Comparative Proteomic Analysis of Rhizoctonia solani Isolates Identifies the Differentially Expressed Proteins with Roles in Virulence.

Sheath blight of rice is a destructive disease that could be calamitous to rice cultivation. The significant objective of this study is to contemplate the proteomic analysis of the high virulent and less virulent isolate of Rhizoctonia solani using a quantitative LC-MS/MS-based proteomic approach to identify the differentially expressed proteins promoting higher virulence. Across several rice-growing regions in Odisha, Eastern India, 58 Rhizoctonia isolates were obtained. All the isolates varied in their pathogenicity. The isolate RS15 was found to be the most virulent and RS22 was identified as the least virulent. The PCR amplification confirmed that the RS15 and RS22 belonged to the Rhizoctonia subgroup of AG1-IA with a specific primer. The proteomic information generated has been deposited in the PRIDE database with PXD023430. The virulent isolate consisted of 48 differentially abundant proteins, out of which 27 proteins had higher abundance, while 21 proteins had lower abundance. The analyzed proteins acquired functionality in fungal development, sporulation, morphology, pathogenicity, detoxification, antifungal activity, essential metabolism and transcriptional activities, protein biosynthesis, glycolysis, phosphorylation and catalytic activities in fungi. A Quantitative Real-Time PCR (qRT-PCR) was used to validate changes in differentially expressed proteins at the mRNA level for selected genes. The abundances of proteins and transcripts were positively correlated. This study provides the role of the proteome in the pathogenicity of R. solani AG1-IA in rice and underpins the mechanism behind the pathogen's virulence in causing sheath blight disease.

Green silver nano-particles: synthesis using rice leaf extract, characterization, efficacy, and non-target effects.

Green synthesis of silver nano-particles (AgNPs) from silver nitrate was carried out using purple-colored rice leaves' extracts containing higher phenols, anthocyanins, and flavonoids. The efficacy of synthesized AgNPs was tested against rice diseases and investigation was carried out to check negative effect of AgNPs on soil microbes. Substantial reduction of total anthocyanins, total phenols, and total flavonoids was observed in reaction mixture during AgNP formation indicating the role of secondary metabolites on AgNP formation and stabilization. Scanning electron microscopy coupled with energy-dispersive spectroscopic images and FTIR spectral analysis of AgNPs confirmed the presence of elemental silver encapped by biomolecules. The optimized reaction parameters for synthesis of AgNPs from silver nitrate were (a) 48 h of incubation, (b) 9:1 (v/v) 1 mM AgNO3:plant extract, and (c) room temperature at 20-30 °C. Zeta potential and hydrodynamic particle sizes of synthesized AgNPs were ranged between - 16.61 to - 29.45 mV and 36-107 nm, respectively, at different time of incubation. AgNPs could control effectively Rhizoctonia solani and Xanthomonas oryzae pv. Oryzae and Helminthosporium oryzae. AgNPs at higher concentration could cause negative effect on microbial biomass carbon and soil enzymes for distant future. But the negative effects of AgNP solution (10% of 1 mM AgNPs) were comparable to commercial fungicide, carbendazim. The synthesized AgNPs with desirable characters were effective against a number of disease-causing pathogens in rice, and it can be recommended as broad-spectrum pesticide.

Natural infection of croton yellow vein mosaic virus and its cognate betasatellite in germplasm of different Crambe spp in India.

Crambe is an important crop grown worldwide for industrial oil and seed meal. Besides the fungal and bacterial diseases, the crop is reported to be infected by tobacco mosaic virus, beet western yellows virus and turnip mosaic virus under experimental condition. Till now, there was no report of natural infection of any begomovirus in this crop. In the present study, a leaf curl disease was observed in germplasm accessions of three species of Crambe (C. abyssinica, C. glabrata and C. hispanica). Based on the symptoms and presence of whitefly population in the field, begomovirus infection was suspected. Molecular characterization through RCA approach, indicated presence of croton yellow vein mosaic virus (CYVMV, KJ747958) and croton yellow vein mosaic betasatellite (CroYVMB, KM229762). Co-agroinoculation of partial dimeric construct of CYVMV with complete dimeric construct of CroYVMB, produced typical leaf curl symptoms in C. abyssinica, whereas, agroinoculation of partial dimeric construct of CYVMV alone could not produce symptoms in the same plant. In contrast, the CYVMV construct alone could produce symptom in Nicotiana benthamiana, a model host for plant virus studies. In N. benthamiana co-inoculation of CroYVMV with CYVMV construct develop more severe symptoms. However, neither the CYVMV construct alone nor the co-inoculation with CroYVMB produce any symptom in Arabidopsis thaliana even with different methods of inoculation. Inoculated Arabidopsis thaliana also did not yield any amplification of the virus as assessed through PCR and rolling circle amplification (RCA). Thus it confirmed that for successful infection in crambe, CYVMV requires betasatellite, while in N. benthamiana, it does not require betasatellite for symptom induction and in Arabidopsis thaliana CYVMV alone or in presence of betasatellite did not replicate and produce any symptom. This study constitutes the first confirmed record of natural infection of a begomovirus in crambe and further confirmed that cognate betasatellite of CYVMV has differential role in infectivity in different hosts.

