Location: Emerging Pests and Pathogens Research
2022 Annual Report
Objectives
Objective 1: Identify genomic resources for development of diagnostics and detection tools for emerging and re-emerging bacterial plant pathogens.
Sub-objective 1.A: Perform comparative genomics of bacterial pathogens.
Sub-objective 1.B: Investigate diversity of soft rot Pectobacteriaceae (SRP).
Objective 2: Characterize biology and virulence factors of bacterial plant pathogens and identify their targets in host plants.
Sub-objective. 2.A: Discover and characterize genes that contribute to disease and/or host adaptation of bacterial soft rot pathogens.
Sub-objective 2.B: Discover and characterize genes involved with interactions between bacterial species.
Sub-objective 2.C: Determine the mechanistic basis of socially affected behaviors in bacteria.
Sub-objective 2.D: Determine the contribution of AlgU to virulence and factors affecting AlgU activity.
Sub-objective 2.E: Characterize the signaling pathways that impact expression of HiVir gene cluster in Pantoea.
Objective 3: Investigate sustainable strategies for control of bacterial plant diseases.
Sub-objective. 3.A: Investigate the role of antimicrobials in tolerance to bacterial soft rot pathogens.
Sub-objective 3.B: Identify genetic markers of soft rot disease tolerance in US Potato Genebank germplasm.
Approach
Soft rot bacteria, such as Dickeya, Pectobacterium, and Pantoae are among the most important pathogens of vegetables, fruits, and ornamentals. Bacterial diseases of potato and onion alone cause more than $60M in losses annually in the U.S. Despite the extensive amount of research available on bacterial plant pathogens, there is a lack of understanding about how bacterial plant pathogens enter and move within crop production systems and to what degree these diseases are caused by endemic populations. Furthermore, some bacterial species are endemic and, in some environments, a single bacterial species can be represented by a number of different strains, some of which are pathogens, and some of which are non-pathogens with beneficial biocontrol activities. Therefore, determining which bacteria are responsible for disease and furthermore how certain bacterial strains become pathogenic is an area of research that warrants further study. To address this, we will use genome sequencing methods to characterize populations bacteria present in diseased crops. The patterns that emerge at the intersection of pathogen diversity and geographical location will provide key insights on disease emergence as well as identify diagnostic markers able to distinguish pathogens from non-pathogens. For some plant diseases, such as bacterial soft rot (potato: Dickeya spp. and Pectobacterium spp.; onion: Pantoea spp.) many bacterial species may be involved, with the pathogens being members of a broader community of plant-associated microbes. Little is known about the relationships and interactions of plant pathogens with the host, the microbial community, and the environment and the impact on disease outcome. We will investigate bacterial communication mechanisms involved in pathogen fitness and formation of complex communities in plants to identify factors critical for disease. For many bacterial soft rot diseases there are no effective management options. For example, there are no commercially available potato or onion cultivars with soft rot resistance, thus management options for these pathogens are very limited. Additionally, there is little known about the specific mechanisms involved in host tolerance or susceptibility. To address this, we will first identify and characterize factors that bacteria use to cause disease and then use that information to guide discovery of bacterial control strategies. Second, we will identify and characterize sources of natural resistance in wild crop relatives to provide information for breeders as well as a source germplasm for breeding resistant varieties. All together this research will lead to improved fundamental understanding of bacterial soft rot disease dynamics and reveal vulnerabilities that can be exploited for control of bacterial plant diseases, helping us work towards the goal of sustainable plant disease management.
Progress Report
Project 8062-21000-042-000D terminated in February 2022 and has been replaced with new project 8062-21000-048-000D. Activities during this reporting period have focused on concluding the old project and laying the foundation for the new project 8062-21000-048-000D.
