Ralstonia solanacearum, the causal agent of bacterial wilt disease, has been found to affect numerous economically important plants. Understanding the molecular basis of resistance, tolerance and susceptibility of plants to pathogens such as R. solanacearum is a major goal of molecular plant pathologists. Prior to this study it was thought that Arabidopsis accession Kil-0 shows gene-for-gene “resistance” to an African Eucalyptus isolate of R. solanacearum, BCCF402. However, a subsequent preliminary study indicated that Kil-0 may exhibit “tolerance” which is defined as the plant’s ability to support high pathogen numbers without displaying disease symptoms or a reduction in host fitness. The aim of this study was to determine if Kil-0 was tolerant to R. solanacearum BCCF402. The bacterial load of R. solanacearum was quantified in accessions Kil-0 and Be-0 using dilution plating and quantitative PCR methods. The cytC gene region was used to quantify R. solanacearum in Arabidopsis plants and the amount of bacterial DNA was normalized to “alien” DNA that was spiked into each sample. High bacterial concentrations of BCCF402 were found in Kil-0 but plants exhibited no wilting symptoms. Additionally, Kil-0 plants inoculated with BCCF402 showed no significant reduction in fitness compared to control Kil-0 plants. In contrast, high bacterial numbers and severe disease symptoms were observed in the susceptible Be-0 plants, whereas Nd1 plants contained a low number of bacteria and no disease symptoms indicative of a resistance response. These results illustrated that Kil-0 is tolerant to R. solanacearum isolate BCCF402. A tool for the visualization of R. solanacearum in Arabidopsis plants was designed. R. solanacearum isolate BCCF402 was tagged with two mCherry-containing plasmids under the constitutive expression of the tac promoter. The expression levels of mCherry were suitable for successful visualization in planta. BCCF402 cells transformed with the mCherry-containing plasmids were not affected in terms of virulence or disease progression compared to wildtype BCCF402 cells. A plasmid loss of 30-35% was observed in mCherry-tagged BCCF402 cells at later stages of Arabidopsis infection. mCherry-tagged BCCF402 was successfully visualized in Kil-0 leaves at early infection stages. / Dissertation (MSc)--University of Pretoria, 2014. / gm2014 / Plant Science / unrestricted

Dissertation (MSc)--University of Pretoria, 2010. / Plant Science / Unrestricted

No description available.

No description available.

Perennial ryegrass (Lolium perenne L.) is a cool-season turfgrass susceptible to low temperature injury. Wilt-based (WB) irrigation is a common practice in scheduling turf irrigation as an alternative to well-watered (WW). Moisture stress has been shown to promote cold hardiness but this has not been investigated in response to WB irrigation. Measurements of 13C isotope discrimination (DELTA) are useful predictors of water use efficiency (WUE), drought resistance, evapotranspiration (ET) and salinity tolerance but the relevance to turfgrass cold hardiness has not been determined. DELTA analyses may enable more efficient screening protocols in breeding for improved cold hardiness. Objectives of this study were to examine perennial ryegrass genotypes in relation to cold hardiness, DELTA and WUE in response to WW and WB irrigation schedules, to compare genetic diversity between top-performing (TP) and bottom-performing (BP) perennial ryegrass genotypes, and to assess the predictive value of DELTA of for cold hardiness. Six genotypes were selected based on turf quality from the most northern NTEP location (Orono, ME) and included three TP (‘All Star 2’, ‘Mach I’ and ‘Sunkissed’) and three BP (‘APR-1234’, ‘Buccaneer’ and ‘WVPB-R-82’) genotypes. ET, yield, WUE, shoot water content, rooting potential, wilting tendency, DELTA and median lethal temperatures (LT50) using whole-plant survival were measured from greenhouse samples grown in weighing lysimeters in 2007 and 2008. Plant measurements in both years were based on sampling conducted at the last cycle after 68-d of irrigation with 100% of ET applied at leaf-roll (WB) versus ET replacement every 4-d (WW). Lower LT50 values were generally associated with low yield, low WUE and low shoot water content, whether the result of irrigation treatment or genotypic variation. TP genotypes demonstrated significantly lower LT50 temperatures (greater cold hardiness) in comparison to BP genotypes in both years. Modest cold hardiness enhancement with WB irrigation was highest for TP genotypes. Wilting tendency and DELTA were not reliable predictors of cold hardiness, although individual TP genotypes exhibited responses distinctly different than some BP genotypes. Further research is needed to investigate the physiological mechanisms of enhanced turfgrass cold hardiness in response to moisture stress.

