Isolation and characterization of Pythium spp. from South Dakota soils under commerial alfalfa production

Alfalfa is a significant crop in South Dakota that provides many different benefits for its growers. South Dakota plants the most acres of alfalfa in the United States. It is used as a protein-rich feed for livestock, a cover crop that protects against soil erosion, and a natural fertilizer because of its ability to fix nitrogen in the soil. However, alfalfa seedlings are susceptible to many diseases. Pythium root and seed rot is one disease known to have devastating effects on alfalfa field establishment and yield. Pythium species are oomycete pathogens that inhabit the soil and remain present and pathogenic as oospores. Pythium diseases of alfalfa cause reduced root systems, plant size, length, and growth rate. Pythium management is centered on fungicidal seed treatments. There have been recent reports of Pythium spp. infecting alfalfa across the world in places like Sudan and China, but current research in South Dakota is needed. In our research, we isolated Pythium spp. from Lake County South Dakota soils under commercial alfalfa production. We also characterized these isolates with a DNA sequencing analysis and evaluated the isolates for fungicide sensitivity. This summer, we will conduct a statewide Pythium disease survey and assess the collected isolates for fungicide sensitivity and pathogenicity towards various commercial lines of alfalfa. This research will provide growers with the information necessary to make educated decisions in order to increase yields and maximize their profits

Plant Defensins: An Innovative Approach to Control Alfalfa Crown Rot

University of Minnesota Ph.D. dissertation. July 2019. Major: Plant Pathology. Advisor: Deborah Samac. 1 computer file (PDF); x, 153 pages.Crown rot is a disease complex that reduces alfalfa (Medicago sativa) stand density and causes substantial losses in productivity in all alfalfa-growing areas. To evaluate plant defensins as a potential control for alfalfa crown rot, defensins were screened for antimicrobial activity. MtDef5, a defensin from Medicago truncatula, displayed high activity against both bacterial and fungal crown rot pathogens in vitro. Agrobacterium-mediated transformation was used to create transgenic lines of alfalfa constitutively expressing MtDef5. Disease bioassays demonstrated increased resistance against fungal and bacterial crown rot pathogens in the transgenic lines expressing MtDef5. Transgenic expression of defensins could be utilized to implement an eco-friendly, protein-based strategy that could provide alfalfa with enhanced resistance against crown rot and reciprocal gains in alfalfa yield. Mini-Tn5-lux mutant strains of Pseudomonas aeruginosa with Tn insertions disrupting outer membrane protective modifications were assessed for sensitivity against plant defensin peptides. Also, these strains were evaluated for lux gene expression in response to sublethal plant defensin exposure. A defensin from M. truncatula, MtDef4, induced dose-dependent gene expression of the aminoarabinose modification of LPS and surface polycation spermidine production operons. A plant pathogen, Pseudomonas syringae pv. syringae was modified through transposon mutagenesis to create mutants that are resistant to in vitro MtDef4 treatments. The transposon insertion site on defensin resistant bacterial mutants was sequenced, and modifications of ribosomal genes were identified to contribute to enhanced resistance to defensin treatments. Therefore, the MtDef4 antibacterial mode of action may also involve inhibition of translation. M. 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. Theses promoters, along with the alfalfa homolog for PR10, were cloned into plant transformation vectors ahead of the beta-glucuronidase (gus) gene. Agrobacterium-mediated transformation was used to create transgenic lines of alfalfa. Quantitative PCR assays were utilized to evaluate pathogen-induced GUS expression. The MtPR10 promoter had greater fold amplifications and greater activity than the MsPR10 and MtPR5 promoters. The MtPR10 promoter is functional in alfalfa for expression of transgenes and up-regulates genes after infection by a wide range of alfalfa pathogens

Plant defensin antibacterial mode of action against Pseudomonas species

Background: Though many plant defensins exhibit antibacterial activity, little is known about their antibacterial mode of action (MOA). Antimicrobial peptides with a characterized MOA induce the expression of multiple bacterial outer membrane modifications, which are required for resistance to these membrane-targeting peptides. Mini-Tn5- lux mutant strains of Pseudomonas aeruginosa with Tn insertions disrupting outer membrane protective modifications were assessed for sensitivity against plant defensin peptides. These transcriptional lux reporter strains were also evaluated for lux gene expression in response to sublethal plant defensin exposure. Also, a plant pathogen, Pseudomonas syringae pv. syringae was modified through transposon mutagenesis to create mutants that are resistant to in vitro MtDef4 treatments. Results: Plant defensins displayed specific and potent antibacterial activity against strains of P. aeruginosa. A defensin from Medicago truncatula, MtDef4, induced dose-dependent gene expression of the aminoarabinose modification of LPS and surface polycation spermidine production operons. The ability for MtDef4 to damage bacterial outer membranes was also verified visually through fluorescent microscopy. Another defensin from M. truncatula, MtDef5, failed to induce lux gene expression and limited outer membrane damage was detected with fluorescent microscopy. The transposon insertion site on MtDef4 resistant P. syringae pv. syringae mutants was sequenced, and modifications of ribosomal genes were identified to contribute to enhanced resistance to plant defensin treatments. Conclusions: MtDef4 damages the outer membrane similar to polymyxin B, which stimulates antimicrobial peptide resistance mechanisms to plant defensins. MtDef5, appears to have a different antibacterial MOA. Additionally, the MtDef4 antibacterial mode of action may also involve inhibition of translation

