Zinc-solubilizing Bacillus spp. in conjunction with chemical fertilizers enhance growth, yield, nutrient content, and zinc biofortification in wheat crop.

Micronutrient deficiency is a serious health issue in resource-poor human populations worldwide, which is responsible for the death of millions of women and underage children in most developing countries. Zinc (Zn) malnutrition in middle- and lower-class families is rampant when daily calorie intake of staple cereals contains extremely low concentrations of micronutrients, especially Zn and Fe. Looking at the importance of the problem, the present investigation aimed to enhance the growth, yield, nutrient status, and biofortification of wheat crop by inoculation of native zinc-solubilizing Bacillus spp. in conjunction with soil-applied fertilizers (NPK) and zinc phosphate in saline soil. In this study, 175 bacterial isolates were recovered from the rhizosphere of wheat grown in the eastern parts of the Indo-Gangetic Plain of India. These isolates were further screened for Zn solubilization potential using sparingly insoluble zinc carbonate (ZnCO3), zinc oxide (ZnO), and zinc phosphate {Zn3(PO4)2} as a source of Zn under in vitro conditions. Of 175 bacterial isolates, 42 were found to solubilize either one or two or all the three insoluble Zn compounds, and subsequently, these isolates were identified based on 16S rRNA gene sequences. Based on zone halo diameter, solubilization efficiency, and amount of solubilized zinc, six potential bacterial strains, i.e., Bacillus altitudinis AJW-3, B. subtilis ABW-30, B. megaterium CHW-22, B. licheniformis MJW-38, Brevibacillus borstelensis CHW-2, and B. xiamenensis BLW-7, were further shortlisted for pot- and field-level evaluation in wheat crop. The results of the present investigation clearly indicated that these inoculants not only increase plant growth but also enhance the yield and yield attributes. Furthermore, bacterial inoculation also enhanced available nutrients and microbial activity in the wheat rhizosphere under pot experiments. It was observed that the application of B. megaterium CHW-22 significantly increased the Zn content in wheat straw and grains along with other nutrients (N, P, K, Fe, Cu, and Mn) followed by B. licheniformis MJW-38 as compared to other inoculants. By and large, similar observations were recorded under field conditions. Interestingly, when comparing the nutrient use efficiency (NUE) of wheat, bacterial inoculants showed their potential in enhancing the NUE in a greater way, which was further confirmed by correlation and principal component analyses. This study apparently provides evidence of Zn biofortification in wheat upon bacterial inoculation in conjunction with chemical fertilizers and zinc phosphate in degraded soil under both nethouse and field conditions.

Arbuscular mycorrhizal fungi-mediated activation of plant defense responses in direct seeded rice (Oryza sativa L.) against root-knot nematode Meloidogyne graminicola.

Rhizosphere is the battlefield of beneficial and harmful (so called phytopathogens) microorganisms. Moreover, these microbial communities are struggling for their existence in the soil and playing key roles in plant growth, mineralization, nutrient cycling and ecosystem functioning. In the last few decades, some consistent pattern have been detected so far that link soil community composition and functions with plant growth and development; however, it has not been studied in detail. AM fungi are model organisms, besides potential role in nutrient cycling; they modulate biochemical pathways directly or indirectly which lead to better plant growth under biotic and abiotic stress conditions. In the present investigations, we have elucidated the AM fungi-mediated activation of plant defense responses against Meloidogyne graminicola causing root-knot disease in direct seeded rice (Oryza sativa L.). The study describes the multifarious effects of Funneliformis mosseae, Rhizophagus fasciculatus, and Rhizophagus intraradices inoculated individually or in combination under glasshouse conditions in rice plants. It was found that F. mosseae, R. fasciculatus and R. intraradices when applied individually or in combination modulated the biochemical and molecular mechanisms in the susceptible and resistant inbred lines of rice. AM inoculation significantly increased various plant growth attributes in plants with simultaneous decrease in the root-knot intensity. Among these, the combined application of F. mosseae, R. fasciculatus, and R. intraradices was found to enhance the accumulation and activities of biomolecules and enzymes related to defense priming as well as antioxidation in the susceptible and resistant inbred lines of rice pre-challenged with M. graminicola. The application of F. mosseae, R. fasciculatus and R. intraradices, induced the key genes involved in plant defense and signaling and it has been demonstrated for the first time. Results of the present investigation advocated that the application of F. mosseae, R. fasciculatus and R. intraradices, particularly a combination of all three, not only helped in the control of root-knot nematodes but also increased plant growth as well as enhances the gene expression in rice. Thus, it proved to be an excellent biocontrol as well as plant growth-promoting agent in rice even when the crop is under biotic stress of the root-knot nematode, M. graminicola.

