Marker-assisted enhancement of bacterial blight (Xanthomonas oryzae pv. oryzae) resistance in a salt-tolerant rice variety for sustaining rice production of tropical islands.

Introduction: Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae is a major disease of rice, specially in the tropical regions of the world. Developing rice varieties with host resistance against the disease is the most effective and economical solution for managing the disease. Methods: Pyramiding resistance genes (Xa4, xa5, xa13,and Xa21) in popular rice varieties using marker-assisted backcross breeding (MABB) has been demonstrated as a cost-effective and sustainable approach for establishing durable BB resistance. Here, we report our successful efforts in introgressing four resistance genes (Xa4, xa5, xa13, and Xa21) from IRBB60 to CARI Dhan 5, a popular salt-tolerant variety developed from a somaclonal variant of Pokkali rice, through functional MABB. Results and discussion: Both BB and coastal salinity are among the major challenges for rice production in tropical island and coastal ecosystems. Plants with four, three, and two gene pyramids were generated, which displayed high levels of resistance to the BB pathogen at the BC3F2 stage. Under controlled salinity microplot environments, the line 131-2-175-1223 identified with the presence of three gene pyramid (Xa21+xa13+xa5) displayed notable resistance across locations and years as well as exhibited a salinity tolerance comparable to the recurrent parent, CARI Dhan 5. Among two BB gene combinations (Xa21+xa13), two lines, 17-1-69-334 and 46-3-95-659, demonstrated resistance across locations and years, as well as salt tolerance and grain production comparable to CARI Dhan 5. Besides salinity tolerance, five lines, 17-1-69-179, 46-3-95-655, 131-2-190-1197, 131-2-175-1209, and 131-2-175-1239, exhibited complete resistance to BB disease. Following multilocation testing, potential lines have been identified that can serve as a prospective candidate for producing varieties for the tropical Andaman and Nicobar Islands and other coastal locations, which are prone to BB and coastal salinity stresses.

Expression profiling and characterization of key RGA involved in lentil Fusarium wilt Race 5 resistance.

Fusarium wilt is a major threat to lentil production in India and worldwide. The presence of evolving virulent races has imposed the necessity of reliable management practices including breeding for resistance using unexplored germplasms. The magnitude of resistance by the plant is determined by rapid recognition of the pathogen and induction of defence genes. Resistance gene analogues have been key factors involved in the recognition and induction of defence response. In the present study, the expression of key RGA previously cloned was determined in three resistant accessions (L65, L83 and L90) and a susceptible accession (L27). The expression was assessed via qPCR at 24, 48 and 72 hpi against virulent race5 (CG-5). All the RGAs differentially transcribed in resistant and susceptible accession showed temporal variation. RGA Lc2, Lc8, Ln1 and Lo6 produced cDNA signals during early infection (24 hpi) predicting its involvement in recognition. LoRGA6 showed significant upregulation in L65 and L83 while downregulating in L27 and the full length of LoRGA6 loci was isolated by 5' and 3' RACE PCR. In-silico characterization revealed LoRGA6 loci code for 912 amino acids long polypeptide with a TIR motif at the N terminal and eight LRR motifs at the C terminal. The tertiary structure revealed a concave pocket-like structure at the LRR domain potentially involved in pathogen effectors interaction. The loci have ADP binding domain and ATPase activity. This has further paved the path for functional analysis of the loci by VIGS to understand the molecular mechanism of resistance.

Quadruplex and q-PCR based diagnostic assay to delineate the major quarantine and other seed-borne fungal pathogens of soybean.

Soybean is one of the most important crops grown worldwide and accounting for significant global trade including transgenic soybean. The crop is attacked by several seed-borne fungal pathogens and some of them are of quarantine concern for India. Keeping in view of the risks associated with movement of soybean seeds, sensitive and reliable molecular diagnostics have been developed for precise and simultaneous detection of three pathogens of quarantine concern for India namely, Diaporthe phaseolorum (stem blight), D. longicolla (seed decay), Peronospora manshurica (downy mildew), along with Macrophomina phaseolina causing dry root rot. The targeted pathogens after isolation from imported transgenic and non-transgenic soybean seeds were identified. Quadruplex and qPCR assays were developed targeting the sequences of different genes such as Histone-3 for detection of D. longicolla and M. phaseolina. The markers DlHisF2&R2 and MpHisF1&R1 produced 265 and 309 bp amplicons for D. longicolla and M. phaseolina, respectively. Actin gene based marker DpActF1&R2 was developed for D. phaseolorum which provided 113 bp amplicon whereas, COX2 based marker PmCoxF2&R2 was developed for P. manshurica with amplified product of 152 bp. During qPCR analysis, these markers proved highly specific and sensitive for detection of these pathogens up to 0.1 pg of template DNA. Quadruplex PCR protocol was also developed by combining these specific markers which could distinguish all the targeted pathogens simultaneously in a single reaction. The developed diagnostic protocols are extremely valuable for quarantine clearance and to ensure the safe transboundary exchange and healthy conservation of germplasm in the National Genebank.

