Melodie L. Putnam

A new species of Phomopsis causing twig dieback of Vaccinium vitis-idaea (lingonberry)

A fungus was discovered causing a progressive twig dieback on stems of Vaccinium vitis-idaea (lingonberry) in Oregon. Both morphological and molecular data suggest that the fungus belongs in Diaporthe/Phomopsis but is distinct from P. vaccinii, cause of a dieback and fruit rot of blueberry and cranberry (Vaccinium spp.). This fungus is described and illustrated as a new species, Phomopsis columnaris. It is distinguished from other species of Phomopsis by the distinctive conidiophores that consist of vertically aligned cells lining the base and sides of the conidiomata. Another species of Phomopsis described on Vaccinium, Phomopsis myrtilli, known from V. myrtillus, is redescribed and illustrated based on authentic herbarium material.

Analysis of genome sequences from plant pathogenic Rhodococcus reveals genetic novelties in virulence loci

Members of Gram-positive Actinobacteria cause economically important diseases to plants. Within the Rhodococcus genus, some members can cause growth deformities and persist as pathogens on a wide range of host plants. The current model predicts that phytopathogenic isolates require a cluster of three loci present on a linear plasmid, with the fas operon central to virulence. The Fas proteins synthesize, modify, and activate a mixture of growth regulating cytokinins, which cause a hormonal imbalance in plants, resulting in abnormal growth. We sequenced and compared the genomes of 20 isolates of Rhodococcus to gain insights into the mechanisms and evolution of virulence in these bacteria. Horizontal gene transfer was identified as critical but limited in the scale of virulence evolution, as few loci are conserved and exclusive to phytopathogenic isolates. Although the fas operon is present in most phytopathogenic isolates, it is absent from phytopathogenic isolate A21d2. Instead, this isolate has a horizontally acquired gene chimera that encodes a novel fusion protein with isopentyltransferase and phosphoribohydrolase domains, predicted to be capable of catalyzing and activating cytokinins, respectively. Cytokinin profiling of the archetypal D188 isolate revealed only one activate cytokinin type that was specifically synthesized in a fas-dependent manner. These results suggest that only the isopentenyladenine cytokinin type is synthesized and necessary for Rhodococcus phytopathogenicity, which is not consistent with the extant model stating that a mixture of cytokinins is necessary for Rhodococcus to cause leafy gall symptoms. In all, data indicate that only four horizontally acquired functions are sufficient to confer the trait of phytopathogenicity to members of the genetically diverse clade of Rhodococcus.

Use of whole genome sequences to develop a molecular phylogenetic framework for Rhodococcus fascians and the Rhodococcus genus

The accurate diagnosis of diseases caused by pathogenic bacteria requires a stable species classification. Rhodococcus fascians is the only documented member of its ill-defined genus that is capable of causing disease on a wide range of agriculturally important plants. Comparisons of genome sequences generated from isolates of Rhodococcus associated with diseased plants revealed a level of genetic diversity consistent with them representing multiple species. To test this, we generated a tree based on more than 1700 homologous sequences from plant-associated isolates of Rhodococcus, and obtained support from additional approaches that measure and cluster based on genome similarities. Results were consistent in supporting the definition of new Rhodococcus species within clades containing phytopathogenic members. We also used the genome sequences, along with other rhodococcal genome sequences to construct a molecular phylogenetic tree as a framework for resolving the Rhodococcus genus. Results indicated that Rhodococcus has the potential for having 20 species and also confirmed a need to revisit the taxonomic groupings within Rhodococcus.

Evaluation of selected methods of plant disease diagnosis

Abstract Plant disease diagnosis has very exacting requirements which present a difficult challenge in a clinical laboratory, which may receive thousands of samples annually comprising perhaps hundreds of different pathogens. Methods used must be reliable and not overly exclusive, since a single plant may be coinfected by more than one pathogen. Modern methods of diagnosis such as microscopy, axenic culture, serology, double-stranded RNA analysis, nucleic acid probes and DNA amplification via the polymerase chain reaction are evaluated relative to the needs of a clinical plant disease laboratory.