Novel Insights into Understanding the Molecular Dialogues between Bipolaroxin and the Gα and Gß Subunits of the Wheat Heterotrimeric G-Protein during Host-Pathogen Interaction.

Spot blotch disease of wheat, caused by the fungus Bipolaris sorokiniana (Sacc.) Shoem., produces several toxins which interact with the plants and thereby increase the blightening of the wheat leaves, and Bipolaroxin is one of them. There is an urgent need to decipher the molecular interaction between wheat and the toxin Bipolaroxin for in-depth understanding of host-pathogen interactions. In the present study, we have developed the three-dimensional structure of G-protein alpha subunit from Triticum aestivum. Molecular docking studies were performed using the active site of the modeled G-protein alpha and cryo-EM structure of beta subunit from T. aestivum and 'Bipolaroxin'. The study of protein-ligand interactions revealed that six H-bonds are mainly formed by Glu29, Ser30, Lys32, and Ala177 of G-alpha with Bipolaroxin. In the beta subunit, the residues of the core beta strand domain participate in the ligand interaction where Lys256, Phe306, and Leu352 formed seven H-bonds with the ligand Bipolaroxin. All-atoms molecular dynamics (MD) simulation studies were conducted for G-alpha and -beta subunit and Bipolaroxin complexes to explore the stability, conformational flexibility, and dynamic behavior of the complex system. In planta studies clearly indicated that application of Bipolaroxin significantly impacted the physio-biochemical pathways in wheat and led to the blightening of leaves in susceptible cultivars as compared to resistant ones. Further, it interacted with the Gα and Gß subunits of G-protein, phenylpropanoid, and MAPK pathways, which is clearly supported by the qPCR results. This study gives deeper insights into understanding the molecular dialogues between Bipolaroxin and the Gα and Gß subunits of the wheat heterotrimeric G-protein during host-pathogen interaction.

A Comparative Analysis of Microbe-Based Technologies Developed at ICAR-NBAIM Against Erysiphe necator Causing Powdery Mildew Disease in Grapes (Vitis vinifera L.).

Globally, Erysiphe necator causing powdery mildew disease in grapevines (Vitis vinifera L.) is the second most important endemic disease, causing huge economic losses every year. At present, the management of powdery mildew in grapes is largely dependent upon the use of chemical fungicides. Grapes are being considered as one of the high pesticide-demanding crops. Looking at the residual impact of toxic chemical pesticides on the environment, animal, and human health, microbe-based strategies for control of powdery mildew is an emerging technique. It offers an environment-friendly, residue-free, and effective yet safer approach to control powdery mildew disease in grapes. The mode of action is relatively diverse as well as specific to different pathosystems. Hence, the aim of this study was to evaluate the microbe-based technologies, i.e., Eco-pesticide®, Bio-Pulse®, and Bio-Care 24® developed at the Plant-Microbe Interaction and Rhizosphere Biology Lab, ICAR-NBAIM, Kushmaur, against grape powdery mildew and to integrate these technologies with a safer fungicide (sulfur) to achieve better disease control under organic systems of viticulture. The experiments were conducted at four different locations, namely, the vineyards of ICAR-NRCG, Rajya Draksha Bagayatdar Sangh (MRDBS), and two farmers' fields at Narayangaon and Junnar in the Pune district of Maharashtra. A significantly lower percent disease index (PDI) was recorded on the leaves of grape plants treated with Eco-Pesticide®/sulfur (22.37) followed by Bio-Pulse®/sulfur (22.62) and Bio-Care 24®/sulfur (24.62) at NRCG. A similar trend was observed with the lowest PDI on bunches of Eco-pesticide® /sulfur-treated plants (24.71) followed by Bio-Pulse®/sulfur (24.94) and Bio-Care®/sulfur (26.77). The application of microbial inoculants singly or in combination with sulfur has a significant positive impact on the qualitative parameters such as pH, total soluble solids (TSS), acidity, berry diameter, and berry length of the grapes at different locations. Among all the treatments, the Bio-Pulse®/sulfur treatment showed the highest yield per vine (15.02 kg), which was on par with the treatment Eco-Pesticide®/sulfur (14.94). When compared with the yield obtained from the untreated control, 2.5 to 3 times more yield was recorded in the plants treated with either of the biopesticides used in combination with sulfur. Even in the case of individual inoculation, the yield per vine was approximately two times higher than the untreated control and water-treated plants across the test locations. Results suggested that microbial technologies not only protect grapevines from powdery mildew but also enhance the quality parameters with increased yield across the test locations.

