Molecular Detection and Identification of Pathogens, Pathotypes, and Genes

Abstract

The accurate, rapid detection and identification of the pathogens is essential for effective management of crucifers’ diseases. The traditional approaches are time consuming, expansive, laborious, and less in accuracy in comparison to molecular approaches like bar-coding markers, qRT-PCR, cDNA sequencing, pathogen enrichment sequencing (Pen seq), and use of sequence repositories. Pen Seq approach has been used to detect Albugo from infected leaves and to identify variations in pathogen genotypes. The distinct lineages that had diverged from each other were also identified. Many putative recombinant regions were identified between lineages. Pen Seq can identify Albugo race structure with phylogenetic lineage. Albugo candida ITS1 DNA has been detected in symptomatic and asymptomatic host tissues. Molecular markers like RAPD and ESTs have been used for identification, detection, and analysis of genetic variability in Alternaria species infecting crucifers. In the genome of Colletotrichum, two effectors (genes), ChELP1 and ChELP2, homologs of LysM have been characterized. Molecular quantification of Erysiphe infection and development in host tissues have been assessed using qPCR assessing host cell entry, area covered by hyphae, conidia, and conidiophores. The expression of genes has been detected in response to powdery mildew infection with a broad range of transcriptional changes conferring susceptibility, resistance, and bimolecular regulations. The phylogenetic relationship of powdery mildew of crucifers with other powdery mildew species and isolates variations have been determined through sequence analysis of ITSrDNA and sequence comparison of DNA encoding 5.85 rDNA, ITS1, and ITS2. DNA (RAPD) finger print approach has been used to distinguish isolates of H. parasitica infecting different Brassica species. The combination of LMD and RNA sequencing of Brassica improves Leptosphaeria detection at the early stage of infection. Sequencing of gene fragments of β-tublin, actin, and ITS region of rDNA from L. biglobosa isolates from different countries could show closer taxonomic similarities between different clades. Genomic prediction tools are valuable approaches in Brassica to assess Leptosphaeria species. Plasmodiophora virulence genes expressed during early stages of colonization of Brassica roots have been identified using SSH and EST analysis. AqPCR-based Plasmodiophora detection and spread in the soils has been developed for country-wide assessment. ITS sequencing and phylogenetic analysis of Pseudocercosporella isolates collected from Brassica species revealed a high degree of genetic similarity. Real-time PCR protocols have been developed to detect and discriminate anastomosis groups (AGs) of R. solani using ribosomal internal transcribed spacer (ITS) regions or β-Tubulin sequences of pathogen from field soils. Phenotypic pipeline has been developed to assess kinetics of disease symptoms caused by Sclerotinia in A. thaliana conferring susceptibility by NLR genes. A simple, rapid, sensitive, and cost effective mRT-PCR protocol has been developed for detection and differentiation of Brassica viruses. Molecular approaches like DAS-ELISA, Rt-PCR, and ELISA can detect, identify, and confirm phylogenetic relationship of Brassica viruses and their strains. Simplex and multiplex PCR assays based on DNA sequence from rITS region and coding and noncoding regions of actin, elongation factors 1-α, glyceraldehydes-3-phosphate dehydrogenase, and tryptophan synthase genes are valuable approaches for identification, detection, and phylogenetic analysis of Verticillium species. Verticillium longisporum isolates have been characterized on the basis of a group-1 Intron in SSU-rRNA gene of pathogen. Isolates from two different species, i.e., V. dahliae and V. albo-atrum have been characterized based on AFLP analysis of rRNA and DNA from mitochondria. Several virulence genes of Xanthomonas have been identified (pig, pigB, DF, EPSs, DSF, cyclic-di-GMP, rpfl, RpfF, RpfG, WspR, xantham, LPs) which regulate production of enzymes for infection and development of black rot in Brassica by multiplication and movement of bacteria in host tissues. Several novel genes for resistance to Xanthomonas in Brassica have been identified by SSH-PCR-based techniques. Molecular identification of different pathotypes, pathogens, elicitors/effectors have been revealed using R genes in the form of isogenic lines of Brassica. The accuracy of qRT-PCR analysis data expression of target genes is affected by factors such as RNA quality, purity, and PCR amplification efficiency, technical, and biological variations. To control these variables and avoid bias in qRT-PCR, selection of appropriate RGs is crucial under multiple stresses. The interplay between multiple pathogens and associated microbiomes requires disentangling of hidden histories in genes and genomics of both hosts and pathogens. R genes homologous DNA fragments in A. thaliana have been identified to use them for cloning and in breeding for resistance against major pathogens of crucifers. The molecular approaches are valuable tools and their efficiency and accuracy depends on proper sampling and cares in using them for correct diagnosis.