Paramjit Singh Minhas

Abiotic Stress Responses and Microbe-Mediated Mitigation in Plants: The Omics Strategies

Abiotic stresses are the foremost limiting factors for agricultural productivity. Crop plants need to cope up adverse external pressure created by environmental and edaphic conditions with their intrinsic biological mechanisms, failing which their growth, development, and productivity suffer. Microorganisms, the most natural inhabitants of diverse environments exhibit enormous metabolic capabilities to mitigate abiotic stresses. Since microbial interactions with plants are an integral part of the living ecosystem, they are believed to be the natural partners that modulate local and systemic mechanisms in plants to offer defense under adverse external conditions. Plant-microbe interactions comprise complex mechanisms within the plant cellular system. Biochemical, molecular and physiological studies are paving the way in understanding the complex but integrated cellular processes. Under the continuous pressure of increasing climatic alterations, it now becomes more imperative to define and interpret plant-microbe relationships in terms of protection against abiotic stresses. At the same time, it also becomes essential to generate deeper insights into the stress-mitigating mechanisms in crop plants for their translation in higher productivity. Multi-omics approaches comprising genomics, transcriptomics, proteomics, metabolomics and phenomics integrate studies on the interaction of plants with microbes and their external environment and generate multi-layered information that can answer what is happening in real-time within the cells. Integration, analysis and decipherization of the big-data can lead to a massive outcome that has significant chance for implementation in the fields. This review summarizes abiotic stresses responses in plants in-terms of biochemical and molecular mechanisms followed by the microbe-mediated stress mitigation phenomenon. We describe the role of multi-omics approaches in generating multi-pronged information to provide a better understanding of plant–microbe interactions that modulate cellular mechanisms in plants under extreme external conditions and help to optimize abiotic stresses. Vigilant amalgamation of these high-throughput approaches supports a higher level of knowledge generation about root-level mechanisms involved in the alleviation of abiotic stresses in organisms.

Acute toxicity, biochemical and histopathological responses of endosulfan in Chanos chanos

This study investigated 96h median lethal concentration of endosulfan (99%, pure α: β ratio of 7:3) by conducting static non-renewable acute toxicity bio-assay in Chanos chanos juvenile with average weight (110±5.65g). Further, the effect of different definitive doses (18.5, 19.5, 20.5, 21.5 and 22.5µg/L) of endosulfan on metabolic, heamato-immunoligcal and histopathological response were probed. Anti-oxidative enzymes CAT, SOD and GST showed significant (p<0.01) increase of activity in the liver, gill and brain during exposure to endosulfan in a dose and time dependent manner. The brain AChE activity showed significant (p<0.01) inhibition from 18.5 to 22.5µg/L exposure of endosulfan than the control group. LDH and MDH activity gradually increased with consequent increasing dose of endosulfan exposure in the liver, gill and brain. Similarly, ALT, AST and G6PDH activities in both liver and gill increased with consequent increases in the dose of endosulfan exposure. Immunological profile such as blood glucose and serum cortisol level significantly enhanced while respiratory burst activity declined with consequent increasing doses of endosulfan exposure. Histopathological alteration in the gill demonstrated curling of secondary lamellae, thickening of primary epithelium, shorting of secondary lamellae, epithelial hyperplasia, fusion of secondary lamellae, aneurism, and collapsed secondary lamellae due to dose dependent exposure of endosulfan. Liver histology illustrated cloudy swelling and necrosis with pyknotic nuclei to the moderate dose of endosulfan, whereas higher dose of endosulfan (21.5µg/L) displayed severe necrosis of hepatic cells. Overall results clearly indicate that acute exposure of endosulfan led to pronounced deleterious alterations on biochemical, heamato-immunological, and histopathological responses of C. chanos juvenile.

Dietary Pyridoxine Protects against Stress and Maintains Immunohaematological Status in Chanos chanos Exposed to Endosulfan.

