Comprehensive genomic analysis of Bacillus subtilis and Bacillus paralicheniformis associated with the pearl millet panicle reveals their antimicrobial potential against important plant pathogens.

BACKGROUND: Plant microbiome confers versatile functional roles to enhance survival fitness as well as productivity. In the present study two pearl millet panicle microbiome member species Bacillus subtilis PBs 12 and Bacillus paralicheniformis PBl 36 found to have beneficial traits including plant growth promotion and broad-spectrum antifungal activity towards taxonomically diverse plant pathogens. Understanding the genomes will assist in devising a bioformulation for crop protection while exploiting their beneficial functional roles. RESULTS: Two potential firmicute species were isolated from pearl millet panicles. Morphological, biochemical, and molecular characterization revealed their identities as Bacillus subtilis PBs 12 and Bacillus paralicheniformis PBl 36. The seed priming assays revealed the ability of both species to enhance plant growth promotion and seedling vigour index. Invitro assays with PBs 12 and PBl 36 showed the antibiosis effect against taxonomically diverse plant pathogens (Magnaporthe grisea; Sclerotium rolfsii; Fusarium solani; Alternaria alternata; Ganoderma sp.) of crops and multipurpose tree species. The whole genome sequence analysis was performed to unveil the genetic potential of these bacteria for plant protection. The complete genomes of PBs 12 and PBl 36 consist of a single circular chromosome with a size of 4.02 and 4.33 Mb and 4,171 and 4,606 genes, with a G + C content of 43.68 and 45.83%, respectively. Comparative Average Nucleotide Identity (ANI) analysis revealed a close similarity of PBs 12 and PBl 36 with other beneficial strains of B. subtilis and B. paralicheniformis and found distant from B. altitudinis, B. amyloliquefaciens, and B. thuringiensis. Functional annotation revealed a majority of pathway classes of PBs 12 (30) and PBl 36 (29) involved in the biosynthesis of secondary metabolites, polyketides, and non-ribosomal peptides, followed by xenobiotic biodegradation and metabolism (21). Furthermore, 14 genomic regions of PBs 12 and 15 of PBl 36 associated with the synthesis of RiPP (Ribosomally synthesized and post-translationally modified peptides), terpenes, cyclic dipeptides (CDPs), type III polyketide synthases (T3PKSs), sactipeptides, lanthipeptides, siderophores, NRPS (Non-Ribosomal Peptide Synthetase), NRP-metallophone, etc. It was discovered that these areas contain between 25,458 and 33,000 secondary metabolite-coding MiBiG clusters which code for a wide range of products, such as antibiotics. The PCR-based screening for the presence of antimicrobial peptide (cyclic lipopeptide) genes in PBs 12 and 36 confirmed their broad-spectrum antifungal potential with the presence of spoVG, bacA, and srfAA AMP genes, which encode antimicrobial compounds such as subtilin, bacylisin, and surfactin. CONCLUSION: The combined in vitro studies and genome analysis highlighted the antifungal potential of pearl millet panicle-associated Bacillus subtilis PBs12 and Bacillus paralicheniformis PBl36. The genetic ability to synthesize several antimicrobial compounds indicated the industrial value of PBs 12 and PBl 36, which shed light on further studies to establish their action as a biostimulant for crop protection.

First report of Neopestalotiopsis clavispora causing cashew leaf blight disease in India.

