Genetic Analysis for Resistance to Sclerotinia Stem Rot, Yield and Its Component Traits in Indian Mustard [Brassica juncea (L.) Czern & Coss.].

Understanding the mode of gene action that controls seed yield and Sclerotinia stem rot resistance in Indian mustard is critical for boosting yield potential. In a line × tester mating design, ten susceptible lines and four resistant testers were used to conduct genetic analysis. The significance of general combining ability (GCA) and specific combining ability (SCA) variances revealed that both additive and non-additive gene actions were involved in the inheritance of Sclerotinia stem rot resistance and yield attributing traits. In addition to 1000-seed weight and number of primary and secondary branches/plant, the genotypes RH 1569 (line) and DRMR 2035 (tester) appeared to be the strongest general combiners for Sclerotinia stem rot resistance. RH 1657 × EC 597317 was the only cross among several that demonstrated a significant desired SCA value for Sclerotinia rot resistance. Regarding SCA effects for yield and component traits, the cross RH 1658 × EC 597328 performed best, with a non-significant but acceptable negative SCA effect for resistance. DRMR 2035, RH 1222-28, RH 1569, RH 1599-41, RH 1657, RH 1658, and EC 597328 are promising genotypes to use as parents in future heterosis breeding and for obtaining populations with high yield potential and greater resistance to Sclerotinia stem rot disease in Indian mustard, based on GCA effects of parents, per se performance, and SCA effects of hybrids. Days to 50% flowering, number of primary branches/plant, main shoot length, and 1000-seed weight all had a high genotypic coefficient of variability (GCV), broad-sense heritability (h2bs), and genetic advance as percent of the mean (GAM) values, as well as significant and desirable correlations and direct effects on seed yield. As a result, these traits have been recognized as the most critical selection criterion for Indian mustard breeding programs.

Genetic and Proteomic Basis of Sclerotinia Stem Rot Resistance in Indian Mustard [Brassica juncea (L.) Czern & Coss.].

Sclerotinia stem rot is one of the utmost important disease of mustard, causing considerable losses in seed yield and oil quality. The study of the genetic and proteomic basis of resistance to this disease is imperative for its effective utilization in developing resistant cultivars. Therefore, the genetic pattern of Sclerotinia stem rot resistance in Indian mustard was studied using six generations (P1, P2, F1, F2, BC1P1, and BC1P2) developed from the crossing of one resistant (RH 1222-28) and two susceptible (EC 766300 and EC 766123) genotypes. Genetic analysis revealed that resistance was governed by duplicate epistasis. Comparative proteome analysis of resistant and susceptible genotypes indicated that peptidyl-prolyl cis-trans isomerase (A0A078IDN6 PPIase) showed high expression in resistant genotype at the early infection stage while its expression was delayed in susceptible genotypes. This study provides important insight to mustard breeders for designing effective breeding programs to develop resistant cultivars against this devastating disease.

First report of Klebsiella aerogenes Inciting Stem Rot of Pearl Millet in Haryana, India.