Objective 1: Identify genomic resources for development of diagnostics and detection tools for emerging and re-emerging bacterial plant pathogens. We continued our efforts to sequence and assemble the genomes of strains of bacteria pathogenic to onions, potatoes and nursery crops. In addition to a performing genome sequencing of number of Pantoea spp. strains, we also closed the genomes of two strains of Dickeya fangzhongdai, a recently described soft rot bacterial pathogen with a broad host range, including pear, orchids, banana, taro, and onions. It has been isolated from diseased plants in Asia, Europe, the Caribbean, and North America. Our lab recently acquired two strains of Dickeya fangzhongdai that were collected from different locations in New York, one was isolated from Chinese evergreen (Aglaonema sp.), and one was isolated from onion (Allium cepa). Together with our colleagues, we have demonstrated that both strains cause soft rot in potatoes and onions. However, one strain is particularly virulent on onions and is able to completely liquefy an onion bulb in several days. We produced and published high-quality closed genomes for both strains. By performing a detailed comparative genomics analysis, we identified hundreds of genomic differences between the two strains, but two were particularly noteworthy. First, the strain which was more virulent had five putative pectinase genes whereas the other had four. Pectinases are used by plant pathogens, such as Dickeya spp., to degrade plant cell walls in order to gain access to nutrients. The disease symptoms in onions (e.g., maceration and tissue collapse) suggest that cell walls are being degraded, so differences between the two strains in cell wall degrading enzymes might explain the observed differences in symptoms. Second, the strain able to cause more disease in onions, possessed a cluster of genes (alt gene cluster) which has previously been shown to provide tolerance of Pantoea sp. to certain thiosulfide compounds produced by onions and other alliums. Experiments are underway to investigate if the additional pectinase genes and/or presence of the alt gene cluster provide an advantage to certain plant pathogenic bacteria. We completed genome sequencing of 88 potato soft rot pathogens that were collected from all major potato growing regions of the United States. One example of the discoveries we are making from these approaches includes discovery of new pathogen species causing disease in U.S. potato production. Our results show that we have identified at least one species of soft rot bacteria that has not been described. This new species was responsible for potato disease in New York and North Dakota. Additionally, in collaboration with Scientists at the Canadian Food Inspection agency, we also determined that this new pathogen was isolated from diseased potato in Canada. We are conducting additional analysis of the genetic relationships between strains found in the U.S. and Canada to determine whether there is evidence of pathogen movement between potato production areas. In work that carries over from the terminating project, we have sequenced and assembled the genomes of three Pantoea spp. These genomes, combined with those produced under the previous project, are being used to assemble a comprehensive database of the genomic potential of pathogenic and non-pathogenic strains of Panotea spp. These genes are being submitted to NCBI’s genome database so that they will be publicly available.
Objective 2: Characterize biology and virulence factors of bacterial plant pathogens and identify their targets in host plants. Previously we constructed a high-density barcoded Transposon library in several Dickeya species and used the libraries to identify genes important for fitness of Dickeya in potato tubers. To identify if common genes are required for fitness of Dickeya sp in other parts of the potato plant, we set up experiments to inoculate the transposon libraries into potato stems. We are currently evaluating and comparing the results with our previous experiments which focused on bacterial genes required for fitness in potato tubers and in vitro conditions. We have selected some of the genes found to be important for fitness of Dickeya in potato tubers and/or stems and are evaluating if the mutants display altered phenotypes that impact the ability of bacteria to cause disease. In planta global transcriptome profiling experiments (RNA-Seq) were performed with Dickeya inoculated in potato stems. We found that Dickeya displays altered gene expression in potato plants that are susceptible to the pathogen compared to potato plants that are tolerant to the pathogen. For instance, we found that when in tolerant potatoes, bacteria showed increased expression of genes that mediate resistance to cationic antimicrobial peptides and genes implicated in a wide variety of physiological functions including nutrient acquisition and host-pathogen interactions in other bacteria disease compared to bacteria that were from susceptible potatoes. We also observed that when in tolerant potatoes, bacteria showed down regulation of genes that encode bacterial factors important for causing disease compared to bacteria that were from susceptible potatoes, suggesting that the tolerant potatoes produce factors that inhibit the ability of the bacteria to cause disease. These experiments provide important insights into how the bacteria responds to the plant environment and provides putative targets to control bacterial soft rot pathogens, like Dickeya.
Plant pathogens express their virulence factors when they are needed in plants. Not all the plant signals that activate bacterial virulence are known. Remarkably, if plants detect an infection, they respond by raising the pH, which interferes with the bacteria’s ability to express their virulence genes. Our experiments have revealed additional insights into the molecular systems controlling the activation of bacterial virulence factors by plant signals, such as pH. This is foundational knowledge that explains some of the most fundamental mechanisms responsible for plant health and disease.
