Sangeeta Gupta

Development and validation of SCAR markers co-segregating with an Agropyron elongatum derived leaf rust resistance gene Lr24 in wheat

SummaryAn Agropyron elongatum-derived leaf rust resistance gene Lr24 located on chromosome 3DL of wheat was tagged with six random amplified polymorphic DNA (RAPD) markers which co-segregated with the gene. The markers were identified in homozygous resistant F2 plants taken from a population segregating for leaf rust resistance generated from a cross between two near-isogenic lines (NILs) differing only for Lr24. Phenotyping was done by inoculating the plants with pathotype 77-5 of Puccinia triticina. To enable gene-specific selection, three RAPD markers (S1302609, S1326615 and OPAB-1388) were successfully converted to polymorphic sequence characterized amplified region (SCAR) markers, amplifying only the critical DNA fragments co-segregating with Lr24. The SCAR markers were validated for specificity to the gene Lr24 in wheat NILs possessing Lr24 in 10 additional genetic backgrounds including the Thatcher NIL, but not to 43 Thatcher NILs possessing designated leaf rust resistance genes other than Lr24. This indicated the potential usefulness of these SCAR markers in marker assisted selection (MAS) and for pyramiding leaf rust resistance genes in wheat.

Broad-spectrum Blast Resistance Gene Pi-kh Cloned from Rice Line Tetep Designated as Pi54

Blast disease caused by Magnaporthe oryzae is one of the important biotic stresses of rice. So far more than 85 blast resistance genes have been identified of these more than 14 have already been cloned. A broad spectrum rice blast resistance gene Pi-kh was cloned from the rice line Tetep. The gene was named Pi-kh based on the earlier reports on its genetic analysis in various rice lines. However, with the advances in molecular genetics and genomics of rice, the Pik locus has now been mapped more precisely. Since there are two reports on the mapping of Pi-kh gene from different rice lines, there is some confusion in the naming of this gene. In this report the name of Pi-kh gene cloned from the rice line Tetep has been designated as per the standard guidelines of Committee on Gene Symbolization, Nomenclature and Linkage (CGSNL) and its physical location on rice chromosome 11, which is ∼2.5 Mbp away from the Pik locus mapped recently. Hence Pi-kh gene cloned from Tetep is now designated as Pi54.

Identification of molecular markers linked to adult plant leaf rust resistance gene Lr48 in wheat and detection of Lr48 in the Thatcher near-isogenic line with gene Lr25

The recessive adult plant resistance (APR) gene Lr48 in wheat was tagged with flanking random amplified polymorphic DNA (RAPD) markers. Markers S336775 in coupling and S3450 in repulsion with Lr48 were identified in wheat line CSP44. Tests of these markers on available Thatcher near-isogenic lines (NILs) detected the likely presence of Lr48 in TcLr25. A test of allelism of APR involving the cross TcLr25xa0×xa0CSP44 indicated that Lr48 was present in both lines. A separate experiment on inheritance of resistance in an F2 population of TcLr25xa0×xa0Agra Local confirmed the presence of a dominant seedling resistance gene (Lr25) and a recessive APR gene (Lr48) in TcLr25. This study demonstrated the value of molecular markers in identifying the presence of masked genes in genetic stocks where direct phenotyping failed to detect their presence.

Molecular Markers Detect Redundancy and Miss-identity in Genetic Stocks with Alien Leaf Rust Resistance Genes Lr32 and Lr28 in Bread Wheat

Ten elite near-isogenic line (NIL) pairs of bread wheat (Triticum aestivum L em Thell) each carrying one of the two alien leaf rust resistance (Lr) genes Lr32 and Lr28, derived from Triticum tauschii and Triticum speltoides, respectively were tested for disease phenotype in controlled conditions. The disease phenotype of the NIL pair detected distinction between the Lr32 donor parent and its derivatives in ten cultivar backgrounds documented as carrying the gene Lr32. The RAPD and SCAR molecular markers identified earlier as linked to Lr32 amplified the critical marker bands identically in eight of the ten NIL pairs as well as the Lr28 donor parent. The critical bands were not amplified in the Lr32 donor parent. A Triticum speltoides specific microsatellite null allele marker located on chromosome 4AL, the genomic region associated with Lr28, expressed in an identical polymorphism as the RAPD and SCAR markers. The PCR product sequenced from a NIL pair revealed 100% homology. It is confirmed that eight of the ten elite Lr32 lines carry the gene Lr28. Molecular marker tools need to be employed to eliminate such miss-identities and reduce redundancy in Indian elite germplasm stocks of wheat possessing the alien Lr genes.