Modulation in Biofertilization and Biofortification of Wheat Crop by Inoculation of Zinc-Solubilizing Rhizobacteria.

Zinc is an important micronutrient needed for the optimum growth and development of plants. Contrary to chemical zinc fertilizers, the use of zinc-solubilizing bacteria is an environmentally friendly option for zinc enrichment in edible parts of crops. This study was conducted with the objective of selecting potential zinc-solubilizing rhizobacteria from the rhizosphere of chickpea grown in soils of eastern Uttar Pradesh and further assessing their impact on the magnitude of zinc assimilation in wheat crops. Among 15 isolates, CRS-9, CRS-17, CRS-30, and CRS-38 produced net soluble zinc in broth to the tune of 6.1, 5.9, 5.63, and 5.6 µg ml-1, respectively, in zinc phosphate with the corresponding pH of 4.48, 5.31, 5.2, and 4.76. However, the bacterial strains CRS-17, CRS-30, CRS-38, and CRS-9 showed maximum zinc phosphate solubilization efficiency of 427.79, 317.39, 253.57, and 237.04%, respectively. The four bacterial isolates were identified as Bacillus glycinifermentans CRS-9, Microbacterium oxydans CRS-17, Paenarthrobacter nicotinovorans CRS-30, and Bacillus tequilensis CRS-38 on the basis of morphological and biochemical studies and 16S rRNA gene sequencing. Bacterial inoculants significantly colonized the roots of wheat plants and formed a biofilm in the root matrix. These strains significantly increased seed germination (%) and vigor indices in wheat grown under glasshouse conditions. After 30 days of sowing of wheat under microcosm conditions, eight zinc transporter (TaZIP) genes were expressed maximally in roots, with concomitant accumulation of higher zinc content in the bacterially treated plant compared to the absolute control. Out of the four strains tested, two bacteria, B. tequilensis CRS-38 and P. nicotinovorans CRS-30, improved seed germination (%), vigor indices (2-2.5 folds), plant biomass, grain yield (2.39 g plant-1), and biofortificated grains (54.25 µg g-1Zn) of wheat. To the best of our knowledge, this may be the first report on the presence of zinc solubilization trait in B. glycinifermentans CRS-9, M. oxydans CRS-17, and P. nicotinovorans CRS-30.

Linking Soil Microbial Diversity to Modern Agriculture Practices: A Review.

Agriculture is a multifarious interface between plants and associated microorganisms. In contemporary agriculture, emphasis is being given to environmentally friendly approaches, particularly in developing countries, to enhance sustainability of the system with the least negative effects on produce quality and quantity. Modern agricultural practices such as extensive tillage, the use of harmful agrochemicals, mono-cropping, etc. have been found to influence soil microbial community structure and soil sustainability. On the other hand, the question of feeding the ever-growing global population while ensuring system sustainability largely remains unanswered. Agriculturally important microorganisms are envisaged to play important roles in various measures to raise a healthy and remunerative crop, including integrated nutrient management, as well as disease and pest management to cut down agrochemicals without compromising the agricultural production. These beneficial microorganisms seem to have every potential to provide an alternative opportunity to overcome the ill effects of various components of traditional agriculture being practiced by and large. Despite an increased awareness of the importance of organically produced food, farmers in developing countries still tend to apply inorganic chemical fertilizers and toxic chemical pesticides beyond the recommended doses. Nutrient uptake enhancement, biocontrol of pests and diseases using microbial inoculants may replace/reduce agrochemicals in agricultural production system. The present review aims to examine and discuss the shift in microbial population structure due to current agricultural practices and focuses on the development of a sustainable agricultural system employing the tremendous untapped potential of the microbial world.