The domesticated tomato Solanum lycopersicum (L.), formerly known as Lycopersicon esculentum is a genetically well-studied crop species with high-density linkage and molecular maps based on crosses done between cultivated tomato and its distant related wild species. Wild tomato species harbor a wealth of resistance to many pathogens that have been introgressed into domesticated tomato for genetic control of diseases and pests and for improvement of many agronomic traits. The wild tomato S. peruvianum (L.) is the source of the Sw-5 gene, characterized and mapped to chromosome 9 of the tomato genome and introgressed into elite tomato germplasm, providing resistance to the tospovirus Tomato spotted wilt virus (TSWV). TSWV has been reported to be a major problem for tomato growers in many parts of the world, which in some cases, has resulted in tomato fields having been abandoned for some time. Additionally, there are reports that new races of TSWV have evolved that overcome Sw-5. TSWV replicates in both, plant cells and in the alimentary canal cells of thrips and then transmitted to plants by this insect acting as a vector. Both, TSWV and thrips have co-evolved to infest and infect more than 1090 plants species in over 100 families, thrips becoming resistant to pesticides and easily escaping by hiding deep in plant parts. World trade has disseminated thrips all over the world and environmental pressures have forced TSWV to recombine its RNA to overcome new resistance.

Strawberries are considered an important crop in California where in 2018 it was in the top 5 valued fruit and vegetable commodities valued at $2.84 billion accounting for 88% of the total U.S. production. Strawberry production can be severely impacted by soilborne pathogens that can affect strawberry roots, crowns and leaves which can result in plant mortality. As much as 50 to 60% mortality can occur in one field. Pathogens responsible for such losses include Colletotrichum acutatum (syn.C. nymphaeae), Macrophomina phaseolina and Verticillium dahliae. With the phaseout of methyl bromide, host resistance and an understanding of host-pathogen interactions can play an important role in control of these diseases. A two-year study was conducted in order to evaluate host resistance of anthracnose in 105 cultivars and elite breeding lines developed by six strawberry breeding programs. Cultivars and elite breeding lines were inoculated using three local isolates in both years. All breeding programs provided genotypes that had a wide range of anthracnose susceptibility ranging from 0 to 100% mortality during both years. In both years an average of 78% of all the plant mortality occurred by 1 January. From the 105 cultivars and elite breeding lines, 30 cultivars were common to both years. Of these 30 cultivars, nine of them differed in their disease susceptibility between experiments by more than 20%. This suggests that several years of field evaluation may be necessary to determine susceptibility to anthracnose. Popular cultivars that represent the spectrum of susceptibility are Monterey (susceptible), Festival (moderately resistant), and Sensation (resistant). A second study was conducted toevaluate pathogen colonization of resistant and susceptible strawberry cultivars, testing interactions among crown and root plant tissue and two sampling timings. These cultivars were challenged with two soilborne pathogens, Macrophomina phaseolinaand Verticillium dahliae,over two years. Existing qPCR protocols for M. phaseolina and V. dahliae were used in order to quantify how much pathogen DNA was detected in crown and root samples. For the 2016-2017 V. dahliae trial there were significant effects for cultivar. Cultivar Benicia had significantly higher pathogen DNA compared to resistant cultivars Marquis, UC-12 and Camino Real. Susceptible cultivar BG 1975 had significantly less pathogen DNA compared to resistant cultivars San Andreas and Petaluma. In the 2017-2018 V. dahliaetrial pathogen DNA amount was not significantly different based on cultivar, plant part colonization, or the sampling period. In the 2017-2018 M. phaseolina trial all three of the fixed factors, cultivars, plant part colonization and sampling period were statistically significant. Cultivar ‘Sweet Ann’ had a significantly higher level of M. phaseolinaDNA in the early vs. the late sampling.

