John J. Weiland

Phylogenetic relationships among plant and animal parasites, and saprotrophs in Aphanomyces (Oomycetes)

Molecular phylogenetic relationships among 12 species of Aphanomyces de Bary (Oomycetes) were analyzed based on 108 ITS sequences of nuclear rDNA. Sequences used in the analyses belonged to the major species currently available in pure culture and GenBank. Bayesian, maximum likelihood, and maximum parsimony analyses support that Aphanomyces constitutes a monophyletic group. Three independent lineages were found: (i) plant parasitic, (ii) animal parasitic, and (iii) saprotrophic or opportunistic parasitic. Sexual reproduction appeared to be critical in plant parasites for survival in soil environments while asexual reproduction seemed to be advantageous for exploiting specialization in animal parasitism. Repeated zoospore emergence seems to be an advantageous property for both plant and animal parasitic modes of life. Growth in unspecific media was generally faster in saprotrophs compared with parasitic species. A number of strains and GenBank sequences were found to be misidentified. It was confirmed molecularly that Aphanomyces piscicida and Aphanomyces invadans appear to be conspecific, and found that Aphanomyces iridis and Aphanomyces euteiches are closely related, if not the same, species. This study has shown a clear evolutionary separation between Aphanomyces species that are plant parasites and those that parasitize animals. Saprotrophic or opportunistic species formed a separate evolutionary lineage except Aphanomyces stellatus whose evolutionary position has not yet been resolved.

Construction of a sugar beet BAC library from a hybrid with diverse traits

A bacterial artificial chromosome (BAC) library of the 750-Mbp sugar beet genome represented in hybrid US H20 was constructed fromHind III-digested DNA, with an average insert size of 120 kbp. US H20 is a variety grown in the eastern United States. It exhibits heterosis for emergence and yield, presumably because of its hybridity between eastern and western US germplasm sources. Filter arrays were used to assess the abundance and distribution of particular nucleotide sequences. An rRNA gene probe found that 1.2% of the library carried sequences similar to these highly repetitive and conserved sequences. A simple sequence repeat element (CA)8 thought to be predominantly distributed throughout centromere regions of all chromosomes was present in 1.7% of clones. For more than half of the 28 randomly chosen expressed sequence tags (ESTs) used as probes, a higher-than-expected number of single-copy hybridization signals was observed. Assuming 6× genome coverage, this suggests that many duplicate genes exist in the beet genome.

Pink eye is an unusual periderm disorder characterized by aberrant suberization : A cytological analysis

Potato tuber pink eye (PE) is a disorder of unknown origin that results in significant postharvest quality deterioration and rot. Little is known about the physiology of PE, including the characteristic tissue autofluorescence that defines the PE syndrome. The objective of this research was to identify the source of PE-induced autofluorescence and PE-related susceptibility to infection. The suberized barrier of the native periderm and cellular characteristics of neighboring parenchyma tissues were investigated to determine their involvement in the PE disorder. The results create a new physiological model describing the disorder and addressing the enigma of PE. Characteristics of the PE model emerge from the following results: (1) the integrity of the suberized barrier of the native periderm was compromised or absent in some surface areas of PE tubers thereby implicating the breakdown of the native periderm and its associated suberin barrier with PE and the susceptibility of PE tubers to pathogen infection; (2) the PE complex was characterized by unusual suberin poly(phenolic) (SPP) accumulations in the cortical parenchyma followed by latent suberin poly(aliphatic) (SPA) accumulations that were generally insufficient to form a complete barrier that was competent to block infections by pathogenic bacteria and fungi; (3) the aberrant absence or compromised integrity of the suberin barrier, including associated waxes, resulted in erratic increased susceptibility to water vapor loss known to cause tuber shrinkage and flaccidity; (4) widespread accumulations of SPP on parenchyma cell walls were the durable source of autofluorescence commonly used to determine the presence of the disorder; (5) the erratic development of unusual internal phellogen and periderm layers that, if complete with SPA, blocked hyphal advancement; (6) combined, the data provide a plausible explanation for PE infection court and rot anomalies as they occur without ingress of a wound opening. Results also demonstrated that neutral red may be used as a sensitive fluorochrome to detect intact hydrophobic areas in hyphae. Collectively, the results provide compelling evidence that the PE disorder includes a physiological basis.ResumenEl ojo rosado (PE) del tubérculo de papa es un desorden de origen desconocido que deviene en un significativo deterioro de la calidad de poscosecha y pudrición. Muy poco se conoce a cerca de la fisiología de este desorden incluyendo la auto fluorescencia del tejido que caracteriza al síndrome de PE. El objetivo de esta investigación fue identificar la fuente de auto fluorescencia inducida por PE y la susceptibilidad a la infección. Se investigaron, la barrera suberizada del peridermo nativo y las características celulares del tejido del parénquima vecino, para determinar su participación en el desorden que produce el PE. Los resultados han creado un nuevo modelo fisiológico que describe el desorden y que señala el enigma de PE. Las características del modelo emergen de los siguientes resultados: (1) la integridad de la barrera suberizada del peridermo nativo estuvo comprometida o ausente en algunas áreas de la superficie de tubérculos afectados implicando una descomposición del peridermo nativo, la integridad de la barrera de suberina asociada, y la susceptibilidad de los tubérculos a la infección por patógenos, (2) el PE estuvo caracterizado por una extraordinaria acumulación de suberina polifenólica (SPF) en el parénquima, seguida por una latente acumulación de suberina polialifática (SPA) que fueron generalmente insuficientes para formar una barrera completa capaz de bloquear infecciones de bacterias y hongos patógenos, (3) la ausencia aberrante o compromiso de la integridad de la barrera de suberina, incluyendo ceras, dio como resultado un aumento errático de la susceptibilidad a la pérdida de vapor de agua, causas conocidas de la reducción y flacidez del tubérculo, (4) la acumulación generalizada de SPF en las paredes de las células del parénquima fue el origen constante de la autofluorescencia, utilizada comúnmente para determinar la presencia del desorden, (5) el desarrollo errático de capas de felógeno interno inusual y capas de peridermo, las cuales cuando se complementan con SPA, bloquean el avance de hifas, (6) combinados los datos, proporcionan una explicación plausible para el ingreso de PE y anomalías de la raíz, como ocurre sin la abertura de una herida de ingreso. Los resultados también han demostrado que se puede utilizar el rojo neutro como un fluorocromo sensible para detectar las áreas hidrofóbicas en hifas. En conjunto, los resultados proporcionan una evidencia precisa de que el PE incluye una base fisiológica.

