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.

Identification of heterotic groups in South-Asian-bred hybrid parents of pearl millet.

KEY MESSAGE: Pearl millet breeding programs can use this heterotic group information on seed and restorer parents to generate new series of pearl millet hybrids having higher yields than the existing hybrids. Five hundred and eighty hybrid parents, 320 R- and 260 B-lines, derived from 6 pearl millet breeding programs in India, genotyped following RAD-GBS (about 0.9 million SNPs) clustered into 12 R- and 7 B-line groups. With few exceptions, hybrid parents of all the breeding programs were found distributed across all the marker-based groups suggesting good diversity in these programs. Three hundred and twenty hybrids generated using 37 (22 R and 15 B) representative parents, evaluated for grain yield at four locations in India, showed significant differences in yield, heterosis, and combining ability. Across all the hybrids, mean mid- and better-parent heterosis for grain yield was 84.0% and 60.5%, respectively. Groups G12 B × G12 R and G10 B × G12 R had highest heterosis of about 10% over best check hybrid Pioneer 86M86. The parents involved in heterotic hybrids were mainly from the groups G4R, G10B, G12B, G12R, and G13B. Based on the heterotic performance and combining ability of groups, 2 B-line (HGB-1 and HGB-2) and 2 R-line (HGR-1 and HGR-2) heterotic groups were identified. Hybrids from HGB-1 × HGR-1 and HGB-2 × HGR-1 showed grain yield heterosis of 10.6 and 9.3%, respectively, over best hybrid check. Results indicated that parental groups can be formed first by molecular markers, which may not predict the best hybrid combination, but it can reveal a practical value of assigning existing and new hybrid pearl millet parental lines into heterotic groups to develop high-yielding hybrids from the different heterotic groups.

Phenotypic Data from Inbred Parents Can Improve Genomic Prediction in Pearl Millet Hybrids.

Pearl millet is a non-model grain and fodder crop adapted to extremely hot and dry environments globally. In India, a great deal of public and private sectors' investment has focused on developing pearl millet single cross hybrids based on the cytoplasmic-genetic male sterility (CMS) system, while in Africa most pearl millet production relies on open pollinated varieties. Pearl millet lines were phenotyped for both the inbred parents and hybrids stage. Many breeding efforts focus on phenotypic selection of inbred parents to generate improved parental lines and hybrids. This study evaluated two genotyping techniques and four genomic selection schemes in pearl millet. Despite the fact that 6× more sequencing data were generated per sample for RAD-seq than for tGBS, tGBS yielded more than 2× as many informative SNPs (defined as those having MAF > 0.05) than RAD-seq. A genomic prediction scheme utilizing only data from hybrids generated prediction accuracies (median) ranging from 0.73-0.74 (1000-grain weight), 0.87-0.89 (days to flowering time), 0.48-0.51 (grain yield) and 0.72-0.73 (plant height). For traits with little to no heterosis, hybrid only and hybrid/inbred prediction schemes performed almost equivalently. For traits with significant mid-parent heterosis, the direct inclusion of phenotypic data from inbred lines significantly (P < 0.05) reduced prediction accuracy when all lines were analyzed together. However, when inbreds and hybrid trait values were both scored relative to the mean trait values for the respective populations, the inclusion of inbred phenotypic datasets moderately improved genomic predictions of the hybrid genomic estimated breeding values. Here we show that modern approaches to genotyping by sequencing can enable genomic selection in pearl millet. While historical pearl millet breeding records include a wealth of phenotypic data from inbred lines, we demonstrate that the naive incorporation of this data into a hybrid breeding program can reduce prediction accuracy, while controlling for the effects of heterosis per se allowed inbred genotype and trait data to improve the accuracy of genomic estimated breeding values for pearl millet hybrids.