Using Wild Relatives of Potato to Illustrate Genetic Control Against the Pathogenic Bacteria Pectobacterium carotovorum

Using Wild Relatives of Potato to Illustrate Genetic Control Against the Pathogenic Bacteria Pectobacterium carotovorum

Summary

Pectobacterium is an enterobacterial plant pathogen that causes wilt, rot and blackleg diseases in a wide variety of hosts and it is found worldwide in agricultural settings. It secretes cell wall degrading enzymes that allows it to digest the plant cell nutrients which ultimately leads to watery, soft decay and maceration of plant tissues.

Pectobacterium causes significant losses each year in potato and potato is vulnerable to infection throughout the production season as well as in storage after harvest. Farmers have very few options for management of Pectobacterium and plant resistance is the best management option. However, identification of resistance genes in potato has been difficult due to the host being tetraploid and due to Pectobacterium resistance being conferred by poorly characterized quantitative trait loci.

We had initially proposed to work with Solanum microdontum and Solanum violaceimarmoratum due their resistance to Pectobacterium. However, we were unable to complete the crosses required for this work due to uncharacterized sterility in these plants lines. Recently, the first inbred-derived F2 population was developed in potato and this population is segregating for Pectobacterium resistance. Therefore, we used this population for our work instead of the initially proposed plant populations and we were able to still reach our objectives. Greenhouse and field grown plants were tested for resistance to initiate a soft rot resistance gene mapping project in potato.

The long term goal of this project is to provide potato breeders with the tools they need to develop soft rot resistant potato varieties.

Objectives/Performance Targets

Hypothesis 1: The trait for resistance against Pectobacterium carotovorum is under genetic control. 

The Jansky lab group at UW-Madison recently created the first inbred line derived F2 population in potato by first crossing DM1-3 and M6 and then self-pollinating a F1 plant. DM1-3 is a diploid Solanum tuberosum line and M6 is a diploid Solanum chacoense line. The F2 population (DM1-3 x M6) contains 181 genetically distinct individuals that have been SNP genotyped with an Illumina Infinium array. Two different versions of this array were used: version 1 contained 8303 SNPs and version 2 has 12,720. After filtering the marker data, 2139 SNPs were polymorphic, positioned on the reference genome, and available for genetic mapping. 

M6 tubers and stems are resistant to Pectobacterium carotovorum WPP14 while like most potato lines, DM1-3 tubers and stems are susceptible. For this assay, we collected stems or petioles from the plants and placed them in test tubes with 10⁸ CFU/ml of bacteria. Many of the M6 stems and petioles retained a green and healthy appearance throughout the 4 day duration of the assay, whereas DM1-3 stems or petioles wilted within two days. We found that as little as 10⁵ CFU/ml of bacteria will kill the stem or petiole section of DM1-3 or of conventional potato and most wild potato species within two to three days.

We then screened petioles from nearly all of the 181 F2 progeny to determine if resistance to P. carotovorum WPP14 segregates in this population. Petioles from multiple greenhouse and field grown plants were screened to determine if the results would be similar across environments. We found that resistance appears to segregate, but there is a significant effect of environment. The phenotypes of the most and least resistant plants were consistent across environments, but intermediate phenotypes were inconsistent across environments. Our results were analyzed as binomial count data with SAS PROC GLIMMIX, treating genotype as a random effect with assistance provided by Dr. Jeff Endelman (Horticulture Department, UW-Madison). We found transgressive segregation, indicating that multiple loci for resistance are segregating in the population.  

Dr. Endelman performed a genome-wide scan was conducted using the 2139 SNP markers and the soft rot phenotype data.  None of the markers had significance scores above the detection threshold, which is not unexpected given the modest heritability, h² = 0.54, of the phenotype data. We are currently repeating this experiment will combine the two datasets to achieve higher heritability (h² > 0.7).

Hypothesis 2: Accession 500036 is only resistant to the species Pectobacterium carotovorum.

We screened M6, the F1 plant, and two of the most resistant and two of the most susceptible F2 lines with four additional Pectobacterium strains that span the range of known Pectobacterium diversity. We found that the resistant lines were resistant to all strains tested when inoculated with 10⁷ CFU/ml, whereas the susceptible lines were susceptible to all strains tested. This result refutes our hypothesis, but suggests that the resistance genes that we are mapping will be useful against multiple Pectobacterium species.

Hypothesis 3: Pectobacterium carotovorum is able to infiltrate through the stems of 500036 but they are not able to cause disease.

We inoculated detached petioles with GFP-labeled P. carotovorum WPP14 to determine if bacterial colonization differed in this assay among resistant and susceptible diploid lines. We examined thin petiole sections cut every 1 cm from the inoculated end of the petiole 12 hours post-inoculation and found that bacteria more frequently colonized the petiole vascular bundles further from the inoculation site in susceptible plants, suggesting that differences in xylem structure or composition affect plant resistance to P. carotovorum (Fig. 1). We also found that the bacteria appeared to adhere to the spiral vessels in the xylem. This suggests that resistant plants restrict bacterial movement in the xylem.

 Hypothesis 4: All the accessions of Solanum microdontum and Solanum violaceimarmoratum, are resistant to Pectobacterium carotovorum

We began characterizing S. microdontum and S. violaceimarmoratum lines for soft rot and stem rot resistance, but because we were unable to construct useful crosses with these lines, we abandoned this line of research. We screened additional accessions of Solanum chacoense and identified lines that vary in soft rot and stem rot resistance, demonstrating that this species is a useful source of resistance, but that not all of lines from any of the accessions tested were resistant.

• Figure 1 GNC13-174

Accomplishments/Milestones

From these results, we made these conclusions:

1. 1. The F1 plant is at least as resistant to carotovorum as its parent, M6. Therefore, at least some of the resistance genes in M6 are likely to be dominant.

1. 1. Some of the F2 progeny were more resistant to carotovorum than the M6 grandparent and the F1 parent, which suggests that multiple soft rot resistance loci are segregating.

1. 1. The resistance from M6 functions against multiple Pectobacterium species, including at least two carotovorum subspecies, P. wasabiae, and P. atrosepticum.

1. 1. Resistant plants may restrict movement of Pectobacterium in plant xylem.

1. 1. Pectobacterium resistance is found in some, but not all, lines derived from various accessions of Solanum chacoense.

Impacts and Contributions/Outcomes

Impacts and Contributions/Outcomes

In the past year, the impacts of this project include:

1. 1. Initiation of a mapping project to identify quantitative trait loci that contribute to soft rot and stem rot resistance in potato.

1. 1. Identification of a possible resistance phenotype, restriction of bacterial movement in the xylem.

1. 1. Identification of additional chacoense lines that resist Pectobacterium carotovorum.

The data described here were used as preliminary data for a USDA-Hatch proposal and a NIFA proposal to continue to map these resistance genes, with the goal of identifying resistance loci and associated molecular markers that can be used by potato breeders to help develop soft rot and stem rot resistant potato varieties.

The results from this work were presented at farmer outreach events in 2014 and at a scientific meeting (American Phytopathological Meeting, Minneapolis MN, Aug 2014).

Current work

We are repeating the resistance assays and will combine the datasets to achieve higher heritability in order to map the resistance QTL. Once this is completed, we intend to prepare and submit a manuscript describing this work to a peer-reviewed journal. 

Collaborators: