Assessment of copper (Cu) nanoparticle for their biocontrol activity against Xanthomonas oryzae pv. oryzae, growth promotion, and physiology of rice (Oryza sativa L.) plants.

Among the various biotic factors that disrupt crop yield, Xanthomonas oryzae pv oryzae (Xoo) is the most ruinous microbe of rice and causes bacterial leaf blight (BLB) disease. The present study focused on the utilization of copper nanoparticles (Cu-NPs) to control BLB. The copper nanosuspension (259.7 nm) prepared using Na-CMC, CuSO4·7H2O, and NaOH showed effectively inhibited Xoo (65.0 µg/ml). The performance of Cu-NPs in vivo showed enhanced plant attributes (127.9% root length and 53.9% shoot length) compared to the control and CuSO4 treated seedling. Furthermore, Cu-NPs treated seedlings showed 23.01% disease incidence (DI) compared to CuSO4 (85.71%) treated and control plants (91.83%). In addition to enhancing the growth parameters and reducing DI, seed priming with Cu-NPs improved the total chlorophyll content to 36.0% compared to the control. The assessment of antioxidant enzymes such as superoxide dismutase (1.9 U), polyphenol oxidase, peroxidase, and phenylalanine ammonia-lyase (two- to three-fold) in roots and shoots of rice plants revealed significant enhancement in Cu-NPs treated seedlings (P < 0.05). The present study suggests that Cu-NPs can be used to control Xoo and enhance rice growth.

Bacteria isolated from e-waste soil enhance plant growth and mobilize trace metals in e-waste-amended soils.

Worldwide accumulation of e-waste poses a major threat to environmental health. However, printed circuit boards contain precious metals, such as gold, and silver, and also contain micronutrient metal elements, such as Fe, Cu, Zn, etc. Therefore, the present study investigated the effects of e-waste-tolerant bacteria (ETB) on promoting plant growth in e-waste-amended soils and mobilizing trace metals into the plants. For this, a total of 18 bacteria were isolated and screened for e-waste tolerance. Screening for plant growth-promoting properties revealed the production of indole-3-acetic acid-like compounds, siderophore production, and phosphate solubilization. Identification based on 16S rRNA gene sequencing revealed that all isolates belonged to the genus Bacillus. Pot experiment revealed that the treated seeds showed the enhancement of chili plants root growth ranging from 106.55 to 208.07% compared to control plants (e-waste) and 0.0 to 47.90% (without e-waste). A similar enhancement was also observed in the shoot length, and size of the leaf compared to e-waste amended control plants. Inoculation of ETB significantly (p < 0.05) mobilized Fe, Zn, Cu, and Ni into chili plants. The identified ETB could be used to mitigate the toxicity posed by the e-waste, enhancing plant growth and mobilization of micronutrients into plants from e-waste.

Bacillus species identified in this study are the potential e-tolerant (PCB) PGP bacteria. Inoculation of e-tolerant bacteria resulted in increased plant growth attributes and biomass index in e-waste amended soil. Bacterial inoculation also showed maximum uptake of Cu, Fe, Zn, and Ni from the e-waste amended soil. This study demonstrated that micronutrients can be fortified/mobilized from e-waste using PGP bacteria.

Effect of zinc-resistant Lysinibacillus species inoculation on growth, physiological properties, and zinc uptake in maize (Zea mays L.).

Soil contamination by heavy metals is one of the major abiotic stresses that cause retarded plant growth and low productivity. Among the heavy metals, excessive accumulations of zinc (Zn) cause toxicity to plants. The toxicity caused by Zn could be managed by application of Zn-tolerant plant growth-promoting (PGP) bacteria. In this study, five Zn-tolerant bacteria (100-400 mg-1 Zn resistant) were selected and identified as Lysinibacillus spp. based on 16S rRNA gene sequencing. The PGP properties of the Lysinibacillus spp. showed the production of indole acetic acid (60.0-84.0 µg/ml) and siderophore, as well as solubilization of potassium. Furthermore, the isolates were evaluated under greenhouse condition with 2 g kg-1 Zn stress and without Zn stress along with control on Zea mays. The results showed that Lysinibacillus spp. coated seeds enhanced plant growth attributes and biomass yield in both conditions compared with control plants. The enhancement of root growth ranged from 49.2 to 148.6% and shoot length from 83.3 to 111.7% under Zn-stressed soils. Also, the inoculated seedlings substantially enhanced chlorophyll a and b, proline, total phenol, and ascorbic acid. The uptake of Zn by maize root ranged from 31.5 to 210.0% compared with control plants. Therefore, this study suggested that the tested Zn-tolerant Lysinibacillus spp. may be used for cultivation of Z. mays in Zn-contaminated agricultural lands.