Cashew (Anacardium occidentale) is an important commercial crop and highly prone to many biotic and abiotic stress. During March 2021, severe leaf blight symptoms were observed in Priyanka variety with 25-30% incidence grown under greenhouse nursery at ICAR-Directorate of Cashew Research (ICAR-DCR), Puttur (12º74'08.92"N; 75º22'97.22"E), Karnataka. Initial symptoms include small, irregular necrotic spots and later, the spots enlarged and covered major portion of the leaf lamina. In severe infection, leaves exhibited coalescing of spots leading to blight appearance. The infected leaves were randomly collected (n=5) and surface sterilized with 1% sodium hypochlorite for 1 min followed by three washes in sterile distilled water (SDW). Samples were plated on PDA plates amended with Rifampicin (40 mg/L) and kept for incubation at 25±2 oC for 5 days (12/12 h dark light period). A white-greyish, aerial, cottony mycelium on upper side with light yellow colour on the reverse side was consistently isolated. The black viscous acervuli were observed after 10-12 days of incubation. The conidia were fusiform, five-celled, versicoloured with three olivaceous brown median cells, two terminal hyaline cells, measured 23.3±2.12 - 28.33±2.7 x 3.6±0.8 - 4.28±0.78 µm (n=30). The apical cells had two to three flexuous, unbranched appendages, and basal appendage was solitary, tubular and unbranched. Morphological and cultural characteristics confirmed the pathogen as Neopestalotiopsis sp. (Maharachchikumbura et al. 2012). Further, two representative isolates (CLB_SCN1 & CLB_SCN2) were subjected for molecular characterization selected for molecular identification based on ITS-rDNA, tef-1α and tub2 gene sequences and phylogenetic analysis. Genomic DNA was isolated from 15 days old cultures and internal transcribed spacer (ITS) of ribosomal DNA (rDNA) (White et al. 1990), translation elongation factor 1α (tef-1α) gene (O'Donnell et al. 1998) and beta tubulin (tub2) using ITS1/ITS4, TEF1/TEF2 and Bt2a/Bt2b (Carbone and Kohn 1999; Glass and Donaldson 1995) were amplified using primer pairs respectively. PCR amplicons were sequenced, and the sequences were deposited in GenBank (accession numbers: ITS: OP880881.1, OP880882.1; tef-1α: OP882579.1, OP882580.1; and tub2: OP882581., OP882582.1). The phylogeny was constructed based on combined ITS, tef-1a, and tub2 regions. Neighbour-Joining (NJ) analysis was conducted and the tree was constructed with the substitution models (branch support was evaluated by 1,000 bootstrap replications). Combined phylogeny confirmed that the sequences shared a common clade with N. clavispora. Hence, morphological, microscopic and molecular characterization confirmed the pathogen as N. clavispora. The pathogenicity test was done on six months old healthy grafts of Priyanka variety (n=9) and repeated thrice. Conidial suspension (2×106 spores/ml) of N. clavispora CLB_SCN1 (15 days old culture) was sprayed on the healthy cashew seedlings, and kept in greenhouse by covering with polythene bags for 24 h (>80 % RH) and maintained under greenhouse condition. The control grafts were inoculated with SDW. The inoculated plants showed blight symptoms after 7-10-day post inoculation and control remained heathy. Re-isolation was done from the symptomatic leaves and identity was confirmed using cultural and molecular studies. Earlier reports showed that, N. clavispora has been reported to cause cardamom leaf blight (Biju et al 2018) and leaf spot disease of plum (Banerjee and Rana 2020). To best of our knowledge, this is the first report of cashew leaf blight disease caused by N. clavispora from India (Farr and Rossman, 2022). Early detection will help farmer in better management and avoiding economic loss caused by N. clavispora.

Stability Analysis and Identification of Superior Hybrids in Pearl Millet [Pennisetum glaucum (L.) R. Br.] Using the Multi Trait Stability Index.

Pearl millet stands as an important staple food and feed for arid and semi-arid regions of India and South Africa. It is also a quick supplier of important micronutrients like Fe and Zn via grain to combat micronutrient deficiencies among people in developing countries. India has notably spearheaded advancements in pearl millet production and productivity through the All India Coordinated Pearl Millet Improvement Project. There were 21 hybrids evaluated over arid and semi-arid ecologies of the western and southern regions of India. AMMI and GGE biplot models were adopted to recommend a specific hybrid for the particular locality. A joint analysis of variation indicated a significant genotype-environment interaction for most of the agronomical and grain micronutrient parameters. Pearson's correlation values dissected the significant and positive correlation among agronomic traits and the negative correlation with grain micronutrient traits. GGE biplot analysis recommended the SHT 106 as a dual-purpose hybrid and SHT 115 as a biofortified hybrid for the grain's Fe and Zn content. SHT 110 and SHT 108 were selected as stable and high grain yield-producing hybrids across all environments and specifically for E1, E2, and E4 as per the Which-Won-Where and What biplot. SHT 109 and SHT 103 hybrids were stable and high dry fodder yield-producing hybrids across all environments. In this study, the Multi-Trait Stability Index (MTSI) was employed to select the most stable and high-performing hybrids for all traits. It selected SHT 120, SHT 106, and SHT 104 for stability and great performance across all environments. These findings underscored the significance of tailored hybrid recommendations and the potential of pearl millet in addressing both food security and malnutrition challenges in various agro-ecological regions.

