1. Unraveling the microbial profile of the rhizosphere of SDS-suppressive soils in Soybean fields
Ali Y. Srour1, Jason Bond1, Leonor Leandro2, Dean Malvick3 and Ahmad Fakhoury Department of Plant, Soil and Agricultural systems, Southern Illinois University, Carbondale IL 62901, USA . 2 Department of Plant Pathology, Iowa state University, Ames IA 50011, USA. 3 Department of Plant Pathology, University of Minnesota, St. Paul MN 55108, USA
Sudden Death Syndrome (SDS) incidence and severity in soybean fields is often found unevenly distributed across the same field. While certain spots in the field are highly conducive to SDS, other regions appear to be naturally suppressive to the disease. The role of microbes and mechanisms involved in SDS control was investigated in different soybean fields in IL, IA and MN in 2010, 2011 and By using highly informative barcodes specific to Bacteria, Fungi, Archaea and Nematodes, and coupling with Illumina Mi-Seq sequencing, we identified key microbial taxa likely to be involved in suppression of SDS. A total of 18,000,000 reads were mapped against the NCBI-NT database and analyzed with MEGAN5 for comparative taxonomic analysis. Our data revealed significant differences in bacterial and fungal community structure and composition between suppressive and conducive soils. At least 20 taxa were found to be attributed to SDS-suppressive soils, however only three genera consisting of Fusarium oxysporum sp. complex, Metacordyceps and Myceliophtora were found to be consistently correlated with SDS suppression. Others such as Proteobacteria, Mycobacteria and Trichoderma sp. were found to dominate in some suppressive soils. This suggests that SDS suppression is dependent on multiple contributors and that their relative abundance plays a key role in determining the incidence of the disease.
Introduction
Microbiota of suppressive and conducive soils
Cluster Analysis
We used several tools to classify OTUs, among these MEGAN was found to more robust and accurate in identifying microbial taxa down to the species level. Moreover MEGAN can compare side by side multiple datasets of a metagenome.
Sudden death Syndrome (SDS) affects Soybean yield by causing root and crown rot. SDS is often found unequally scattered across the same field which might be attributed to soil biome either suppressing or promoting the effect of the pathogen Fusarium virguliforme. In order to gain insight into the mechanism by which SDS is suppressed we conducted a metagenomic analysis of a large number of samples collected from suppressive soils over three years.
Hierarchical clustering analysis (UPGMA tree) of all nodes of the taxon profile using Bray-Curtis measure showed suppressive soils from IL and IA to cluster together more than its respective conducive soil originating in the same state.
Conducive
Suppressive
Sampling
Illinois samples formed 6 datasets, each comprising an average of million reads. A taxonomic profile was created for each dataset at different ranks of NCBI taxonomy. Read counts were assigned to each taxon.
Comparison of suppressive to conducive soils in Illinois pointed to a number of taxa that might be involved in disease suppression mainly : F. oxyporum, Metacordyceyps, Myceliophtora, Trichoderma and Purpureocillium.
Soil samples were collected from fields in Illinois, Minnesota and Iowa with a history of SDS over three years.
At least 10 fields per state were selected each year. 20 soil probes were taken within SDS hotspots, and another 10 probes were taken outside the hotspot >10 ft beyond symptoms edge.
Conclusions
In this study we were able to identify potential players in SDS suppressive soils. F. oxypsorum dominated the list in IL and IA.
The fungus metacordyceps chamydosporia parasitizes nematode eggs and has become one of the most promising biological control agents (BCAs) for plant-parasitic nematodes.
Myceliophtora thermophila is an ascomycete that expresses laccases and cellulases, hence its importance in soil fertility and bioremediation.
Purpureocillium lilacinum is a known fungal endophyte that protects plants against a wide variety of stressors and pathogens and promotes plant growth.
Trichoderma species are know BCA and have been tested.
While this study identified key players in SDS suppression across IL, IA and Mn, it is important to note that the combination of these players and relative abundance is essential to induce suppression in an SDS infested soybean field.
Methods
Conducive
Suppressive
Metagenomics workflow consisted of:
Total DNA extraction from both suppressive and conducive soils
Targeted amplification of DNA for each microbial kingdom
Next Gen Sequencing of amplicons
De novo assembly and sequence clustering into operational taxonomic units (OTU)
Blastn against NCBI database
Taxonomy classification and statistical analysis
Analysis of Iowa samples revealed two important players seen in Illinois F. oxysporum and Myceliophtora thermophila. A new player that might be associated with SDS suppression is Mycobacterium.
Soil Microbial Community
DNA extraction PCR
Amplicon Sequencing (MiSeq)
Blastn
(NCBI-nt database)
OTU assignment
Taxon classification and phylogenetic analysis
Suppressive
Conducive
Minnesota suppressive soils pointed mainly to Trichoderma harzianum and Trichoderma brevicompactum as potential agents in SDS suppression.
Notably Fusarium solani sp. complex was found in all conducive soils.
All the above observations are based on the lowest common ancestor (LCA) assigned in the phylogenetic tree.
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