The amelioration effect of water‐soluble vitamin pyridoxine against stress was evaluated in milkfish, Chanos chanos exposed to endosulfan. Two hundred and twenty‐five fish were distributed randomly into five treatments, each with three replicates. Four isocaloric and isonitrogenous diets with graded levels of pyridoxine feed were as follows: normal water and fed with control diet (En0/PY0); endosulfan‐treated water and fed with control diet (En/PY0); and endosulfan‐treated water and fed with 50 (En/PY 50 mg/kg), 75 (En/PY 75 mg/kg) and 100 mg/kg (En/PY 100 mg/kg) pyridoxine‐supplemented feed. The endosulfan in treated water was maintained at a level of 1/40th of LC50 (0.52 ppb). The effect of dietary pyridoxine supplementation was studied in terms of antioxidative enzymes (catalase, superoxide dismutase, glutathione‐S‐transferase), stress markers [heat‐shock protein 70, caspase‐3, cortisol, acetylcholine esterase (AChE), blood glucose], immunohaematological parameters (total protein, albumin, globulin and A/G ratio, nitroblue tetrazolium, RBC, WBC, Hb), gill histopathology and a subsequent challenge study with Vibrio parahaemolyticus. The antioxidative enzymes, stress markers, albumin and A/G ratio were significantly (p < 0.01) elevated, brain AChE and immunohaematological parameters were significantly (p < 0.01) decreased, and chromosome aberration and gill histopathology were also altered due to endosulfan exposure. The relative survival % was reduced due to the combined effect of endosulfan stress and bacterial challenge. Fish fed the diet supplemented with pyridoxine at 75 and 100 mg/kg was found to restore the studied parameter towards normal compared with control and indicated protection against endosulfan‐induced stress significantly (p < 0.01). Results obtained in the present study indicate that the supplementation of 75 and 100 mg/kg of pyridoxine in the diet has a definitive role in the mitigation of the endosulfan‐induced stress in milkfish, C. chanos fingerlings.

Dietary lecithin potentiates thermal tolerance and cellular stress protection of milk fish (Chanos Chanos) reared under low dose endosulfan-induced stress.

Endosulfan is an organochlorine pesticide commonly found in aquatic environments that has been found to reduce thermal tolerance of fish. Lipotropes such as the food additive, Lecithin has been shown to improve thermal tolerance in fish species. This study was conducted to evaluate the role of lipotropes (lecithin) for enhancing the thermal tolerance of Chanos chanos reared under sublethal low dose endosulfan-induced stress. Two hundred and twenty-five fish were distributed randomly into five treatments, each with three replicates. Four isocaloric and isonitrogenous diets were prepared with graded levels of lecithin: normal water and fed with control diet (En0/L0), endosulfan-treated water and fed with control diet (En/L0), endosulfan-treated water and fed with 1% (En/L1%), 1.5% (En/L 1.5%) and 2% (En/L 2%) lecithin supplemented feed. The endosulfan in treated water was maintained at the level of 1/40th of LC50 (0.52ppb). At the end of the five weeks, critical temperature maxima (CTmax), lethal temperature maxima (LTmax), critical temperature minima (CTmin) and lethal temperature minima (LTmin) were Determined. There was a significant (P<0.01) effect of dietary lecithin on temperature tolerance (CTmax, LTmax, CTmin and LTmin) of the groups fed with 1, 1.5 and 2% lecithin-supplemented diet compared to control and endosulfan-exposed groups. Positive correlations were observed between CT max and LTmax (R(2)=0.934) as well as between CTmin and LTmin (R(2)=0.9313). At the end of the thermal tolerance study, endosulfan-induced changes in cellular stress enzymes (Catalase, SOD and GST in liver and gill and neurotansmitter enzyme, brain AChE) were significantly (p<0.01) improved by dietary lecithin. We herein report the role of lecithin in enhancing the thermal tolerance and protection against cellular stress in fish exposed to an organochlorine pesticide.

Temperature Impacts the Development and Survival of Common Cutworm (Spodoptera litura): Simulation and Visualization of Potential Population Growth in India under Warmer Temperatures through Life Cycle Modelling and Spatial Mapping