Development of PCR-based assays to diagnose the major fungal pathogens infecting pulse crops, potential for germplasm health certification and quarantine processing.

Plant diseases cause serious economic losses of agriculture production worldwide. Rapid, accurate and reliable diagnostic methods are required to alleviate the detection of fungal plant pathogens to prevent their spread and achieve effective management. This study was aimed to develop fast, reliable and highly sensitive diagnostics to detect fungal plant pathogens for quarantine processing, safe exchange and conservation of germplasms of pulse crops. Multiplex and real time PCR assays were developed for detection of Rhizoctonia solani, Macrophomina phaseolina, Ascochyta rabiei, Alternaria alternata, A. tenuissima, Fusarium oxysporum f. sp. ciceris, Sclerotium (Athelia) rolfsii, Sclerotinia sclerotiorum, Pseudocercospora cruenta and Cercospora canescens causing various diseases in pulse crops. Twenty-two sets of primers from various genomic regions such as cytochrome oxidase subunit (COX 1), internal transcribed spacer region (ITS), translation elongation factor-1 alpha (TEF-1α), large subunit (LSU), small subunit (SSU) and ß-tubulin as well as two SCAR primers from RAPD profile were designed. The developed markers proved to be species-specific and validated against other fungal plant pathogens associated with pulses for cross-reactivity. The markers proved highly sensitive during conventional and qPCR analysis. Duplex PCR assays for R. solani and M. phaseolina; C. canescens and P. cruenta; A. alternata and A. tenuissima; and a quadruplex PCR assay for A. rabiei, S. sclerotiorum, S. rolfsii and F. oxysporum f. sp. ciceris were developed and validated for simultaneous detection of these pathogens in a single reaction. The assays developed in the present study were able to detect and identify major fungal plant pathogens causing disease in pulse crops.

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.

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.

Genetic diversity analysis of fungal pathogen Bipolaris sorghicola infecting Sorghum bicolor in India.

Bipolaris sorghicola (Lefebvre and Sherwin) is a well known and economically important seed-borne pathogen with the specific species of sorghum (Sorghum bicolor [L] Moench) as host. Thirty-two strains were obtained from different geographical area of sorghum growing places in India. Molecular characterization using three marker systems i.e., universal rice primers (URP), inter simple sequence repeat (ISSR) and random amplified polymorphic DNA (RAPD) was carried out. Molecular marker work revealed differences along with geographical origin clustering of various B. sorghicola strains which could not be revealed through conventional method of characterization. Out of 13 URPs, 20 ISSR and 50 RAPD primers screened, 8 primers each from URP and ISSR, and 10 primers from RAPD marker were found to result in reproducible banding pattern. One hundred per cent of polymorphic bands was recorded in all three molecular markers. Total number of bands was recorded 1986 with average of 248.25 in URP marker, and 2026 bands with average of 253.25 in ISSR marker and 2158 bands with average of 215.80 in RAPD markers. Maximum heterozygosity (Hn) was revealed by URP 17R (0.40), ISSR 10 (0.41) and RAPD marker OPC-5 (0.34). The polymorphism information content (PIC) values ranged between 5.89 to 8.28 in URP, 4.57 to 8.79 in ISSR and 4.44 to 9.64 in RAPD marker profiles. Maximum cophenetic correlation was found in URP (r = 0.910) followed by ISSR (r = 0.904) and RAPD (r = 0.870). The combined analysis of all three marker systems showed high cophenetic correlation (r = 0.911), which indicated a very good fit of the data for genetic diversity analysis. To best of our knowledge, this is a first report of genetic characterization of B. sorghicola. Hence, combined use of three marker systems would be more sensitive and reliable in characterizing genetic variability in B. sorghicola strains.

Modern spectroscopic technique in the characterization of biosensitive macrocyclic Schiff base ligand and its complexes: inhibitory activity against plantpathogenic fungi.

Complexes of the type [M(L)Cl2], where M = Co(II), Ni(II) and Cu(II) have been synthesized with a macrocyclic Schiff base ligand (1,4,5,7,10,11,12,15-octaaza,5,11,16,18-tetraphenyl, 3,4,12,13-tetramethyl cyclo-octadecane) derived from Schiff base (obtained by the condensation of 4-aminoantipyrine and dibenzoyl methane) and ethylenediamine. The ligand was characterized on the basis of elemental analysis, IR, (1)H NMR, EI Mass and molecular modeling studies while the complexes were characterized by elemental analysis, molar conductance measurements, magnetic susceptibility measurements, IR, electronic and EPR spectral studies. All the complexes are non-electrolyte in nature. The covalency factor (ß) and coefficient factor (α) suggest the covalent nature of the complexes. The ligand and its metal complexes have shown antifungal activity with their LD50 values determined by probit analysis against two economically important fungal plant pathogens i.e. Macrophomina phaseolina and Fusarium solani.