Molecular insights and omics-based understanding of plant-microbe interactions under drought stress.

The detrimental effects of adverse environmental conditions are always challenging and remain a major concern for plant development and production worldwide. Plants deal with such constraints by physiological, biochemical, and morphological adaptations as well as acquiring mutual support of beneficial microorganisms. As many stress-responsive traits of plants are influenced by microbial activities, plants have developed a sophisticated interaction with microbes to cope with adverse environmental conditions. The production of numerous bioactive metabolites by rhizospheric, endo-, or epiphytic microorganisms can directly or indirectly alter the root system architecture, foliage production, and defense responses. Although plant-microbe interactions have been shown to improve nutrient uptake and stress resilience in plants, the underlying mechanisms are not fully understood. "Multi-omics" application supported by genomics, transcriptomics, and metabolomics has been quite useful to investigate and understand the biochemical, physiological, and molecular aspects of plant-microbe interactions under drought stress conditions. The present review explores various microbe-mediated mechanisms for drought stress resilience in plants. In addition, plant adaptation to drought stress is discussed, and insights into the latest molecular techniques and approaches available to improve drought-stress resilience are provided.

Y12F mutation in Pseudomonas plecoglossicida S7 lipase enhances its thermal and pH stability for industrial applications: a combination of in silico and in vitro study.

Appropriate amino acid substitutions are critical for protein engineering to redesign catalytic properties of industrially important enzymes like lipases. The present study aimed for improving the environmental stability of lipase from Pseudomonas plecoglossicida S7 through site-directed mutagenesis driven by computational studies. lipA gene was amplified and sequenced. Both wild type (WT) and mutant type (MT) lipase genes were expressed into the pET SUMO system. The expressed proteins were purified and characterized for pH and thermostability. The lipase gene belonged to subfamily I.1 lipase. Molecular dynamics revealed that Y12F-palmitic acid complex had a greater binding affinity (-6.3 Kcal/mol) than WT (-6.0 Kcal/mol) complex. Interestingly, MDS showed that the binding affinity of WT-complex (-130.314 ± 15.11 KJ/mol) was more than mutant complex (-108.405 ± 69.376 KJ/mol) with a marked increase in the electrostatic energy of mutant (-26.969 ± 12.646 KJ/mol) as compared to WT (-15.082 ± 13.802 KJ/mol). Y12F mutant yielded 1.27 folds increase in lipase activity at 55 °C as compared to the purified WT protein. Also, Y12F mutant showed increased activity (~ 1.2 folds each) at both pH 6 and 10. P. plecoglossicida S7. Y12F mutation altered the kinetic parameters of MT (Km- 1.38 mM, Vmax- 22.32 µM/min) as compared to WT (Km- 1.52 mM, Vmax- 29.76 µM/min) thus increasing the binding affinity of mutant lipase. Y12F mutant lipase with better pH and thermal stability can be used in biocatalysis.

Multi-substrate sequential optimization, characterization and immobilization of lipase produced by Pseudomonas plecoglossicida S7.

Lipases are important biocatalysts having the third largest global demand after amylases and proteases. In the present study, we have screened 56 potential lipolytic Pseudomonas strains for their lipolytic activity. Pseudomonas plecoglossicida S7 showed highest lipase production with specific activity of 70 U/mg. Statistical optimizations using Plackett Burman design and response surface methodology evaluated fourteen different media supplements including various oilcakes, carbon sources, nitrogen sources, and metal ions which led to a 2.23-fold (156.23 U/mg) increase in lipase activity. Further, inoculum size optimization increased the overall lipase activity by 2.81-folds. The lipase was active over a range of 30-50° C with a pH range (7-10). The enzyme was tolerant to various solvents like chloroform, methanol, 1-butanol, acetonitrile, and dichloromethane and retained 60% of its activity in the presence of sodium dodecyl sulfate (0.5% w/v). The enzyme was immobilized onto Ca-alginate beads which increased thermal (20-60 °C) and pH stability (5-10). The purified enzyme could successfully remove sesame oil stains and degraded upto 25.2% of diesel contaminated soil. These properties of the lipase will help in its applicability in detergent formulations, wastewater treatments, and biodegradation of oil in the environment.

Genetic Diversity and Anti-Oxidative Potential of Streptomyces spp. Isolated from Unexplored Niches of Meghalaya, India.

Streptomyces is genetically and functionally diverse genus known to produce a wide array of phenolics and flavonoids with significant biotechnological applications. 52 isolates belonging to 26 species of Streptomyces collected from Meghalaya, India were analyzed for their genetic diversity using BOX-PCR. Significant inter- and intra- generic diversity was observed among the Streptomyces isolates especially those belonging to S. cacaoi, S. lavendulae, S. olivochromogenes, S. aureus, S. flavovirens. During bioactivity screening of the isolates, S. rectiviolaceus MJM72 recorded the highest DPPH activity (77.13 ± 0.91%) whereas S. antimycoticus MSCA162 showed excellent ABTS radical scavenging activity (99.65 ± 0.41%). On the other hand, S. novaecaesareae MJM58 had the highest (756.4 ± 7.38 µg GAE g-1 fresh weight) phenolic content while S. rectiviolaceus MJM72 was recorded with the highest flavonoid content (69.3 ± 0.12 µg QE g-1 fresh weight). As compared to total flavonoid content, total phenolic content had a stronger correlation with antioxidant activities. HPLC analysis of five selected isolates showed presence of gallic acid and pyrocatechol as predominant phenolics. In case of flavonoids, three isolates showed presence of rutin with S. rochei MSCA130 having the highest rutin content (0.95 µg g-1 fresh weight). The results of this study showed high genetic diversity and antioxidant potential among the Streptomyces isolates.

Colorimetric loop-mediated isothermal amplification assay for detection and ecological monitoring of Sarocladium oryzae, an important seed-borne pathogen of rice.

Accurate and timely disease detection plays a critical role in achieving sustainable crop protection. Globally, rice has been a staple crop for centuries plagued by the diseases that greatly hamper its productivity. Sheath rot, an emerging disease of rice caused by the seed-borne pathogen Sarocladium oryzae, has reportedly caused heavy losses to agricultural produce in recent years. Our study has led to the development and validation of a LAMP assay for early detection of S. oryzae, the causal agent of sheath rot from the live-infected tissues, seeds, weeds, and environmental samples. The assay could detect as low as 1.6 fg/µl of the pathogen in 15 min. The assay was implemented to bio-surveil the presence of this pathogen by testing it on three weed species (Echinochloa colona, Echinochloa crus-galli, and Cyperus teneriffae) growing around the rice fields. The results showed the presence of the pathogen in two of the weed species viz. E. colona and E. crus-galli. The assay was used to test 13 different rice varieties for the presence of S. oryzae in seeds. In total, three of the varieties did not show the presence of S. oryzae in their seeds while the rest were found to harbor the pathogen. The developed assay can effectively be used to detect and screen the presence of S. oryzae in live samples including seeds and field soil.

Deciphering the genetic and functional diversity of cultivable bacteria from chasmophytic pigweed (Chenopodium album) from Tsomoriri, Ladakh, India.

Chasmophytes are a group of diverse plants growing on cracks and crevices of rocks. They survive under nutrient and water-limited conditions. Microorganisms associated with chasmophytes may play a critical role in their survival. In the present study, 263 bacterial isolates were obtained from chasmophytic wild Chenopodium collected from Tsomoriri, Ladakh. Members of Enterobacter, Pseudomonas, Pantoea, and Alcaligenes comprised ~ 90% of the Gram-negative bacteria, while among Gram-positive, Bacillus, Solibacillus, Fictibacillus, Microbacterium, and Micrococcus were most abundant. When evaluated for various plant growth-promoting traits, 36 bacteria could solubilize insoluble phosphate, 10 bacteria could release potassium from silicate minerals, and 25 bacteria could solubilize ZnO, while 124 bacteria produced siderophores. ACC deaminase activity was present in 31 isolates, while 46 bacteria could produce IAA (10.40-232.0 µg/mL). Furthermore, more than 64% of the isolates could grow at 50 °C, while ~ 60% could grow at 4 °C. Similarly, ~ 50% isolates were able to grow with > 1.7 M NaCl and ~ 70% could grow under high osmolarity (~ 67 mOsmol/L). The ability of these microorganisms to grow under such a wide range of temperature, salinity, and osmolarity offers adaptive advantage to colonize plants surviving under harsh environmental conditions. A large number (30-49%) of these isolates could produce acids from various sugars and sugar alcohols which is crucial to release mineral nutrients trapped in the rocks. The results indicated that genetically and functionally diverse microflora associated with wild Chenopodium might be helping these plants to effectively mine nutrients and water under extreme conditions. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03278-0.