Evolutionary transitions between beneficial and phytopathogenic Rhodococcus challenge disease management

Understanding how bacteria affect plant health is crucial for developing sustainable crop production systems. We coupled ecological sampling and genome sequencing to characterize the population genetic history of Rhodococcus and the distribution patterns of virulence plasmids in isolates from nurseries. Analysis of chromosome sequences shows that plants host multiple lineages of Rhodococcus, and suggested that these bacteria are transmitted due to independent introductions, reservoir populations, and point source outbreaks. We demonstrate that isolates lacking virulence genes promote beneficial plant growth, and that the acquisition of a virulence plasmid is sufficient to transition beneficial symbionts to phytopathogens. This evolutionary transition, along with the distribution patterns of plasmids, reveals the impact of horizontal gene transfer in rapidly generating new pathogenic lineages and provides an alternative explanation for pathogen transmission patterns. Results also uncovered a misdiagnosed epidemic that implicated beneficial Rhodococcus bacteria as pathogens of pistachio. The misdiagnosis perpetuated the unnecessary removal of trees and exacerbated economic losses.

Eradication of potato wart disease from Maryland

Potato wart disease, caused bySynchytrium endobioticum (Schilb.) Perc., is a disease of quarantine significance due to the production of persistent resting spores and lack of effective chemical control measures. The disease was present in limited areas of Pennsylvania, Maryland and West Virginia in 1918–1920 and was thought to have been eradicated by 1974. A recent survey in Maryland to verify eradication in that state had revealed the presence of resting spores of the fungus in one home garden in Allegany County. A subsequent delimiting soil sample survey was conducted in and around the old quarantine area in Allegany County. None of the soil samples from 176 home gardens surveyed were positive forS. endobioticum resting spores. Bioassays with susceptible potato cultivars were conducted for five consecutive years at the site from which spores had been recovered. During three of the five years, environmental conditions did not preclude development of disease, yet no symptoms were obtained. According to the USDA APHIS guidelines for eradication of potato wart disease, the spores at the Allegany County, Maryland site are declared nonviable and Maryland is considered to be free of the potato wart pathogen.

Molecular characterization of a new tymovirus from Diascia ornamental plants.

Two tymoviruses were identified in plants of Diascia × hybrida ‘Sun Chimes™ Coral’ that exhibited chlorotic mottling and reduced growth. A strain of Nemesia ring necrosis virus (NeRNV) designated NeRNV-WA was detected in symptomatic plants; the deduced amino acid sequence is virtually identical to that of the previously reported NeRNV-Nf from Nemesia fruticosa. Sequence analysis also revealed the presence of a new tymovirus, and the entire genomic sequence of this virus was determined. The genome of 6,290 nucleotides was organized into three potential open reading frames (ORFs) typical of viruses in the genus Tymovirus. Based on sequence identity to tymovirus sequences, ORFs I to III encoded the replicase, movement protein and coat protein, respectively. Amino acid sequence identities to those of NeRNV-Nf were 84.8, 50.3 and 94.8%, respectively. The 5′-untranslated region could potentially form four hairpin structures. Secondary structure analysis of the 3′-terminus showed that the RNA can form a transfer-RNA-like structure that has an anticodon specific for histidine. Only 77.9% nucleotide identity was found when complete genomic sequences of this tymovirus from diascia and NeRNV-Nf were compared. The name Diascia yellow mottle virus (DiaYMV) is proposed for this new tymovirus.

Complete nucleotide sequence of an isolate of coleus vein necrosis virus from verbena.

