James M. Crosslin

Whole genome characterization of Potato virus Y isolates collected in the western USA and their comparison to isolates from Europe and Canada

Summary.Potato virus Y (PVY) is a serious potato pathogen that affects potato seed and commercial production crops. In recent decades, novel PVY strains have been described that cause necrotic symptoms on tobacco foliage and/or potato tubers. The major PVY strains that affect potato include PVYO and PVYN, which have distinct serotypes that can be differentiated by immunoassay. Other economically important strain variants are derived from recombination events, including variants that cause tuber necrotic symptoms (PVYNTN) and PVYO serotypes that cause tobacco veinal necrosis (PVYN-W, PVYN:O). Although the PVYNTN and PVYN-W variants were first reported in Europe, apparently similar strains have been appearing in North America. Confirmation of the existence of these recombinant strains in North America is important, as is whether they spread from a common source or were derived by independent recombination. Whole genome sequencing can be used to positively identify strain variants and begin to address the issue of origins. Symptomology, serology, RT-PCR, and partial sequencing of the coat protein region were used to identify isolates of the PVYNTN, PVYN, PVYNA-N, and PVYN:O for whole-genome sequencing. Sequencing confirmed the presence of PVYNTN and PVYN isolates that were >99% identical to European sequences deposited in GenBank in the 1990’s. Sequences of the PVYNA-N and PVYN:O types were 99.0% and 99.5% identical to known sequences, respectively. There was no indication that recombinant strains PVYNTN or PVYN:O had different parental origins than recombinant strains previously sequenced. This is the first confirmation by whole-genome sequencing that “European”-type strain variants of PVYN and PVYNTN are present in North America, and the first reported full-length sequence of a tuber necrotic isolate of PVYN:O.

Genetic diversity of the ordinary strain of Potato virus Y (PVY) and origin of recombinant PVY strains.

The ordinary strain of Potato virus Y (PVY), PVY(O), causes mild mosaic in tobacco and induces necrosis and severe stunting in potato cultivars carrying the Ny gene. A novel substrain of PVY(O) was recently reported, PVY(O)-O5, which is spreading in the United States and is distinguished from other PVY(O) isolates serologically (i.e., reacting to the otherwise PVY(N)-specific monoclonal antibody 1F5). To characterize this new PVY(O)-O5 subgroup and address possible reasons for its continued spread, we conducted a molecular study of PVY(O) and PVY(O)-O5 isolates from a North American collection of PVY through whole-genome sequencing and phylogenetic analysis. In all, 44 PVY(O) isolates were sequenced, including 31 from the previously defined PVY(O)-O5 group, and subjected to whole-genome analysis. PVY(O)-O5 isolates formed a separate lineage within the PVY(O) genome cluster in the whole-genome phylogenetic tree and represented a novel evolutionary lineage of PVY from potato. On the other hand, the PVY(O) sequences separated into at least two distinct lineages on the whole-genome phylogenetic tree. To shed light on the origin of the three most common PVY recombinants, a more detailed phylogenetic analysis of a sequence fragment, nucleotides 2,406 to 5,821, that is present in all recombinant and nonrecombinant PVY(O) genomes was conducted. The analysis revealed that PVY(N:O) and PVY(N-Wi) recombinants acquired their PVY(O) segments from two separate PVY(O) lineages, whereas the PVY(NTN) recombinant acquired its PVY(O) segment from the same lineage as PVY(N:O). These data suggest that PVY(N:O) and PVY(N-Wi) recombinants originated from two separate recombination events involving two different PVY(O) parental genomes, whereas the PVY(NTN) recombinants likely originated from the PVY(N:O) genome via additional recombination events.

High resolution melting analysis of the cytochrome oxidase I gene identifies three haplotypes of the potato psyllid in the United States.

ABSTRACT The potato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Triozidae), is a vector of the bacterium “Candidatus Liberibacter solanacearum,” the putative causal agent of potato zebra chip disease that has seriously affected the potato industry in the Central and Southwestern United States for the past decade. The 2011 potato growing season saw the first report of zebra chip disease in Washington, Oregon, and Idaho; however, B. cockerelli has been recorded in this region every season at least for the past 7 yr. Studies were conducted to determine the relationship between psyllids collected from the Pacific Northwest potatoes in 2011 and those from the Southwestern and Central United States. High resolution melting analysis of the B. cockerelli mitochondrial Cytochrome C Oxidase subunit I-like gene was conducted on over 450 psyllids collected from numerous locations across the Central and Western United States. Results suggest that at least three potato psyllid haplotypes exist in the United States, correlating to the Central, Western, and Northwestern United States geographical regions. The high resolution melting analysis results were subsequently supported by DNA sequencing data.

