Rajesh Kumar Singh

Optimization of media components for chitinase production by chickpea rhizosphere associated Lysinibacillus fusiformis B‐CM18

Chitinase producing strain B‐CM18 was isolated from chickpea rhizosphere and identified as Lysinibacillus fusiformis B‐CM18. It showed in vitro antifungal activity against a wide range of fungal plant pathogens and was found to produce several PGPR activities. Further, a multivariate response surface methodology was used to evaluate the effects of different factors on chitinolytic activity and optimizing enzyme production. A central composite design was employed to achieve the highest chitinase production at optimum values of the process variables, viz., temperature (20–45 °C), sodium chloride (2–7%), starch (0.1–1%) and yeast extract (0.1–1%), added in the minimal medium supplemented with colloidal chitin (1–10%; w:w). The fit of the model (R2 = 0.5692) was found to be significant. The production medium to achieve the highest chitinase production (101 U ml−1) was composed of the minimal medium composed of chitin (6.09%), NaCl (4.5%), starch (0.55%) and yeast extract (0.55%) with temperature (32.5 °C). The results show that the optimization strategy led to an increase in chitinase production by 56.1‐fold. The molecular mass of the chitinase was estimated to be 20 kDa by anion exchange and gel filtration chromatography. Further, purified chitinase showed strong antifungal activity against test pathogens. Overall, these results may serve as a base line data for enhancing the chitinolytic potential of bacterial antagonists for bio‐management of chickpea pathogens.

Multifarious plant growth promoting characteristics of chickpea rhizosphere associated Bacilli help to suppress soil-borne pathogens

Wilt and root rot are the major constraints in chickpea production and very difficult to manage through agrochemicals. Hence, for an ecofriendly and biological management, 240 strains of Bacillus and Bacillus derived genera were isolated from chickpea rhizosphere, further narrowed down to 14 strains on the basis of in vitro production of indole acetic acid, siderophore, phosphate solubilization, hydrolytic enzymes and were evaluated for antagonism against chickpea pathogens (Fusarium oxysporum f. sp. ciceri race 1, F. solani and Macrophomina phaseolina). The strains were identified on the basis of physiological characters and 16S RNA gene sequencing. The genotypic comparisons of strains were determined by BOX-polymerase chain reaction profiles and amplified rDNA restriction analysis. These isolates were evaluated in greenhouse assay in which B. subtilis (B-CM191, B-CV235, B-CL-122) proved to be effective in reducing wilt incidence and significant enhancement in growth (root and shoot length) and dry matter of chickpea plants. PCR amplification of bacillomycin (bmyB) and β-glucanase genes suggests that amplified genes from the Bacillus could have a role to further define the diversity, ecology, and biocontrol activities in the suppression of soil-borne pathogens.

Isolation and characterization of siderophore producing antagonistic rhizobacteria against Rhizoctonia solani

Plant protection through siderophore producing rhizobacteria (SPR) has emerged as a sustainable approach for crop health management. In present study, 220 bacteria isolated from tomato rhizosphere were screened for in vitro antagonistic activity against Rhizoctonia solani AG‐4. Nine potent antagonistic strains viz., Alcaligenes sp. (MUN1, MB21, and MPF37), Enterobacter sp. (MPM1), Pseudomonas sp. (M10A and MB65), P. aeruginosa (MPF14 and MB123) and P. fluorescens (MPF47) were identified on the basis of physiological characters and 16S rDNA sequencing. These strains were able to produce hydrolytic enzymes, hydrogen cyanide, indole acetic acid, although, only few strains were able to solubilize phosphate. Two strains (MB123 and MPF47) showed significant disease reduction in glasshouse conditions were further evaluated under field conditions using three different application methods. Application of P. fluorescens (MPF47) in nursery as soil mix + seedling root treatments prior to transplantation resulted in significant disease reduction compared to control. Total chlorophyll and available iron were significantly higher in the MPF47 treated plants in contrast to infected control. In conclusion, siderophore producing bacteria MPF47 have strong biocontrol abilities and its application as soil mix + seedling root treatments provided strong shield to plant roots against R. solani and could be used for effective bio‐management of pathogen.

Characterization of antagonistic‐potential of two Bacillus strains and their biocontrol activity against Rhizoctonia solani in tomato

To investigate the biocontrol mechanism of two antagonistic Bacillus strains (Bacillus subtilis MB14 and Bacillus amyloliquefaciens MB101), three in vitro antagonism assays were screened and the results were concluded that both strains inhibited Rhizoctonia solani growth in a similar manner by dual culture assay, but the maximum percent of inhibition only resulted with MB101 by volatile and diffusible metabolite assays. Moreover, cell free supernatant (CFS) of MB101 also showed significant (p > 0.05) growth inhibition as compared to MB14, when 10 and 20% CFS mix with the growth medium of R. solani. After in vitro-validation, both strains were evaluated under greenhouse and the results concluded that strain MB101 had significant biocontrol potential as compared to MB14. Strain MB101 was enhanced the plant height, biomass and chlorophyll content of tomato plant through a higher degree of root colonization. In field trials, strain MB101 showed higher lessening in root rot symptoms with significant fruit yield as compare to strain MB14 and infected control. Next to the field study, the presence of four antibiotic genes (srfAA, fenD, ituC, and bmyB) also concluded the antifungal nature of both Bacillus strains. Phylogenetic analysis of protein sequences revealed a close relatedness of three genes (srfAA, fenD, and ituC) with earlier reported sequences of B. subtilis and B. amyloliquefaciens. However, bmyB showed heterogeneity in among both strains (MB14 and MB101) and it may be concluded that higher degree of antagonism, root colonization and different antibiotic producing genes may play an important role in biocontrol mechanism of strain MB101.