Yes / The significant role of plants can be observed through the dependency of animals and humans on them. Oxygen, materials, food and the beauty of the world are contributed by plants. Climate change, the decrease in pollinators, and plant diseases are causing a significant decline in both quality and coverage ratio of the plants and crops on a global scale. In developed countries, above 80 percent of rural production is produced by sharecropping. However, due to widespread diseases in plants, yields are reported to have declined by more than a half. These diseases are identified and diagnosed by the agricultural and forestry department. Manual inspection on a large area of fields requires a huge amount of time and effort, thereby reduces the effectiveness significantly. To counter this problem, we propose an automatic disease detection and classification method in radish fields by using a camera attached to an unmanned aerial vehicle (UAV) to capture high quality images from the fields and analyze them by extracting both color and texture features, then we used K-means clustering to filter radish regions and feeds them into a fine-tuned GoogleNet to detect Fusarium wilt of radish efficiently at early stage and allow the authorities to take timely action which ensures the food safety for current and future generations. / Supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries(IPET) through Agri-Bio Industry Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs(MAFRA) (316033-04-2-338 SB030).

In the United States corn is one of the leading agricultural products and one of the top exports. The majority of U.S corn is grown in the Midwestern region of the U.S. known as the Corn Belt where the bacterial disease Stewart's Wilt reduces crop yield. Pantoea stewartii subsp. stewartii (Pss) is transmitted into corn via the corn flea beetle insect vector, Chaetocnema pulicaria. As the beetle feeds on the corn plant leaves, Pss deposited in beetle feces enter the leaf through lesions. The early stage of Pss infection begins in the mesophyll apoplast of the corn leaf where a type III secretion system (T3SS) and its associated effectors induce water soaking (WS) and nutrient release. Ultimately, Pss will enter the plant xylem apoplast (will be referred to as the xylem) and use quorum sensing (QS) to initiate a lifestyle shift. Within the xylem, Pss grows to high cell density and secretes exopolysaccharide (EPS), forming a biofilm which eventually obstructs water transport, leading to wilting and necrosis. Previous Tn-Seq experiments provided insights into genes that are essential for in planta survival, including the master transcriptional regulator, Leucine-responsive Regulatory Protein (Lrp). To better understand the role of Lrp when Pss inhabits the xylem, RNA-Seq experiments comparing Pss wild-type and ∆lrp strains grown in planta were conducted to ascertain differential gene expression. The RNA-Seq data was further analyzed using DESeq2 and validated using qRT-PCR methods. Following validation, the Pss genome was annotated using Blast2GO software and genes upregulated and downregulated by Lrp were linked with biological processes. Lrp was found to be involved in regulating capsule biosynthesis and nitrogen-associated assimilation and metabolism during Pss survival in the xylem. This provides further insight into how Pss contends with harmful host defense compounds and extracts scarce nutrients present in the in planta xylem environment. A corn xylem fluid extraction method was developed that has enabled more physiologically relevant growth experiments to be conducted in vitro. Extracted xylem fluid was used to grow Pss wild-type and ∆lrp mutant strains as monocultures to observe any differences in growth patterns in different growth media. When grown separately in xylem fluid or Luria-Bertani (LB) medium, the Pss wild-type and ∆lrp mutant strains grew at similar rates and to final cell densities . The Pss ∆lrp mutant strain greatly outcompeted the wild type when grown together in LB medium. However, when the two Pss strains were growth together in xylem fluid, a shift in relative competition was observed, providing evidence of the wild type slightly outcompeting the ∆lrp mutant. Analysis of the composition of extracted xylem fluid through metabolomics will help define the nutrients specifically utilized by Pss in planta. Altogether, the outcome of these research projects was to provide pertinent discoveries to contribute to understanding the mechanisms used by Pss to survive in the corn xylem environment. Broadly, increased understanding of Pss pathogenesis may translate to understanding pathogenesis mechanisms in other bacterial wilt-disease causing plant pathogens. / Master of Science / Corn is a significant agricultural product and export in the United States. This important crop is used as a food source for humans, a primary nutrient source of livestock, and a major ingredient for corn-based industries manufacturing commodities such as culinary additives, biofuels, and preservatives. Certain bacteria are greatly beneficial to plants, able to increase their overall health and growth, while other bacteria share a more insidious relationship with plants and cause disease. The research discussed in this thesis focuses on the bacterial pathogen Pantoea stewartii subspecies stewartii (Pss), the causal agent of Stewart's wilt disease in corn. Pss grows inside the plant xylem (vascular tissues which distribute water throughout the plant) and forms a biofilm that causes plant wilt leading to lower crop yield and even plant death. Previous research on Pss identified important genes for successful Pss survival inside the corn plant xylem. One of those genes codes for the Leucine-responsive Regulatory Protein (Lrp). Using a combination of experimental (RNA-Seq) and computational (bioinformatics) analyses, Lrp was found to control other genes related in biological process important for living inside the plant, necessary for the metabolism of available nutrients and production the protect slime layer within biofilm. By better understanding the key bacterial genes needed for Pss to grow inside the xylem, new disease intervention strategies can be developed to disrupt these genes and impede the ability of the bacterium to infect the plant. A second part of this research project was to develop a method for extracting corn xylem fluid from the plant. Using this extracted xylem fluid, experiments could be conducted in the laboratory to study Pss growth in more detail. The original strain of Pss (wild type) was grown separately and in combination with a Pss mutant lacking the Lrp gene in the extracted xylem fluid. Both strains grew similarly in the xylem fluid, but the wild type slightly outcompeted the mutant strain when they were grown in competition. Future work in the lab will use extracted xylem fluid to determine its precise nutrient composition and the development of synthetic xylem fluid that will enable a more detailed analysis of mechanisms used by Pss to grow in the xylem. Work on Pss serves as a model for the study of other bacterial wilt-disease causing pathogens.

