Genomics and Management of Fusarium Root Rot of Field Peas

Dry Pea or field pea (Pisum sativum L.) is an important cool season legume crop grown in the United States. Field peas are vulnerable to many diseases of which, soil borne diseases including wilt and root rot are of major economic importance and can cause significant reduction in yield. There is a dearth of satisfactory methods for control of root rot and no varieties with complete resistance to Fusarium root rot are currently available. Root rot disease was found to be prevalent in all the major pea growing counties of North Dakota surveyed in 2004, 2005, 2010 and 2011. Fusarium species were the most frequently isolated fungal species from the infected pea roots of which, F. oxysporum and F. avenaceum were the most common. 21 Field pea varieties were screened for resistance against F. avenaceum and F. solani f. sp. pisi, the Fusarium species traditionally associated with root rots of field pea in growth chamber experiments and field trials. Low levels of resistance were detected in a few cultivars but no variety was found to be completely resistant to any of the pathogens tested. Efficiency of precipitated calcium carbonate (PCC) in controlling Fusarium species most commonly associated with root rots was evaluated under in vitro and field conditions. Significant reduction in spore production, spore germination, and dry mycelial weight of Fusarium spp. were detected on PCC amended media in laboratory studies. In greenhouse and field experiments significant reduction in root rot disease severity was observed with PCC application compared to control. Fungal gene expression in artificially infected field pea roots and F. graminearum grown in culture was assessed using the Illumina mRNA-Seq technology. A total of 613 F. graminearum genes were found to be differentially expressed in planta on pea. Functional classes associated with amino acid metabolism, nitrogen metabolism, extracellular polysaccharide degradation, detoxification by degradation and defense related proteins were found to be significantly enriched in the up-regulated gene set as determined using FunCatDB. Expression of four up-regulated genes was confirmed by RT-PCR to validate the inferences from the sequencing results

Transcriptome dynamics reveal different patterns of gene regulation in resistant and susceptible common bean–<i>Heterodera glycines</i> interactions.

<p>MA-plots displaying the log₂ fold change in the expression plotted against mean of normalized counts in a. resistant genotype inoculated vs. resistant genotype non-inoculated (RI vs. RC) b. susceptible genotype inoculated vs. susceptible genotype non-inoculated (SI vs. SC). Y-axes indicate fold change values (p<0.05) and x-axes indicate mean of normalized counts. Differentially expressed genes (DEGs) are shown in green, black and red indicating down-regulated, no change and up-regulated genes respectively.</p

List of primers to amplify the selected genes for qPCR analysis.

<p>List of primers to amplify the selected genes for qPCR analysis.</p

Quantitative RT-PCR (qPCR) validation of the relative expression levels of genes selected from the RNA-seq analysis of common bean–<i>Heterodera glycines</i> interaction.

<p>Expression profiles of 10 genes (color coded) as determined by qPCR and RNA-seq. The signal intensity of each transcript was normalized using Actin11. The x-axis indicates two groups of comparisons: a. resistant genotype inoculated vs. resistant genotype non-inoculated (RI vs. RC). b. susceptible genotype inoculated vs. susceptible genotype non-inoculated (SI vs SC). The y-axis shows the fold change increase/decrease in expression level of the genes.</p

Expression analysis of differentially expressed genes in response to common bean–<i>Heterodera glycines</i> interaction.

<p>Heat maps are arranged according to their hierarchical clustering based on expression pattern of the genes. From left to right, the four columns respectively show the expression of genes in the non-inoculated susceptible genotype (SC), the non-inoculated resistant genotype (RC), the SCN inoculated resistant genotype (RI) and the inoculated susceptible genotype (SI). Green, black and red indicate low, no change and high expression levels of genes, respectively.</p

Comparative Transcriptome Analysis of Resistant and Susceptible Common Bean Genotypes in Response to Soybean Cyst Nematode Infection