Salt-Tolerant Compatible Microbial Inoculants Modulate Physio-Biochemical Responses Enhance Plant Growth, Zn Biofortification and Yield of Wheat Grown in Saline-Sodic Soil.

A wide range of root-associated mutualistic microorganisms have been successfully applied and documented in the past for growth promotion, biofertilization, biofortification and biotic and abiotic stress amelioration in major crops. These microorganisms include nitrogen fixers, nutrient mobilizers, bio-remediators and bio-control agents. The present study aimed to demonstrate the impact of salt-tolerant compatible microbial inoculants on plant growth; Zn biofortification and yield of wheat (Triticum aestivum L.) crops grown in saline-sodic soil and insight of the mechanisms involved therein are being shared through this paper. Field experiments were conducted to evaluate the effects of Trichoderma harzianum UBSTH-501 and Bacillus amyloliquefaciens B-16 on wheat grown in saline-sodic soil at Research Farm, ICAR-Indian Institute of Seed Sciences, Kushmaur, India. The population of rhizosphere-associated microorganisms changed dramatically upon inoculation of the test microbes in the wheat rhizosphere. The co-inoculation induced a significant accumulation of proline and total soluble sugar in wheat at 30, 60, 90 and 120 days after sowing as compared to the uninoculated control. Upon quantitative estimation of organic solutes and antioxidant enzymes, these were found to have increased significantly in co-inoculated plants under salt-stressed conditions. The application of microbial inoculants enhanced the salt tolerance level significantly in wheat plants grown in saline-sodic soil. A significant increase in the uptake and translocation of potassium (K+) and calcium (Ca2+) was observed in wheat co-inoculated with the microbial inoculants, while a significant reduction in sodium (Na+) content was recorded in plants treated with both the bio-agents when compared with the respective uninoculated control plants. Results clearly indicated that significantly higher expression of TaHKT-1 and TaNHX1 in the roots enhances salt tolerance effectively by maintaining the Na+/K+ balance in the plant tissue. It was also observed that co-inoculation of the test inoculants increased the expression of ZIP transporters (2-3.5-folds) which ultimately led to increased biofortification of Zn in wheat grown in saline-sodic soil. Results suggested that co-inoculation of T. harzianum UBSTH-501 and B. amyloliquefaciens B-16 not only increased plant growth but also improved total grain yield along with a reduction in seedling mortality in the early stages of crop growth. In general, the present investigation demonstrated the feasibility of using salt-tolerant rhizosphere microbes for plant growth promotion and provides insights into plant-microbe interactions to ameliorate salt stress and increase Zn bio-fortification in wheat.

Lesson from Ecotoxicity: Revisiting the Microbial Lipopeptides for the Management of Emerging Diseases for Crop Protection.

