Seed Endophytic Bacteria of Pearl Millet (Pennisetum glaucum L.) Promote Seedling Development and Defend Against a Fungal Phytopathogen

Seed endophytic bacteria (SEB) are primary symbionts that play crucial roles in plant growth and development. The present study reports the isolation of seven culturable SEB including Kosakonia cowanii (KAS1), Bacillus subtilis (KAS2), Bacillus tequilensis (KAS3), Pantoea stewartii (KAS4), Paenibacillus dendritiformis (KAS5), Pseudomonas aeruginosa (KAS6), and Bacillus velezensis (KAS7) in pearl millet seeds. All the isolates were characterized for their plant growth promoting activities. Most of the SEB also inhibited the growth of tested fungal phytopathogens in dual plate culture. Removal of these SEB from seeds compromised the growth and development of seedlings, however, re-inoculation with the SEB (Kosakonia cowanii, Pantoea stewartii, and Pseudomonas aeruginosa) restored the growth and development of seedlings significantly. Fluorescence microscopy showed inter and intracellular colonization of SEB in root parenchyma and root hair cells. Lipopeptides were extracted from all three Bacillus spp. which showed strong antifungal activity against tested fungal pathogens. Antifungal lipopeptide genes were also screened in Bacillus spp. After lipopeptide treatment, live-dead staining with fluorescence microscopy along with bright-field and scanning electron microscopy (SEM) revealed structural deformation and cell death in Fusarium mycelia and spores. Furthermore, the development of pores in the membrane and leakages of protoplasmic substances from cells and ultimately death of hyphae and spores were also confirmed. In microcosm assays, treatment of seeds with Bacillus subtilis or application of its lipopeptide alone significantly protected seedlings from Fusarium sp. infection.

Pan-Genome of Novel Pantoea stewartii subsp. indologenes Reveals Genes Involved in Onion Pathogenicity and Evidence of Lateral Gene Transfer

Pantoea stewartii subsp. indologenes (Psi) is a causative agent of leafspot on foxtail millet and pearl millet; however, novel strains were recently identified that are pathogenic on onions. Our recent host range evaluation study identified two pathovars; P. stewartii subsp. indologenes pv. cepacicola pv. nov. and P. stewartii subsp. indologenes pv. setariae pv. nov. that are pathogenic on onions and millets or on millets only, respectively. In the current study, we developed a pan-genome using the whole genome sequencing of newly identified/classified Psi strains from both pathovars [pv. cepacicola (n = 4) and pv. setariae (n = 13)]. The full spectrum of the pan-genome contained 7030 genes. Among these, 3546 (present in genomes of all 17 strains) were the core genes that were a subset of 3682 soft-core genes (present in ≥16 strains). The accessory genome included 1308 shell genes and 2040 cloud genes (present in ≤2 strains). The pan-genome showed a clear linear progression with >6000 genes, suggesting that the pan-genome of Psi is open. Comparative phylogenetic analysis showed differences in phylogenetic clustering of Pantoea spp. using PAVs/wgMLST approach in comparison with core genome SNPs-based phylogeny. Further, we conducted a horizontal gene transfer (HGT) study using Psi strains from both pathovars along with strains from other Pantoea species, namely, P. stewartii subsp. stewartii LMG 2715T, P. ananatis LMG 2665T, P. agglomerans LMG L15, and P. allii LMG 24248T. A total of 317 HGT events among four Pantoea species were identified with most gene transfer events occurring between Psi pv. cepacicola and Psi pv. setariae. Pan-GWAS analysis predicted a total of 154 genes, including seven gene-clusters, which were associated with the pathogenicity phenotype (necrosis on seedling) on onions. One of the gene-clusters contained 11 genes with known functions and was found to be chromosomally located.