Bacteria alter their behavior depending on their genetic relationship and proximity to other cells. In most cases, bacterial cells try to balance the benefit of cooperation with the cost of competition. One of the ways that they do this is by moving closer to or farther away from other cells. We found that populations of soft rot pathogens change their spatial arrangement to enhance cooperative digestion of plant nutrients. This is a significant discovery that shows that bacterial cells cooperate with each other to digest and consume nutrients that plants provide.
A gene cluster consisting of 11 genes, designated the HiVir gene cluster, is present in pathogenic strains of Pantoea species known to cause onion center rot. The signals that activate this important gene cluster are not known. To identify the environmental signals and regulatory pathways that induce the HiVir genes we have started several high-throughput (‘omics) studies of the HiVir gene cluster in Pantoea. We established conditions that induce expression of HiVir for several strains of Pantoea spp. Separately we evaluated growth conditions in different media, both inducing and non-inducing conditions.These experiments will be completed and described in future reports.
Objective 3: Investigate sustainable strategies for control of bacterial plant diseases. We purchased purified peptides that we hypothesized would exhibit bactericidal or bacteriostatic activity against bacterial plant pathogens. We set up assays with various concentrations of the peptides and found that the peptides did not inhibit growth of the plant pathogenic bacteria. Studies are in progress to evaluate the ability of the peptides to inhibit virulence related phenotypes such as motility and pectinase activity of bacteria. Efforts were also spent consulting with collaborators and designing experiments to overexpress plant peptides and putative bacterial toxins in plants to enable testing of the molecules on growth of bacterial plant pathogens in planta.
This year marked our first test of hybrid potato plants carrying resistance to soft rot disease. In our previous project, we identified wild potato plant relatives that carry resistance to bacterial soft rot. We collaborated with scientist at the U.S. Potato GeneBank to produce three families of hybrid plants made by crossing the resistant wild potato plant with a sensitive domesticated variety. We tested the offspring and found that the resistant trait was inherited and distributed among the offspring. We will repeat this study and use the results to map the location of genes responsible so that the genes can be used to improve disease resistance in potatoes grown in the U.S.
Accomplishments
1. Disease symptoms caused by Dickeya fangzhongdai vary by strain. Dickeya fangzhongdai (Dfa) is an emerging bacterial pathogen that can cause devasting disease symptoms of several important crops. Unfortunately, its host range is poorly understood, so management is difficult. To better understand this important pathogen, USDA scientists in Ithaca, New York, performed a detailed comparison of two strains of Dfa that were isolated in different regions of New York State. For this study, the strains were inoculated into potato tubers and onion bulbs and disease progression and symptoms were compared. One of the strains produced far more severe symptoms far more quickly than the other. The genomes of both strains were sequenced and compared. This study revealed that the more aggressive bacterial strain contains extra copies of genes that produce cell wall degrading enzymes and a gene cluster that helps bacteria survive in onion tissue. These results provide knowledge that can be used to guide development of new strategies to prevent emerging plant diseases.
2. An assay to detect viruses in dormant potato tubers. Potato is one of the most valuable food crops grown in the United States. Because potatoes are produced by planting tubers from the previous year’s crop, diseases and pests of potatoes can spread rapidly causing significant losses. For viruses, potato seed certification relies primarily on visual inspection of fields during the growing season followed by post-harvest grow outs. However, because of the emergence of new viruses that do not always display symptoms and a desire for potato seed growers to obtain seed certification results earlier, there is a need for improving potato tuber testing. ARS scientists in Ithaca, New York, and collaborators optimized a high throughput diagnostic assay for viral pathogens in dormant potato tubers. For the assay, plant material is taken from harvested potato tubers and pressed onto a paper card. Nucleic acid is extracted and the pathogens are detected using molecular methods. This assay provides earlier detection of pathogens of potatoes with an accuracy of ninety-five percent when compared to current testing methods, providing a robust alternative to current seed certification testing allowing growers and buyers to receive data earlier. We anticipate that the assay will be used for subsequent testing of a wide array of pathogens that are a current or future interest of potato seed certification.
