Barbara J. Christ

Mapping genes for resistance to Verticillium albo-atrum in tetraploid and diploid potato populations using haplotype association tests and genetic linkage analysis

AbstractVerticillium wilt disease of potato is caused predominantly by Verticillium albo-atrum and V. dahliae. StVe1 —a putative QTL for resistance against V. dahliae —was previously mapped to potato chromosome 9. To develop allele-specific, SNP-based markers within the locus, the StVe1 fragment from a set of 30 North American potato cultivars was analyzed. Three distinct and highly diverse haplotypes can be distinguished at the StVe1 locus. These were detected in 97%, 33%, and 10% of the cultivars analyzed. We tested for haplotype association and for genetic linkage between the StVe1 haplotypes and resistance of tetraploid potato to V. albo-atrum. Moreover, field resistance was assessed in diploid populations with known molecular linkage maps in order to identify novel QTLs. Resistance QTLs against V. albo-atrum were detected on four chromosomes (2, 6, 9, and 12) at the diploid level, with one QTL on chromosome 2 contributing over 40% to the total phenotypic variation of the trait. At the tetraploid level, a significant association between the StVe1-839-C haplotype and susceptibility to the disease was detected, suggesting that resistance-related genes directed against V. albo-atrum and V. dahliae are located in the same genomic region of chromosome 9. However, on the basis of the present analysis, we cannot determine whether these genes are closely linked or if a single gene provides resistance against both Verticillium species. To assess the usefulness of the StVe1-839-C haplotype for marker-assisted selection, we subjected the resistance data to Bayesian analysis, and calculated positive (0.65) and negative (0.75) predictive values, and overall predictive accuracy (0.72). Our results indicate that tagging of additional genes for resistance to Verticillium with molecular markers will be required for efficient marker-assisted selection.

Phenotypic stability of resistance to late blight in potato clones evaluated at eight sites in the United States

Changes in the fungal pathogenPhytophthora infestans in the United States pose a significant threat to potato production. Sources of resistance to these new genotypes of P.infestans need to be identified for potato breeders to have parental materials for crossing, and the phenotypic stability of late blight resistance in these potato clones needs to be determined. Sixteen potato clones which reportedly have some resistance to late blight were evaluated at eight locations: Florida (FL), Maine (ME), Michigan (MI), Minnesota (MN), North Dakota (ND), New York (NY), Pennsylvania (PA) and Wisconsin (WI) in 1996. Percent infected foliage was recorded at approximately weekly intervals following the onset of the disease at each location. Area under the disease progress curve (AUDPC) was calculated. Clones were ranked for mean AUDPC within location and the nonparametric stability statistics, mean absolute rank differences and variance of the ranks, were analyzed for phenotypic stability. Neither of these statistics was significant, indicating a lack of genotype x environment interaction on the rankings of these clones across locations in 1996. The four clones with lowest AUDPC scores were U.S. clones AWN86514-2, B0692-4, B0718-3 and B0767-2. These clones should be useful parental materials for breeders seeking to incorporate genes for late blight resistance into potatoes.

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.

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.

Foliar resistance to late blight in potato clones evaluated in national trials in 1997

Changes in the oomycetePhytophthora infestans in the United States and other parts of the world pose a significant threat to potato production. A continual evaluation of potato clones for resistance to late blight is necessary to identify clones with resistance and to monitor the stability of resistance in light of the emergence of new and more aggressive strains of this pathogen. Twentytwo potato clones (10 cultivars and 12 selections) were evaluated in 1997 for late blight resistance at seven U.S. locations. Seven late blight differentials (R1R2R3R4, R1R2R4, R1R3R4 R3, R8 R10, and Rmulti) were also included in the test at five of these locations. The US-8 strain of P.infestans was present at all locations. Percent infected foliage was recorded at approximately weekly intervals following the onset of disease. Area under the disease progress curve (AUDPC) was calculated. The nonparametric stability statistics mean absolute rank differences (Si(1)) and variances of the ranks (Si(2)) were used to analyze phenotypic stability. Although neither of these statistics was significant for individual clones, both of these statistics were significant when summed over clones, indicating the importance of genotype × environment interactions on the rankings of these clones across locations. The most late blight-resistant and susceptible clones were the most stable; clones in the intermediate ranges were most subject to rank changes due to genotype × environment interactions. The most late blight-resistant clones were AWN86514-2, B0692-4, B0718-3, and B0767-2. The most susceptible clones were B0811-13, B1004-8, Nor-Donna, and Krantz. AUDPC was very low for the late blight differentials R8 and Rmulti, moderately low for R10 and very high for the remaining differentials. This study is important in characterizing the reaction of potato clones to new strains of P.infestans.

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.

Influence of potato cultivars on the effectiveness of fungicide control of early blight

Three potato cultivars differing in degree of susceptibility to early blight were grown under crop management practices typical for Pennsylvania in 1987 and 1988. There were three experimental treatments: no fungicides, mancozeb with weekly applications initiated at 7 or 8 weeks after planting (early treatment), and weekly applications initiated at first symptoms of disease (late treatment). The no-fungicide control treatment had significantly higher AUDPC values than either treatment with fungicides and the no-fungicide treatment had significantly lower yield in 1987 but not in 1988. Tubers from the no-fungicide control had lower specific gravity. Norchip, the cultivar most susceptible to early blight examined in this study, responded the most in yield increases by the increased fungicide applications.CompendioTres cultivares de papa, con diferentes grados de susceptibilidad al tizón temprano, fueron cultivados en 1987 y 1988 bajo prácticas de cultivo tÍpicas para Pennsylvania. El experimento comprendió tres tratamientos: testigo sin fungicidas, mancozeb con aplicaciones semanales iniciadas siete u ocho semanas después de la siembra (tratamiento temprano) y mancozeb con aplicaciones semanales iniciadas al aparecer los primeros síntomas de la enfermedad (tratamiento tardío). El tratamineto testigo, sin fungicidas, tuvo valores de AUDPC (Area Under Disease Progress Curve) significativamente mayores que los tratamientos con fungicidas. El mismo tratamiento tuvo en 1987 rendimientos significativamente menores, lo que no ocurrió en 1988. Los tubérculos del testigo sin fungicidas tuvieron una gravedad específica más baja. Norchip, el más susceptible al tizón temprano de los cultivares estudiados, fue el que más incrementó sus rendimientos con el aumento en las aplicaciones del fungicida.

Colonization of rotation crops and weeds by the potato black dot pathogenColletotrichum coccodes

The rotation crops wheat, barley, oat, maize, soybean, rye, yellow mustard, alfalfa, and spring canola and weeds eastern black nightshade, velvetleaf, timothy grass, orchard grass, and Giant foxtail common to potato-growing areas in North America were used to study the host range ofColletotrichum coccodes, the causal agent of potato black dot. The fungus was isolated from nine of 14 rotation crops and weeds that were inoculated: yellow mustard, soybean, spring canola, alfalfa, oat, eastern black nightshade, velvetleaf, giant foxtail, and timothy grass. In all, colonization was highest in black nightshades (87%) and velvetleaf (80%). Among the rotation crops, colonization was highest on yellow mustard (59%) followed by spring canola (33%) and soybean (30%).Colletotrichum coccodes was not isolated from wheat, barley, rye, maize, or orchard grass. The results indicated that crops used for rotation with potato should be selected carefully to prevent the increase ofC. coccodes inoculum in the soil and that weeds may help maintain viable inoculum ofC. coccodes in the absence of potato. Based on these results we recommend that wheat, barley, maize, or rye be used in rotation with potato in areas whereC. coccodes is present in high levels in the soil.

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.