Vegetatively propagated crops are particularly prone to virus infection, especially those that can be transmitted mechanically or by vectors that flourish in commercial production environments. Numerous viruses and viroids have been reported in Verbena hybrida [17]. A V. hybrida ‘Taylor Town Red’ plant exhibiting mottling, necrosis, and low vigor was received at the Oregon State University Plant Diagnostic Clinic from Illinois and tested positive for the presence of double-stranded RNA (dsRNA). The dsRNA was cloned and sequenced and was found to be nearly identical to a two-kilobase sequence of coleus vein necrosis virus (CVNV, Genbank DQ915963), a newly discovered carlavirus [14]. This communication reports the complete sequence of the verbena isolate of CVNV hereafter referred to as CVNV-V and provides data on biological properties of the virus including mechanical transfer to herbaceous indicators and identification of an insect vector. Virus material

Loop-Mediated Isothermal Amplification and Polymerase Chain Reaction Methods for Specific and Rapid Detection of Rhodococcus fascians

Rhodococcus fascians is a phytopathogenic actinobacterium which causes leafy galls and other plant distortions that result in economically significant losses to nurseries producing ornamental plants. Traditional assays for detection and identification are time-consuming and laborious. We developed a rapid polymerase chain reaction (PCR) diagnostic assay based on two primer pairs, p450 and fas, which target the fasA and fasD genes, respectively, that are essential for pathogenicity. We also developed a faster, more convenient, loop-mediated isothermal amplification (LAMP) assay targeting the fasR gene, which regulates expression of virulence genes. Both assays were evaluated for sensitivity and specificity in vitro and in planta. The p450 and fas primers amplified DNA only from pure cultures of pathogenic reference isolates of R. fascians. Nonpathogenic isolates and 51 other plant-associated bacteria were not amplified. The PCR primers correctly detected pathogenic R. fascians from 73 of 75 (97%) bacterial strains isolated from naturally infected plants. The PCR assay correctly discriminated between pathogenic R. fascians and other bacteria in 132 of 139 (95%) naturally infected plants, and in 34 of 34 (100%) artificially inoculated plants. The fas primers were slightly more accurate than the p450 primers. The LAMP assay accurately detected pathogenic R. fascians in 26 of 28 (93%) naturally infected plants and did not react with 23 asymptomatic plants. The LAMP primers also amplified product for DNA extracts of 40 of 41 bacterial strains isolated from plants with leafy galls. The detection limit of both the PCR and LAMP assays was approximately 103 CFU/30-μl reaction. These new tools allow fast, reliable, and accurate detection of R. fascians in vitro and in planta. The LAMP assay in particular is a significant advancement in rapid R. fascians diagnostics, and enables those with limited laboratory facilities to confirm the presence of this pathogen in infected plants.

Phlyctema vagabunda Isolated from Coin Canker of Ash Trees in Michigan

The coelomycetous fungus Phlyctema vagabunda Desm. (teleomorph Neofabraea alba (E.J. Guthrie) Verkley, synonym Pezicula alba E.J. Guthrie) is associated with a serious canker disease of cultivated ash trees in Michigan. Four- to five-year-old trees of Fraxinus americana cv. Autumn Purple and F. pennsylvanica cvs. Champ Tree, Cimmaron, and Urbanite had cankers that were smooth, round, brownish yellow, approximately 2 to 4 cm in diameter with distinct reddish, cracked margins. Immersed, eventually erumpent, unilocular acervuli developed in the central portions of these cankers. The same fungus was isolated both from the conidia as well as from the margin of the canker. The internal transcribed spacer (ITS) (AY064704) and beta-tubulin (AY064702) sequences were identical to sequences identified in GenBank as Pezicula alba from apple (1), and the morphology was consistent with Phlyctema vagabunda as well (2). P. vagabunda has been studied primarily as the cause of Bulls eye canker of apple (1). P. vagabunda under its numerous synonyms has been reported on various hardwood and herbaceous hosts from temperate regions around the world, including the United States. However, it has not been reported previously on species of Fraxinus. A specimen and culture from the ash cankers in Michigan have been deposited (BPI 841384 and CBS 109875). References: (1) S. N. De Jong et al. Mycol. Res. 105:658, 2001. (2) B. C. Sutton. The Coelomycetes. CMI, Kew, Surrey, UK, 1980.