Understanding the biological role of PqqB in Pseudomonas stutzeri using molecular dynamics simulation approach.

Phosphate solubilization is an important and widely studied plant growth promoting trait exhibited by many bacteria. Pyrroloquinoline quinone (PQQ), a redox cofactor of methanol and glucose dehydrogenases has been well established as essential for phosphate solubilization. PQQ operon has been well studied in growth promoting rhizobacteria like Pseudomonas spp., Gluconobacter oxydans, Klebsiella pneumoniae, etc. However, the role of PqqB is quite ambiguous as its functional role has been contradicted in many studies. In the present study, we selected Pseudomonas stutzeri - a well-known P solubilizing bacterium as a representative species of the Pseudomonas genus on the basis of phylogenetic and statistical analyses of PqqB proteins. A 3 D model was generated for this protein. Docking of PqqB with PQQ showed good interaction with a theoretical binding affinity of -7.4 kcal/mol. On the other hand, docking of PqqC with 3a-(2-amino-2-carboxy-ethyl)-4,5-dioxo-4,5,6,7,8,9-hexahydro-quinoline-7,9-dicarboxylic acid (AHQQ, immediate precursor of PQQ) showed strong interaction (-10.4 kcal/mol) but the same was low with PQQ (-6.4 kcal/mol). Molecular dynamic simulation of both the complexes showed stable conformation. The binding energy of PqqB-PQQ complex (-182.710 ± 16.585 kJ/mol) was greater than PqqC-PQQ complex (-166.114 ± 12.027 kJ/mol). The results clearly indicated that kinetically there is a possibility that after cyclization of AHQQ to PQQ by PqqC, PQQ can be taken up by PqqB and transported to periplasm for the oxidation of glucose. To the best of our knowledge, this is the first attempt to understand the biological role of PqqB on the basis of molecular interactions and dynamics.Communicated by Ramaswamy H. Sarma.

Bioactive antifungal metabolites produced by Streptomyces amritsarensis V31 help to control diverse phytopathogenic fungi.

Actinomycetes due to their unique repertoire of antimicrobial secondary metabolites can be an eco-friendly and sustainable alternative to agrochemicals to control plant pathogens. In the present study, antifungal activity of twenty different actinomycetes was evaluated via dual culture plate assay against six different phytopathogens, viz., Alternaria alternata, Aspergillus flavus, Fusarium oxysporum f. sp. lycopersici, Sarocladium oryzae, Sclerotinia sclerotiorum, and Rhizoctonia solani. Two potential isolates, Streptomyces amritsarensis V31 and Kribella karoonensis MSCA185 showing high antifungal activity against all six fungal pathogens, were further evaluated after extraction of bioactive metabolites in different solvents. Metabolite extracted from S. amritsarensis V31 in different solvents inhibited Rhizoctonia solani (7.5-65%), Alternaria alternata (5.5-52.7%), Aspergillus flavus (8-30.7%), Fusarium oxysporum f. sp. lycopersici (25-44%), Sarocladium oryzae (11-55.5%), and Sclerotinia sclerotiorum (29.7-40.5%); 1000 D diluted methanolic extract of S. amritsarensis V31 showed growth inhibition against R. solani (23.3%), A. flavus (7.7%), F. oxysporum (22.2%), S. oryzae (16.7%), and S. sclerotiorum (19.0%). Metabolite extracts of S. amritsarensis V31 significantly reduced the incidence of rice sheath blight both as preventive and curative sprays. Chemical profiling of the metabolites in DMSO extract of S. amritsarensis V31 revealed 6-amino-5-nitrosopyrimidine-2,4-diol as the predominant compound present. It was evident from the LC-MS analyses that S. amritsarensis V31 produced a mixture of potential antifungal compounds which inhibited the growth of different phytopathogenic fungi. The results of this study indicated that metabolite extracts of S. amritsarensis V31 can be exploited as a bio-fungicide to control phytopathogenic fungi.