Morpho-biochemical characterization and molecular marker based genetic diversity of pearl millet (Pennisetum glaucum (L.) R. Br.).

Pearl millet is a key food for millions living in semi-arid and arid regions and is a main diet for poorer populations. The genetic diversity existing in the pearl millet germplasm can be used to improve the micronutrient content and grain yield. Effective and organized exploitation of diversity at morphological and DNA levels is the strategy for any crop improvement program. In this study, the genetic diversity of 48 pearl millet genotypes was evaluated for eight morphological traits and eleven biochemical characters. All genotypes were also characterized using twelve SSR and six SRAP markers to evaluate genetic diversity. The significant mean difference between morphological and biochemical traits were detected. The productive tillers per plant varied from 2.65 to 7.60 with a mean of 4.80. The grain yield of genotypes varied more than 3× from 15.85 g (ICMR 07222) to 56.75 g (Nandi 75) with an average of 29.54 g per plant. Higher levels of protein, iron, and zinc contents were found to be present in ICMR 12555 (20.6%), ICMR 08666 (77.38 ppm), and IC 139900 (55.48 ppm), respectively, during the experiment. Substantial variability was observed for grain calcium as it ranged from 100.00 ppm (ICMR 10222) to 256.00 ppm (ICMR 12888). The top eight nutrient-dense genotypes flowered in 34-74 days and had 5.71-9.39 g 1,000 grain weight. Genotype ICMR 08666 was superior for Fe, Zn, K and P. The inter-genotype similarity coefficient at the genetic level, generated using DNA markers, ranged from 0.616 to 0.877 with a mean of 0.743. A combination of morpho-biochemical traits and DNA markers based diversity may help to differentiate the genotypes and diverse genotypes can be used in breeding programs to improve the mineral content in pearl millet.

Stage specific comparative transcriptomic analysis to reveal gene networks regulating iron and zinc content in pearl millet [Pennisetum glaucum (L.) R. Br.].

Pearl millet is an important staple food crop of poor people and excels all other cereals due to its unique features of resilience to adverse climatic conditions. It is rich in micronutrients like iron and zinc and amenable for focused breeding for these micronutrients along with high yield. Hence, this is a key to alleviate malnutrition and ensure nutritional security. This study was conducted to identify and validate candidate genes governing grain iron and zinc content enabling the desired modifications in the genotypes. Transcriptome sequencing using ION S5 Next Generation Sequencer generated 43.5 million sequence reads resulting in 83,721 transcripts with N50 of 597 bp and 84.35% of transcripts matched with the pearl millet genome assembly. The genotypes having high iron and zinc showed differential gene expression during different stages. Of which, 155 were up-regulated and 251 were down-regulated while during flowering stage and milking stage 349 and 378 transcripts were differentially expressed, respectively. Gene annotation and GO term showed the presence of transcripts involved in metabolic activities associated with uptake and transport of iron and zinc. Information generated will help in gaining insights into iron and zinc metabolism and develop genotypes with high yield, grain iron and zinc content.

Pearl Millet: A Climate-Resilient Nutricereal for Mitigating Hidden Hunger and Provide Nutritional Security.