Correction: Use of molecular markers in identification and characterization of resistance to rice blast in India.

[This corrects the article DOI: 10.1371/journal.pone.0176236.].

Use of molecular markers in identification and characterization of resistance to rice blast in India.

Rice blast disease caused by Magnaporthe oryzae is one of the most destructive disease causing huge losses to rice yield in different parts of the world. Therefore, an attempt has been made to find out the resistance by screening and studying the genetic diversity of eighty released rice varieties by National Rice Research Institute, Cuttack (NRVs) using molecular markers linked to twelve major blast resistance (R) genes viz Pib, Piz, Piz-t, Pik, Pik-p, Pikm Pik-h, Pita/Pita-2, Pi2, Pi9, Pi1 and Pi5. Out of which, nineteen varieties (23.75%) showed resistance, twenty one were moderately resistant (26.25%) while remaining forty varieties (50%) showed susceptible in uniform blast nursery. Rice varieties possessing blast resistance genes varied from four to twelve and the frequencies of the resistance genes ranged from 0 to 100%. The cluster analysis grouped the eighty NRVs into two major clusters at 63% level of genetic similarity coefficient. The PIC value for seventeen markers varied from 0 to 0.37 at an average of 0.20. Out of seventeen markers, only five markers, 195R-1, Pi9-i, Pita3, YL155/YL87 and 40N23r corresponded to three broad spectrum R genes viz. Pi9, Pita/Pita2 and Pi5 were found to be significantly associated with the blast disease with explaining phenotypic variance from 3.5% to 7.7%. The population structure analysis and PCoA divided the entire 80 NRVs into two sub-groups. The outcome of this study would help to formulate strategies for improving rice blast resistance through genetic studies, plant-pathogen interaction, identification of novel R genes, development of new resistant varieties through marker-assisted breeding for improving rice blast resistance in India and worldwide.

Molecular Evidence for Occurrence of Tomato leaf curl New Delhi virus in Ash Gourd (Benincasa hispida) Germplasm Showing a Severe Yellow Stunt Disease in India.

An evaluation of 70 accessions of ash gourd germplasm grown at National Bureau of Plant Genetic Resources, New Delhi, India during Kharif season (2010) showed natural occurrence of a yellow stunt disease in three accessions (IC554690, IC036330 and Pusa Ujjwal). A set of begomovirus specific primers used in PCR gave expected amplicon from all the symptomatic plants; however no betasatellite was detected. Complete genome of the begomovirus (DNA-A and DNA-B), amplified through rolling circle amplification, was cloned and sequenced. The begomovirus under study shared high sequence identities to different isolates of Tomato leaf curl New Delhi virus (ToLCNDV) and clustered with them. Among those isolates, the DNA-A and DNA-B of the present begomovirus isolate showed highest 99.6 and 96.8 % sequence identities, respectively with an isolate reported on pumpkin from India (DNA-A: AM286433, DNA-B: AM286435). Based on the sequence analysis, the begomovirus obtained from ash gourd was considered as an isolate of ToLCNDV. Thus, the present findings constitute the first report of occurrence of a new yellow stunt disease in ash gourd from India and demonstrated the association of ToLCNDV with the symptomatic samples. Occurrence of ToLCNDV in ash gourd germplasm not only adds up a new cucurbitaceous host of this virus but also raises the concern about the perpetuation of this virus in absence of its main host tomato and thus has an epidemiological relevance for understanding the rapid spread of this virus in tomato and other hosts in Indian sub-continent.