Pearl millet [Pennisetum glaucum (L.) R. Br. Syn. Pennisetum americanum (L.) Leeke] is the oldest and widely cultivated millet in Asian and African countries, mostly grown over low fertile soils in more than 40 countries covering an area of 312.00 lakh hectares (FAOSTAT 2017). In Haryana, crop was grown over an area of 4.30 lakh hectares during Kharif 2019. Pearl millet is prone to many fungal and bacterial diseases. During 2018 to 2020, a new devastating diseas exhibiting stem rot like symptoms was observed in pearl millet growing regions in Indian state of Haryana. The isolated disease causing agent was a bacterium, where 16S rDNA-based nucleotide sequence deposited in NCBI GenBank (Accession nos. MZ433194.1) conferred its nearness to Klebsiella aerogenes (Hormaeche and Edwards 1960) Tindall et al. 2017. Further, DNA gyrase genomic sequence (NCBI Accession nos. MZ707528.1) also stayed its high homology to K. aerogenes. Klebsiella usually known to cause diseases in humans and animals, and also has been found inciting different kind of rots in different plantations viz. top rot in maize (Huang Min et al. 2016). Pearl millet is susceptible to minor bacterial diseases viz. bacterial leaf streak (Xanthomonas campestris), bacterial leaf spot (Pseudomonas syringae) and leaf stripe (P. avenae). Earlier, among the plant pathogenic bacterial entirety, only Erwinia chrysanthemi is known to cause stem rot diseases in sorghum (Saxena et al. 1991) amongst different types of millet. Extensive disease survey of pearl millet growing regions (Hisar, Bhiwani, Rewari, Mohindergarh and Bawal districts of Haryana having an altitude of 215, 225, 245, 262 and 266 m, respectively) in rainy seasons of 2019 and 2020 revealed the prevalence of typical stem rot disease, representing up to 70% disease incidence in the infected fields. The pieces of symptomatic stem of different plants were collected from two locations (Hisar and Bhiwani) and associated organism was isolated following the techniques of Janse (2005). The resulting growth of bacterial cultures were further purified on nutrient agar (NA) media using streak plate technique where colony growth of both the isolates were observed as morphotypes. The resulting bacteria were gram-negative and rod-shaped. Colonies were round and creamish white on NA. Isolated morphotypes were positive for indole production, methyl red, Voges Proskauer's test, citrate utilization, arabinose, mannitol, rhamnose and sucrose, whereas negative for glucose, adonitol, lactose and sorbitol tests. Biochemical tests were performed following standard methods (Holt et al. 1994). Molecular analysis of both isolates was performed using two sets of primers (universal 16S rRNA gene and genus-specific gyrA gene). The gyrA fragment (F: 5'-CGCGTACTATACGCCATGAACGTA-3'; R: 5'-ACCGTTGATCACTTCGGTCAGG-3') has been adopted as Klebsiella genus-specific gene (Brisse and Verhoef 2001). The quality and quantity of the isolated genomic DNA were analyzed using NanoDrop-2000 (Thermo Fisher Scientific, USA) and resolved in 1% (w/v) agarose gel. Thereafter, visualized in gel documentation to confirm a single band of high-molecular-weight DNA. The fragment 16S rDNA was amplified using 27F and 1492R primers, where a single discrete PCR amplicon of 1500 bp was observed in 1% (w/v) agarose gel. Similarly, the gyrA gene was amplified using 09510F and 09510R primers that conferred a single discrete band of 400 bp. The forward and reverse DNA sequencing reaction of purified PCR amplicons (16S rDNA and gyrA) was carried out using BDT v3.1 Cycle sequencing kit on a genetic analyzer to generate gene sequences. The consensus sequences of both gene were generated from forward and reverse sequences data using aligner software. The obtained sequences of both genes were compared with the available nucleotide sequences in the NCBI using the blast 2.2.9 system (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch). The sequenced PCR amplicons showed up to 100% similarity with Klebsiella aerogenes 16s RNA nucleotide sequences (Accession nos. NR102493.2, MT373521.1; MF682950.1; MF462979.1 etc.). The bacterium also showed high nucleotide homology to K. aerogenes gyrA gene sequences (Accession nos. LR607333.1; CP035466.1; CP049600.1 etc.). The molecular phylogenetic analysis was done by the maximum likelihood method based on the Tamura-Nei model, and 1000 replicates for bootstrap testing in MEGA 7.0 software. The analysis involved 16 nucleotide sequences and evolutionary distances were computed. The 16s RNA based phylogenetic tree raised using MEGA7 (Kumar et al. 2016) elucidates that Klebsiella aerogenes Hisar formed a cluster with three K. aerogenes strains (Accession nos. MZ577128.1, MT373521.1 and MT 373520.1), whereas K. aerogenes Bhiwani displayed higher homology to NCBI sequences viz. MF682950.1, MT355368.1, MW331687.1and LC515412.1. Bacterial suspension was prepared by suspending bacterial cells into sterile water and cell density was adjusted to 1×107 colony forming unit/ml. For pathogenicity, leaf whorl inoculation (10 ml suspension/ whorl) was done on 15 days old seedlings of pearl millet genotype 7042S raised under controlled conditions (Temperature 35±2°C and more than 80% Relative Humidity). The pathogenicity was proved under field conditions as well. Initial symptoms were observed 4-5 days after inoculation as long streaks on leaves. Soon a spike in number of these leaf streaks was observed. Thereafter, water-soaked lesions appeared on the stem at 20-25 days after inoculation which later on turned brown to black. Severely diseased plants were dead, exhibiting hollowing of the stem and drying of leaves. The infected stem pith disintegrated and showed slimy rot symptoms and the pearl millet clumps toppled down. The rotten stems of both inoculations were again cut in to small pieces and the reisolated bacterium showed exactly the same morphological, biochemical and molecular characteristics. To our knowledge, this is the first report of stem rot of pearl millet incited by K. aerogenes in south-western regions of Haryana, India. Because the stem rot caused by K. aerogenes poses a significant threat to pearl millet cultivation, further research on biology, epidemiology and management choices is needed.