Pearl millet [Pennisetum glaucum (L.) R. Br.] is the sixth most important cereal crop after rice, wheat, maize, barley and sorghum. It is widely grown on 30 million ha in the arid and semi-arid tropical regions of Asia and Africa, accounting for almost half of the global millet production. Climate change affects crop production by directly influencing biophysical factors such as plant and animal growth along with the various areas associated with food processing and distribution. Assessment of the effects of global climate changes on agriculture can be helpful to anticipate and adapt farming to maximize the agricultural production more effectively. Pearl millet being a climate-resilient crop is important to minimize the adverse effects of climate change and has the potential to increase income and food security of farming communities in arid regions. Pearl millet has a deep root system and can survive in a wide range of ecological conditions under water scarcity. It has high photosynthetic efficiency with an excellent productivity and growth in low nutrient soil conditions and is less reliant on chemical fertilizers. These attributes have made it a crop of choice for cultivation in arid and semi-arid regions of the world; however, fewer efforts have been made to study the climate-resilient features of pearl millet in comparison to the other major cereals. Several hybrids and varieties of pearl millet were developed during the past 50 years in India by both the public and private sectors. Pearl millet is also nutritionally superior and rich in micronutrients such as iron and zinc and can mitigate malnutrition and hidden hunger. Inclusion of minimum standards for micronutrients-grain iron and zinc content in the cultivar release policy-is the first of its kind step taken in pearl millet anywhere in the world, which can lead toward enhanced food and nutritional security. The availability of high-quality whole-genome sequencing and re-sequencing information of several lines may aid genomic dissection of stress tolerance and provide a good opportunity to further exploit the nutritional and climate-resilient attributes of pearl millet. Hence, more efforts should be put into its genetic enhancement and improvement in inheritance to exploit it in a better way. Thus, pearl millet is the next-generation crop holding the potential of nutritional richness and the climate resilience and efforts must be targeted to develop nutritionally dense hybrids/varieties tolerant to drought using different omics approaches.

Performance and Stability of Pearl Millet Varieties for Grain Yield and Micronutrients in Arid and Semi-Arid Regions of India.

Pearl millet [Pennisetum glaucum (L.) R. Br.] is grown under both arid and semi-arid conditions in India, where other cereals are hard to grow. Pearl millet cultivars, hybrids, and OPVs (open pollinated varieties) are tested and released by the All India Coordinated Research Project on Pearl Millet (AICRP-PM) across three zones (A1, A, and B) that are classified based on rainfall pattern. Except in locations with extreme weather conditions, hybrids dominate pearl millet growing areas, which can be attributed to hybrid vigor and the active role of the private sector. The importance of OPVs cannot be ruled out, owing to wider adaptation, lower input cost, and timely seed availability to subsidiary farmers cultivating this crop. This study was conducted to scrutinize the presently used test locations for evaluation of pearl millet OPVs across India, identify the best OPVs across locations, and determine the variation in grain Fe and Zn contents across locations in these regions. Six varieties were evaluated across 20 locations in A1 and A (pooled as A) and B zones along with three common checks and additional three zonal adapted checks in the respective zones during the 2019 rainy season. Recorded data on yield and quality traits were analyzed using genotype main effects and genotype × environment interaction biplot method. The genotype × environment (G × E) interaction was found to be highly significant for all the grain yield and agronomic traits and for both micronutrients (iron and zinc). However, genotypic effect (G) was four (productive tillers) to 49 (grain Fe content) times that of G × E interaction effect for various traits across zones that show the flexibility of OPVs. Ananthapuramu is the ideal test site for selecting pearl millet cultivars effectively for adaptation across India, while Ananthapuramu, Perumallapalle, and Gurugram can also be used as initial testing locations. OPVs MP 599 and MP 600 are identified as ideal genotypes, because they showed higher grain and fodder yields and stability compared with other cultivars. Iron and zinc concentration showed highly significant positive correlation (across environment = 0.83; p < 0.01), indicating possibility of simultaneous effective selection for both traits. Three common checks were found to be significantly low yielders than the test entries or zonal checks in individual zones and across India, indicating the potential of genetic improvement through OPVs.