Leaf rust of wheat caused by Puccinia triticina has significant impact on wheat production worldwide. Effective and quick detection methodologies are required to mitigate yield loss and time constraints associated with monitoring and management of leaf rust of wheat. In the present study, detection of P. triticina has been simplified by developing a rapid, reliable, efficient and visual colorimetric method i.e., loop mediated isothermal amplification of DNA (LAMP). Based on in silico analysis of P. triticina genome, PTS68, a simple sequence repeat was found highly specific to leaf rust fungus. A marker (PtRA68) was developed and its specificity was validated through PCR technique which gave a unique and sharp band of 919 bp in P. triticina pathotypes only. A novel gene amplification method LAMP which enables visual detection of pathogen by naked eye was developed for leaf rust pathogen. A set of six primers was designed from specific region of P. triticina and conditions were optimised to complete the observation process in 60 minutes at 65o C. The assay developed in the study could detect presence of P. triticina on wheat at 24 hpi (pre-symptomatic stage) which was much earlier than PCR without requiring thermal cycler. Sensitivity of LAMP assay developed in the study was 100 fg which was more sensitive than conventional PCR (50 pg) and equivalent to qPCR (100 fg). The protocol developed in the study was utilized for detection of leaf rust infected samples collected from different wheat fields. LAMP based colorimetric detection assay showed sky blue color in positive reaction and violet color in negative reaction after addition of 120 μM hydroxyl napthol blue (HNB) solution to reaction mixture. Similarly, 0.6 mg Ethidium bromide/ml was added to LAMP products, placed on transilluminator to witness full brightness in positive reaction and no such brightness could be seen in negative reaction mixture. Further, LAMP products spread in a ladder like banding pattern in gel electrophoresis. Our assay is significantly faster than the conventional methods used in the identification of P. triticina. The assay developed in the study shall be very much useful in the development of diagnostic kit for monitoring disease, creation of prediction model and efficient management of disease.
Aims: To characterize the geo-distinct isolates of Ustilaginoidea virens for morpho-molecular and mating-type locus diversity. Methods and Results: Sixty-one isolates of U. virens collected from Southern India exhibited significant diversity in mycelial width (3Á45-5Á50 µm), colony colour (yellow, pale yellow, and white), and growth pattern (thick leather mat, raised-fluffy, flat-fluffy, and raised). Field-borne chlamydospores of each isolate were significantly smaller in size (3Á34-5Á26 µm 2 ) compared to those formed on culture media (18Á6-100Á89 µm 2 ). The phylogenetic study based on internal transcribed sequences revealed two clusters; however, most isolates (n = 54) were grouped in cluster-I, indicating common ancestral origin. We also identified 42 haplotypes; among them, Hap_3 has the highest number of isolates (n = 19). Mating-type locus (MAT1) analysis revealed all sixty-one isolates as heterothallic, wherein 37 and 24 isolates belonging to MAT1-1-1 and MAT1-2-1 heterothallic mating types, respectively. The microsynteny analysis of MAT1 loci of one of the Indian strain (Uv-Gvt) along with Uv-8b (China) strain revealed synteny conservation at MAT1 locus, which is flanked by conserved genes SLA2 and a hypothetical protein in the upstream and APN2, COX12 and APC5 in the downstream of the locus. Conclusions: Morpho-molecular study revealed the significant diversity among geo-distinct isolates, and MAT1 loci analysis indicated the distribution of heterothallic mating types in south Indian paddy fields. And also, complete synteny conservation between Indian and Chinese strain was observed at the MAT1 locus. Significance and Impact of the Study: This is the first report describing the sexuality of Indian strains of the U. virens, which would help better understand the genetic diversity of the U. virens prevailing in Southern India and aid in developing resistant rice cultivars against this pathogen population.
Stripe rust caused by Puccinia striiformis f. sp.
tritici (Pst) is one of the most devastating diseases of
wheat (Triticum spp.) worldwide. Indian isolates were
characterised based on their phenotypic reaction on differential hosts carrying
different Yr genes. Based on virulence/avirulence structure,
isolates were characterised into ten different pathotypes viz.
70S0-2, 67S64, 70S4, 66S0, 70S64, 66S64-1, 38S102, 47S102, 46S119, and 78S84.
These Indian pathotypes of P. striiformis f. sp.
tritici and 38 pathotypes of other rust species (P.
graminis tritici and P. triticina) were used in
this study to analyze their molecular phylogenetic relationship. The nucleotides
of rDNA-ITS, partial β-tubulin and ketopantoate
reductase genes of all the pathotypes were sequenced directly after
PCR. Based on sequence data of rDNA-ITS and β-tubulin, three
phylogenetic groups corresponding to three different species of
Puccinia were obtained. Asian isolates formed a distinct
evolutionary lineage than from those derived from USA. The sequence similarity
of Indian pathotypes with other Asian (China and Iran) isolates indicated the
same origin of pathotypes. The results will allow rapid identification of Indian
P.striiformis f. sp. tritici pathotypes
causing stripe rust in wheat, assist in making predictions regarding potential
rust pathotypes, and identifying sources of resistance to the disease in
advance.
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