Bacterial and Fungal composition of Sorghum bicolor: a metagenomics and transcriptomics analysis using next-generation sequencing

Abstract

Sorghum crop has become attractive to breeders due to its drought tolerance, and many uses including a human food source, animal feed, industrial fibre and bioenergy crop. Sorghum, like any other plant, is a host to a variety of microbes that can have neutral, negative or positive effects on the plant. While the majority of microorganisms are beneficial, pathogens colonize plant tissues and overwhelm its defence mechanisms. This colonization is a direct threat to the sorghum productivity. The development of microbiome-based approaches for sustainable crop productivity and yield is hindered by a lack of understanding of the main biotic factors affecting the crop microbiome. Metabarcoding has proven to be a valuable tool which has been widely used for characterizing the microbial diversity and composition of different environments and has been utilized in many research endeavours. This study analyses the relationship between the microbiota and their response to natural pathogen infection in sorghum disease groups (R, MR, S and HS) and identifies the most dominant pathogen in the highly susceptible disease group. The study also, assesses the spore viability through the use of the automated cell counter and confirms Fusarium graminearum (dominant pathogen linked to the HS disease group) through sequencing of the marker genes, to subsequently characterize pathways likely to be involved in pathogen infection resistance. To achieve the objectives, a combination of 16S rRNA (V3/V4 regions) and ITS (ITS1/ITS4) of the internal transcribed spacer regions were amplified and sequenced using NGS technologies to study the microbiota in response to natural infection. Additionally, comparative transcriptional analysis of sorghum RILs in response to Fusarium graminearum infection was conducted through RNA-Seq. Upon natural infection, the foliar symptoms assessment of the RILs was conducted and four disease groups; resistant (R), moderately resistant (MR), susceptible (S) and highly susceptible (HS) were designated. The results of the present metabarcoding study indicate that resistant sorghum leaves (R group) supported a large diversity of fungal and bacterial microbes. The genera Methylorubrum, Enterobacter and Sphingomonas with reported plant growth promoting traits were more abundant and highly enriched in the R and MR group, with members of the latter genus significantly enriched in the R group. The resistant fungal group had a majority of OTUs showing similarity to well-known plant growth-promoting fungal genus including Papiliotrema (Tremellaceae family), which are known biocontrol agents. The yeast Hannaella was also highly linked with the resistant plants. Some Hannaella species are known to produce indole acetic acid (IAA) for promoting plant growth. Metabarcoding was also used to assess the major potential disease-causing taxa associated with the highly diseased group. It identified fungal pathogenic species, that have not previously been identified as pathogens of sorghum such as Ascochyta paspali and Ustilago kamerunensis (which are known pathogenic fungi of grass species) and were associated with the susceptible disease groups (S and HS). These analyses revealed the potential sorghum fungal pathogen Epicoccum sorghinum, and was highly linked with the S disease group. It further expanded the identification of a reportedly economically importance species causing sorghum related diseases Fusarium graminearum (anamorph Gibberella zeae). This species has also been identified in this study to be highly associated with the RILs showing major disease symptoms. Fusarium graminearum a significant pathogen in winter cereals and maize has been associated with stalk rot of sorghum and sorghum grain mould. The presence of Fusarium graminearum in sorghum can be a toxicological risk, since this species has the potential to produce mycotoxins. It was further shown that natural pathogen infection results in distinct foliar microbial communities in sorghum RILs. The co-occurrence taxa represented by Tremellomycetes and Dothiomycetes fungal classes and Bacillaceae and Sphingomonadaceae bacterial family had more central roles in the network. The modules which are located centrally on the network have been expected to play important ‘topological roles’ in interconnecting pairs of other fungal and bacterial taxa in the symbiont–symbiont co-occurrence network. These taxa having a central role, are considered to be keystone microbes, and have been suggested to be drivers of microbiome structure and functioning. The results of bacterial and fungal community composition, community co-occurrences further suggested the importance of keystone taxa which may disproportionately shape the structure of foliar microbiomes. The foliar disease symptom assessments revealed that sorghum RIL 131 was highly diseased and RIL 103 did not show any visible