Evaluating the imazethapyr herbicide mediated regulation of phenol and glutathione metabolism and antioxidant activity in lentil seedlings.

The imidazolinone group of herbicides generally work for controlling weeds by limiting the synthesis of the aceto-hydroxy-acid enzyme, which is linked to the biosynthesis of branched-chain amino acids in plant cells. The herbicide imazethapyr is from the class and the active ingredient of this herbicide is the same as other herbicides Contour, Hammer, Overtop, Passport, Pivot, Pursuit, Pursuit Plus, and Resolve. It is commonly used for controlling weeds in soybeans, alfalfa hay, corn, rice, peanuts, etc. Generally, the herbicide imazethapyr is safe and non-toxic for target crops and environmentally friendly when it is used at low concentration levels. Even though crops are extremely susceptible to herbicide treatment at the seedling stage, there have been no observations of its higher dose on lentils (Lens culinaris Medik.) at that stage. The current study reports the consequence of imazethapyr treatment on phenolic acid and flavonoid contents along with the antioxidant activity of the phenolic extract. Imazethapyr treatment significantly increased the activities of several antioxidant enzymes, including phenylalanine ammonia lyase (PAL), phenol oxidase (POD), glutathione reductase (GR), and glutathione-s-transferase (GST), in lentil seedlings at doses of 0 RFD, 0.5 RFD, 1 RFD, 1.25 RFD, 1.5 RFD, and 2 RFD. Application of imazethapyr resulted in the 3.2 to 26.31 and 4.57-27.85% increase in mean phenolic acid and flavonoid content, respectively, over control. However, the consequent fold increase in mean antioxidant activity under 2, 2- diphenylpicrylhdrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assay system was in the range of 1.17-1.85 and 1.47-2.03%. Mean PAL and POD activities increased by 1.63 to 3.66 and 1.71 to 3.35-fold, respectively, in agreement with the rise in phenolic compounds, indicating that these enzyme's activities were modulated in response to herbicide treatment. Following herbicide treatments, the mean thiol content also increased significantly in corroboration with the enhancement in GR activity in a dose-dependent approach. A similar increase in GST activity was also observed with increasing herbicide dose.

MAPK signaling pathway orchestrates and fine-tunes the pathogenicity of Colletotrichum falcatum.

Colletotrichum falcatum is the causal organism of red rot, the most devastating disease of sugarcane. Mitogen-activated protein kinase (MAPK) signaling pathway plays pivotal role in coordinating the process of pathogenesis. We identified eighteen proteins implicated in MAPK signaling pathway in C. falcatum, through nanoLCMS/MS based proteomics approach. Twelve of these proteins were the part of core MAPK signaling pathway, whereas remaining proteins were indirectly implicated in MAPK signaling. Majority of these proteins had enhanced abundance in C. falcatum samples cultured with host sugarcane stalks. To validate the findings, core MAPK pathway genes (MAPKKK-NSY1, MAPK 17-MAPK17, MAPKKK 5-MAPKKK5, MAPK-HOG1B, MAPKKK-MCK1/STE11, MAPK-MST50/STE50, MAPKK-SEK1, MAPKK-MEK1/MST7/STE7, MAPKK-MKK2/STE7, MAPKKK-MST11/STE11, MAPK 5-MPK5, and MAPK-MPK-C) were analyzed by qPCR to confirm the real-time expression in C. falcatum samples cultured with host sugarcane stalks. The results of qPCR-based expression of genes were largely in agreement with the findings of proteomics. String association networks of MAPKK- MEK1/MST7/STE7, and MAPK- MPK-C revealed strong association with plenty of assorted proteins implicated in the process of pathogenesis/virulence. This is the novel and first large scale study of MAPK proteins in C. falcatum, responsible for red rot epidemics of sugarcane various countries. KEY MESSAGE: Our findings demonstrate the pivotal role of MAPK proteins in orchestrating the pathogenicity of Colletotrichum falcatum, responsible devastating red rot disease of sugarcane. SIGNIFICANCE: Our findings are novel and the first large scale study demonstrating the pivotal role of MAPK proteins in C. falcatum, responsible devastating red rot disease of sugarcane. The study will be useful for future researchers in terms of manipulating the fungal pathogenicity through genome editing.

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.

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.

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.

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.