Occurrence, identification and pathogenicity of Fusarium species associated with bakanae disease of basmati rice in India

The incidence of bakanae disease of rice was studied in the basmati growing states of Punjab, Haryana, Uttar Pradesh and Uttarakhand in India. Maximum disease incidence (20xa0%) was observed from the Karnal district of Haryana and Bulandshar, Ghaziabad and Mathura districts of Uttar Pradesh in rice variety Pusa Basmati 1121 and Pusa Basmati 1509. Disease was also observed in other popular basmati rice varieties of the country including Pusa Basmati 6, Pusa 2511, CSR 30, Pakistani Basmati and Sarbati with the incidence of 0.5xa0% to 10xa0%. One hundred and twenty-six isolates of Fusarium spp. were isolated from symptomatic plants and were characterized morphologically and 42 isolates were characterized at the molecular level. Out of 42 isolates characterized based on translation elongation factor 1 α (TEF- 1α), 41 were identified as Fusarium fujikuroi. Virulence analysis performed on rice variety Pusa Basmati 1509 categorized the isolates as moderately virulent (37.3xa0%), virulent (34.1xa0%) and highly virulent (28.6xa0%). Results indicated that F. fujikuroi is predominant in symptomatic diseased plants and a management programme should be developed by considering the prevalence and virulence of the pathogen in the area of intended cultivation.

Microbial detoxification of pathotoxin produced by spot blotch pathogen Bipolaris sorokiniana infecting wheat

Bipolaris sorokiniana causes spot blotch in wheat and barley. The pathogen produces toxin (BS-toxin), which is a sesquiterpenoid belonging to eremophilane family. Isolates of Trichoderma spp., Chaetomium globosum and Pseudomonas fluorescens were tested for detoxification of BS-toxin amended in semi-synthetic medium at different concentrations. All the antagonists showed mycelial growth in toxin amended medium but their growth was less in comparison to growth in normal medium. The growth of biocontrol agents decreased with increasing concentration of toxin. Two isolates of C. globosum (Cg1 and Cg2), T.viride (TV5-2) and Pseudomonas fluorescens produced 4.9, 2.9, 3.6xa0g mycelium and 5.5 × 105xa0cfu /ml, respectively exhibiting 50% or less reduction in growth in BS-toxin amended medium at 1,000xa0ppm concentration. The biocontrol agents also reduced the severity of toxin-induced symptoms and electrolyte leakage from the wheat leaf tissues. Among the microbes tested, maximum reduction in electrolyte leakage was observed in C. globosum (Cg2) treated toxin samples. The spectral analysis also showed a remarkable decrease in optical density of Cg2 treated toxin at 294xa0nm. High Performance Liquid Chromatography (HPLC) analysis showed almost complete degradation of BS-toxin in C. globosum (Cg2) treated samples.

Gene-based analysis of Puccinia species and development of PCR-based marker to detect Puccinia striiformis f. sp. tritici causing yellow rust of wheat

Stripe rust is considered as the current major rust disease affecting winter cereal production across the world. A quick, reliable PCR-based marker was developed here to detect, identify and rapidly monitor Puccinia striiformis f. sp. tritici (Pst) in wheat-growing areas. Three respective sets of primers, designed from β-tubulin, squalene monooxygenase and ketopantoate reductase genes selected from the full genome of Puccinia striiformis f. sp. tritici, amplified sequences of 239, 358 and 1518xa0bp, respectively, in Pst pathotypes. A fragment of 1518xa0bp unique to Pst pathotypes was amplified using primer set PstKeto F1_30/Pst KetoR1_1547 and distinguished the pathogen clearly from different Puccinia spp. and other fungal pathogens. The detection limit of the marker (KetoPstRA1500, accession no. KU240073) by conventional PCR assay was 10xa0pg. This marker could detect the pathogen in the host before symptom expression. The sensitivity and utility of the marker were further enhanced in a qPCR-based assay that was developed with a newly designed primer set PstKeto F1_1246/Pst KetoR1_1547, which amplified a product of 302xa0bp and detected as little as 10xa0fg of DNA. This PCR/qPCR based marker is suitable for studying cultivar resistance, which requires accurate quantification of the pathogen in diseased host tissue.

