Viral Reservoir Capacity of Wild Prunus Alternative Hosts of Plum Pox Virus Through Multiple Cycles of Transmission and Dormancy.

Plum pox virus (PPV) is a significant pathogen of Prunus worldwide and is known for having a broad experimental host range. Many of these hosts represent epidemiological risks as potential wild viral reservoirs. A comparative study of the PPV reservoir capacity of three commonly found native North American species, western choke cherry (Prunus virginiana var. demissa), black cherry (Prunus serotina), and American plum (Prunus americana) was conducted. Pennsylvania isolates of PPV-D were transmitted from the original host peach (Prunus persica cv. GF305) to all three species. Viral accumulation and transmission rates to alternative hosts and peach were monitored over the course of five vegetative growth and cold induced dormancy (CID) cycles. The three alternative host species demonstrated differences in their ability to maintain PPV-D and the likelihood of transmission to additional alternative hosts or back transmission to peach. Western choke cherry had low (5.8%) initial infection levels, PPV-D was not transmissible to additional western choke cherry, and transmission of PPV-D from western choke cherry to peach was only possible before the first CID cycle. Black cherry had intermediate initial infection levels (26.6%) but did not maintain high infection levels after repeated CID cycles. Conversely, American plum had a high level (50%) of initial infection that was not significantly different from initial infection in peach (72.2%) and maintained moderate levels (15 to 25%) of infection and PPV-D transmission to both American plum and peach through all five cycles of CID. Our results indicate that American plum has the greatest potential to act as a reservoir host for Pennsylvania isolates of PPV-D.

Reassortment of Genome Segments Creates Stable Lineages Among Strains of Orchid Fleck Virus Infecting Citrus in Mexico.

The genus Dichorhavirus contains viruses with bipartite, negative-sense, single-stranded RNA genomes that are transmitted by flat mites to hosts that include orchids, coffee, the genus Clerodendrum, and citrus. A dichorhavirus infecting citrus in Mexico is classified as a citrus strain of orchid fleck virus (OFV-Cit). We previously used RNA sequencing technologies on OFV-Cit samples from Mexico to develop an OFV-Cit-specific reverse transcription PCR (RT-PCR) assay. During assay validation, OFV-Cit-specific RT-PCR failed to produce an amplicon from some samples with clear symptoms of OFV-Cit. Characterization of this virus revealed that dichorhavirus-like particles were found in the nucleus. High-throughput sequencing of small RNAs from these citrus plants revealed a novel citrus strain of OFV, OFV-Cit2. Sequence comparisons with known orchid and citrus strains of OFV showed variation in the protein products encoded by genome segment 1 (RNA1). Strains of OFV clustered together based on host of origin, whether orchid or citrus, and were clearly separated from other dichorhaviruses described from infected citrus in Brazil. The variation in RNA1 between the original (now OFV-Cit1) and the new (OFV-Cit2) strain was not observed with genome segment 2 (RNA2), but instead, a common RNA2 molecule was shared among strains of OFV-Cit1 and -Cit2, a situation strikingly similar to OFV infecting orchids. We also collected mites at the affected groves, identified them as Brevipalpus californicus sensu stricto, and confirmed that they were infected by OFV-Cit1 or with both OFV-Cit1 and -Cit2. OFV-Cit1 and -Cit2 have coexisted at the same site in Toliman, Queretaro, Mexico since 2012. OFV strain-specific diagnostic tests were developed.

Partial Proteome of the Corynetoxin-Producing Gram-Positive Bacterium, Rathayibacter toxicus.

Rathayibacter toxicus is a Gram-positive bacterium that is the causative agent of annual ryegrass toxicity (ARGT), a disease that causes devastating losses in the Australian livestock industry. R. toxicus exhibits a complex life cycle, using the nematode Anguina funesta as a physical vector to carry it up to the seed head of the host plant. ARGT is caused by a tunicamycin-like corynetoxin that is produced in R. toxicus-infected seed galls. We analyzed protein expression in R. toxicus under stationary growth phase conditions to obtain a more complete understanding of the biology of this organism and identify potential targets for immunoassay development. A total of 323 unique proteins were identified, including those with putative roles in secondary metabolism and pathogenicity. The proteome analysis for this complex phytopathogenic Gram-positive bacterium will facilitate in the characterization of proteins necessary for host colonization and toxin production, and assist in the development of diagnostic assays. Data are available via ProteomeXchange with identifier PXD004238.

