Fine-scale mapping of the microbiome on phylloplane and spermoplane of aromatic and non-aromatic rice genotypes.

Adaxial, abaxial phylloplane (leaf), and spermoplane (seed) are proximal yet contrasting habitats for a microbiota that needs to be adequately explored. Here, we proposed novel methods to decipher the adaxial/abaxial-phylloplane and spermoplane-microbiomes. Comparison of 22 meta barcoded-NGS datasets (size of total data set-1980.48 Mb) enabled us to fine-map the microbiome of the rice foliar niche, which encompasses the lower, middle, top leaf as well panicle. Here, the total- and the cultivable-microbiome profiling revealed 157 genera representing ten phyla and 87 genera from 4 bacterial phyla, respectively, with a predominance of Proteobacteria and Actinobacteria. Interestingly, more bacterial communities (124-genera) preferred the abaxial than the adaxial phylloplane (104-genera) and spermoplane (67-genera) for colonization. The microbiome profiles were nearly identical on the aromatic (125-genera) and non-aromatic rice (116-genera) with high representation of Pantoea, Methylobacterium, Curtobacterium, Sphingopyxis, and Microbacterium. The culturomics investigation confirmed the abundance of Pantoea, Chryseobacterium, Pseudomonas, Acinetobacter, Sphingobacterium, and Exiguobacterium. One hundred bacterial isolates characterized and identified by polyphasic-taxonomic tools revealed the dominance of Acinetobacter, Chryseobacterium, Enterobacter, Massilia, Pantoea, Pseudomonas, and Stenotrophomonas on adaxial/abaxial-phylloplane and spermoplane. The study culminated in identifying hitherto unexplored bacterial communities on the adaxial/abaxial phylloplane and spermoplane of rice that can be harnessed for microbiome-assisted rice cultivation in the future.

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.