Xinshun Qu

The host range of Spongospora subterranea f. sp. subterranea in the United States

This study was performed to determine the host range ofSpongospora subterranea f. sp.subterranea on common crops and weeds in the northeastern United States. Seedlings of the plants were grown in nutrient solutions and inoculated with spore balls ofS. subterranea. The roots were microscopically examined for the presence of plasmodia or zoosporangia 14 days after inoculation. The plants were then transferred to a greenhouse and grown in a soilless mix. The roots were examined for the presence of symptoms and spore balls after 4 months. Of 26 species within 10 families from monocotyledons and dicotyledons tested, 16 species were found to be susceptible toS. subterranea. Twelve species were newly recorded hosts forS. subterranea. Gall symptoms were observed on the roots of six species and spore balls were found on three species. Evidence is presented for the first time that galls and spore balls ofS. subterranea might form on non-Solanaceous species. This investigation is important for the cultural management of potato powdery scab disease because there currently are no effective controls.

Putative EPHX1 Enzyme Activity Is Related with Risk of Lung and Upper Aerodigestive Tract Cancers: A Comprehensive Meta-Analysis

Background EPHX1 is a key enzyme in metabolizing some exogenous carcinogens such as products of cigarette-smoking. Two functional polymorphisms in the EPHX1 gene, Tyr113His and His139Arg can alter the enzyme activity, suggesting their possible association with carcinogenesis risk, particularly of some tobacco-related cancers. Methodology/Principal Findings A comprehensive systematic review and meta-analysis was performed of available studies on these two polymorphisms and cancer risk published up to November 2010, consisting of 84 studies (31144 cases and 42439 controls) for Tyr113His and 77 studies (28496 cases and 38506 controls) for His139Arg primarily focused on lung cancer, upper aerodigestive tract (UADT) cancers (including oral, pharynx, larynx and esophagus cancers), colorectal cancer or adenoma, bladder cancer and breast cancer. Results showed that Y113H low activity allele (H) was significantly associated with decreased risk of lung cancer (OR = 0.88, 95%CI = 0.80–0.96) and UADT cancers (OR = 0.86, 95%CI = 0.77–0.97) and H139R high activity allele (R) with increased risk of lung cancer (OR = 1.18, 95%CI = 1.04–1.33) but not of UADT cancers (OR = 1.05, 95%CI = 0.93–1.17). Pooled analysis of lung and UADT cancers revealed that low EPHX1 enzyme activity, predicted by the combination of Y113H and H139R showed decreased risk of these cancers (OR = 0.83, 95%CI = 0.75–0.93) whereas high EPHX1 activity increased risk of the cancers (OR = 1.20, 95%CI = 0.98–1.46). Furthermore, modest difference for the risk of lung and UADT cancers was found between cigarette smokers and nonsmokers both in single SNP analyses (low activity allele H: OR = 0.77/0.85 for smokers/nonsmokers; high activity allele R: OR = 1.20/1.09 for smokers/nonsmokers) and in combined double SNP analyses (putative low activity: OR = 0.73/0.88 for smokers/nonsmokers; putative high activity: OR = 1.02/0.93 for smokers/ nonsmokers). Conclusions/Significance Putative low EPHX1 enzyme activity may have a potential protective effect on tobacco-related carcinogenesis of lung and UADT cancers, whereas putative high EPHX1 activity may have a harmful effect. Moreover, cigarette-smoking status may influence the association of EPHX1 enzyme activity and the related cancer risk.

Detection and quantification ofSpongospora subterranea f. sp.subterranea by PCR in host tissue and naturally infested soils