Rathayibacter agropyri (non O'Gara 1916) comb. nov., nom. rev., isolated from western wheatgrass (Pascopyrum smithii).

Aplanobacter agropyri was first described in 1915 by O'Gara and later transferred to the genus Corynebacterium by Burkholder in 1948 but it was not included in the Approved Lists of Bacterial Names in 1980 and, consequently, is not recognized as a validly published species. In the 1980s, bacteria resembling Corynebacterium agropyri were isolated from plant samples stored at the Washington State Mycological Herbarium and from a diseased wheatgrass plant collected in Cardwell, Montana, USA. In the framework of this study, eight additional isolates were recovered from the same herbarium plant samples in 2011. The isolates are slow-growing, yellow-pigmented, Gram-stain-positive and coryneform. The peptidoglycan is type B2γ containing diaminobutyric acid, alanine, glycine and glutamic acid, the cell-wall sugars are rhamnose and mannose, the major respiratory quinone is MK-10, and the major fatty acids are anteiso-15 : 0, anteiso 17 : 0 and iso-16 : 0, all of which are typical of the genus Rathayibacter. Phylogenetic analysis of 16S rRNA gene sequences placed the strains in the genus Rathayibacter and distinguished them from the six other described species of Rathayibacter. DNA-DNA hybridization confirmed that the strains were members of a novel species. Based on phenotypic, chemotaxonomic and phylogenetic characterization, it appears that strains CA-1 to CA-12 represent a novel species, and the name Rathayibacter agropyri (non O'Gara 1916) comb. nov., nom. rev. is proposed; the type strain is CA-4T (=DSM 104101T;=ATCC TSD-78T).

Rathayibacter toxicus, Other Rathayibacter Species Inducing Bacterial Head Blight of Grasses, and the Potential for Livestock Poisonings.

Rathayibacter toxicus, a Select Agent in the United States, is one of six recognized species in the genus Rathayibacter and the best known due to its association with annual ryegrass toxicity, which occurs only in parts of Australia. The Rathayibacter species are unusual among phytopathogenic bacteria in that they are transmitted by anguinid seed gall nematodes and produce extracellular polysaccharides in infected plants resulting in bacteriosis diseases with common names such as yellow slime and bacterial head blight. R. toxicus is distinguished from the other species by producing corynetoxins in infected plants; toxin production is associated with infection by a bacteriophage. These toxins cause grazing animals feeding on infected plants to develop convulsions and abnormal gate, which is referred to as "staggers," and often results in death of affected animals. R. toxicus is the only recognized Rathayibacter species to produce toxin, although reports of livestock deaths in the United States suggest a closely related toxigenic species may be present. A closely related but undescribed species, Rathayibacter sp. EV, originally isolated from Ehrharta villosa var. villosa in South Africa, is suspected of producing toxin. Many of the diseases caused by Rathayibacter species occur in arid areas and the extracellular polysaccharide they produce is believed to aid in their survival between crops. For example, R. "agropyri" was isolated from infected plant material after being stored for 50 years in a herbarium. Similarly, the anguinid vectors associated with these bacteria form seed galls in infected plants and are capable of surviving for very long periods of time under dry conditions. The addition of R. toxicus to the list of Select Agents has raised concern over its potential introduction and a realization that current diagnostic methods are inadequate to distinguish among Rathayibacter species. In addition, little is known about the Rathayibacter species and their seed gall nematode vectors present in the United States.

History and Diversity of Citrus leprosis virus Recorded in Herbarium Specimens.