Evaluation and characterization of new α-L-rhamnosidase-producing yeast strains.

A total of thirty yeast strains were isolated from a whey beverage and screened for α-L-rhamnosidase enzyme production. Of these, only four isolates were capable of producing the α-L-rhamnosidase enzyme by hydrolyzing naringin. Scanning electron microscopy images showed that the morphology of the yeast isolate (isolate No. 84) producing the greatest enzyme, changed from oval to filamentous in the presence of naringin. On the basis of morphological and molecular characterization (ITS sequencing), these four isolates were identified as Clavispora lusitaniae-84, Clavispora lusitaniae-B82, Candida sp.-86 and Candida hyderabadensis-S82). Fermentation parameters and the biochemical characterization of the α-L-rhamnosidase-producing yeast isolates were studied based on carbon substrate utilization profiles using BIOLOG phenotype microarray plates. Intra-species genetic diversity among the isolates was evaluated by whole genome analysis with repetitive DNA sequences (ERIC, REP and BOX) based DNA fingerprinting. On the basis of these results, it was found that these isolates of yeast producing L-rhamnosidase have a great potential application for beverage quality enhancement, and can build a strong foundation of α-L-rhamnosidase-producing yeast strains in the debittering of citrus juice.

Performance studies of free-living tomato (Lycopersicon exculentum L.) rhizospheric Bacillus for their multiple plant growth promoting activity

The present investigation is based on the study of the diversity of Bacillus spp. isolated from tomato rhizosphere and their evaluation as plant growth promoter. A total of twenty eight rhizospheric gram positive rod-shaped bacteria (DPNSB-1 to DPNSB-28) were isolated on nutrient agar and confirmed as Bacillus spp. on the basis of phenotypic characterization. Carbon source utilization pattern based on BIOLOG and 16S rDNA-RFLP analysis of these isolates using three tetra cutter restriction enzymes (AluI, HaeIII and MspI) was employed for diversity studies. All the 28 strains deliberated with multiple plant growth promotion (PGP) attributes such as production of Indole acetic acid (IAA), siderophore, ammonia, HCN and phosphate solubilization. The diversity pattern followed amongst isolates was based on carbon utilization profiling, which revealed 9 distinct clusters ranging from 10 to 50%, whereas RFLP based resulted into pattern 5 determined distinct clusters with a range of 10 to 70%. However, all the isolates were endowed with variable range of PGP activities. Among them, DPNSB-2, DPNSB-11 and DPNSB-28 produced significant levels of IAA production 503.65, 687.94 and 845.28 µg mg -1 respectively. While 19 (67%) isolates were capable of solubilizing phosphate, 13 (46%) isolates produced siderophore, 21 (75%) isolates produced Ammonia and 20 (71%) were produced HCN. The positive isolates appeared attractive for exploring their plant growth- promoting activity and may be useful for control of Fusarium wilt in tomato field.

Integrated genomics, physiology and breeding approaches for improving nitrogen use efficiency in potato: translating knowledge from other crops

Potato plays a key role in global food and nutritional security. Potato is an N fertiliser-responsive crop, producing high tuber yields. However, excessive use of N can result in environmental damage and high production costs, hence improving nitrogen use efficiency (NUE) of potato plants is one of the sustainable options to address these issues and increase yield. Advanced efforts have been undertaken to improve NUE in other plants like Arabidopsis, rice, wheat and maize through molecular and physiological approaches. Conversely, in potato, NUE studies have predominantly focussed on agronomy or soil management, except for a few researchers who have measured gene expression and proteins relevant to N uptake or metabolism. The focus of this review is to adapt knowledge gained from other plants to inform investigation of N metabolism and associated traits in potato with the aim of improving potato NUE using integrated genomics, physiology and breeding methods.

Genomics Approaches for Improving Nitrogen Use Efficiency in Potato

Increasing global food production to feed rapidly growing populations where cultivable land area is limited is a serious challenge. Moreover, increasing production costs, with high fertilizer input costs, particularly using nitrogen (N), and degrading soil health are the major concerns when enhancing the sustainable agricultural food production. Potato, being the major non-cereal food crop globally, is a heavy N fertilizer feeder crop. In the past several agricultural best management practices have been discussed regarding N management in potato crop production through the intervention of agronomy and soil science. However, unlike the advances in other model plants and cereals, the application of molecular genomics tools is lacking in potato, hence it is not possible to enhance plant genetic potential with better nitrogen use efficiency (NUE). Better N-use efficient plants can be grown with less fertilizer input and also on poor soil. This chapter highlights the application of novel genomics tools to improve NUE in potato through the discovery of novel genes and markers for applications in molecular breeding methods and gene manipulation (transgenic) techniques.