Douglas G. Luster

Phytophthora Database: A Forensic Database Supporting the Identification and Monitoring of Phytophthora

Phytophthora spp. represent a serious threat to agricultural and ecological systems. Many novel Phytophthora spp. have been reported in recent years, which is indicative of our limited understanding of the ecology and diversity of Phytophthora spp. in nature. Systematic cataloging of genotypic and phenotypic information on isolates of previously described species serves as a baseline for identification, classification, and risk assessment of new Phytophthora isolates. The Phytophthora Database (PD) was established to catalog such data in a web-accessible and searchable format. To support the identification of new Phytophthora isolates via comparison of their sequences at one or more loci with the corresponding sequences derived from the isolates archived in the PD, we generated and deposited sequence data from more than 1,500 isolates representing the known diversity in the genus. Data search and analysis tools in the PD include BLAST, Phyloviewer (a program for building phylogenetic trees using sequences of selected isolates), and Virtual Gel (a program for generating expected restriction patterns for given sequences). The PD also provides a customized means of storing and sharing data via the web. The PD serves as a model that easily can be adopted to develop databases for other important pathogen groups.

Plum Pox in North America: Identification of Aphid Vectors and a Potential Role for Fruit in Virus Spread

ABSTRACT Thirteen aphid species were tested for their ability to transmit Pennsylvania isolates of Plum pox virus (PPV) collected in Columbia (PENN-3), Franklin (PENN-4), and York (PENN-7) Counties, PA. Four species, Aphis fabae, A. spiraecola, Brachycaudus persicae, and Myzus persicae, consistently transmitted PPV in preliminary transmission tests. Two species, Metopolophium dirhodum and Rhopalosiphum padi, were occasional inefficient vectors. Toxoptera citricida, from Florida, also was an effective vector but it does not occur in major stone-fruit-growing states. Species not transmitting PPV in parallel tests included Acyrthosiphon pisum, Aphis glycines, Aulacorthum solani, Macrosiphum euphorbiae, Rhopalosiphum maidis, and Sitobion avenae. When given a 3-day probing access period simultaneously on PPV-infected peach seedlings and healthy peach seedlings, Myzus persicae, Aphis spiraecola, A. fabae, and B. persicae transmitted PPV to 63, 31, 38, and 32% of the healthy peach seedlings, respectively. When given a similar probing period on PPV-infected peach fruit and healthy peach seedlings, the same aphid species transmitted PPV to 50, 35, 0, and 0% of seedlings, respectively. Results support the hypothesis of secondary PPV spread by indigenous aphids in Pennsylvania, and suggest that PPV-infected fruit has the potential to function as a virus source for long-distance dispersal.

Expression of NEP1 by Fusarium oxysporum f. sp. erythroxyli after gene replacement and overexpression using polyethylene glycol-mediated transformation

ABSTRACT The necrosis inducing extracellular protein Nep1 is produced by Fusarium oxysporum f. sp. erythroxyli in liquid culture. NEP1, the Nep1 protein structural gene, was disrupted in F. oxysporum f. sp. erythroxyli isolate EN-4 by gene replacement using polyethylene glycol (PEG)-mediated transformation. NEP1 disruption was verified by polymerase chain reaction (PCR), Southern blot, and northern blot analysis. NEP1-disrupted transformants failed to produce Nep1 in liquid culture. NEP1 disruption did not affect the pathogenicity of isolate EN-4 toward Erythroxylum coca. Transformation of isolate EN-4 with construct pPB-FO11-45 carrying NEP1 between the trpC promoter and terminator resulted in increased production of Nep1 in potato dextrose broth plus 1% casamino acids or Czapek-Dox broth plus 1% casamino acids but not in potato dextrose broth alone. Transformation of EN-4 with construct pPB-FO11-45 was verified by PCR and Southern blot analysis. Overexpression of NEP1 was confirmed by northern blot and Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. NEP1-overexpressing transformant 15 produced 64 to 128 times as much Nep1 as EN-4 wild type when grown in shake cultures. Transformants overexpressing Nep1 in liquid culture were no more or less pathogenic toward E. coca than wild-type isolates. Nep1 was not detected in E. coca seedlings infected with NEP1-overexpressing transformants or with EN-4 wild type. In large-scale fermentations of NEP1-overexpressing transformant 15, the amount of secreted protein including Nep1 was 15.1 times that of the wild-type EN-4, providing a ready source of Nep1 for future study.