Microorganisms area treasure in terms of theproduction of various bioactive compounds which are being explored in different arenas of applied sciences. In agriculture, microbes and their bioactive compounds are being utilized in growth promotion and health promotion withnutrient fortification and its acquisition. Exhaustive explorations are unraveling the vast diversity of microbialcompounds with their potential usage in solving multiferous problems incrop production. Lipopeptides are one of such microbial compounds which havestrong antimicrobial properties against different plant pathogens. These compounds are reported to be produced by bacteria, cyanobacteria, fungi, and few other microorganisms; however, genus Bacillus alone produces a majority of diverse lipopeptides. Lipopeptides are low molecular weight compounds which havemultiple industrial roles apart from being usedas biosurfactants and antimicrobials. In plant protection, lipopeptides have wide prospects owing totheirpore-forming ability in pathogens, siderophore activity, biofilm inhibition, and dislodging activity, preventing colonization bypathogens, antiviral activity, etc. Microbes with lipopeptides that haveall these actions are good biocontrol agents. Exploring these antimicrobial compounds could widen the vistasof biological pest control for existing and emerging plant pathogens. The broader diversity and strong antimicrobial behavior of lipopeptides could be a boon for dealing withcomplex pathosystems and controlling diseases of greater economic importance. Understanding which and how these compounds modulate the synthesis and production of defense-related biomolecules in the plants is a key question-the answer of whichneeds in-depth investigation. The present reviewprovides a comprehensive picture of important lipopeptides produced by plant microbiome, their isolation, characterization, mechanisms of disease control, behavior against phytopathogens to understand different aspects of antagonism, and potential prospects for future explorations as antimicrobial agents. Understanding and exploring the antimicrobial lipopeptides from bacteria and fungi could also open upan entire new arena of biopesticides for effective control of devastating plant diseases.

Trichoderma harzianum- and Methyl Jasmonate-Induced Resistance to Bipolaris sorokiniana Through Enhanced Phenylpropanoid Activities in Bread Wheat (Triticum aestivum L.).

The aim of the present study was to evaluate the impact of Trichoderma harzianum UBSTH-501- and methyl jasmonate-induced systemic resistance and their integration on the spot blotch pathogen, Bipolaris sorokiniana through enhanced phenylpropanoid activities in bread wheat (Triticum aestivum L.). It was found that the application of MeJA (>100 mg L-1) inhibits the germination of B. sorokiniana spores under controlled laboratory conditions. To assess the effect of MeJA (150 mg L-1) in combination with the biocontrol agent T. harzianum UBSTH-501 in vivo, a green house experiment was conducted. For this, biocontrol agent T. harzianum UBSTH-501 was applied as seed treatment, whereas MeJA (150 mg L-1) was applied 5 days prior to pathogen inoculation. Results indicated that application of MeJA (150 mg L-1) did not affect the root colonization of wheat by T. harzianum UBSTH-501 in the rhizosphere. The combined application of T. harzianum UBSTH-501 and MeJA also enhanced indole acetic acid production in the rhizosphere (4.92 µg g-1 of soil) which in turn helps in plant growth and development. Further, the combined application found to enhance the activities of defense related enzymes viz. catalase (5.92 EU min-1 g-1 fresh wt.), ascorbate peroxidase [µmol ascorbate oxidized (mg prot)-1 min-1], phenylalanine ammonia lyase (102.25 µmol cinnamic acid h-1 mg-1 fresh wt.) and peroxidase (6.95 Unit mg-1 min-1 fresh wt.) significantly in the plants under treatment which was further confirmed by assessing the transcript level of PAL and peroxidase genes using semi-quantitative PCR approach. The results showed manifold increase in salicylic acid (SA) along with enhanced accumulation of total free phenolics, ferulic acid, caffeic acid, coumaric acid, and chlorogenic acid in the leaves of the plants treated with the biocontrol agent alone or in combination with MeJA. A significant decrease in the disease severity (17.46%) and area under disease progress curve (630.32) were also observed in the treatments with biocontrol agent and MeJA in combination as compared to B. sorokiniana alone treated plant (56.95% and 945.50, respectively). Up-regulation of phenylpropanoid cascades in response to exogenous application of MeJA and the biocontrol agent was observed. It was depicted from the results that PAL is the primary route for lignin production in wheat which reduces cell wall disruption and tissue disintegration and increases suberization and lignification of the plant cell as seen by Scanning Electron microphotographs. These results clearly indicated that exogenous application of MeJA with T. harzianum inducing JA- and/or SA-dependent defense signaling after pathogen challenge may increase the resistance to spot blotch by stimulating enzymatic activities and the accumulation of phenolic compounds in a cooperative manner. This study apparently provides the evidence of biochemical cross-talk and physiological responses in wheat following MeJA and biocontrol agent treatment during the bio-trophic infection.