<div><p>Soybean cyst nematode (SCN; <i>Heterodera glycines</i> Ichinohe) reproduces on the roots of common bean (<i>Phaseolus vulgaris</i> L.) and can cause reductions in plant growth and seed yield. The molecular changes in common bean roots caused by SCN infection are unknown. Identification of genetic factors associated with SCN resistance could help in development of improved bean varieties with high SCN resistance. Gene expression profiling was conducted on common bean roots infected by SCN HG type 0 using next generation RNA sequencing technology. Two pinto bean genotypes, PI533561 and GTS-900, resistant and susceptible to SCN infection, respectively, were used as RNA sources eight days post inoculation. Total reads generated ranged between ~ 3.2 and 5.7 million per library and were mapped to the common bean reference genome. Approximately 70–90% of filtered RNA-seq reads uniquely mapped to the reference genome. In the inoculated roots of resistant genotype PI533561, a total of 353 genes were differentially expressed with 154 up-regulated genes and 199 down-regulated genes when compared to the transcriptome of non- inoculated roots. On the other hand, 990 genes were differentially expressed in SCN-inoculated roots of susceptible genotype GTS-900 with 406 up-regulated and 584 down-regulated genes when compared to non-inoculated roots. Genes encoding nucleotide-binding site leucine-rich repeat resistance (NLR) proteins, WRKY transcription factors, pathogenesis-related (PR) proteins and heat shock proteins involved in diverse biological processes were differentially expressed in both resistant and susceptible genotypes. Overall, suppression of the photosystem was observed in both the responses. Furthermore, RNA-seq results were validated through quantitative real time PCR. This is the first report describing genes/transcripts involved in SCN-common bean interaction and the results will have important implications for further characterization of SCN resistance genes in common bean.</p></div

Identification and Characterization of Fusarium spp. Associated with Root Rots of Field Pea in North Dakota

Root rots are a major concern in field pea production in North Dakota. However, it is unclear which pathogens are involved in causing these diseases. This report brings together findings from surveys conducted over four years (2004, 2005, 2008, and 2009). The 2004 and 2005 surveys were mainly aimed at establishing the importance of pea root rot in North Dakota and providing an indication of the most prevalent root rot pathogens. The 2008 and 2009 surveys involved thorough evaluation of root rot incidence and severity, and included isolations and characterization of Fusarium species associated with the root rots. Greater mean root rot incidence and severity were observed in 2009 compared to three previous years. Fusarium species were the most frequently isolated fungal species from infected pea roots, of which F. oxysporum (66.7 and 94.7 % of the fields) and F. avenaceum (71.8 and 89.5 % of the fields) were most commonly isolated in 2008 and 2009, respectively. Pathogenicity tests showed that all nine Fusarium species isolated from symptomatic roots were capable of causing root rot of pea, and isolates of F. avenaceum were the most virulent at causing root rot. Significant differences in virulence were observed among F. avenaceum isolates. The prevalence of F. avenaceum on roots of field peas, and the ability of isolates of this species to cause severe root rot, emphasizes the possibility of this pathogen to emerge as a potential risk under the current cropping practices for pulse crops in North Dakota, and potentially in other regions with similar growing conditions

Classification of differentially expressed genes based on biological processes in a common bean genotype resistant to <i>Heterodera glycines</i>.

<p>Significant gene ontology categories (p < 0.05) along with the number of genes involved are indicated for differentially expressed genes. RU = resistant upregulated; RD = resistant downregulated.</p

Expression analysis of differentially expressed kinases in response to common bean–<i>Heterodera glycines</i> interaction.

<p>Heat maps are arranged according to their hierarchical clustering based on expression pattern of the genes. From left to right, the four columns respectively show the expression of genes in the non-inoculated resistant genotype (RC), the SCN inoculated resistant genotype (RI), the non-inoculated susceptible genotype (SC) and the inoculated susceptible genotype (SI). Green, black and red indicate low, no change and high expression levels of genes, respectively.</p

Expression analysis of differentially expressed transcription factors in response to common bean–<i>Heterodera glycines</i> interaction.

<p>Heat maps are arranged according to their hierarchical clustering based on expression pattern of the genes. From left to right, the four columns respectively show the expression of genes in the non-inoculated resistant genotype (RC), the inoculated resistant genotype (RI), the non- inoculated susceptible genotype (SC) and the inoculated susceptible genotype (SI). Green, black and red indicate low, no change and high expression levels of genes, respectively.</p