Enzymology of ferric chelate reduction at the root plasma membrane

Abstract Our research has focused on the characterization and purification of plasma membrane reductases of root cells that are involved in ferric chelate reduction in the rhizosphere of “Fe‐efficient”; tomato (Lycopersicon esculentum Mill). Fe chelate reductase activity in plasma membranes from Fe‐deficient plants exhibited Michaells‐Menten kinetics with regard to the substrate Fe3+ citrate. The kinetic data, as well as sensitivity of Fe chelate reduction to the protease trypsin, confirmed the essential enzymatic nature of the tomato root plasma membrane Fe chelate reductase. Differential enzyme rates obtained upon altering the sequence of addition of reactants in vitro suggested that the root plasma membrane Fe chelate reductase may require an ordered substrate binding mechanism for optimal activity. Partial inhibition of enzyme activity by catalase added to reaction mixtures suggested some participation by hydrogen peroxide to Fe chelate reduction in vitro. Electrophoretic separation of proteins from t...

Plant pathogen culture collections: it takes a village to preserve these resources vital to the advancement of agricultural security and plant pathology.

ABSTRACT Plant pathogen culture collections are essential resources in our fight against plant disease and for connecting discoveries of the present with established knowledge of the past. However, available infrastructure in support of culture collections is in serious need of improvement, and we continually face the risk of losing many of these collections. As novel and reemerging plant pathogens threaten agriculture, their timely identification and monitoring depends on rapid access to cultures representing the known diversity of plant pathogens along with genotypic, phenotypic, and epidemiological data associated with them. Archiving such data in a format that can be easily accessed and searched is essential for rapid assessment of potential risk and can help track the change and movement of pathogens. The underexplored pathogen diversity in nature further underscores the importance of cataloguing pathogen cultures. Realizing the potential of pathogen genomics as a foundation for developing effective disease control also hinges on how effectively we use the sequenced isolate as a reference to understand the genetic and phenotypic diversity within a pathogen species. In this letter, we propose a number of measures for improving pathogen culture collections.

Kenyan Isolates of Puccinia graminis f. sp. tritici from 2008 to 2014: Virulence to SrTmp in the Ug99 Race Group and Implications for Breeding Programs

Frequent emergence of new variants in the Puccinia graminis f. sp. tritici Ug99 race group in Kenya has made pathogen survey a priority. We analyzed 140 isolates from 78 P. graminis f. sp. tritici samples collected in Kenya between 2008 and 2014 and identified six races, including three not detected prior to 2013. Genotypic analysis of 20 isolates from 2013 and 2014 collections showed that the new races TTHST, TTKTK, and TTKTT belong to the Ug99 race group. International advanced breeding lines were evaluated against an isolate of TTKTT (Sr31, Sr24, and SrTmp virulence) at the seedling stage. From 169 advanced lines from Kenya, 23% of lines with resistance to races TTKSK and TTKST were susceptible to TTKTT and, from two North American regional nurseries, 44 and 91% of resistant lines were susceptible. Three lines with combined resistance genes were developed to facilitate pathogen monitoring and race identification. These results indicate the increasing virulence and variability in the Kenyan P. graminis f. sp. tritici population and reveal vulnerabilities of elite germplasm to new races.

Simple sequence repeat markers useful for sorghum downy mildew (Peronosclerospora sorghi) and related species