Earthworm Grazed-Trichoderma harzianum Biofortified Spent Mushroom Substrates Modulate Accumulation of Natural Antioxidants and Bio-Fortification of Mineral Nutrients in Tomato.

The present investigation was aimed at evaluating the impact of earthworm grazed and Trichoderma harzianum biofortified spent mushroom substrate (SMS) on natural antioxidant and nutritional properties of tomato. Results of the investigation reveal that earthworm grazing and T. harzianum bio-fortification led to significant improvement in the physico-chemical properties of fresh SMS and its application increased the accumulation of natural antioxidants and mineral content in tomato as compared to either T. harzianum biofortified SMS or fresh SMS. In particular, the earthworm grazed, T. harzianum biofortified SMS (EGTHB-SMS) was found to inhibit lipid peroxidation and protein oxidation with significant increase in total polyphenol and flavonoid content in tomato. Further, it increased Fe2+/Fe3+ chelating activity, superoxide anion radical scavenging activity compared to other treatments. The results thus suggest an augmented elicitation of natural antioxidant properties in tomato treated with EGTHB-SMS, resulting in a higher radical scavenging activity, that is highly desirable for human health. In addition, the use of SMS to enhance the nutritional value of tomato fruits becomes an environment friendly approach in sustainable crop production.

Bio-protective microbial agents from rhizosphere eco-systems trigger plant defense responses provide protection against sheath blight disease in rice (Oryza sativa L.).

Sheath blight of rice (Oryza sativa L.) caused by Rhizoctonia solani is a major disease and attempts are being made to develop microbe based technologies for biocontrol of this pathogen. However, the mechanisms of biocontrol are not fully understood and still require indepth study in the backdrop of emerging concepts in biological systems. The present investigation was aimed at deciphering the mechanisms of biocontrol of sheath blight of rice employing Pseudomonas fluorescens and Trichoderma harzianum as model agents for biocontrol. Initially 25, 5 and 5 strains of P. fluorescens, T. viride and T. harzianum, respectively, were screened for their biocontrol potential. Out of which, six strains with higher value of percent inhibition of fungal mycelium in dual plate assay were selected. The role of P. fluorescens, T. viride and T. harzianum were investigated in induction and bioaccumulation of natural antioxidants, defence-related biomolecules and other changes in plant which lead not only to growth promotion but also protection from pathogenic stress conditions in rice. The two most promising strains, P. fluorescens PF-08 and T. harzianum UBSTH-501 selected on the basis of in planta evaluation, when applied individually or in combination, significantly enhanced the accumulation of defence-related biomolecules, enzymes and exhibited biocontrol potential against R. solani. A modified/newly developed delivery system was applied for the first time in the experiments involving inoculation of plants with both bioagents, viz. P. fluorescens PF-08 and T. harzianum UBSTH-501. Results suggested that application of P. fluorescens PF-08 and T. harzianum UBSTH-501 alone or in combination, not only helps in control of the disease but also increases plant growth along with reduction in application of toxic chemical pesticides.

Metals sorption from aqueous solutions by Kluyveromyces marxianus: process optimization, equilibrium modeling and chemical characterization.

The dead Kluyveromyces marxianus biomass, a fermentation industry waste, was used to explore its sorption potential for lead, mercury, arsenic, cobalt, and cadmium as a function of pH, biosorbent dosage, contact time, agitation speed, and initial metal concentration. The equilibrium data fitted the Langmuir model better for cobalt and cadmium, but Freundlich isotherm for all metals tested. At equilibrium, the maximum uptake capacity (Qmax) was highest for lead followed by mercury, arsenic, cobalt, and cadmium. The RL values ranged between 0-1, indicating favorable sorption of all test metals by the biosorbent. The maximum Kf value of Pb showed its efficient removal from the solution. However, multi-metal analysis depicted that sorption of all metals decreased except Pb. The potentiometric titration of biosorbent revealed the presence of functional groups viz. amines, carboxylic acids, phosphates, and sulfhydryl group involved in heavy metal sorption. The extent of contribution of functional groups and lipids to biosorption was in the order: carboxylic>lipids>amines>phosphates. Blocking of sulfhydryl group did not have any significant effect on metal sorption.