Alok Kumar Srivastava

Bacterial xylanases: biology to biotechnology

In this review, a comprehensive discussion exclusively on bacterial xylanases; their gene organization; different factors and conditions affecting enzyme yield and activity; and their commercial application have been deliberated in the light of recent research findings and extensive information mining. Improved understanding of biological properties and genetics of bacterial xylanase will enable exploitation of these enzymes for many more ingenious biotechnological and industrial applications.

Trichoderma for climate resilient agriculture

Climate change is one of the biggest challenges of the twenty-first century for sustainable agricultural production. Several reports highlighted the need for better agricultural practices and use of eco-friendly methods for sustainable crop production under such situations. In this context, Trichoderma species could be a model fungus to sustain crop productivity. Currently, these are widely used as inoculants for biocontrol, biofertilization, and phytostimulation. They are reported to improve photosynthetic efficiency, enhance nutrient uptake and increase nitrogen use efficiency in crops. Moreover, they can be used to produce bio-energy, facilitate plants for adaptation and mitigate adverse effect of climate change. The technological advancement in high throughput DNA sequencing and biotechnology provided deep insight into the complex and diverse biotic interactions established in nature by Trichoderma spp. and efforts are being made to translate this knowledge to enhance crop growth, resistance to disease and tolerance to abiotic stresses under field conditions. The discovery of several traits and genes that are involved in the beneficial effects of Trichoderma spp. has resulted in better understanding of the performance of bioinoculants in the field, and will lead to more efficient use of these strains and possibly to their improvement by genetic modification. The present mini-review is an effort to elucidate the molecular basis of plant growth promotion and defence activation by Trichoderma spp. to garner broad perspectives regarding their functioning and applicability for climate resilient agriculture.

Nanodiagnostics for plant pathogens

Rapid detection technologies with high sensitivity and selectivity for plant pathogens are essential to prevent disease spread and minimize losses to assure optimal productivity and food security. Traditional laboratory techniques such as microscopy and culture are time-consuming, labour intensive and require complex sample handling. Immunological and molecular techniques have advanced but have some issues related to rapidity, signal strength and instrumentation. The integration of immunological and molecular diagnostics with nanotechnology systems offers an option where all detection steps can be accommodated on a portable miniaturized device for rapid and accurate detection of plant pathogens. The sensitive nature of functionalized nanoparticles can be used to design phytopathogen detection devices with smart sensing capabilities for field use. This review summarizes the current status and future prospects of nanotechnology for detection and diagnosis of plant pathogens.

Mating type genes and genetic markers to decipher intraspecific variability among Fusarium udum isolates from pigeonpea.

To ascertain the variability in Fusarium udum (Fu) isolates associated with pigeonpea wilt is a difficult task, if based solely on morphological and cultural characters. In this respect, the robustness of five different genetic marker viz., random amplified polymorphic DNA (RAPD), enterobacterial repetitive intergenic consensus (ERIC), BOX elements, mating type locus, and microsatellite markers were employed to decipher intra‐specific variability in Fu isolates. All techniques yielded intra‐specific polymorphism, but different levels of discrimination were obtained. RAPD‐PCR was more discriminatory, enabling the detection of thirteen variants among twenty Fu isolates. By microsatellite, ERIC‐ and BOX‐PCR fingerprinting, the isolates were categorized in seven, five, and two clusters, respectively. Cluster analysis of the combined data also showed that the Fu isolates were grouped into ten clusters, sharing 50–100% similarity. The occurrence of both mating types in Fu isolates is reported for the first time in this study. All examined isolates harbored one of the two mating‐type idiomorphs, but never both, which suggests a heterothallic mating system of sexual reproduction among them. Information obtained from comparing results of different molecular marker systems should be useful to organize the genetic variability and ideally, will improve disease management practices by identifying sources of inoculum and isolate characteristics.