Amelioration effect of salt-tolerant plant growth-promoting bacteria on growth and physiological properties of rice (Oryza sativa) under salt-stressed conditions.

For sustainable agriculture in saline soil, extensive exploitation of salt-tolerant plant growth-promoting (PGP) bacteria and other symbiotic bacteria is required. This study was carried out to evaluate the efficiency of native salt-tolerant rice rhizobacteria for plant growth promotion under salt stress. A total of 188 bacteria were screened for assessing salt-tolerant capacity and nine isolates tolerating 12% NaCl (w/v) concentration were selected. Biochemical and molecular identification revealed that the salt-tolerant bacteria belonged to Bacillus sp, Exiguobacterium sp, Enterobacter sp, Lysinibacillus sp, Stenotrophomonas sp, Microbacterium sp, and Achromobacter sp. The increase in NaCl concentration from 2 to 4% decreases the PGP activities such as IAA production, P solubilization, K solubilization, and nitrate reduction. The effects of inoculation of salt-tolerant bacteria on the growth and different physiological properties of rice (Oryza sativa) were studied. It was found that the salinity affected the root and shoot length of the control plants; however, bacterial inoculant were found to effectively promote the growth of paddy under salinity stress. Further, bacterial inoculants substantially enhanced total chlorophyll, proline, total phenol, and oxidative damage such as electrolyte leakage and membrane stability index under salt stress. This study suggests that salt-tolerant PGP bacteria may be used for cultivation of O. sativa in salinized agricultural lands.

In Silico and In Vitro Analyses of Glucosamine and Indole Acetaldehyde Inhibit Pathogenic Regulator Gene phcA of Ralstonia solanacearum, a Causative Agent of Bacterial Wilt of Tomato.

In this study, medicinal plant (Solanum surattense)-associated bacteria were isolated and their extracellular secondary metabolites were extracted. Dual-plate application of crude secondary metabolites proved that SSL2I and SSL5 had a good inhibitory activity against Ralstonia solanacearum. These biocontrol bacteria were identified as Bacillus subtilis and Bacillus velezensis by 16S rRNA gene sequencing analysis. The crude extracts of secondary metabolites were identified based on high-resolution liquid chromatography/mass spectrometry (HR-LCMS) analysis. On the basis of HR-LCMS analysis, we selected the compounds such as glucosamine and indole acetaldehyde for in silico analysis and inhibition of pathogenic gene of phcA from R. solanacearum. The specificity of identified pathogenic gene of R. solanacearum and its cytoplasmic localization were identified by BLASTP and PSORTB bioinformatics tools. The protein-protein interaction between the identified secondary metabolites and pathogenic gene revealed that the compound had antagonistic potential against pathogenic gene of phcA. Furthermore, the synthetic forms of the above metabolites showed that both the compounds had the ability to inhibit R. solanacearum under in vitro condition. On the basis of in silico and in vitro analyses, it was concluded that medicinal plant-associated Bacillus spp. could be used as a biocontrol agent in managing wilt disease caused by R. solanacearum.

Characterisation of antagonistic Bacillus paralicheniformis (strain EAL) by LC-MS, antimicrobial peptide genes, and ISR determinants.