Pan-genome of Novel Pantoea stewartii subsp. indologenes Reveal Genes Involved in Onion Pathogenicity and Evidence of Lateral Gene Transfer

Pantoea stewartii subsp. indologenes (Psi) is a causative agent of leafspot of foxtail millet and pearl millet; however, novel strains were recently identified that are pathogenic on onion. Our recent host range evaluation study identified two pathovars; P. stewartii subsp. indologenes pv. cepacicola pv. nov. and P. stewartii subsp. indologenes pv. setariae pv. nov. that are pathogenic on onion and millets or on millets only, respectively. In the current study we developed a pan-genome using the whole genome sequencing of newly identified/classified Psi strains from both pathovars [pv. cepacicola (n= 4) and pv. setariae (n=13)]. The full spectrum of the pan-genome contained 7,030 genes. Among these, 3,546 (present in genomes of all 17 strains) were the core genes that were a subset of 3,682 soft-core genes (present in ≥16 strains). The accessory genome included 1,308 shell genes and 2,040 cloud genes (present in ≤ 2 strains). The pan-genome showed a clear liner progression with >6,000 genes, suggesting the pan-genome of Psi is open. Comparative phylogenetic analysis showed differences in phylogenetic clustering of Pantoea spp. using PAVs/wgMLST approach in comparison to core genome SNP-based phylogeny. Further, we conducted a horizontal gene transfer (HGT) study including four other Pantoea species namely, P. stewartii subsp. stewartii LMG 2715T, P. ananatis LMG 2665T, P. agglomerans LMG L15, and P. allii LMG 24248T. A total of 317 HGT events among four Pantoea species were identified with most gene transfers observed between Psi pv. cepacicola and Psi pv. setariae. Pan-GWAS analysis predicted a total of 154 genes including seven cluster of genes associated with the pathogenicity phenotype on onion. One of the clusters contain 11 genes with known functions and are found to be chromosomally located.

First report of pearl millet bacterial leaf blight caused by Pantoea stewartii subspecies indologenes in India

Pearl millet (Cenchrus americanus L.) field-grown plants of cv. 7042S shown unusual water-soaked lesions on leaf tips spreading towards the leaf base from Manasagangothri region (12.31°N 76.61°E), Karnataka, a southern Indian state during March 2020. Later those infected plants showed extensive necrosis and typical leaf blight symptoms with 70% disease incidence and 59% severity. Surface sterilized (3 x 3 mm) infected leaf tissues were crushed in 1mL sterile distilled water and streaked onto nutrient agar media. Bright-yellowish, circular, mucoid single bacterial colonies (PPi-M1) with regular margin were recovered after 24 hours of incubation at 28oC, and the same bacterial colonies were used for further biochemical and molecular characterization. The isolate, PPi-M1 found as gram-negative rods, gelatin, starch hydrolysis negative, and catalase, indole production positive. The partial sequence of 16S rRNA gene (primers: 27F/1492R) of the isolate PPi-M1 was amplified, sequenced, and curated sequence submitted to NCBI GenBank (accession number: MN808555). In nucleotide BLAST search for homologous sequences, 99.5% nucleotide matching similarity (1410bp) was observed with other Pantoea stewartii subspecies indologenes strains (MF163274; NR_104928) at NCBI database indicating that our isolate PPi-M1 belongs to this species. In Phylogenetic analysis using the Maximum Likelihood method and Tamura Nei model (1993), PPi-M1 formed a distinct cluster with other Pantoea stewartii strains with bootstrap value >95 and it was distant from P. allii, P. ananatis, P. agglomerans, and P. dispera. Besides, the subspecies-specific PCR assay and subsequent sequencing of galE and recA genes (primers: 3614galE/3614galEc; 3614recA/3614recAc; 372 and 223 bp) also confirmed the identity of the isolate as Pantoea stewartii subspecies indologenes. Further, the pathogenicity test was performed in-planta on 21 days old seedlings of pearl millet cv. CO-10. The bacterial suspension of isolate PPi-M1 (1x108 CFU/ml) was used for inoculation by leaf clipping method (Ke et al. 2017). All the inoculated plants (n=4 leaves per plant; 15 plants) maintained under greenhouse conditions (Temp: 27-29oC; RH: 80-85%) except mock (sterile water inoculation) shown similar water-soaked lesions from the cut end of the leaf, with a definite spreading margin and a typical leaf blight symptom in 8 dpi, as observed in the field. Re-isolated bacterial colonies from infected leaves shared similar morphological characters and molecular identity with inoculated culture, thus proving Koch’s postulates. This pearl millet leaf blight causing bacterial strain PPi-M1 was deposited in the National Agriculturally Important Microbial Culture Collection, Mau, India (accession no.: NAIMCC-B-02508). Previously, P. stewartii was reported to cause leaf blight and rot diseases on rice and maize (Kini et al. 2016; Roper et al. 2011), also the international seed federation has instigated the phytosanitary measures highlighting its true seed transmission ability (Pataky et al. 2003). This study will supplement future pearl millet breeding programs, and to our knowledge, this is the first report of P. s. subsp. indologenes inciting pearl millet leaf blight disease in India.

