Isolation and evaluation of bacterial endophytes against Sclerospora graminicola (Sacc.) Schroet, the causal of pearl millet downy mildew

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.

A Jacalin-like lectin-domain-containing protein of Sclerospora graminicola act as an apoplastic virulence effector in plant–oomycete interactions

SUMMARYThe plant extracellular space, including the apoplast and plasma membrane, is the initial site of plant– pathogen interactions. Pathogens deliver numerous secreted proteins, called effectors, into this region to suppress plant immunity and establish infection. Downy mildew caused by the oomycete pathogen Sclerospora graminicola (Sg) is an economically important disease of Poaceae crops including foxtail millet (Setaria italica). We previously reported the genome sequence of Sg and showed that the Jacalin-related lectin (JRL) gene family has significantly expanded in this lineage. However, the biological functions of JRL proteins remained unknown. Here, we show that JRL from S. graminicola (SgJRL) functions as an apoplastic virulence effector. We identified eight SgJRLs via protein mass spectrometry analysis of extracellular fluid from S. graminicola-inoculated foxtail millet leaves. SgJRLs consist of a Jacalin-like lectin domain and an N-terminal putative secretion signal, and SgJRL expression is induced by Sg infection. Heterologous expression of three SgJRLs with N-terminal secretion signal peptides in Nicotiana benthamiana enhanced the virulence of the pathogen Phytophthora palmivora inoculated onto the same leaves. Of the three SgJRLs, SG06536 fused with GFP localized to the apoplastic space in N. benthamiana leaves. INF1-mediated induction of defense-related genes was suppressed by co-expression of SG06536-GFP. These findings suggest that JRLs are novel apoplastic effectors that contribute to pathogenicity by suppressing plant defense responses.

Transcriptome Profiling Analysis Reveals Co-Regulation of Hormone Pathways in Foxtail Millet during Sclerospora graminicola Infection

Sclerospora graminicola (Sacc.) Schroeter is a biotrophic pathogen of foxtail millet (Setaria italica) and increasingly impacts crop production. We explored the main factors for symptoms such as dwarfing of diseased plants and the “hedgehog panicle” by determining panicle characteristics of varieties infected with S. graminicola and analyzing the endogenous hormone-related genes in leaves of Jingu 21. Results indicated that different varieties infected by S. graminicola exhibited various symptoms. Transcriptome analysis revealed that the ent-copalyl diphosphate synthetase (CPS) encoded by Seita.2G144900 and ent-kaurene synthase (KS) encoded by Seita.2G144400 were up-regulated 4.7-fold and 2.8-fold, respectively. Results showed that the biosynthesis of gibberellin might be increased, but the gibberellin signal transduction pathway might be blocked. The abscisic acid (ABA) 8′-hydroxylase encoded by Seita.6G181300 was continuously up-regulated by 4.2-fold, 2.7-fold, 14.3-fold, and 12.9-fold from TG1 to TG4 stage, respectively. Seita.2G144900 and Seita.2G144400 increased 79-fold and 51-fold, respectively, at the panicle development stage, promoting the formation of a “hedgehog panicle”. Jasmonic acid-related synthesis enzymes LOX2s, AOS, and AOC were up-regulated at the early stage of infection, indicating that jasmonic acid played an essential role in early response to S. graminicola infection. The expression of YUC-related genes of the auxin synthesis was lower than that of the control at TG3 and TG4 stages, but the amidase encoded by Seita.2G313400 was up-regulated by more than 30-fold, indicating that the main biosynthesis pathway of auxin had changed. The results suggest that there was co-regulation of the hormone pathways during the infection of foxtail millet by S. graminicola.

Bioimaging structural signatures of the oomycete pathogen Sclerospora graminicola in pearl millet using different microscopic techniques

In this case study, the mycelium growth of Sclerospora graminicola in the infected tissues of pearl millet and the process of sporulation and liberation of sporangia and zoospores were observed using four different microscopic techniques. The cotton blue-stained samples observed under light microscope revealed the formation of zoospores with germ tubes, appressoria and initiation of haustorium into the host cells, while the environmental scanning electron microscopy showed the rapid emergence of sporangiophores with dispersed sporangia around the stomata. For fluorescence microscopy, the infected leaf samples were stained with Fluorescent Brightener 28 and Calcofluor White, which react with β-glucans present in the mycelial walls, sporangiophores and sporangia. Calcoflour White was found to be the most suitable for studying the structural morphology of the pathogen. Therefore, samples observed by confocal laser scanning microscopy (CLSM) were pre-treated with Calcofluor White, as well as with Syto-13 that can stain the cell nuclei. Among the four microscopic techniques, CLSM is ideal for observing live host-pathogen interaction and studying the developmental processes of the pathogen in the host tissues. The use of different microscopic bioimaging techniques to study pathogenesis will enhance our understanding of the morphological features and development of the infectious propagules in the host.

Inheritance and Allelic Relationship Among Downy Mildew Resistance Genes in Pearl Millet

Pearl millet downy mildew (DM), caused by Sclerospora graminicola, is of serious economic concern to pearl millet farmers in the major crop-growing areas of the world. To study the inheritance and allelic relationship among genes governing resistance to this disease, three DM-resistant pearl millet lines (834B, IP 18294-P1, and IP 18298-P1) and one susceptible line (81B) were selected on the basis of disease reaction under greenhouse conditions against two isolates of S. graminicola (Sg 526-1 and Sg 542-1). Three resistant parents were crossed with the susceptible parent to generate F1, F2, and backcross BC1P1 (susceptible parent × F1) and BC1P2 (resistant parent × F1) generations for inheritance study. To carry out a test for allelism, the three resistant parents were crossed with each other to generate F1 and F2 generations. The different generations of these crosses were screened for disease reaction against two isolates (Sg 526-1 and Sg 542-1) by artificial inoculation under greenhouse conditions. The segregation pattern of resistance in the F2 and corresponding backcross generations revealed that resistance to DM is controlled by a single dominant gene in 834B and IP 18294-P1 and by two dominant genes in IP 18298-P1. A test for allelism inferred that a single dominant gene for resistance in 834B is nonallelic to that which governs resistance in IP 18294-1, whereas one of the two dominant genes for DM resistance in IP 18298-P1 against the test isolates is allelic to the gene for DM resistance in 834B and a second gene is allelic to the resistance gene present in IP 18294-P1.