Microscope Assisted Uni-algal isolation through Dilution (MAU-D): a simple modified technique for tapping diverse cyanobacteria.

Cyanobacteria are oxygenic photosynthetic microorganisms known for their agricultural and industrial importance. Unavailability of efficient and fast isolation and purification methods of cyanobacteria has impeded our understanding of cyanobacterial diversity. A number of techniques for isolation and purification of cyanobacteria are available, but most of them are cumbersome as well as time-consuming. In the present study, we modified and validated a uni-algal isolation technique named as Microscope Assisted Uni-algal isolation through Dilution (MAU-D) which used dilution of mixed algal population on slide and isolation of single type of cyanobacterial cells using light microscope. Using this technique, we obtained 81 cyanobacterial isolates belonging to various species from 19 different genera from soil and water samples collected from rice fields of Uttar Pradesh, India. This technique also resulted in isolation of six distinct genera, viz., Cyanobacterium, Toxopsis, Desertifilum, Chroococcidiopsis, Halomicronema, and Alkalinema, which were previously not reported from rice fields of India. Hence, the MAU-D technique presents a simple, comparatively fast method of isolation and purification of cyanobacteria which can help to isolate those cyanobacteria which are difficult to isolate through routine sub-culturing. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02890-w.

DNA barcoding of phytopathogens for disease diagnostics and bio-surveillance.

DNA barcoding has proven to be a versatile tool for plant disease diagnostics in the genomics era. As the mass parallel and next generation sequencing techniques gained importance, the role of specific barcodes came under immense scrutiny. Identification and accurate classification of phytopathogens need a universal approach which has been the main application area of the concept of barcode. The present review entails a detailed description of the present status of barcode application in plant disease diagnostics. A case study on the application of Internal Transcribed Spacer (ITS) as barcode for Aspergillus and Fusarium spp. sheds light on the requirement of other potential candidates as barcodes for accurate identification. The challenges faced while barcoding novel pathogens have also been discussed with a comprehensive outline of integrating more recent technologies like meta-barcoding and genome skimming for detecting plant pathogens.

A rapid colorimetric LAMP assay for detection of Rhizoctonia solani AG-1 IA causing sheath blight of rice.

Rhizoctonia solani is one of the most devastating pathogens. R. solani AG-1 IA causes sheath blight in rice, maize, and other Gramineous plants. Accurate identification is essential for the effective management of this pathogen. In the present study, a set of four primers were designed viz. RSPG1, RSPG2, RSPG4, and RSPG5 for polygalacturonase (PG) gene, an important virulence factor in phytopathogenic fungi. All four primer sets showed specific amplification of 300 bp (RSPG1F/R), 375 bp (RSPG2F/R), 500 bp (RSPG4F/R) and 336 bp (RSPG5F/R) amplicons. q-PCR detection using each primer sets could detect up to 10 pg of DNA. We also designed six primers (RS_pg_F3_1/RS_pg_B3_1, RS_pg_FIP_1.1/RS-pg_BIP_1.1, and RS_pg_LF_1/RS_pg_LB_1) for PG gene. Further, a colorimetric LAMP assay developed yielded visual confirmation of the pathogen within 45 min of sample collection when coupled with rapid high throughput template preparation method (rHTTP) from infected samples. The sensitivity of the LAMP assay was as low as 1.65 fg/µl of template DNA and could effectively detect R. solani AG-1 IA from diseased plant tissues and soil samples. The LAMP assay was highly specific for R. solani as it did not show any amplification with other AG groups of R. solani and closely related fungal and bacterial outgroups. This study will help in designing an effective point of care diagnostic method for early monitoring of R. solani and thereby planning timely preventive measures against the pathogen.