Pantoea stewartii (bacterial wilt of maize).

Economic losses in maize due to P. stewartii subsp. stewartii have been inconsequential in North America for the past 50 years except for a few, small sporadic outbreaks and a few extensive epidemics on susceptible sweetcorn hybrids (Pepper, 1967; Anderson, 1986; Anderson and Buzzell, 1986; Pataky et al., 1996; Pataky et al., 2000b). The lack of economic importance of this disease in North America is due primarily to adequate levels of resistance incorporated into maize hybrids that are grown where the disease occurs. Stewart's wilt caused substantial economic losses in the 1930s before the development of resistant cultivars (Pepper, 1967). Severe losses due to Stewart's wilt were reported in Italy in the 1940s and the disease reoccurred there as an important problem in the 1980s (Anon., 1983; Mazzucchi, 1984).

Revisiting Substrate Specificity Concept in Microbial Screening Methodologies for Fish Waste Management

Microbial and enzymatic processing is an attractive area for production of valuable byproducts from fish waste. Functional screening methodologies for the purpose are still based on activities in non-specific substrates, and concept of substrate specificity is not yet validated. Therefore, reliability of using non-specific substrate for the purpose was checked. Results revealed the existence of a limited number of mutually inclusive positive isolates in non-specific and specific substrate based assays (13% for fish proteolysis and 22% for fish lipolysis), with no significant positive correlations (P>0.05). Further, using non-specific substrates in screening assays missed 57.1% and 53.33% of fish proteolytic and fish lipolytic microbes respectively, signifying the use of same substrates. Beyond methodological perspectives, the paper forms the first report on fish proteolytic activity of Bacillus tropicus, Bacillus vallismortis, Paenibacillus alvei, Staphylococcus epidermidis and Staphylococcus hominis. Similarly, fish oil hydrolyzing capacities of B. tropicus, Cronobacter sakazakii, P. alvei, Paenibacillus pinisoli, Pantoea stewartii, S. hominis and Staphylococcus caprae are recorded for the first time. Further, the paper points out 6 and 3 potential microbial species producing > 1 protease units/ml and >1 enzymatic index for fish proteolytic and lipolytic activities, without any optimization, warranting future use in fish waste management.

Identification of two novel pathovars of Pantoea stewartii subsp. indologenes affecting Allium sp. and millets

Pantoea stewartii subsp. indologenes is a causative agent of leafspot of foxtail millet and pearl millet; however, novel strains were recently identified that are pathogenic on onion. We phenotypically and genotypically characterized seventeen Pantoea stewartii subsp. indologenes strains from onion and other sources (pearl millet, foxtail millet, guar pulse, verbena and corn). Based on the host range evaluation, we propose two pathovars P. stewartii subsp. indologenes pv. cepacicola pv. nov. and P. stewartii subsp. indologenes pv. setariae pv. nov. Pantoea stewartii subsp. indologenes pv. cepacicola pv. nov. causes symptoms on Allium species (leek, onion, chive and Japanese bunching onion) and also on foxtail millet, pearl millet and oat. However, P. stewartii subsp. indologenes pv. setariae pv. nov can only infect the members of Poaceae (foxtail millet, pearl millet and oat). Our study also showed that P. stewartii subsp. indologenes pathovars and P. stewartii subsp. stewartii share high genotypic relatedness and cannot be differentiated by dDDH and ANI values. Although, the newly proposed pathovars are not clearly distinguishable by their fatty acid and methyl esterase profiles, and substrate utilization patterns, a fatty acid (unknown with retention time: 10.9525) and few metabolites (3 methyl glucose, Na butyrate and fusidic acid) can be potentially used to distinguish them. We also report the distribution of previously known pathogenicity (HiVir, hrcC) and virulence (alt) factors of Pantoea sp. in the new pathovars. The impact of these new pathovars in the center rot pathosystem of onion is yet to be determined.