Pantoea stewartii subsp. stewartii is a bacterium that causes Stewart’s wilt disease in corn plants. The bacteria are transmitted to the plants via an insect vector, the corn flea beetle Chaetocnema pulicaria. Once in the plant, the bacteria migrate to the xylem and grow to high cell densities, forming a biofilm by secreting excess capsular exopolysaccharide, which blocks water transport and causes wilting. The timing of virulence factor synthesis is regulated by the cell-density dependent quorum sensing (QS) system. Such temporal regulation is crucial in establishing infection and is orchestrated by the QS-dependent transcriptional regulator EsaR. EsaR represses expression of capsular exopolysaccharide at low cell densities. At high cell densities, an acylated homoserine lactone (AHL) molecule produced during growth by the cognate AHL-synthase EsaI accumulates. The AHL binds to and inactivates EsaR, causing derepression of capsule production. EsaR is a member of the LuxR family of QS-dependent transcriptional factors. Most LuxR homologs are unstable and/or insoluble in the absence of AHL which has hindered structural studies. Chapter Two describes the changes in the structure of EsaR due to binding of AHL ligand as determined through biochemical methods. EsaR was found to be stable and retain its multimeric state in the absence or presence of AHL, but intra- and inter-domain changes occurred that affect its DNA-binding capacity. Apart from repressing expression of capsule at low cell-densities, EsaR represses its own expression and activates production of a small RNA, EsaS, with unknown function. In Chapter Three a proteomic approach was used to identify an additional 30 QS-controlled proteins. Genes encoding three of these proteins are directly regulated by EsaR and the EsaR binding sites in the respective promoters were defined. In Chapter Four, a high-throughput RNA-Seq method identified even more genes in the QS regulon that the proteomic approach overlooked. RNA-Seq analysis of rRNA-depleted RNA from two strains of P. stewartii was used as a screen to help identify 11 promoters, subsequently shown to be directly regulated by EsaR in vitro. Most of the genes controlled by QS grouped into three major physiological responses, capsule & cell wall production, surface motility & adhesion and stress response. In Chapter Five, the role of two QS regulated genes, dkgA (encoding 2, 5-diketo-D-gluconate) and lrhA (encoding a repressor of chemotaxis, adhesion and motility), in plant virulence were examined. These studies have better characterized the QS regulator EsaR and its interaction with the AHL ligand, and shown that QS has a more global response in P. stewartii than previously recognized. Further characterization of the genes identified in this study could facilitate identification of factors crucial in plant pathogenesis or insect-vector symbiosis and aid in the development of molecular-based approaches for possible disease intervention. / Ph. D.