First infectious clone of the propagatively transmitted Oat blue dwarf virus

Oat blue dwarf virus (OBDV) is a small, phloem-limited marafivirus that replicates in its leafhopper vector. We have developed complete cDNA clones of OBDV from which infectious transcripts may be derived––the first such clones for any propagatively transmitted plant virus. Prior to clone construction, the reported sequences of the 5′ and 3′ ends were confirmed using 5′ RACE, primer extension, and ligation-anchored PCR. Using vascular puncture of maize seeds with capped transcripts, multiple clones were shown to be infectious at an average rate of 24.3% (range 14–36%). Aster leafhoppers successfully transmitted OBDV to oats and barley after feeding on detached, infected maize leaves. Proteins and RNAs consistent in size with those expected in OBDV infection were detected in young leaves via western and northern blotting, respectively. One construct, pOBDV-2r, was designated as the reference clone. An infectious clone of OBDV will be valuable in examining the interaction of this virus with both its insect and plant hosts.

Presence of a polyA tail at the 3' end of maize rayado fino virus RNA

Maize rayado fino virus (MRFV) is distinct from other marafiviruses in that its genome reportedly lacks a poly(A) tail at the 3’ terminus. We now show that the MRFV genome is indeed polyadenylated.

Linear-motion tattoo machine and prefabricated needle sets for the delivery of plant viruses by vascular puncture inoculation

Vascular puncture inoculation (VPI) of plant viruses previously has been conducted either manually or by use of a commercial engraving tool and laboratory-fabricated needle arrays. In an effort to improve this technique, a linear-motion tattoo machine driving industry-standard needle arrays was tested as a means of delivering plant viruses into maize and small grain seed embryos. The new method was applied in the successful transmission of maize rayado fino virus (MRFV), the type member of the genus Marafivirus, from an archived sample to maize. Subsequent transfer of MRFV from the sap of an infected plant using the method produced an average infection rate in maize of 70% (range 39–93%). Maize, oat, and triticale were successfully infected with oat blue dwarf virus (OBDV) using the method; similar infection rates were observed between maize seeds inoculated with the tattoo machine and those inoculated with the engraving machine when using prefabricated needle arrays. No infection was obtained in repeated tests with barley yellow dwarf virus (BYDV-PAV) or cereal yellow dwarf virus (CYDV-RPV) using either sap or RNA from infectious cloned cDNA. Replacement of the original engraving-tool with a linear-motion tattoo machine in VPI provides greater flexibility and convenience in a quiet, readily-available instrument, while improving reproducibility through the use of prefabricated needle arrays.

Infectious Maize rayado fino virus from Cloned cDNA

A full-length cDNA clone was produced from a U.S. isolate of Maize rayado fino virus (MRFV), the type member of the genus Marafivirus within the family Tymoviridae. Infectivity of transcripts derived from cDNA clones was demonstrated by infection of maize plants and protoplasts, as well as by transmission via the known leafhopper vectors Dalbulus maidis and Graminella nigrifrons that transmit the virus in a persistent-propagative manner. Infection of maize plants through vascular puncture inoculation of seed with transcript RNA resulted in the induction of fine stipple stripe symptoms typical of those produced by wild-type MRFV and a frequency of infection comparable with that of the wild type. Northern and Western blotting confirmed the production of MRFV-specific RNAs and proteins in infected plants and protoplasts. An unanticipated increase in subgenomic RNA synthesis over levels in infected plants was observed in protoplasts infected with either wild-type or cloned virus. A conserved cleavage site motif previously demonstrated to function in both Oat blue dwarf virus capsid protein and tymoviral nonstructural protein processing was identified near the amino terminus of the MRFV replicase polyprotein, suggesting that cleavage at this site also may occur.