Department of Plant Pathology
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The Department of Plant Pathology at the University of Minnesota has a strong research and teaching emphasis in disease resistance, molecular genetics and genomics, control of diseases caused by biotic pathogens, wood deterioration, effect of air pollution on plants, biological control, ecology and evolution of plant-associated microbes, and in the physiology and molecular biology of plant-microbe interactions. The department also has a strong outreach emphasis through the extension service, plant disease clinic, and other avenues.
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Item Antibacterial Activity of Plant Defensins Against Alfalfa Crown Rot Pathogens(2017) Sathoff, Andrew; Velivelli, Siva; Shah, Dilip; Samac, Deborah AAlfalfa (Medicago sativa) is the fourth most widely grown crop in the United States. Alfalfa crown rot is a disease complex that severely decreases alfalfa stand density and productivity in all alfalfa-producing areas. Currently, there are no viable methods of disease control. Plant defensins are small cationic antimicrobial peptides with a conserved signature of cysteines. The in vitro and in planta antifungal activity of plant defensins has been extensively studied. However, their antibacterial activity has been less well characterized. Defensins have a γ-core motif, a cluster of cationic and hydrophobic residues, which is essential for antimicrobial activity. The γ-core motifs of five synthetic defensins were tested for antibacterial activity against the bacterial pathogens in the alfalfa crown rot disease complex. Full length defensins, expressed using a Pichia pastoris expression system, were tested to compare antibacterial activity. A spread plate method was used to quantify antibacterial activity of defensins. Bacteria were grown out to an OD600 value of 0.1, and a 200 μL culture was incubated with shaking for 3 hours with concentrations of defensin peptide up to 30 μg/mL. The bacteria were serially diluted, and 100 μL was plated on to NBY plates. After 48 hours of incubation, the bacterial colonies were counted. The amount of defensin needed to inhibit growth of pathogen strains by 50% (IC50) was calculated. The core motif of MtDef4 was shown to be the most effective truncated peptide with IC50 values of 3.4 μM against Pseudomonas syringae pv. syringae and 4.52 μM against Xanthomonas alfalfae. Also, the corresponding full length MtDef4 peptide was found to be active against P. syringae pv. syringae and X. alfalfae with IC50 values of 0.43 μM and 0.68 μM, respectively. These experiments show the previously overlooked high biological activity of plant defensins against bacterial pathogens. Additionally, these results indicate that the γ-core-motif can be used to predict biological activity of the full-length defensin, and that transgenic expression of plant defensins in alfalfa has the potential to lead to improved crown rot resistance.Item Antimicrobial Activity of Brassica rapa Nectar Lipid Transfer Protein(2017) Sathoff, Andrew E; Samac, Deborah A; Holl, Catherine; Schmidt, Tony; Carter, ClayAntimicrobial peptides (AMPs) provide an ancient, innate immunity conserved in all multicellular organisms. In plants, there are several large families of AMPs defined by sequence similarity. The nonspecific lipid transfer protein (LTP) family is defined by a conserved signature of eight cysteines and has a compact structure with a lipid-binding hydrophobic cavity. The antimicrobial activity of LTPs varies greatly among plant species. An LTP from Brassica rapa (variety R-o-18) nectar was tested for antimicrobial activity. In a 96-well microplate, each well contained half strength potato dextrose broth, approximately 2000 spores, and concentrations of LTP peptide up to 300 μg/mL in a total volume of 100 μL. After 48 hours of incubation at 25 C in the dark, absorbance of the wells was measured at 595 nm on a microplate reader to quantify the inhibition of fungal growth. The amount of LTP needed to inhibit growth of pathogen strains by 50% (IC50) was calculated. This Brassica LTP was most effective against Trichoderma and Bipolaris oryzae with IC50 values of 0.78 μM and 1.71 μM, respectively. Additionally, both Colletotrichum trifolii and Alternaria solani had IC50 values of less than 4.0 μM. The activity of this Brassica LTP at such low biological values indicates that it is a potent defense protein. These results suggest that transgenic expression of antimicrobial LTPs has the potential to lead to improved broad-spectrum disease resistanceItem Aurora Sporealis 1924 - 2006 Personal Name and Subject Index(2008-10-28) Tertell, Susan M.; Van Why, Carol B.Comprehensive index of personal names and subjects found