Plant defensins inhibit growth of pathogens in the alfalfa crown rot disease complex

Poster presentation at the 2016 American Phytopathological Society Annual Meeting.Alfalfa 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

Transgenic expression of Medicago truncatula PR10 and PR5 promoters in alfalfa shows pathogen induced up-regulation of transgene expression

Poster presentation at the 2016 North American Alfalfa Improvement Conference.Genetic 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

Antibacterial Activity of Plant Defensins Against Alfalfa Crown Rot Pathogens

Poster presentation at the 2017 meeting of the American Society for Microbiology.Alfalfa (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

Antimicrobial Activity of Brassica rapa Nectar Lipid Transfer Protein

Poster presentation at the 2017 Mycological Society of America Annual Meeting.Antimicrobial 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 resistanc

Appendix 8, Decay of Cerium-144

As part of an earlier program of investigation in this laboratory, studies were made of the gamma ray spectrum and the beta ray spectrum of cerium-144. In the present work, seme coincidence studies were made on one of the beta groups appearing in the cerium-144 decay and on the gamma rays appearing in the deexcitations from the energy levels of praseodymium-144. Sources of cerium-144 were prepared frcm carrier free radioactive cerium-144 as supplied by the Oak Ridge National Laboratory. The sample material was more than two years old at the time of preparation of sources. No additional chemical purification was attempted. Sources for use in the beta crystal spectrometer were mounted on thin Formvar film on spectrometer ring mounts. The gamma ray spectrum of cerium-144 in the energy range 20 kev to 180 kev is shown in Figure 1. This spectrum was determined using a 2-inch by 2-inch NaI(Tl) crystal. The pulse spectrum was analyzed by a Radiation Instrument Development Laboratory (RIDL) 200 channel analyzer. The spectrum gives clear evidence of gamma ray peaks at 34 {+-} 3 kev and 134 {+-} 2 kev. A rather broad peak at 80 kev is observed. An indication of a gamma ray group of energy near 100 kev is also shown. The resolution of the detecting assembly was 9.8 percent at 662 kev. The uncorrected relative intensities of the three groups of 34, 80 and 134 kev are 95, 35, and 100, respectively. These intensities are for the gamma radiation exclusive of internal conversion. Gamma-gamma coincidence measurements were made using two of the 2-inch by 2-inch sodium iodide crystals placed at 90 degrees to one another, Ganmma radiation of a particular energy was selected by means of a single channel analyzer and the 200 channel analyzer was used to analyze any coincident pulses frcm detector number two. Insertion of a 1.6 m/sec delay in channel one gave direct observation of random coincidences under identical experimental conditions. Coincidence spectra were taken setting the single channel analyzer for 34 {+-} 2, 80 {+-} 2, 100 {+-} 2 kev gamma radiation. Five hour true and random counts were taken at each setting. Figure 2 shows the spectrum of gamma radiation of cerium-144-praseodymium-144 in coincidence with the 34 kev gamma radiation. There is evidence for coincident radiation of 34, 55, 82 and 101 kev energy with only weak coincidences at 135 kev energy. Figure 3 shows the gamma ray spectrum of cerium-144-prasedymium-144 in coincidence with the 80 kev gamma radiation. Here the expected coincidences with radiation of energy about 35 kev is observed as well as coincidences with gamma radiation of about 54 kev energy. There are no indications of coincidences with gamma radiation of energy greater than 54 kev and less than 180 kev (channels 100 to 199 are not shown). Figure 4 shows the gamma ray spectrum of cerium-144-praseodymium-144 in coincidence with the 100 kev gamma radiation. Here coincidences are observed with radiation of about 34 kev energy (channels 100 to 199 are not shown). No coincidences were observed between gamma rays of 34 kev and gamma rays of energy less than 180 kev (channel 100 to 199 are not shown) except for relatively weak coincidences vith 34 kev

Management Strategies and Distribution of Aphanomyces Root Rot of Alfalfa (Medicago sativa), a continuing threat to forage production in the United States

Alfalfa (Medicago sativa) is one of several legumes that is affected by Aphanomyces root rot (ARR) caused by Aphanomyces euteiches. Symptoms of ARR on alfalfa seedlings include a yellow-grey discolouration of roots, rotting and loss of lateral roots, stunted growth, chlorotic foliage and reduction of nitrogen-producing nodules on roots. Infection can also occur on adult plants leading to loss of lateral roots and nodules. At the seedling stage, ARR decreases alfalfa stand establishment, and field longevity is reduced when adult plants are infected. A. euteiches is an oomycete pathogen that has motile zoospores and thick-walled oospores that can survive for many years in soil. Two races are currently recognized by pathogenicity on differential alfalfa check cultivars. Most alfalfa cultivars contain race 1 resistance, but there is an increasing development of cultivars with resistance to race 2. Management strategies include planting resistant cultivars, avoiding planting in fields with poor drainage and rotating crops with nonhost plants