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Sorghum (Sorghum bicolor [L.] Moench) is one of the top ten cereal crops in the world and is grown for fodder and seed purposes. During the fall of 2019 to 2022, a disease causing small to long streaks on leaves was observed in sorghum fields of Hisar (29° 9' 6.6996'' N, 75° 43' 16.0428'' E), Rohtak (28° 53' 43.8540'' N, 76° 36' 23.8068'' E) and Mohindergarh (28° 16' 6.0492'' N, 76° 9' 3.3552'' E) regions of Haryana between July and October. The reddish brown streaks were observed in the interveinal spaces of upper and lower leaves. The disease incidence reached 20-30% of plants in affected fields. The diseased leaf tissues were disinfected with 70% alcohol and placed in a tube with sterile water. After 30 minutes, 100 µl of the suspension was inoculated onto nutrient agar medium, incubated at 28 ± 2°C for three days, and a pure culture was obtained by restreaking on nutrient agar (Janse, 2005). The rod-shaped gram-negative bacterium with round, cream to white colonies was positive for methyl red, citrate utilization, urease activity, and glucose, lactose, sorbitol, rhamnose and sucrose fermentation tests. The genomic DNA of the bacterial suspension was extracted and 16S rDNA was amplified using universal 27F/1492R primers (Marchesi et al. 1998), resulting in tentative identification as Klebsiella sp. It was further confirmed with PCR amplification of Klebsiella specific primers (F:5ʹ-CGCGTACTATACGCCATGAACGTA-3ʹ; R:5ʹ-ACCGTTGATCACTTCGGTCAGG-3ʹ) for gyrA gene (Brisse and Verhoef 2001). Discrete PCR amplicons of 1,500 (16S rDNA) and 300 bp (gyrA) were observed in a 1% (w/v) agarose gel. Forward and reverse DNA sequencing of both amplicons of the Hisar isolate (VMKV101) was carried out using a BDT v3.1 Cycle sequencing kit and consensus sequences were generated by using the program SeqMan Ultra (DNASTAR Lasergene). Sequences of the PCR products were deposited in GenBank with accession numbers MZ569433 (16S rDNA) and OP390080 (gyrA). The 16S rDNA sequence was 97.32% similar to K. variicola strain 13450 (CP026013; 1,450/1,490 bp) and the gyrA sequence had 99.66% similarity to K. variicola strain FH-1 (CP054254; 297/298 bp). A 16S RNA-based phylogenetic tree done by MEGA11 (Tamura et al. 2021) using the Maximum Likelihood method showed that strain VMKV101 clustered with K. variicola type strain F2R9. The complete bacterial genome (GCA025629215), sequenced by the Ion GeneStudio S5 system using Ion 530 chips (Thermo Fisher Scientific), was 99.03% identical by average nucleotide identity (ANI) to the type genome (CP045783) of Klebsiella variicola, with 87.8% genome coverage. For pathogenicity testing, a bacterial suspension (10 ml, 1×107 colony forming units/ml) was injected into the whole whorl after mechanical injury on 15-20 days old seedlings of the susceptible genotype HC-171, then plants were incubated at 35 ± 2°C, >80% relative humidity. Control plants were injected with sterile distilled water. Initial symptoms were observed on leaves of inoculated plants after 5 to 7 days as narrow, small longitudinal reddish brown streaks. As the disease progressed, the streaks on the leaf blade increased in number and size maintaining the reddish brown color. These streaks had slightly wavy margins and were surrounded by bright yellow halos. After 15 to 20 days, the streaks were 0.5 to 2.0 mm wide and 1.0 to 5.0 cm long, occasionally up to 10.0 cm long on both side of the leaves. Over time, neighboring streaks coalesced to form large necrotic areas. All inoculated plants exhibited identical symptoms. No symptoms were observed on control leaves. The reisolated bacterium from diseased sorghum leaves showed exactly the same morphological, biochemical and 16S RNA and gyrA molecular characteristics. To our knowledge, this is the first report of K. variicola causing a leaf streak disease on sorghum. Klebsiella species primarily cause diseases in humans and animals, but K. variicola has been found to incite banana soft rot (Fan et al. 2015) and K. aerogenes to cause stem rot in pearl millet (Malik et al. 2021). Differences of prevalence, spread and control between K. variicola and two other bacteria (Xanthomonas vasicola pv. holcicola causing Bacterial leaf streak; Paraburkholderia andropogonis causing Bacterial leaf stripe) causing leaf streak diseases on sorghum need to be determined. The identification of Klebsiella leaf streak disease lays the groundwork for future investigations into epidemiology and management of K. variicola on sorghum.

Nanotechnology emerged as a revolutionary technology in various fields of applied sciences, such as biomedical engineering and food technology. The pivotal roles of nanocompounds have been explored in various fields, such as food protection, preservation, and enhancement of shelf life. In this sequence, metallic nanoparticles (MNPs) are proven to be useful in developing products with antimicrobial activity and subsequently improve the shelf life of agrifoods. The major application of MNPs has been observed in the packaging industry due to the combining ability of biopolymers with MNPs. In recent years, various metal nanoparticles have been explored to formulate various active food packaging materials. However, the method of production and the need for risk evaluation are still a topic of discussion among researchers around the world. In general, MNPs are synthesized by various chemical and physical means, which may pose variable health risks. To overcome such issues, the green synthesis of MNPs using microbial and plant extracts has been proposed by various researchers. In this review, we aimed at exploring the green synthesis of MNPs, their properties and characterization, various ways of utilizing MNPs to extend their shelf life, and, most importantly, the risk associated with these along with their quality and safety considerations.

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: 5ʹ-CGCGTACTATACGCCATGAACGTA-3ʹ; R: 5ʹ-ACCGTTGATCACTTCGGTCAGG-3ʹ) 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.

Background Pearl millet remains prone to many diseases; among them downy mildew caused by Sclerospora graminicola (Sacc.) Schroet is economically more important. The use of endophytic bacteria for management of downy mildew of pearl millet as eco-friendly approach is increasing attention as sustainable alternative to pesticides. The objective of the present study was to isolate endophytic bacteria from roots of pearl millet cultivars and assess for biocontrol activity against Sclerospora graminicola. Results Thirty pearl millet root bacterial endophytes (PMRBEs) were isolated and screened in vitro for biocontrol activities such as: siderophore production, hydrogen cyanide (HCN) production and 1-amino cyclopropane-1-carboxylate (ACC) deaminase activity. Sixteen isolates possessed siderophore production potential, 3 isolates were found to be HCN producers, and 30% of the bacterial endophytes showed a good growth on ACC supplemented plates. On the basis of biocontrol activities, promising endophyte PMRBE6 was selected for seed treatment as well as a foliar spray to manage downy mildew of pearl millet in screen house experiment. The isolate PMRBE6 was found to be effective in managing downy mildew disease. Grain yield, test weight, plant height and average number of productive tillers were found to be maximum on inoculation of seeds of different pearl millet cultivars with PMRBE6, and the results were statistically significant as compared to control. Conclusions On the basis of biochemical characterization and partial 16S rRNA sequencing, the isolate PMRBE6 was identified as Bacillus subtilis strain PD4 (Accession no. MN400209). Pearl millet root bacterial endophyte (PMRBE6) exhibiting biocontrol activities could be exploited in friendly, sustainable organic agriculture.