Computational identification and antifungal bioassay reveals phytosterols as potential inhibitor of Alternaria arborescens.

Alternaria arborescens is a major pathogen for crops like tomato, tangerine and so on and its control is mostly dependent on the application of chemical agents. Plants as the sources of natural products are very attractive option for developing eco-friendly and natural antifungal agents. In this study, we modeled three-dimensional structure of chorismate synthase (CS) enzyme from A. arborescens. Docking studies of phytosterols, namely, γ-sitosterol and ß-sitosterol, with CS showed them to be potential inhibitor of CS. To explore the stability and conformational flexibility of all the AaCS complex systems, molecular dynamics simulations were performed. None of the putative inhibitors as well as ß- and γ-sitosterol showed interaction with the FMNH2 binding pocket of the tomato CS (major host of A. arborescens) indicating their suitability as antifungal compounds inhibiting the shikimate pathway without causing any harm to the host. An in vivo antifungal bioassay showed a significant reduction in fungal growth in the presence of ß-sitosterol (500 ppm) which resulted in ∼23% and ∼17% reduction in fungal fresh and dry weight, respectively, at 8 days after inoculation. This study provides experimental evidence establishing natural sterols like ß-sitosterol can be useful in curbing A. arborescens damage in an eco-friendly manner.Communicated by Ramaswamy H. Sarma.

Sustainable use of the spent mushroom substrate of Pleurotus florida for production of lignocellulolytic enzymes.

Spent mushroom substrate (SMS), a major byproduct of the mushroom industry, is a lignocellulosic biomass, which contains approximately 57-74.3% of holocellulose fraction. This study was aimed at utilizing SMS of Pleurotus florida for recovery of lignocellulolytic enzymes and sugars and also as a substrate for production of cellulolytic enzymes using different isolates of Trichoderma and Aspergillus under solid-state fermentation (SSF). SMS of P. florida extracts contained significant amounts of laccase (3,015.8 ± 29.5 U/g SMS) and xylanase (1,187.9 ± 12 U/g SMS) activity. Crystallinity pattern and chemical changes in SMS revealed that SMS had a lower crystallinity index (34.2%) as compared with the raw biomass (37.8%), which, in turn, helps in enhancing the accessibility of cellulolytic enzymes to holocellulose. Among the isolates, Trichoderma longibrachiatum A-01 showed maximum activity of endoglucanase (220.4 ± 5.9 U/mg), exoglucanase (78.5 ± 3.2 U/mg) and xylanase (1,550.4 ± 11.6 U/mg) while Aspergillus aculeatus C-08 showed maximum activity of cellobiase (113.9 ± 3.9 U/mg). Extraction with sodium citrate buffer (pH 4.8) showed maximum cellulolytic enzyme activity as compared with other solvents tested. Partial purification of endoglucanase, exoglucanase, xylanase, and cellobiase resulted in 56.3% (1,112.5 U/mg), 48.4% (212.5 U/mg), 44% (4,492.3 U/mg), and 62% (705.0 U/mg) yield with an increase by 5.2-, 4.5-, 4.1-, and 5.0-fold as compared with crude extract. The results reveal that SMS from P. florida could be a potential and cost-effective substrate for production of cellulolytic enzymes from T. longibrachiatum A-01 and A. aculeatus C-08.

Rapid high throughput template preparation (rHTTP) method: a novel cost effective method of direct PCR for a wide range of plants.