disease symptoms and were subsequently used for transcriptomic analysis. Gene expression patterns were studied between the identified RIL that did not show visible symptoms (resistant RIL no 103) and the RIL that showed major disease symptoms (susceptible RIL no 131). Fusarium graminearum the dominant potential pathogen found in this study to be associated with the highly susceptible plants was used to inoculate RILs at seedling stage in a greenhouse and samples were collected in triplicates at 24 hours post infection (hpi), 48 hpi, 7 days post infection (dpi) and 14 dpi. Prior to that, ITS and UBC genes confirmed the identity of Fusarium graminearum, and the automated haemocytometer confirmed the cell/spore viability. Using RNA-Seq analysis it was shown that the resistant RIL had defence related pathways from early response (24- 48 hpi) to late response (7-14 dpi). And the more the infection progressed, the more the defence related genes were up-regulated in terms of fragments per kilobase of exon model per million reads mapped (FPKM) and False Discovery Rate (FDR ≤ 0.05) values. Transcriptome time series expression profiling was used to characterize the plant response to Fusarium graminearum with the Dirichlet Process Gaussian Process mixture model software (DPGP) in susceptible and resistant RILs. The susceptible RIL (number 131) transcriptional response upon Fusarium graminearum infection presented differences of the closely related clustered expression profiles across all timepoints in both RILs. Group 2 exclusively clustered the genes encoding the sesquiterpene metabolism pathway, which is one of the major physiological change occurring in response to fungal infection and has been previously reported to produce the mycotoxins associated with Fusarium head blight (FHB) of cereals. This pathway presented an increase from the initial infection phase to the late infection phase in group 4, the genes encoding starch sucrose, metabolism and cyanoamino acid pathways presented a pattern that had a sharp decline from 48 hpi -14 dpi (at a later stage of infection). This could suggest that, as the time progresses in the susceptible RIL the pathways which are important in plant defence declines at a late infection stage. Group 3 presented a pattern increase of the 5-lipoxygenase (LOX 5) gene expressed from 48 hpi-14 dpi timepoints. The loss and silencing of LOX5 function have in the past described to be linked with enhanced disease resistance. In this study the LOX5 was expressed and this could suggest that LOX5 might have a function as a susceptibility factor in disease caused by Fusarium graminearum in sorghum RILs. CBL-interacting protein kinase 6 (CIPK6) gene was also associated with this group. This gene has been associated with negative regulation of immune response to Pseudomonas syringae in Arabidopsis as plants overexpressing CIPK6 were more susceptible to Pseudomonas syringae. Transcriptional response of a resistant RIL (number 103) to infection with Fusarium graminearum presented an increase in genes encoding metabolic and biosynthesis of metabolites pathways in group 1 and group 4 at early infection phase and a sharp decline in the late infection phase. An increase in the genes encoding pathways in earlier infection state could suggest the establishment of a beneficial energy balance for defence. Additionally, genes encoding phenylpropanoid (PAL), galactose and glycolysis pathway were amongst the genes increased at early stages of infection in group 1. Sugar can play a significant role in resistance to fungal pathogens through phenylpropanoid metabolism stimulation, and previous studies showed that the phenylpropanoid pathway could play a role in resistance of wheat to Fusarium graminearum and deoxynivalenol. Overall, this study represents a first step in understanding the molecular mechanisms involved in resistance to Fusarium graminearum. This analysis has also identified the reported beneficial microbes and defence related genes and pathways. Together, the current findings suggest that different ‘resident’ consortia found in naturally infected and uninfected sorghum plants may be viable biocontrol and plant-growth promoting targets. Cultivation studies may shed light on the nature of the putative symbiotic relationships between bacteria and fungi. These results have consequences for crop breeding, and the analysis of microbial diversity and community composition can be useful biomarkers for assessing disease status in plants. The transcriptome and metabarcoding data generated will help guide further research to develop novel strategies for management of disease in sorghum RILs through the integrative approach considering both beneficial microbes and defence related genes. This provides the baseline information and will positively impact in the development of Fusarium graminearum resistant genotypes in future through the integration/incorporation of beneficial microorganisms (bacteria and fungi) and resistant genes in breeding strategies.