Leprosis refers to two diseases of citrus that present similar necrotic local lesions, often surrounded by chlorotic haloes on citrus. Two distinct viruses are associated with this disease, one that produces particles primarily in the nucleus of infected plant cells (Citrus leprosis virus nuclear type [CiLV-N]; Dichorhavirus) and another type that produces particles in the cytoplasm of infected plant cells (Citrus leprosis virus cytoplasmic type [CiLV-C]; Cilevirus). Both forms are transmitted by Brevipalpid mites and have bipartite, single-stranded, RNA genomes. CiLV-C and CiLV-N are present in South and Central America and as far north as parts of Mexico. Although leprosis disease was originally described from Florida, it disappeared from there in the 1960s. The United States Department of Agriculture-Agricultural Research Service maintains preserved citrus specimens identified at inspection stations 50 or more years ago with symptoms of citrus leprosis. We isolated RNA from these samples and performed degradome sequencing. We obtained nearly full-length genome sequences of both a typical CiLV-C isolate intercepted from Argentina in 1967 and a distinct CiLV-N isolate obtained in Florida in 1948. The latter is a novel form of CiLV-N, not known to exist anywhere in the world today. We have also documented the previously unreported presence of CiLV-N in Mexico in the mid-20th century.

Identification and Molecular Characterization of Nuclear Citrus leprosis virus, a Member of the Proposed Dichorhavirus Genus Infecting Multiple Citrus Species in Mexico.

Citrus leprosis is one of the most destructive diseases of Citrus spp. and is associated with two unrelated virus groups that produce particles primarily in either the cytoplasm or nucleus of infected plant cells. Symptoms of leprosis, including chlorotic spots surrounded by yellow haloes on leaves and necrotic spots on twigs and fruit, were observed on leprosis-affected mandarin and navel sweet orange trees in the state of Querétaro, Mexico. Serological and molecular assays showed that the cytoplasmic types of Citrus leprosis virus (CiLV-C) often associated with leprosis symptomatic tissues were absent. However, using transmission electron microscopy, bullet-shaped rhabdovirus-like virions were observed in the nuclei and cytoplasm of the citrus leprosis-infected leaf tissues. An analysis of small RNA populations from symptomatic tissue was carried out to determine the genome sequence of the rhabdovirus-like particles observed in the citrus leprosis samples. The complete genome sequence showed that the nuclear type of CiLV (CiLV-N) present in the samples consisted of two negative-sense RNAs: 6,268-nucleotide (nt)-long RNA1 and 5,847-nt-long RNA2, excluding the poly(A) tails. CiLV-N had a genome organization identical to that of Orchid fleck virus (OFV), with the exception of shorter 5' untranslated regions in RNA1 (53 versus 205 nt) and RNA2 (34 versus 182 nt). Phylogenetic trees constructed with the amino acid sequences of the nucleocapsid (N) and glycoproteins (G) and the RNA polymerase (L protein) showed that CiLV-N clusters with OFV. Furthermore, phylogenetic analyses of N protein established CiLV-N as a member of the proposed genus Dichorhavirus. Reverse-transcription polymerase chain reaction primers for the detection of CiLV-N were designed based on the sequence of the N gene and the assay was optimized and tested to detect the presence of CiLV-N in both diseased and symptom-free plants.

Role Bending: Complex Relationships Between Viruses, Hosts, and Vectors Related to Citrus Leprosis, an Emerging Disease.

Citrus leprosis complex is an emerging disease in the Americas, associated with two unrelated taxa of viruses distributed in South, Central, and North America. The cytoplasmic viruses are Citrus leprosis virus C (CiLV-C), Citrus leprosis virus C2 (CiLV-C2), and Hibiscus green spot virus 2, and the nuclear viruses are Citrus leprosis virus N (CiLV-N) and Citrus necrotic spot virus. These viruses cause local lesion infections in all known hosts, with no natural systemic host identified to date. All leprosis viruses were believed to be transmitted by one species of mite, Brevipalpus phoenicis. However, mites collected from CiLV-C and CiLV-N infected citrus groves in Mexico were identified as B. yothersi and B. californicus sensu lato, respectively, and only B. yothersi was detected from CiLV-C2 and CiLV-N mixed infections in the Orinoco regions of Colombia. Phylogenetic analysis of the helicase, RNA-dependent RNA polymerase 2 domains and p24 gene amino acid sequences of cytoplasmic leprosis viruses showed a close relationship with recently deposited mosquito-borne negevirus sequences. Here, we present evidence that both cytoplasmic and nuclear viruses seem to replicate in viruliferous Brevipalpus species. The possible replication in the mite vector and the close relationship with mosquito borne negeviruses are consistent with the concept that members of the genus Cilevirus and Higrevirus originated in mites and citrus may play the role of mite virus vector.

