Nanocatalytic Interface to Decode the Phytovolatile Language for Latent Crop Diagnosis in Future Farms.

Crop diseases cause the release of volatiles. Here, the use of an SnO2-based chemoresistive sensor for early diagnosis has been attempted. Ionone is one of the signature volatiles released by the enzymatic and nonenzymatic cleavage of carotene at the latent stage of some biotic stresses. To our knowledge, this is the first attempt at sensing volatiles with multiple oxidation sites, i.e., ionone (4 oxidation sites), from the phytovolatile library, to derive stronger signals at minimum concentrations. Further, the sensitivity was enhanced on an interdigitated electrode by the addition of platinum as the dopant for a favorable space charge layer and for surface island formation for reactive interface sites. The mechanistic influence of oxygen vacancy formation was studied through detailed density functional theory (DFT) calculations and reactive oxygen-assisted enhanced binding through X-ray photoelectron spectroscopy (XPS) analysis.

Phenotypic variability, race profiling and molecular diversity analysis of Indian populations of Fusarium oxysporum f. sp. lentis causing lentil wilt.

Wilt (Fusarium oxysporum f. sp. lentis; Fol) is one of the major diseases of lentil worldwide. Two hundred and thirty-five isolates of the pathogen collected from 8 states of India showed substantial variations in morphological characters such as colony texture and pattern, pigmentation and growth rate. The isolates were grouped as slow (47 isolates), medium (118 isolates) and fast (70 isolates) growing. The macroconidia and microconidia (3.0-77.5 × 1.3-8.8 µm for macroconidia and 1.8-22.5 × 0.8-8.0 µm for microconidia for length × width) were variable in size and considering the morphological features, the populations were grouped into 12 categories. Seventy representative isolates based on their morphological variability and place of origin were selected for further study. A set of 10 differential genotypes was identified for virulence analysis and based on virulence patterns on these 10 genotypes, 70 Fol isolates were grouped into 7 races. Random amplified polymorphic DNA (RAPD), universal rice primers (URPs), inter simple sequence repeats (ISSR) and sequence-related amplified polymorphism (SRAP) were used for genetic diversity analysis. URPs, ISSR and SRAP markers gave 100% polymorphism while RAPD gave 98.9% polymorphism. The isolates were grouped into seven clusters at genetic similarities ranging from 21 to 80% using unweighted paired group method with arithmetic average analysis. The major clusters include the populations from northern and central regions of India in distinct groups. All these three markers proved suitable for diversity analysis, but their combined use was better to resolve the area specific grouping of the isolates. The sequences of rDNA ITS and TEF-1α genes of the representative isolates were analysed. Phylogenetic analysis of ITS region grouped the isolates into two major clades representing various races. In TEF-1α analysis, the isolates were grouped into two major clades with 28 isolates into one clade and 4 remaining isolates in another clade. The molecular groups partially correspond to the lentil growing regions of the isolates and races of the pathogen.

Development of multiplex PCR assay for detection of Alternaria brassicae, A. brassicicola and Xanthomonas campestris pv. campestris in crucifers.

Among biotic stresses, Alternaria leaf spots caused by Alternaria brassicae and A. brassicicola and black rot caused by Xanthomonas campestris pv. campestris are major limiting factors in brassica cultivation across the world. Because of seed-borne nature of these pathogens primarily, disease-free conservation as well as exchange of brassica seeds at domestic as well as international level are major challenges. To facilitate disease-free conservation and transboundary movement of brassica germplasm, a highly specific and sensitive method was developed for simultaneous detection of these pathogens. A set of primers namely, AbeABC1F and AbeABC1R based on ABC transporter (Atr1) gene for A. brassicae, Aba28sF and Aba28sR based on SSR marker was developed for A. brassicicola as well as rpf gene-based primers namely, rpfH_F and rpfH_R for X. campestris pv. campestris were used for multiplex PCR. The specific bands of 586, 201 and 304 bp were obtained in multiplex PCR assay for A. brassicae, A. brassicicola and X. campestris pv. campestris, respectively. Therefore, the developed multiplex PCR protocol could be utilized for a reliable diagnosis of these pathogens to facilitate safe conservation, exchange of seeds to the researchers and also by seed certification agencies for ensuring quality seed availability to farmers.