in the Aurora Sporealis magazines issued by the Plant Pathology Department from 1924-2006, accounting for spelling errors, name changes and nicknames.Item Characterization of the bacterial stem blight pathogen of alfalfa, Pseudomonas syringae pv. syringae ALF3(2014) Samac, Deborah A; Studholme, David J; Ao, SamadanglaBacterial stem blight of alfalfa occurs sporadically in the central and western U.S. Yield losses of up to 50% of the first harvest can occur with some cultivars. Developing resistant cultivars is hampered by lack of information on the pathogen and a standard test for evaluating plant germplasm. Bacteria producing a fluorescent pigment were isolated on King’s B agar from alfalfa with symptoms of bacterial stem blight from near Cheyenne, WY. The strain ALF3 was tentatively identified as Pseudomonas syringae pv. syringae based on 16S rDNA sequence and PCR amplification of syrB for lipodepsinonapeptide toxin production. Multilocus sequence analysis indicated that ALF3 falls within a clade containing strains of P. syringae pv. syringae with closest affinity to FF5 from pear. Comparison of a draft whole-genome sequence of ALF3 further confirmed that ALF3 most closely resembles FF5 (~96% sequence identity) and P. syringae pv. aptata DSM50252 from beet. Approximately 60 genes were unique to ALF3, including several predicted genes in the T3SS cluster such as a type III helper protein HrpZ (Pto) and phage-associated genes. ALF3 was highly pathogenic to snapbean pods but caused only mild symptoms on leaves of snapbean, pear, and sugarbeet. A standardized method for evaluating disease resistance in alfalfa was developed. Cultivars with fall dormancy ratings of 1 and 2 had higher percentages of resistant plants than cultivars with fall dormancy ratings of 8-11.Item Identification of markers associated with race-specific resistance to Aphanomyces root rot in alfalfa, Poster at the 2017 APS meeting(2017) Samac, Deborah; Bucciarelli, Bruna; Dornbusch, Melinda; Miller, Susan; Yu, Long-XiAphanomyces root rot, caused by Aphanomyces euteiches, is one of the most important diseases of alfalfa in the United States. Two races of the pathogen are recognized and although most cultivars are resistant to race 1, fewer have resistance to race 2, the predominant race in North America. Molecular markers are needed to facilitate breeding for resistance and to clarify race/resistance gene structure. Resistant and susceptible seedlings were identified from three resistant cultivars, WAPH1, WAPH5 and 53V52, and used as parents to produce F1 populations. Severity of symptoms corresponded with amount of pathogen DNA and oospore density in roots. Race-specific resistance involves a hypersensitive response of individual epidermal or cortical cells upon pathogen attack followed by suberization of cells surrounding the stele and strong autofluorescence in cortical cells, indicating the presence of phenolic compounds. Segregation ratios of F1 populations suggested that resistance to race 1 in WAPH1 is conditioned by a single gene but resistance to race 1 is multigenic in WAPH5 and 53V52, and resistance to race 2 is multigenic in all three cultivars. Segregation for resistance to seven strains of A. euteiches in 70 F1 full-sib plants derived from 53V52 suggested the presence of clustered resistance genes and multiple race types. Identification of resistance gene loci is in progress using genotyping by sequencing and genetic mapping of F1 populations.Item Plant defensins inhibit growth of pathogens in the alfalfa crown rot disease complex(2016) Sathoff, Andrew E; Samac, Deborah AAlfalfa crown rot is a disease complex that severely limits alfalfa stand density and productivity in all alfalfa-producing areas. Currently, there are no viable methods of control. Plant defensins are small cationic antimicrobial peptides with a conserved signature of cysteines. Defensins have a γ-core motif, a cluster of positively charged residues, which is essential for antimicrobial activity. The γ-core motifs of five synthetic defensins were tested for antimicrobial activity against the pathogens in the alfalfa crown rot disease complex. In a 96-well microplate, each well contained half strength potato dextrose broth, approximately 2000 spores, and concentrations of defensin peptide up to 30 μg/mL in a total volume of 100 μL. After 48 hours of incubation at 25 C in the dark, absorbance of the wells was measured at 595 nm on a microplate reader to quantify the inhibition of fungal growth. The amount of defensin needed to inhibit growth of pathogen strains by 50% (EC50) was calculated. The γ-core motif of MtDef4 was shown to be the most effective peptide with EC50 values of 5.3 μM against Phoma medicaginis and 6.9 μM against Fusarium oxysporum f.sp. medicaginis. In