A polymerase chain reaction (PCR) assay using primers SsF and SsR designed from the internal transcribed spacer (ITS) regions ofSpongospora subterranea f. sp.subterranea was developed for the specific identification and quantification ofS. subterranea. These primers amplified a 434 bp product from DNA ofS. subterranea spore balls, but not from DNA of healthy potato, common scab tuber, and taxonomically related plasmodiophorids. This PCR assay was successfully used for the detection ofS. subterranea in naturally infected symptomatic and asymptomatic potato tubers.Spongospora subterranea in other infected symptomless host plants was detected by PCR. The PCR assay was modified with improved soil DNA extraction methods to detectS. subterranea in soil. The assay was sensitive, and one spore ball per gram of soil could be detected. Following the design of a heterologous competitor DNA template from the sequence of λDNA, a competitive PCR assay for the quantification ofS. subterranea in soil was developed and provided accurate quantification in the range of 1 to 104 spore balls per 0.25 g of soil. In a preliminary survey of naturally infested field soil samples, spore ball concentrations were estimated to vary from ca 0 to 3600 spore balls per 0.25-g soil sample by this competitive PCR assay. The spore ball levels were compared with the powdery scab disease incidence of potatoes in these fields, and a correlationship between spore ball levels and subsequent disease incidence was found. The PCR assays developed in this investigation can be routinely used to detect and quantifyS. subterranea in diseased plant tissue, asymptomatic plant tissue, and infested soil.ResumenLa prueba de reacción en cadena de la polimerasa (PCR) utilizando los “primers” SsF y SsR, diseñados a partir de las regiones transmitidas internamente del espaciador (ITS) deS. subterránea f. sp.subterranea, ha sido desarrollada para la identificación específica y cuantificación deSpongospora subterranea. Estos “primers” amplificaron el producto 434 bp del DNA de las masas de esporas deS. subterranea pero no del DNA de papa sana, o de tubérculos con sarna común y plasmodiophoridos taxonómicamente relacionados. Esta prueba de PCR ha sido utilizada exitosamente para la detección deS. subterranea en tubérculos naturalmente infectados, sintomáticos y asintomáticos. También se ha detectado a través del PCR,S. subterranea en otros huéspedes infectados que no presentaban síntomas. La prueba PCR ha sido modificada con métodos mejorados de extracción de DNA del suelo para detectarS. subterranea. La prueba es tan sensible que pude detectar una espora por gramo de suelo. Después del diseño de una muestra competitiva heteróloga de DNA de la secuencia de λDNA se desarrolló una prueba competitiva de PCR para la cuantificaciónS. subterranea en el suelo, la misma que proporcionó una cuantificación segura en el rango de 1 a 104 masas de esporas por 0.25 gramos de suelo. En un estudio preliminar de muestras de suelo infestado naturalmente, se estimó que la concentración varió de c. 0 a 3600 masas de esporas por 0.25 gramos de muestra de suelo por medio de esta prueba de PCR. El nivel de masas de esporas se comparó con la incidencia de sarna polvorienta en estos campos de papa y se encontró una correlación entre los niveles de masas de esporas e incidencia de la enfermedad en una siguiente campaña. Las pruebas de PCR desarrolladas en esta investigación pueden ser rutinariamente utilizadas para detectar y cuantificarS. subterranea en el tejido de plantas enfermas, tejido de plantas asintomáticas y suelo infestado.

Multiplex real‐time PCR (TaqMan) assay for the simultaneous detection and discrimination of potato powdery and common scab diseases and pathogens

Aims:  To develop a multiplex real‐time PCR assay using TaqMan probes for the simultaneous detection and discrimination of potato powdery scab and common scab, two potato tuber diseases with similar symptoms, and the causal pathogens Spongospora subterranea and plant pathogenic Streptomyces spp.

Using the TxtAB operon to quantify pathogenic Streptomyces in potato tubers and soil.

The phytotoxin thaxtomin, produced by plant pathogenic Streptomyces species, is the only known pathogenicity determinant for common scab diseases of potato and other root and tuber crops. Genes encoding thaxtomin synthetase (txtAB) are found on a pathogenicity island characteristic of genetically diverse plant pathogenic Streptomyces species. In this study, an SYBR Green quantitative real-time polymerase chain reaction (PCR) assay using primers designed to anneal to the txtAB operon of Streptomyces was developed to quantify pathogenic bacterial populations in potatoes and soil. The real-time PCR assay was specific for pathogenic Streptomyces strains. The detection limit of the assay was 10 fg of the target DNA, or one genome equivalent. Cycle threshold (Ct) values were linearly correlated with the concentration of the target DNA (correlation coefficient R(2) = 0.99) and were not affected by the presence of plant DNA extracts, indicating the usefulness of the assay for quantitative analyses of the pathogenic bacteria in plant tissues. The amount of pathogenic Streptomyces DNA in total DNA extracts from 1 g asymptomatic and symptomatic tubers was quantified using the assay and ranged from 10(1) to 10(6) pg. A standard curve was established to quantify pathogenic Streptomyces in soil. Using the standard curve, numbers of pathogenic Streptomyces colony forming units were extrapolated to range from 10(3) to 10(6) per gram of soil from potato fields where common scab was found. This real-time PCR assay using primers designed from the txtAB operon allows rapid, accurate, and cost effective quantification of pathogenic Streptomyces strains in potato tubers and in soil.

Genetic variation and phylogeny ofSpongospora subterranea f.sp.subterranea based on ribosomal DNA sequence analysis