Diversity analysis of different Diaporthe (Phomopsis) species and development of molecular marker to identify quarantine important species Phomopsis phaseolorum.

The genus Diaporthe Nitschke (Phomopsis Sacc. & Harter) infect various agricultural and horticultural important crops and cause diseases such as damping off, leaf spots, blights, canker, dieback, wilt, root and fruit rots. P. vexans, P. helianthi and P. phaseolorum are the important species within genus causing huge yield and economic loss. Being primarily seed borne it also hinders import and export of germplasm and seeds. Therefore, extensive characterization is required to diagnose and manage the disease. Seventeen isolates collected from ITCC, IARI and ICAR-NBPGR belonging to eight species were morphological and molecularly characterized and diversity was analyzed. Several morphological and cultural characters were studied and analyzed. Due to lack of sufficient morphological variation to identify/differentiate species, molecular characterization using house-keeping genes, internal transcriber spacer (ITS) was carried out. ITS produced amplicon of ~ 600 bp in the isolates of Phomopsis and phylogenetic tree obtained revealed that isolates of a species belonging same geographic region had more sequence similarity than isolates belonging to different geographic regions this might be due to population adaption under varied environments. Development of EF-1alpha-based marker specific to P. phaseolorum helps in easily detection of pathogen in quarantine stations. In addition, species of Phomopsis were previously named based on host association which has led to misidentification and proliferation of species. Cross pathogenicity of isolates on three important hosts, brinjal, soybean and chilli revealed its broad host range and naming only basis of host association is unjustified.

DNA barcode, multiplex PCR and qPCR assay for diagnosis of pathogens infecting pulse crops to facilitate safe exchange and healthy conservation of germplasm.

The DNA barcodes were developed from ITS region for the identification of fungal plant pathogens namely, Alternaria alternata and A. tenuissima both causing leaf spots, Ascochyta rabiei causing Ascochyta blight, Fusarium oxysporum f. sp. ciceris causing wilt, Macrophomina phaseolina causing dry root rot, Rhizoctonia solani causing web blight and wet root rot, Sclerotium (Athelia) rolfsii causing collar rot, Sclerotinia sclerotiorum causing stem rot and Cercospora canescens and Pseudocercospora cruenta both causing leaf spots in pulse crops. Barcode compliance for A. alternata (DBTPQ001-18), A. tenuissima (DBTPQ002-18), A. rabiei (DBTPQ003-18), F. oxysporum f. sp. ciceris (DBTPQ004-18), M. phaseolina (DBTPQ005-18), R. solani (DBTPQ006-18), S. rolfsii (DBTPQ007-18), S. sclerotiorum (DBTPQ008-18), C. canescens (DBTPQ009-18) and P. cruenta (DBTPQ029-20) have been generated based on the Barcode of Life Data System (BOLD) system. In addition to ITS, other genomic regions were also explored and on the basis of sequence variation they were ranked as TEF-α > SSU > LSU > ß-tubulin. These genes could be considered for secondary barcode and phylogenetic relatedness. ITS-based markers for the detection of A. alternata (BAA2aF and BAA2aR) and R. solani (BRS17cF and BRS17cR) were developed which provided 400 bp and 220 bp amplicons, respectively. While, for F. oxysporum f. sp. ciceris, COX1-based marker (FOCox1F and FOCox3R) was developed which amplified 150 bp. The markers proved highly specific and sensitive with detection limit of 0.0001 ng of template DNA using qPCR and simultaneously detected these three pathogens. The DNA barcodes and diagnostics developed are suitable for quick and reliable detection of these pathogens during quarantine processing and field diagnostics.