DEVELOPMENT OF CONVENTIONAL AND REAL TIME PCR ASSAY FOR THE RAPID DETECTION AND QUANTIFICATION OF A BIOCONTROL AGENT, CHAETOMIUM GLOBOSUM

Chaetomium globosum is a potential biocontrol agent against various seed and soil-borne pathogens. To ensure proper use of C. globosum in agriculture, accurate data is essential for population monitoring. A PCR-based marker has been developed for detection of this biocontrol agent, which will help to detect the fungus at the place of its application. Out of twelve URP primers tested against 15 isolates of C. globosum and other Chaetomium species, URP 2R amplified a monomorphic band of 1,900 bp only in C. globosum isolates. This amplicon was cloned and sequenced, and based on the sequence obtained, four primer sets were designed, one of which in PCR assays amplified a region (SCAR; SCCgRA1900) of the expected size (1.9 kb) in C. globosum isolates. The specific marker also detected the presence of C. globosum in soil, roots and leaves. The detection limit of marker in conventional PCR assay was 75 pg. The sensitivity and usefulness of SCAR marker was further enhanced by developing qPCR using the primer set SCCgQF/SCCgQR designed from SCCgRA1900, which detected as much as 1 pg of DNA (4.83x105 copy number of target DNA). The initial population of C. globosum in terms of target DNA in C. globosum-amended soil was equivalent to 2.5x108 copy number/g soil (0.51 ng target DNA/g soil) which increased approximately 10 times after 15 days of application i.e., 2x109copy number/g soil (3.1 ng/g soil). However, with, Bipolaris sorokiniana the quantity of C. globosum target DNA increased slowly reaching 4.32 x108 copy number/g soil after 15 days. Conventional PCR-based detection using SCAR marker and subsequent qPCR provided a rapid and reliable tool for efficient detection and monitoring of C. globosum at the site of its application.

Protein mapping of Chaetomium globosum, a potential biological control agent through proteomics approach

Chaetomium globosum is a ubiquitous filamentous fungus having biological control properties. The potential isolates mycoparasitize the pathogen and produce antifungal metabolites which suppress the growth of pathogenic fungi. A proteomics approach was undertaken to separate and identify proteins from a mycoparasitic strain Cg1 of C. globosum under normal and heat shock conditions in order to identify differentially expressed proteins. We developed and standardized the procedure for extraction of total proteins and 2D gel electrophoresis, which resulted in profiling of more than 100 protein spots. 48 proteins were identified by a combination of matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF) and liquid chromatography mass spectrometry (LCMS/MS). Out of total proteins identified, 79xa0% were hypothetical proteins and 21xa0% proteins were functionally characterized. Out of total 79xa0% hypothetical proteins 24xa0% proteins matched with C. globosum while 18xa0% proteins matched with Aspergillus spp., 13xa0% with Coprinopsis cinerea, 10xa0% with Giberrella zaea, 8xa0% with Magnaporthe grisea and 5xa0% with Neurospora crassa and Lodderomyces elonisporus. Some of the functionally characterized proteins included MAP kinase, maltose permease, GTP binding protein, dyenin heavy chain, HET- C2, vacuolar Dig A protein, polyketide synthase, peptide prolyl cis trans isomerase and translation elongation factor. This study has generated a protein reference map for Chaetomium globosum, and being the first report on proteomics studies would greatly help to unravel biocontrol mechanism and its survival under heat stress conditions.

Pathogenicity, Ecology and Genetic Diversity of the Fusarium spp. Associated with an Emerging Bakanae Disease of Rice ( Oryza sativa L.) in India

Bakanae disease is one of the emerging diseases of rice (Oryza sativa L.). Gibberella fujikuroi species complex were detected in popularly grown rice varieties of India with infection percentage ranging from 1 to 24 %. Pathogenicity test of Fusarium spp. was performed in susceptible rice variety Pusa 1121, which showed reduced seed germination and possessed varying ability to cause symptoms. On the basis of internal transcribed spacer (ITS) and Translation Elongation Factor (TEF), three Fusarium spp., viz. F. verticillioides, F. fujikuroi and F. proliferatum were found associated with bakanae disease of rice in India. Maximum numbers of slender and chlorotic leaves were produced by F. fujikuroi (90 %), whereas crown rot and stem rot was produced by F. verticillioides (50 %). F. proliferatum produced both elongation and rotting symptoms. Information on the bakanae disease, its distribution, characterization and identification in India could be helpful for the development of management strategies.