BACKGROUND: Conventional plant DNA isolation methods are complex, time consuming and require technical expertise. These limitations were overcome using the DNA isolation kits which, however significantly add to the research costs. Hence the present study was aimed to develop a high throughput, rapid and inexpensive method of PCR ready DNA template preparation from plant materials. METHODS: Concentration of SDS in lysis buffer, amount of starting material, period and temperature for lysis were optimized for obtaining PCR ready templates from plant materials. The method was tested using RAPD and ITS specific primers for different plant species like rice, wheat, mustard, pea, soybean, pigeonpea, tomato, maize, march lilly, bougainvillea, Indian blanket flower, nerium, petunia, purple pirouette petunia, moses-in-the-cradle, golden cane palm, duranta, periwinkle, chrysanthemum and two xerophytes viz. Dipterygium glaucum and Crotaleria burhia. SSR markers RM18398 and RM26108 showed successful amplification in rice varieties Improved Pusa Basmati 1 and KS Dev 12. The effectiveness of the method was tested using fresh as well as 1 year old tissues. The storability of the lysate was also tested. RESULTS: In this report, we developed a novel method called rapid high throughput template preparation (rHTTP) method to prepare PCR ready DNA templates. Most striking feature of this technique is that it can be done anywhere where water can be boiled by any means. Using rHTTP method, PCR ready templates can be prepared in just 10 min. Robust and reproducible amplification for all the test plants were recorded with RAPD, plant ITS primers and SSR markers following this method. rHTTP methods works well for both fresh as well as old plant tissues. The lysates had a shelf life of 1 month when stored at 4 °C and 3 days when stored at room temperature. CONCLUSIONS: rHTTP method has several advantages over the other protocols like ease of execution, no requirement of tissue grinding/liquid nitrogen/hazardous chemicals and above all, equally effective for both fresh and old samples. Using this method, costs per prep comes down ~ 10-50 times as compared to most commercial kits. This method can be used for on-field experiments like molecular diagnostics, varietal identification etc.

noxB-based marker for Alternaria spp.: a new diagnostic marker for specific and early detection in crop plants.

Alternaria species are a major plant pathogen and their precise detection and identification is crucial for effective management. In the present study, a polymerase chain reaction (PCR)-based diagnostic technique has been developed for detection of Alternaria species. Four primers were designed for four genes viz. noxB, AMK1, AKT3 and NIK1. In gradient PCR, only the primer sets for noxB gene showed specific amplicon of ~ 200 bp in all the isolates of Alternaria, while no amplification was observed in related fungal species such as Ulocladium botrytis, Ulocladium consortiale, Stemphylium vesicarium, Cochliobolus tuberculatus, Curvularia prasadii, and Bipolaris sorokiniana. The noxB primer set was used as diagnostic marker to discriminate and diagnose Alternaria species in nine different crop plants. Real-time assay revealed that the primer set was able to detect Alternaria noxB genes in leaves with no characteristic visible symptoms. Through real-time PCR, the noxB gene of Alternaria could be detected even in 0.5 ng of host DNA. This is the first report of noxB gene for molecular detection of Alternaria spp.

Genome-Wide Analysis of Microsatellites in Alternaria arborescens and Elucidation of the Function of Polyketide Synthase (PksJ).

Microsatellites or simple sequence repeats (SSRs) have been the most widely applied class of molecular markers used in genetic studies, having applications in genetic conservation, population studies, as well as diagnostics of fungi. Mining and analysis of SSRs of the whole genome sequence have been carried out in this study for the fungus Alternaria arborescens causing early blight of tomato and well known for producing mycotoxins like alternariol (AOH), alternariol monomethyl ether (AME), etc. A total of 4097 microsatellites were identified in A. Arborescens genome. Contig 1 was identified as the most SSR-rich region which was further analyzed to correlate the presence of SSRs with different biological processes. A total of 246 putative genes were predicted in this study and KEGG pathway analysis of 155 predicted genes indicated that SSRs can be linked with important metabolic pathways, molecular functioning, signal transduction, and cellular processes. The prediction of fungal mycotoxin inducer gene Polyketide synthase (PksJ) linked with SSR in this study may be a potential candidate participating in oncogenic signal transduction in human. Our study is the first report of PksJ gene in A. arborescens, a precursor of AOH and AME.