A previously undescribed potyvirus isolated and characterized from arborescent Brugmansia.

A suspected virus disease was identified from an arborescent Brugmansia x candida Pers. (syn. Datura candida Pers.) tree. The causal agent was aphid transmissible at low rates. Viral particles were purified from infected tobacco tissue, analyzed, and purified virions were inoculated into healthy tobacco plants to recreate the symptoms. The virions had a mean length of 720-729 nm, and infected cells contained inclusion bodies typical of potyvirus infections. Analysis of infected tissues and purified virions with a panel of potyvirus-specific antibodies confirmed identification as a potyvirus. Viral host range, dilution end point, thermal tolerance and aphid transmission characteristics were examined. The viral genome (9761 nt) is typical of potyviruses, with the closest related potyvirus being pepper mottle virus, at 72 % nt sequence identity. Based on conventions for naming novel potyviruses, the virus was determined to be a member of a previously undescribed species, tentatively named "Brugmansia mosaic virus" (BruMV).

Molecular, ultrastructural, and biological characterization of Pennsylvania isolates of Plum pox virus.

Plum pox virus (PPV) was identified in Pennsylvania in 1999. The outbreak was limited to a four-county region in southern Pennsylvania. Initial serological and molecular characterization indicated that the isolates in Pennsylvania belong to the D strain of PPV. The Pennsylvania isolates were characterized by sequence analysis, electron microscopy, host range, and vector transmission to determine how these isolates related to their previously studied European counterparts. Genetically, Pennsylvania (PPV-Penn) isolates were more closely related to each other than to any other PPV-D strains, and isolates from the United States, Canada, and Chile were more closely related to each other than to European isolates. The PPV-Penn isolates exist as two clades, suggesting the possibility of multiple introductions. Electron microscopy analysis of PPV-Penn isolates, including cytopathological studies, indicated that the virions were similar to other Potyvirus spp. PPV-Penn isolates had a herbaceous host range similar to that of European D isolates. There were distinct differences in the transmission efficiencies of the two PPV-Penn isolates using Myzus persicae and Aphis spiraecola as vectors; however, both PPV-Penn isolates were transmitted by M. persicae more efficiently than a European D isolate but less efficiently than a European M isolate.

Evolution of the U.S. Biological Select Agent Rathayibacter toxicus.

Rathayibacter toxicus is a species of Gram-positive, corynetoxin-producing bacteria that causes annual ryegrass toxicity, a disease often fatal to grazing animals. A phylogenomic approach was employed to model the evolution of R. toxicus to explain the low genetic diversity observed among isolates collected during a 30-year period of sampling in three regions of Australia, gain insight into the taxonomy of Rathayibacter, and provide a framework for studying these bacteria. Analyses of a data set of more than 100 sequenced Rathayibacter genomes indicated that Rathayibacter forms nine species-level groups. R. toxicus is the most genetically distant, and evidence suggested that this species experienced a dramatic event in its evolution. Its genome is significantly reduced in size but is colinear to those of sister species. Moreover, R. toxicus has low intergroup genomic diversity and almost no intragroup genomic diversity between ecologically separated isolates. R. toxicus is the only species of the genus that encodes a clustered regularly interspaced short palindromic repeat (CRISPR) locus and that is known to host a bacteriophage parasite. The spacers, which represent a chronological history of infections, were characterized for information on past events. We propose a three-stage process that emphasizes the importance of the bacteriophage and CRISPR in the genome reduction and low genetic diversity of the R. toxicus species.IMPORTANCERathayibacter toxicus is a toxin-producing species found in Australia and is often fatal to grazing animals. The threat of introduction of the species into the United States led to its inclusion in the Federal Select Agent Program, which makes R. toxicus a highly regulated species. This work provides novel insights into the evolution of R. toxicusR. toxicus is the only species in the genus to have acquired a CRISPR adaptive immune system to protect against bacteriophages. Results suggest that coexistence with the bacteriophage NCPPB3778 led to the massive shrinkage of the R. toxicus genome, species divergence, and the maintenance of low genetic diversity in extant bacterial groups. This work contributes to an understanding of the evolution and ecology of an agriculturally important species of bacteria.