Plants have their own defense mechanisms such as induced systemic resistance (ISR) and systemic-acquired resistance. Bacillus spp. are familiar biocontrol agents that trigger ISR against various phytopathogens by eliciting various metabolites and producing defense enzyme in the host plant. In this study, B. paralicheniformis (strain EAL) was isolated from the medicinal plant Enicostema axillare. Butanol extract of B. paralicheniformis showed potential antagonism against Fusarium oxysporum compared to control well (sterile distilled water) A liquid chromatography mass spectrometry analysis showed 80 different compounds. Among the 80 compounds, we selected citrulline, carnitine, and indole-3-ethanol based on mass-to-charge ratio, database difference, and resolution of mass spectrum. The synthetic form of the above compounds showed biocontrol activity against F. oxysporum under in vitro condition in combination, not as individual compounds. However, the PCR amplification of 11 antimicrobial peptide genes showed that none of the genes amplified in the strain. B. paralicheniformis inoculation challenged with F. oxysporum on tomato plants enhanced production of defense enzymes such as peroxidase (POD), superoxide dismutase (SOD), phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO), and proline compared to control plants (without inoculation of B. paralicheniformis) at significant level (p < 0.005). Stem of tomato plants expressed higher POD (2.2-fold), SOD (2.2-fold), PPO (1.9-fold), and PAL (1.3-fold) contents followed by the leaf and root. Elevated proline accumulation was observed in the leaf (1.8-fold) of tomato plants. Thus, results clearly showed potentiality of B. paralicheniformis (EAL) in activation of antioxidant defense enzyme against F. oxysporum-infected tomato plants and prevention of oxidative damage though hydroxyl radicals scavenging activities that suppress the occurrence of wilt diseases.

Effect of copper-resistant Stenotrophomonas maltophilia on maize (Zea mays) growth, physiological properties, and copper accumulation: potential for phytoremediation into biofortification.

In this study, Cu-tolerant PGP bacteria were isolated from the contaminated soils of Tapi (Surat, Gujarat, India). From a set of 118 bacteria isolated from the contaminated soil, the isolate RBTS7 was found to be efficient in tolerating 0.3 g (w/v) Cu. The isolate was identified as Stenotrophomonas maltophilia, based on biochemical and 16S rRNA gene sequencing. Further, the isolate was also found to produce indole acetic acid (140 µg/ml) and siderophore, and solubilize potassium. Inoculation study was carried out in the presence and absence of Cu in the greenhouse. The results revealed that S. maltophilia enhanced plant growth and biomasses compared to control. In addition to plant growth attributes, the isolate also enhanced chlorophyll a and b (434.1 and 496.7%) contents and antioxidant properties such as proline (168.2%), total phenolic compounds (33.5%), and ascorbic acid oxidase (62.3%) compared to control with Cu and without Cu. Inoculation of S. maltophilia + Cu enhanced the uptake of Cu in maize root (77.4%) and stem (112.0%) compared to Cu-stressed control. The results clearly indicated the inoculation of S. maltophilia reduced the toxicity of Cu and in turn enhanced the plant growth and mobilization of Cu to the plant parts.

Amelioration effect of chromium-tolerant bacteria on growth, physiological properties and chromium mobilization in chickpea (Cicer arietinum) under chromium stress.

In this study, chromium (Cr)-tolerant bacteria were test for their efficiency in alleviating Cr stress in Cicer arietinum plants. On the basis of 16S rRNA gene analysis, the isolates were identified belonging to genus Stenotrophomonas maltophilia, Bacillus thuringiensis B. cereus, and B. subtilis. The strains produced a considerable amount of indole-3-acetic acid in a medium supplemented with tryptophan. The strains also showed siderophore production (S2VWR5 and S3VKR17), phosphorus production (S1VKR11, S3VKR2, S3VKR16, and S2VWR5), and potassium solubilization (S3VKR2, S2VWR5, and S3VKR17). Furthermore, the strains were evaluated in pot experiments to assess the growth promotion of C. arietinum in the presence of chromium salts. Bacterization improved higher root and shoot length considerably to 6.25%-60.41% and 11.3%-59.6% over the control. The plants also showed increase in their fresh weight and dry weight in response to inoculation with Cr-tolerant strains. The accumulation of Cr was higher in roots compared to shoots in both control and inoculated plants, indicating phytostabilization of Cr by C. arietinum. However, phytostabilization was found to be improved manifold in inoculated plants. Apart from the plant attributes, the amendment of soil with Cr and Cr-tolerant bacteria significantly increased the content of total chlorophyll and carotenoids, suggesting the inoculant's role in protecting plants from deleterious effects. This work suggests that the combined activity of Cr-tolerant and plant growth-promoting (PGP) properties of the tested strains could be exploited for bioremediation of Cr and to enhance the C. arietinum cultivation in Cr-contaminated soils.