The common cutworm, Spodoptera litura, has become a major pest of soybean (Glycine max) throughout its Indian range. With a changing climate, there is the potential for this insect to become an increasingly severe pest in certain regions due to increased habitat suitability. To examine this possibility, we developed temperature-based phenology model for S. litura, by constructing thermal reaction norms for cohorts of single life stages, at both constant and fluctuating temperatures within the ecologically relevant range (15–38°C) for its development. Life table parameters were estimated stochastically using cohort updating and rate summation approach. The model was implemented in the geographic information system to examine the potential future pest status of S. litura using temperature change projections from SRES A1B climate change scenario for the year 2050. The changes were visualized by means of three spatial indices demonstrating the risks for establishment, number of generations per year and pest abundance according to the temperature conditions. The results revealed that the development rate as a function of temperature increased linearly for all the immature stages of S. litura until approximately 34–36°C, after which it became non-linear. The extreme temperature of 38°C was found lethal to larval and pupal stages of S. litura wherein no development to the next stage occurred. Females could lay no eggs at the extreme low (15°C) and high (> 35°C) test temperatures, demonstrating the importance of optimum temperature in determining the suitability of climate for the mating and reproduction in S. litura. The risk mapping predicts that due to temperature increase under future climate change, much of the soybean areas in Indian states like Madhya Pradesh, Maharashtra and Rajasthan, will become suitable for S. litura establishment and increased pest activity, indicating the expansion of the suitable and favourable areas over time. This has serious implication in terms of soybean production since these areas produce approximately 95% of the total soybeans in India. As the present model results are based on temperature only, and the effects of other abiotic and biotic factors determining the pest population dynamics were excluded, it presents only the potential population growth parameters for S. litura. However, if combined with the field observations, the model results could certainly contribute to gaining insight into the field dynamics of S. litura.

Identification of SNP in HSP90AB1 and its Association with the Relative Thermotolerance and Milk Production Traits in Indian Dairy Cattle

Heat shock proteins (Hsp) play crucial role in cellular thermotolerance and heat stress response. In the present work, Allele specific PCR (AS-PCR) was standardized to detect the nucleotide polymorphism within the HSP90AB1 gene (SNP g.4338T>C) in Indian breeds of dairy cattle. The identified genotypes were associated with relative thermotolerance in terms of physiological parameters and milk production traits. The results of the experiments revealed that the genotype frequency of CC, CT, and TT for Sahiwal were 0.05, 0.78, and 0.17, respectively, and in Frieswal, the frequencies were 0.20, 0.70, and 0.10, respectively. The average rectal temperature (ART) and average respiration rates (ARR) were recorded during peak summer stress and heat tolerance coefficient (HTC) was calculated. The association studies indicated that TT genotypes had significantly (P < 0.01) higher HTC and lower ARR values than CT and CC in both the breeds. The TT genotype animals also had better production parameter in terms of total milk yield (TMY) (P < 0.01). These findings may partly suggest the role of HSP90AB1 polymorphisms in the regulation of heat stress response and consequent effect on production traits. Nevertheless, involvement of other regulatory mechanisms cannot be overruled.

Comparative conventional and phenomics approaches to assess symbiotic effectiveness of Bradyrhizobia strains in soybean ( Glycine max L. Merrill) to drought

Symbiotic effectiveness of rhizobitoxine (Rtx)-producing strains of Bradyrhizobium spp. in soybean (cultivar NRC-37/Ahilya-4) under limited soil moisture conditions was evaluated using phenomics tools such as infrared(IR) thermal and visible imaging. Red, green and blue (RGB) colour pixels were standardized to analyse a total of 1017 IR thermal and 692 visible images. Plants inoculated with the Rtx-producing strains B. elkanii USDA-61 and USDA-94 and successive inoculation by B. diazoefficiens USDA-110 resulted in cooler canopy temperatures and increased canopy greenness. The results of the image analysis of plants inoculated with Rtx-producing strains were correlated with effective nodulation, improved photosynthesis, plant nitrogen status and yield parameters. Principal component analysis (PCA) revealed the reliability of the phenomics approach over conventional destructive approaches in assessing the symbiotic effectiveness of Bradyrhizobium strains in soybean plants under watered (87.41–89.96%) and water-stressed (90.54–94.21%) conditions. Multivariate cluster analysis (MCA) revealed two distinct clusters denoting effective (Rtx) and ineffective (non-Rtx) Bradyrhizobium inoculation treatments in soybean. Furthermore, correlation analysis showed that this phenotyping approach is a dependable alternative for screening drought tolerant genotypes or drought resilience symbiosis. This is the first report on the application of non-invasive phenomics techniques, particularly RGB-based image analysis, in assessing plant-microbe symbiotic interactions to impart abiotic stress tolerance.