addition, MtDef4 had activity against Pseudomonas syringae pv. syringae and Xanthomonas alfalfae subsp. alfalfae but not the oomycete Aphanomyces euteiches in in vitro assays. These results indicate that transgenic expression of plant defensins in alfalfa has the potential to lead to improved crown rot resistance.Item Transgenic expression of Medicago truncatula PR10 and PR5 promoters in alfalfa shows pathogen induced up-regulation of transgene expression(2016) Sathoff, Andrew E; Samac, Deborah AGenetic modification of alfalfa for introduction of novel traits requires promoters for controlling gene expression. Promoters that are constitutively activated for expression of genes that enhance disease resistance pose a great energy load on the plant and exert a strong selective pressure on the pathogens. Promoters that are induced upon pathogen invasion are needed for engineering plants with disease resistance. Medicago truncatula promoter regions of pathogenesis-related (PR) genes, PR5 and PR10, were identified as being highly up-regulated during the initial stages of infection by root and foliar pathogens. These promoters were PCR amplified and cloned into plant transformation vectors ahead of the β-glucuronidase (gus) gene. Agrobacterium mediated transformation was used to create transgenic lines of alfalfa (cultivar Regen SY27x). The transgenic plants were stained for GUS activity. In uninoculated plants, GUS activity was primarily seen in the root vascular tissues. No activity was observed in uninoculated leaves. With fungal pathogen infection, staining was greatly enhanced and allowed for stain visualized in the leaves. Quantitative PCR assays were done to quantify pathogen-induced GUS expression, as well as expression of PR5 and PR10 in infected leaves. RNA was extracted from symptomatic infected leaves after inoculation and converted to cDNA. Using specific primers, transcript accumulation was compared between cDNA from mock inoculated and inoculated plant tissue. In plants with the PR10:GUS or PR5:GUS constructs, GUS transcripts accumulated 41- to 378-fold over the mock inoculated plants at 7 days after inoculation with Phoma medicaginis, depending on the plant line. GUS transcripts were also strongly up-regulated in response to Colletotrichum trifolii and Pseudomonas syringae pv. syringae. Consistently, the PR10 promoter had greater fold amplifications and greater activity than the PR5 promoter. In response to P. medicaginis, transcripts of the PR10 gene were up-regulated 31- to 221-fold at 7 days after inoculation and transcripts of the PR5 gene were up-regulated 44- to 60-fold. These experiments show that the M. truncatula PR10 promoter is functional in alfalfa for expression of transgenes and up-regulates genes after infection by a range of alfalfa pathogens.Item Variances in nutritient content and yield of alfalfa protein concentrate processed with five mthods from high yielding biomass type alfalfa(2017) Coburn, Jessica; Wells, Scott; Samac, Deborah AThe demand for protein is growing with increased populations and world affluence. A sustainable and affordable protein source is needed to support the growing aquaculture industry worldwide. Alfalfa produces high levels of protein and provides numerous environmental services, potentially making it an ideal feedstock in aquaculture. This project evaluates five methods of alfalfa protein concentrate (APC) coagulation for yield and nutritional content. A biomass-type alfalfa was harvested at full flower with a flail mower, fresh material homogenized in a laboratory blender, juice expressed by hand, and protein coagulated using five methods. The methods were: (1) heating in an 80°C water bath for 30 minutes, (2) freezing the juice at -15°C, (3) lowering the pH of the juice to 4.0 with hydrochloric acid, (4) lowering the pH of juice chilled in refrigeration with chilled hydrochloric acid, (5) first, raising the pH of the juice to 10.0 with sodium hydroxide, letting it set for 15 minutes, then lowering the pH down to 4.0 with hydrochloric acid. The temperature and pH based methods resulted in significant differences in protein yield and concentration. While the acid based coagulation methods resulted in the highest yield and lowest fiber content, temperature methods resulted in higher concentrations of protein. Extraction methods also impacted crude fat, sugar, and specific amino acids. Acid coagulation resulted in decreased fiber and fat content, which may be beneficial for aquaculture uses. Heat treatment resulted in increased concentration of limiting amino acids and lower sugar content compared to the other methods. These findings show that APC producers can adjust production methods for a specialized APC product meant for specific diets or uses.