Characterization of medicinal plant-associated biocontrol Bacillus subtilis (SSL2) by liquid chromatography-mass spectrometry and evaluation of compounds by in silico and in vitro methods.

This study explores the antimicrobial properties of bioactive secondary metabolites extracted from the medicinal plant (Solanum surattense)-associated Bacillus subtilis strain SSL2. The secondary metabolites were extracted from B. subtilis (SSL2) using ethyl acetate, acetone, butanol, chloroform and methanol solvents. The crude extract was tested against two wilt causing pathogens: Ralstonia solanacearum and Fusarium oxysporum. The results revealed that the ethyl acetate extract has maximum inhibition against both the pathogens tested in this study. Furthermore, liquid chromatography-mass spectrometry (LC-MS) analysis of ethyl acetate extract identified 80 different compounds based on mass-to-charge ratio, database difference, resolution of mass spectrum and so on. Among the 80 compounds, citrulline (m/z = 158.0917), chloramphenicol (m/z = 195.075) and carnitine (m/z 162.11) were further selected based on m/z ratio for in silico and in vitro analyses. The in silico analysis revealed that citrulline, chloramphenicol and carnitine inhibited the virulent genes phcA (R. solanacearum) and ste12 (F. oxysporum). Further, under in vitro condition, citrulline and chloramphenicol were found to inhibit the growth of R. solanacearum and F. oxysporum. On the basis of the biocontrol activity of B. subtilis (SSL2) in in silico and in vitro conditions, the bacteria could be used as a biocontrol agent against both bacterial and fungal wilt-causing pathogens. However, this needs to be tested in pot studies or field conditions before being used as biocontrol agents.Communicated by Ramaswamy H. Sarma.

Phytoextraction of iron from contaminated soils by inoculation of iron-tolerant plant growth-promoting bacteria in Brassica juncea L. Czern.

Iron (Fe) is one of the essential micronutrients for all living organisms. Despite its abundance in most of the contaminated soil, it is usually in unavailable forms. The unavailable form of Fe could be mobilized to plants by the use of microorganisms. This study was carried out to show that the Fe-contaminated field soils could be used to accumulate Fe in the plant parts using bacterial inoculation. For this, from a set of bacterial isolates, four Fe-tolerant bacteria were selected and identified based on 16S rRNA gene sequencing. The Fe-tolerant bacteria belonged to the genus Bacillus toyonensis (MG430287), Rhodococcus hoagii (MG432495), Lysinibacillus mangiferihumi (MG432492), and Lysinibacillus fusiformis (MG430290). Screening of plant growth-promoting properties of these isolates revealed that all isolates were able to produce indole acetic acid (50.0-84.0 µg/ml), siderophore, and potassium solubilization (except R. hoagii). Pot assay using Fe-contaminated ((8.07-8.35 g kg-1) soils River Directorate of India) revealed that Fe-tolerant bacteria enhanced the growth of Brassica juncea and its biomass. Besides the improved plant growth, the inoculated plants also showed an overall percentage increase in the uptake of iron in root, stem, and leaf (57.91-128.31%) compared with uninoculated plants. In addition to enhanced plant growth attributes, the isolates also improved the total chlorophyll content and antioxidant properties such as total phenol, proline, and ascorbic acid oxidase. Thus, the results clearly indicated that these isolates could be used as a bioinoculant to improve the sequestration of Fe from the contaminated soils and alleviation of Fe stress in plants.