Isolation and characterization of biosurfactant producing Bacillus sp. from diesel fuel-contaminated site

Among hydrocarbon pollutants, diesel oil is a complex mixture of alkanes and aromatic compounds which are often encountered as soil contaminants leaking from storage tanks and pipelines or as result of accidental spillage. One of the best ecofriendly approaches is to restore contaminated soil by using microorganisms able to degrade those toxic compounds in a bioremediation process. In the present study, nineteen bacteria were isolated by enrichment culture technique from diesel spilled soil collected from electric generator shed of NBAIM, Mau. All the isolates were subjected to screening for lipase production and twelve isolates were found to be positive for lipase. When the isolates were screened for biosurfactant production using CTAB-methylene blue agar plates, only one isolate viz. 2NBDSH3 was found positive which was found to be phylogenetically closely related with Bacillus flexus. Despite having low emulsification index, the bacterium could degrade 88.6% of diesel oil in soil. Biosurfactant from the isolate was extracted and characterized through infra-red spectroscopy which indicated its possible lipopeptide nature which was further supported by strong absorption in UV range in the UV-Vis spectrum. The results of the present study indicated that the isolate either does not produce any bioemulsifier or produces very low amount of emulsifier rather it produces a lipopeptide biosurfactant which helps in degradation of diesel oil by lowering the surface tension. The bacterium thus isolated and characterized can serve as a promising solution for ecofriendly remediation of bacterium diesel contaminated soils.

Nanotechnology for the Detection and Diagnosis of Plant Pathogens

Rapid detection technologies with high sensitivity and selectivity for plant pathogens are essential to prevent disease spread with minimal loss to crop production and food quality assurance. Traditional laboratory techniques such as microscopic and cultural techniques are time-consuming and require complex sample handling. Immunological and molecular techniques are advanced but have some issues related to rapidity and signal strength. In this context, integration of immunological and molecular diagnostics with nanotechnology systems offers an alternative where all detection steps are done by a portable miniaturized device for rapid and accurate identification of plant pathogens. Further, nanomaterial synthesis by utilizing functionalized metal nanoparticles as a sensing component offer several desirable features required for pathogen detection. The sensitive nature of functionalized nanoparticles can be utilized to design phytopathogen detection devices with smart sensing capabilities for field use. This chapter provides an overview of the application of nanotechnology in the field of microbial diagnostics with special focus on plant pathogens.

Identification, characterization and phylogenetic analysis of antifungal Trichoderma from tomato rhizosphere

The use of Trichoderma isolates with efficient antagonistic activity represents a potentially effective and alternative disease management strategy to replace health hazardous chemical control. In this context, twenty isolates were obtained from tomato rhizosphere and evaluated by their antagonistic activity against four fungal pathogens (Fusarium oxysporum f. sp. lycopersici, Alternaria alternata, Colletotrichum gloeosporoides and Rhizoctonia solani). The production of extracellular cell wall degrading enzymes of tested isolates was also measured. All the isolates significantly reduced the mycelial growth of tested pathogens but the amount of growth reduction varied significantly as well. There was a positive correlation between the antagonistic capacity of Trichoderma isolates towards fungal pathogens and their lytic enzyme production. The Trichoderma isolates were initially sorted according to morphology and based on the translation elongation factor 1-α gene sequence similarity, the isolates were designated as Trichoderma harzianum, T. koningii, T. asperellum, T. virens and T. viride. PCA analysis explained 31.53, 61.95, 62.22 and 60.25% genetic variation among Trichoderma isolates based on RAPD, REP-, ERIC- and BOX element analysis, respectively. ERG-1 gene, encoding a squalene epoxidase has been used for the first time for diversity analysis of antagonistic Trichoderma from tomato rhizosphere. Phylogenetic analysis of ERG-1 gene sequences revealed close relatedness of ERG-1sequences with earlier reported sequences of Hypocrea lixii, T. arundinaceum and T. reesei. However, ERG-1 gene also showed heterogeneity among some antagonistic isolates and indicated the possibility of occurrence of squalene epoxidase driven triterpene biosynthesis as an alternative biocontrol mechanism in Trichoderma species.