Complete Genome Sequence of Rathayibacter toxicus Phage NCPPB3778.

The phage NCPPB3778 was isolated from Rathayibacter toxicus strain CS14, and the genomic DNA was sequenced. The genome is similar to siphoviruses, consisting of 44,520 bases including 77 predicted open reading frames. Portions of the genome are annotated as typical phage proteins, but much of the genome sequence is unique from other bacteriophages.

Host Adaptation of Soybean Dwarf Virus Following Serial Passages on Pea (Pisum sativum) and Soybean (Glycine max).

Soybean Dwarf Virus (SbDV) is an important plant pathogen, causing economic losses in soybean. In North America, indigenous strains of SbDV mainly infect clover, with occasional outbreaks in soybean. To evaluate the risk of a US clover strain of SbDV adapting to other plant hosts, the clover isolate SbDV-MD6 was serially transmitted to pea and soybean by aphid vectors. Sequence analysis of SbDV-MD6 from pea and soybean passages identified 11 non-synonymous mutations in soybean, and six mutations in pea. Increasing virus titers with each sequential transmission indicated that SbDV-MD6 was able to adapt to the plant host. However, aphid transmission efficiency on soybean decreased until the virus was no longer transmissible. Our results clearly demonstrated that the clover strain of SbDV-MD6 is able to adapt to soybean crops. However, mutations that improve replication and/or movement may have trade-off effects resulting in decreased vector transmission.

Whole genome sequence of two Rathayibacter toxicus strains reveals a tunicamycin biosynthetic cluster similar to Streptomyces chartreusis.

Rathayibacter toxicus is a forage grass associated Gram-positive bacterium of major concern to food safety and agriculture. This species is listed by USDA-APHIS as a plant pathogen select agent because it produces a tunicamycin-like toxin that is lethal to livestock and may be vectored by nematode species native to the U.S. The complete genomes of two strains of R. toxicus, including the type strain FH-79, were sequenced and analyzed in comparison with all available, complete R. toxicus genomes. Genome sizes ranged from 2,343,780 to 2,394,755 nucleotides, with 2079 to 2137 predicted open reading frames; all four strains showed remarkable synteny over nearly the entire genome, with only a small transposed region. A cluster of genes with similarity to the tunicamycin biosynthetic cluster from Streptomyces chartreusis was identified. The tunicamycin gene cluster (TGC) in R. toxicus contained 14 genes in two transcriptional units, with all of the functional elements for tunicamycin biosynthesis present. The TGC had a significantly lower GC content (52%) than the rest of the genome (61.5%), suggesting that the TGC may have originated from a horizontal transfer event. Further analysis indicated numerous remnants of other potential horizontal transfer events are present in the genome. In addition to the TGC, genes potentially associated with carotenoid and exopolysaccharide production, bacteriocins and secondary metabolites were identified. A CRISPR array is evident. There were relatively few plant-associated cell-wall hydrolyzing enzymes, but there were numerous secreted serine proteases that share sequence homology to the pathogenicity-associated protein Pat-1 of Clavibacter michiganensis. Overall, the genome provides clear insight into the possible mechanisms for toxin production in R. toxicus, providing a basis for future genetic approaches.

Characterization of a Brome mosaic virus strain and its use as a vector for gene silencing in monocotyledonous hosts.

Virus-induced gene silencing (VIGS) is used to analyze gene function in dicotyledonous plants but less so in monocotyledonous plants (particularly rice and corn), partially due to the limited number of virus expression vectors available. Here, we report the cloning and modification for VIGS of a virus from Festuca arundinacea Schreb. (tall fescue) that caused systemic mosaic symptoms on barley, rice, and a specific cultivar of maize (Va35) under greenhouse conditions. Through sequencing, the virus was determined to be a strain of Brome mosaic virus (BMV). The virus was named F-BMV (F for Festuca), and genetic determinants that controlled the systemic infection of rice were mapped to RNAs 1 and 2 of the tripartite genome. cDNA from RNA 3 of the Russian strain of BMV (R-BMV) was modified to accept inserts from foreign genes. Coinoculation of RNAs 1 and 2 from F-BMV and RNA 3 from R-BMV expressing a portion of a plant gene to leaves of barley, rice, and maize plants resulted in visual silencing-like phenotypes. The visual phenotypes were correlated with decreased target host transcript levels in the corresponding leaves. The VIGS visual phenotype varied from maintained during silencing of actin 1 transcript expression to transient with incomplete penetration through affected tissue during silencing of phytoene desaturase expression. F-BMV RNA 3 was modified to allow greater accumulation of virus while minimizing virus pathogenicity. The modified vector C-BMV(A/G) (C for chimeric) was shown to be useful for VIGS. These BMV vectors will be useful for analysis of gene function in rice and maize for which no VIGS system is reported.

