haryana state of India, designated as IC-76 was characterized for in vitro plant growth-promoting (PGP) properties and further evaluated under greenhouse, on-station and on-farm field conditions for PGP activity in chickpea. The isolate IC-76 produced indole acetic acid, siderophore, hydrocyanic acid, cellulase, protease, and ß-1,3-glucanase. When the bacterium was evaluated individually for their PGP potential in the greenhouse on chickpea and in combination with five Streptomyces sp. (strains CAI-24, CAI-121, CAI-127, KAI-32, and KAI-90; demonstrated earlier as biocontrol potential against Fusarium wilt disease in chickpea), the traits, including nodule number and weight, shoot, and root weight, pod number and weight, seed number and weight, available phosphorus and  % organic carbon were found significantly, enhanced over un-inoculated control. In the on-station and on-farm field conditions, IC-76 significantly enhanced nodule number and weight, shoot, and root weight, stover and grain yield and total dry matter. In the rhizosphere (0-15 cm soil), the bacterium also significantly enhanced the total nitrogen, available phosphorus and  % organic carbon. The sequence of 16S rDNA gene of the IC-76 was matched with Pseudomonas geniculata in BLAST analysis. This study demonstrates that IC-76 has the potential for PGP in chickpea.

Plant growth-promoting activities of Streptomyces spp. in sorghum and rice.

Five strains of Streptomyces (CAI-24, CAI-121, CAI-127, KAI-32 and KAI-90) were earlier reported by us as biological control agents against Fusarium wilt of chickpea caused by Fusarium oxysporum f. sp. ciceri (FOC). In the present study, the Streptomyces were characterized for enzymatic activities, physiological traits and further evaluated in greenhouse and field for their plant growth promotion (PGP) of sorghum and rice. All the Streptomyces produced lipase, ß-1-3-glucanase and chitinase (except CAI-121 and CAI-127), grew in NaCl concentrations of up to 6%, at pH values between 5 and 13 and temperatures between 20 and 40°C and were highly sensitive to Thiram, Benlate, Captan, Benomyl and Radonil at field application level. When the Streptomyces were evaluated in the greenhouse on sorghum all the isolates significantly enhanced all the agronomic traits over the control. In the field, on rice, the Streptomyces significantly enhanced stover yield (up to 25%; except CAI-24), grain yield (up to 10%), total dry matter (up to 18%; except CAI-24) and root length, volume and dry weight (up to 15%, 36% and 55%, respectively, except CAI-24) over the control. In the rhizosphere soil, the Streptomyces significantly enhanced microbial biomass carbon (except CAI-24), nitrogen, dehydrogenase (except CAI-24), total N, available P and organic carbon (up to 41%, 52%, 75%, 122%, 53% and 13%, respectively) over the control. This study demonstrates that the selected Streptomyces which were antagonistic to FOC also have PGP properties.