Functional and phylogenetic diversity of cultivable rhizobacterial endophytes of sorghum [Sorghum bicolor (L.) Moench]

A diverse group of bacteria colonize the exo- and endo-rhizospheres of sorghum and play a critical role in its tolerance to drought and other abiotic stresses. Two hundred and eighty endophytic bacteria were isolated from the surface-sterilized roots of four sorghum cultivars that were grown on three soil types at three different phenological stages of growth. The isolates were subjected to in vitro screening for their plant growth promoting traits. Out of 280 isolates, 70 could produce Indole 3-Acetic Acid (IAA), 28 showed N-fixation, 28 could solubilize phosphate, 24 had ACC deaminase activity and 13 isolates were able to produce siderophores. Functional diversity grouping of the isolates indicated one isolate having five PGP traits and two isolates having four PGP traits; two and 29 isolates having three and two PGP traits, respectively. Among the thirty-four isolates that possessed multiple PGP traits, 19 and 17 isolates were able to produce significant quantities of IAA in the presence and absence of l-tryptophan, an inducer. Eight isolates possessed high levels of ACC deaminase activity. PCR–RFLP of the 16Sr RNA gene revealed a distinct clustering and considerable genetic diversity among these functionally characterized isolates. The 16S rRNA gene based identification of the isolates of single and multiple PGP traits revealed phylogenetic dominance of Firmicutes; Acinetobacter, Bacillus, Enterobacter, Geobacillus, Lysinibacillus, Microbacterium, Ochrobactrum, Paenibacillus and Pseudomonas were the major genera present in the endo-rhizosphere of sorghum. Results of this study are constructive in selection of effective rhizobacterial endophytes or consortia for drought stress alleviation in sorghum.

Genetic variation in physiological responses of mungbeans (Vigna radiata (L.) Wilczek) to drought

Mungbean is a relatively drought tolerant leguminous crop with a short life cycle. Using leaf water loss (LWL) as a screen for drought tolerance, two mungbean genotypes exhibiting more than two–fold variation in leaf water loss were explored for the genetic variation in their physiological and molecular responses to drought. Efficient stomatal regulation together with better photosynthetic capacity constituted an important trait combination for drought adaptation in water saving low LWL genotype. The stomatal closure under drought was accompanied with a concomitant down-regulation of farnesyl transferase gene. However, cooler canopy temperature, a well branched root system coupled with a relatively higher proline accumulation in water spending high LWL genotype constituted another set of adaptive traits operating when exposed to deficit soil moisture conditions. We report drought induced down-regulation of proline dehydrogenase and the presence of 118 base pair intron in this gene. The high seed yield of low LWL genotype despite a hotter canopy might be attributed to higher net assimilation and quantum yield recorded under drought in this genotype. Thus, these interlinked features contribute to adaptive mechanisms of mungbeans which is widely grown in harsh environments exposed to drought and high temperatures.

Influence of Moisture Stress on Growth, Development, Physiological Process and Quality of Fruits and Vegetables and Its Management Strategies

According to world estimates, only 9 % of the area is conducive for crop production. Abiotic stresses like temperature, water, radiation and hazardous chemicals/pollutants are responsible for major reduction in agricultural production. Fruits and vegetables can be potentially exposed to numerous abiotic stresses during production and distribution. Water, an integral part of living systems, is ecologically important because it is a major force in shaping climatic patterns and is biochemically important because it is a necessary component in physiological processes. Plant acclimation to drought stress depends on the process like osmotic adjustment and osmoregulation. Among hydrophilic proteins, several LEA proteins including dehydrins accumulate in various plant parts during the process of osmotic adjustment. ABA signalling pathway plays a vital role in plant stress responses. Moisture stress has also been shown to induce phenolic accumulation through up-regulation of phenylalanine ammonia lyase (PAL) and proline accumulation. Reactive oxygen species (ROS) levels increase when plants are exposed to abiotic stress conditions. Carbon metabolism and the levels of specific sugars are severely affected by abiotic stress. Deficit irrigation (DI) has effects on fruit maturation and ripening. Shallow-rooted vegetables are known to be sensitive to water deficiency. Some of the basic methods to collect and conserve water are harvesting of rainwater, development of catchment area and storing run-off water for recycling and construction of waterways. Crop rotation, conservation agriculture, cover crops, strip cropping, mulching, contour bunds, contour drains, terraces, contour furrow, stabilisation structures, soil conservation and water retention through use of vegetation are some of the agronomical measures used to conserve moisture. Systems biology approach which integrates the series of -omics, i.e. phenomics, genomics, transcriptomics, proteomics and metabolomics, is getting roots on moisture stress alleviation and evolving new breeding strategies. Growth regulator applications can also potentially enhance stress resistance. Plant growth-promoting rhizobacteria (PGPR) and fungi (mycorrhizae) can facilitate plant growth directly by facilitating the uptake of nutrients from the environment, by influencing phytohormone production and by enzymatic lowering of plant ethylene levels.