Synthesis of minus-strand copies of a viral transgene during viral infections of transgenic plants.

Viral transgenes designed to provide resistance to specific plant viruses frequently consist of the coat protein gene and a contiguous 3' untranslated region (3'UTR) of viral origin. In many RNA viruses the viral 3'UTR establishes a recognition and initiation site for viral RNA replication. Thus the transgenic transcript may contain a functional virus replication site. Experiments were designed to determine if a challenging virus would recognize this replication site on a nuclear derived transcript and synthesize the complementary RNA. These data demonstrate that upon infection by a virus that recognizes the viral replication site, a full-length complement of the transgenic transcript is produced. In these experiments the replication complex of Brome Mosaic bromovirus recognized the transgenic transcript derived from a Cowpea Chlorotic Mottle bromovirus transgene. The resulting RNA may contribute to RNA recombination events.

Advances in molecular-based diagnostics in meeting crop biosecurity and phytosanitary issues.

Awareness of crop biosecurity and phytosanitation has been heightened since 9/11 and the unresolved anthrax releases in October 2001. Crops are highly vulnerable to accidental or deliberate introductions of crop pathogens from outside U.S. borders. Strategic thinking about protection against deliberate or accidental release of a plant pathogen is an urgent priority. Rapid detection will be the key to success. This review summarizes recent progress in the development of rapid real-time PCR protocols and evaluates their effectiveness in a proposed nationwide network of diagnostic laboratories that will facilitate rapid diagnostics and improved communication.

Specific detection and quantification of Plum pox virus by real-time fluorescent reverse transcription-PCR.

Plum pox virus (PPV), a destructive and economically devastating pathogen of Prunus species, was recently discovered in Pennsylvania and Canada. Current containment efforts involve eradication of infected trees based on ELISA surveys, which are laborious and less sensitive than PCR-based techniques. A real-time, fluorescent, reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for the detection of PPV in the Smart Cycler (Cepheid). The methods developed are reproducible, specific to PPV, and sensitive enough to consistently detect PPV transcripts at the 10-20 fg level. The assay is more sensitive than either ELISA or traditional PCR followed by visualization with ethidium-bromide. PPV was detected from multiple hosts and from multiple Prunus tissues (leaf, stem, bud, and root). A dilution series using an in vitro synthesized transcript containing the target sequence as a standard demonstrated that the assay was effective for quantitation of viral template. The real-time PCR assay is a valuable tool for PPV detection and liter quantification in field or laboratory settings.

Metagenomic search strategies for interactions among plants and multiple microbes.

Plants harbor multiple microbes. Metagenomics can facilitate understanding of the significance, for the plant, of the microbes, and of the interactions among them. However, current approaches to metagenomic analysis of plants are computationally time consuming. Efforts to speed the discovery process include improvement of computational speed, condensing the sequencing reads into smaller datasets before BLAST searches, simplifying the target database of BLAST searches, and flipping the roles of metagenomic and reference datasets. The latter is exemplified by the e-probe diagnostic nucleic acid analysis approach originally devised for improving analysis during plant quarantine.

Population of endogenous pararetrovirus genomes in carrizo citrange.

The complete genome sequences of three related endogenous pararetroviruses (EPRVs) were obtained by 454 sequencing of nucleic acid extracts from Carrizo citrange, used as a citrus rootstock. Numerous homologous sequences have been found in the sweet orange genome. The new EPRVs are most closely related to petunia vein-clearing virus.