The nuclear rDNA regions of the two internal transcribed spacers (ITS1 and ITS2) and 5.8S rRNA gene from 52 field isolates ofSpongospora subterranea f.sp.subterranea obtained from the British Isles and North America were polymerase chain reaction-amplified, sequenced, and assessed for genetic variation. Two genetically distinct groups (I and II) were identified based on the ITS sequence diversity among the isolates, representing 34.6% and 65.4% of the isolates, respectively. British Isles isolates occurred in groups I and II, whereas North American isolates belonged only to group II. The British Isles groups ofS. subterranea were associated with particular potato cultivars. The full-length small-subunit rRNA gene ofS. subterranea was sequenced and analyzed by both neighbor-joining and parsimony methods to clarify the taxonomic position of this pathogen. The results of phylogenetic analysis showed thatS. subterranea grouped together with other species of plasmodiphorids, and this group clustered with the phylum Cercozoa, an assemblage of filose and reticulose amoebae and phylogenetically related zooflagellates. The recognition of the existence of different genetic groups withinS. subterranea will be important for the design of plant-breeding programs and in testing for plant resistance.ResumenLas regiones rADN nuclear de los dos espaciadores transcrites internos (ITS1 e ITS2) y el gen 5.8S rARN de 52 aislamientos de campo deSpongospora subterranea f.sp.subterranea obtenidos de las Islas Británicas y de Norteamérica fueron amplificadas por reacción en cadena de la polimerasa, secuenciadas y evaluadas para variación genética. Se identificaron dos grupos genéticamente diferentes (I y II) en base de la diversidad de frecuencia ITS entre los aislamientos, lo que representa el 34.6% y el 65.4% de los aislamientos respectivamente. En los aislamientos de las Islas Británicas se encontró los grupos I y II, mientras que los aislamientos de Norteamérica pertenecían sólo al grupo II. Los grupos deS. subterranea de las Islas Británicas estuvieron asociados con cultivares especiales de papa. El tamaño complete de la sub-unidad del gen rARN deS. subterranea fue secuenciado y analizado por los métodos “neighborjoining y parsimony”, con el objeto de aclarar la posición taxonómica de este patógeno. El resultado del análisis filogenético demostró queS. subterranea junto con otras especie de plasmodiophoridos está relacionado con el phylum Cercozoa que incluye un conjunto de amebas filiformes y zooflagelados filogenéticamente relacionados. El reconocimiento de la existencia de diferentes grupos genéticos dentro deS. subterranea será importante para el diseño de programas de mejoramiento y pruebas de resistencia de la planta.

Qualitative real-time PCR SYBR ® Green detection of Petri disease fungi

Real-time PCR provides a fast, reliable, and cost-effective method for detecting the presence or absence of Petri disease fungi in grapevines. The primer pairs, Pmo1f + Pmo2r, and Pac1f + Pac2r, were designed for species and genus-specific amplification of Phaeomoniella chlamydospora and Phaeoacremonium spp. respectively, using realtime PCR with SYBR® Green. The primers were specific and showed no primer-primer dimers until after 35 cycles. Pa. chlamydospora was detected in roots, shoots, and young trunks of drill-inoculated vines. Phaeoacremonium was detected in trunk cross-sections of naturally infected vines from which Phaeoacremonium aleophilum had been isolated. The protocol presented here can be adapted to provide a reliable detection system for research and industry.

Field efficacy of nonpathogenic Streptomyces species against potato common scab

The primary objective of these experiments was to reduce pathogenicity and virulence of endemic soil pathogenic Streptomyces strains that cause potato common scab (CS) using nonpathogenic Streptomyces strains to suppress CS in a field situation.

In Vitro Culture of the Obligate Parasite Spongospora subterranea (Cercozoa; Plasmodiophorida) Associated with Root‐Inducing Transferred‐DNA Transformed Potato Hairy Roots

ABSTRACT. Spongospora subterranea is a soil‐borne, obligate parasitic protist that causes powdery scab of potatoes. In this study, an in vitro culture system was developed for the maintenance and proliferation of the protist in potato hairy roots. The hairy roots of potato were induced in vitro with Agrobacterium rhizogenes. Cystosori of S. subterranea from potato scab lesions were surface disinfested and used to inoculate potato hairy roots. Plasmodia, zoosporangia, and cystosori were observed microscopically in the hairy roots within 6 wk after inoculation, indicating the completion of the life cycle of S. subterranea in vitro. This is the first in vitro culture system for S. subterranea, and will be a valuable tool to study fundamental and practical aspects of the biology of the parasite.

Single Cystosorus Isolate Production and Restriction Fragment Length Polymorphism Characterization of the Obligate Biotroph Spongospora subterranea f. sp. subterranea

ABSTRACT Spongospora subterranea f. sp. subterranea causes powdery scab in potatoes and is distributed worldwide. Genetic studies of this pathogen have been hampered due, in part, to its obligate parasitism and the lack of molecular markers for this pathogen. In this investigation, a single cystosorus inoculation technique was developed to produce large amounts of S. subterranea f. sp. subterranea plasmodia or zoosporangia in eastern black nightshade (Solanum ptycanthum) roots from which DNA was extracted. Cryopreservation of zoosporangia was used for long-term storage of the isolates. S. subterranea f. sp. subterranea-specific restriction fragment length polymorphism (RFLP) markers were developed from randomly amplified polymorphic DNA (RAPD) fragments. Cystosori of S. subterranea f. sp. subterranea were used for RAPD assays and putative pathogen-specific RAPD fragments were cloned and sequenced. The fragments were screened for specificity by Southern hybridization and subsequent DNA sequence BLAST search. Four polymorphic S. subterranea f. sp. subterranea-specific probes containing repetitive elements, and one containing single copy DNA were identified. These RFLP probes were then used to analyze 24 single cystosorus isolates derived from eight geographic locations in the United States and Canada. Genetic variation was recorded among, but not within, geographic locations. Cluster analysis separated the isolates into two major groups: group I included isolates originating from western North America, with the exception of those from Colorado, and group II included isolates originating from eastern North America and from Colorado. The techniques developed in this study, i.e., production of single cystosorus isolates of S. subterranea f. sp. subterranea and development of RFLP markers for this pathogen, provide methods to further study the genetic structure of S. subterranea f. sp. subterranea.