BackgroundA recent outbreak of sorghum downy mildew in Texas has led to the discovery of both metalaxyl resistance and a new pathotype in the causal organism, Peronosclerospora sorghi. These observations and the difficulty in resolving among phylogenetically related downy mildew pathogens dramatically point out the need for simply scored markers in order to differentiate among isolates and species, and to study the population structure within these obligate oomycetes. Here we present the initial results from the use of a biotin capture method to discover, clone and develop PCR primers that permit the use of simple sequence repeats (microsatellites) to detect differences at the DNA level.ResultsAmong the 55 primers pairs designed from clones from pathotype 3 of P. sorghi, 36 flanked microsatellite loci containing simple repeats, including 28 (55%) with dinucleotide repeats and 6 (11%) with trinucleotide repeats. A total of 22 microsatellites with CA/AC or GT/TG repeats were the most abundant (40%) and GA/AG or CT/TC types contribute 15% in our collection. When used to amplify DNA from 19 isolates from P. sorghi, as well as from 5 related species that cause downy mildew on other hosts, the number of different bands detected for each SSR primer pair using a LI-COR- DNA Analyzer ranged from two to eight. Successful cross-amplification for 12 primer pairs studied in detail using DNA from downy mildews that attack maize (P. maydis & P. philippinensis), sugar cane (P. sacchari), pearl millet (Sclerospora graminicola) and rose (Peronospora sparsa) indicate that the flanking regions are conserved in all these species. A total of 15 SSR amplicons unique to P. philippinensis (one of the potential threats to US maize production) were detected, and these have potential for development of diagnostic tests. A total of 260 alleles were obtained using 54 microsatellites primer combinations, with an average of 4.8 polymorphic markers per SSR across 34 Peronosclerospora, Peronospora and Sclerospora spp isolates studied. Cluster analysis by UPGMA as well as principal coordinate analysis (PCA) grouped the 34 isolates into three distinct groups (all 19 isolates of Peronosclerospora sorghi in cluster I, five isolates of P. maydis and three isolates of P. sacchari in cluster II and five isolates of Sclerospora graminicola in cluster III).ConclusionTo our knowledge, this is the first attempt to extensively develop SSR markers from Peronosclerospora genomic DNA. The newly developed SSR markers can be readily used to distinguish isolates within several species of the oomycetes that cause downy mildew diseases. Also, microsatellite fragments likely include retrotransposon regions of DNA and these sequences can serve as useful genetic markers for strain identification, due to their degree of variability and their widespread occurrence among sorghum, maize, sugarcane, pearl millet and rose downy mildew isolates.

Identification, characterization, and relatedness of luteovirus isolates from forage legumes.

ABSTRACT Virus isolates from forage legumes collected from eight different states were identified as luteoviruses closely related to soybean dwarf luteovirus dwarfing (SbDV-D) and yellowing (SbDV-Y) described in Japan. All isolates produced reddened leaf margins in subterranean clover and were transmitted in a persistent manner by Acrythosiphon pisum, but not by Aulacorthum solani. Specific monoclonal antibodies raised against SbDV-Y were differentially reactive with endemic isolates. Immunoblots probed with a SbDV-D polyclonal antiserum showed single 26-kDa coat protein bands, confirming close serological relatedness to SbDV. Analyses of genomic and subgenomic double-stranded RNAs and northern blot analyses confirmed genomic relatedness to SbDV. Based on our results, we conclude that the U.S. luteovirus isolates studied comprise a strain or strains of the soybean dwarf virus that have clovers as common hosts and the pea aphid as a common vector.

An Assessment Model for Rating High-Threat Crop Pathogens

ABSTRACT Natural, accidental, and deliberate introductions of nonindigenous crop pathogens have become increasingly recognized as threats to the U.S. economy. Given the large number of pathogens that could be introduced, development of rapid detection methods and control strategies for every potential agent would be extremely difficult and costly. Thus, to ensure the most effective direction of resources a list of high-threat pathogens is needed. We address development of a pathogen threat assessment model based on the analytic hierarchy process (AHP) that can be applied world-wide, using the United States as an illustrative example. Previously, the AHP has been shown to work well for strategic planning and risk assessment. Using the collective knowledge of subject matter expert panels incorporated into commercial decision-making software, 17 biological and economic criteria were determined and given weights for assessing the threat of accidental or deliberately introduced pathogens. The rating model can be applied by experts on particular crops to develop threat lists, especially those of high priority, based on the current knowledge of individual diseases.

Development of simple sequence repeat markers for the soybean rust fungus, Phakopsora pachyrhizi

Twenty‐four simple sequence repeat markers were developed for Phakopsora pachyrhizi, a fungal pathogen of soybean (Glycine max) and other legumes. All 24 of the loci were evaluated on 28 isolates of P. pachyrhizi. Twenty‐one loci were polymorphic, with allelic diversity ranging from two to eight alleles, and null alleles were observed for eight of the 24 loci. A preliminary screen with the closely related species, P. meibomiae, indicated that these primer pairs are specific to P. pachyrhizi.