Comparison of molecular and phenetic typing methods to assess diversity of selected members of the genus Bacillus

A comparison of different molecular typing methods viz. ERIC-PCR, BOX-PCR and ARDRA along with carbohydrate utilization pattern was carried out to analyze their discriminatory power and suitability for assessing diversity of selected Bacillus isolates. ERIC-PCR generated 61 bands ranging from 0.56 to 3.9 kb while BOX-PCR resulted in 127 bands ranging from 0.16 to 3.9 kb. Restriction analysis of 16S rDNA with AluI and HaeIII produced 56 and 67 bands ranging from 0.14 to 0.54 and 0.12 to 0.96, respectively. Clustering of isolates based on the ERIC, BOX and ARDRA pattern clearly showed the superiority of the former two methods to reveal the intrageneric and intraspecific diversity. Carbohydrate utilization pattern showed that most preferred sugar was Fructose while Xylose, Rhamnose and D-Arabinose were least preferred by the isolates used for the study. Clustering based on carbohydrate utilization was also able to differentiate among the isolates which showed 100% similarity based on ARDRA profiles. This study clearly shows that typing methods exploiting the repetitive elements distributed over the genome are more useful for assessing genetic diversity. Moreover, metabolic diversity of the bacterial groups may also be useful instead of using single locus specific marker systems for revealing the diversity.

Deciphering the salinity adaptation mechanism in Penicilliopsis clavariiformis AP, a rare salt tolerant fungus from mangrove.

Penicilliopsis clavariiformis AP, a rare salt tolerant fungus reported for the first time from India was identified through polyphasic taxonomy. Scanning electron microscopy showed that the fungus has unique features such as biverticillate penicilli bearing masses of oval to ellipsoidal conidia. The fungus has been characterized for salt tolerance and to understand the relevance of central carbon metabolism in salt stress adaptation. It showed optimal growth at 24 °C and able to tolerate up to 10% (w/v) NaCl. To understand the mechanism of adaptation to high salinity, activities of the key enzymes regulating glycolysis, pentose phosphate pathway, and tricarboxylic acid cycle were investigated under normal (0% NaCl) and saline stress environment (10% NaCl). The results revealed a re‐routing of carbon metabolism away from glycolysis to the pentose phosphate pathway (PPP), served as a cellular stress‐resistance mechanism in fungi under saline environment. The detection and significant expression of fungus genes (Hsp98, Hsp60, HTB, and RHO) under saline stress suggest that these halotolerance conferring genes from the fungus could have a role in fungus protection and adaptation under saline environment. Overall, the present findings indicate that the rearrangement of the metabolic fluxes distribution and stress related genes play an important role in cell survival and adaptation under saline environment.

Genetic diversity, mating types and phylogenetic analysis of Indian races of Fusarium oxysporum f. sp. ciceris from chickpea

Abstract The present study describes the comparative analysis of five genetic markers viz., random amplified polymorphic DNA (RAPD), enterobacterial repetitive intergenic consensus (ERIC), BOX-elements, mating type (MAT) locus and microsatellites for genetic analysis of virulent isolates of Fusarium oxysporum f. sp. ciceri (FOC) representing seven races from chickpea. Phylogenetic analysis of translation elongation factor 1-α and internal transcribed spacer region separated all the FOC isolates into two major clades. Majority of the isolates (FOC 63, FOC 33, FOC 40, FOC 100, FOC 6, FOC 22, FOC 31, FOC 79 and NDFOC 98) representing race 1, 2, 5 and 6 grouped in clade I, while isolates (FOC 90, FOC 108 and FOC 88) belonging to race 3, 4 and 7 were clustered in clade II. Isolates (FOC 33, FOC 40, FOC 17 and FOC 100) representing race 2 had MAT-2 loci, while race 1 isolates (FOC 63, FOC 72 and FOC 76) contained MAT-1 loci only. The principal component analysis (PCA) of RAPD, ERIC, BOX and microsatellite marker data explained 39.94, 39.98, 42.04 and 62.59% of the total variation among test isolates, respectively. Furthermore, there was no correlation existed between genetic diversity, virulence, race compositions or geographic origin of the isolates. Overall, these findings will assist in better understanding of the genetic variability and ideally, will improve disease management practices.