Genotype-Specific Antioxidant Responses and Assessment of Resistance Against Sclerotinia sclerotiorum Causing Sclerotinia Rot in Indian Mustard.

Productivity of Indian mustard, an important oilseed crop of India, is affected by several pathogens. Among them, the hemibiotroph Sclerotinia sclerotiorum, which causes sclerotinia rot disease, is the most devastating fungal pathogen causing up to 90% yield losses. The availability of host resistance is the only efficient approach to control and understand the host-pathogen interaction. Therefore, the present investigation was carried out using six Indian mustard genotypes with contrasting behavior towards sclerotinia rot to study the antioxidant resistance mechanism against S. sclerotiorum. The plants at post-flowering stage were inoculated with five-day-old pure culture of S. sclerotiorum using artificial stem inoculation method. Disease evaluation revealed significant genotypic differences for mean lesion length among the tested genotypes, where genotype DRMR 2035 was found highly resistant, while genotypes RH 1569 and RH 1633 were found highly susceptible. The resistant genotypes had more phenolics and higher activities of peroxidase, catalase and polyphenol oxidase which provide them more efficient and strong antioxidant systems as compared with susceptible genotypes. Studies of antioxidative mechanisms validate the results of disease responses.

Morphological and molecular diversity of Sclerotinia sclerotiorum infecting Indian mustard.

Fourteen isolates of Sclerotinia sclerotiorum were collected from different locations of mustard growing regions of India and were studied for cultural, morphological and molecular variability at CCS HAU, Hisar. Variability was observed for colony colour, type of growth, diameter of mycelial growth, sclerotia initiation, number and pattern of sclerotia formation among the isolates. Mycelial growth and sclerotia initiation were faster in Bhiwani isolate as compared to others. Bhiwani isolate was found to be the most diverse and had least similarity with Chhanibari isolate on the basis of molecular variability. Hence, morphological and cultural variability observed in the present investigation is by and large strongly correlated to molecular marker based variability.

Physicochemical, morphological, thermal and pasting characteristics of starches from moth bean (Vigna aconitifolia) cultivars grown in India: an underutilized crop.

This is a first kind of study on genotype diversity of starches of Moth Bean an underutilized pulse of India. Physicochemical properties like amylose content (7.8-21.4%), swelling power (11-13.5 g/g), solubility (5.9-9.0%) of starches were observed to differ significantly among the six moth bean starches. Swelling power of all the moth bean starches was observed to increase in the temperature range of 55-95 °C. Scanning electron microscopy indicated polyhedral, irregular shape of granule. X-ray diffraction studies indicated a 'C' type crystalline structure and the starches differed significantly in relative crystallinity (17-34%) which affected significantly retro gradation tendencies of the starches. Peak viscosity of starches varied significantly and ranged between 4580 and 5087 cP. Resistant starch content of starches also varied significantly among the cultivars and ranged between 57.3 and 75.6%.