Carrot Purple Leaf: A New Spiroplasmal Disease Associated with Carrots in Washington State

During the growing seasons of 2003 and 2004, a disease occurred in several carrot crops in south central Washington with symptoms suggestive of infection by phytopathogenic mollicutes (phytoplasmas and spiroplasmas). In the fall, many affected carrot plants exhibited extensive purple or yellow-purple leaf discoloration, general stunting of shoots and taproots, and formation of bunchy, fibrous secondary roots. For detection of the putative causal agents, polymerase chain reaction (PCR) assays were performed using primers specific to phytoplasmas as well as primers specific to plant-pathogenic spiroplasmas. Restriction fragment length polymorphism (RFLP) analyses of PCR-amplified 16S rDNA sequences revealed that about 81% of affected plants showing dark purple or yellow-purple leaf symptoms tested positive for Spiroplasma citri. Of affected plants showing mild purple discoloration of leaf margins, 18% tested positive for a phytoplasma strain belonging to the clover proliferation group (16SrVI), subgroup 16SrVI-A, and 11% for another phytoplasma strain belonging to the aster yellows group (16SrI), subgroup 16SrI-A. Nucleotide sequence analysis of cloned 16S rDNA confirmed the phytoplasma group affiliations. Some symptomatic plants were co-infected with S. citri and either aster yellows phytoplasma or clover proliferation group phytoplasma. To our knowledge, this is the first documentation of spiroplasma infection of carrot in the United States.

The Occurrence of PVYO, PVYN, and PVYN:O Strains of Potato virus Y in Certified Potato Seed Lot Trials in Washington and Oregon

Totals of 960 and 286 certified potato seed lots from locations across North America were planted in trials in Washington and Oregon, respectively, in 2001 to 2003 and tested for strains of Potato virus Y (PVY). The incidence of PVYO-infected lots averaged 16.4 and 25.9% in the Washington and Oregon trials, respectively. There was a general trend of increasing incidence of the PVYO, PVYN:O, and PVYN strains during this period, as evidenced by more infected cultivars, sites of seed origin, and number of seed growers providing infected seed lots. In particular, there was a dramatic increase in seed lots with the PVYN:O strain from 2002 to 2003. PVYN:O, in contrast to PVYO, which only causes yield reduction, also causes internal and external damage to tubers, making them unmarketable. In 2003, PVYN:O occurred in seed lots originating in eight states and three Canadian provinces. The increased incidence of PVYN:O was likely due to the difficulty in differentiating this strain from PVYO. The prevalence of PVY in potato seed lots documented herein poses a threat to potato production in the United States and suggests that current measures to reduce the incidence of this virus are inadequate.

Phenotypic and Etiological Differences Between Psyllid Yellows and Zebra Chip Diseases of Potato

Both potato psyllid yellows and zebra chip (ZC) potato diseases are associated with the potato psyllid, Bactericera cockerelli (Sulc). Aboveground plant symptoms of both diseases are similar but there is a difference in symptoms in potato tubers. ZC has recently been associated with a new species of the bacterium liberibacter, ‘Candidatus Liberibacter solanacearum’, also known as ‘Ca. Liberibacter psyllaurous’. Mechanisms by which the potato psyllid might cause either ZC or potato psyllid yellows symptoms are not understood. Insect transmission studies were conducted to demonstrate psyllid vectoring of both diseases and to compare symptoms and development of the two diseases. Potato plants were exposed to both liberibacter-free and liberibacter-carrying potato psyllids and later evaluated for plant and tuber symptoms. These plants and tubers were then tested for liberibacter by polymerase chain reaction (PCR). In addition, potato plants exhibiting severe psyllid yellows/ZC-like symptoms were collected from a commercial potato field heavily infested with the potato psyllid and tested for liberibacter. PCR detected ‘Ca. Liberibacter solanacearum’ in ZC symptomatic plants and tubers resulting from exposure to liberibacter-carrying psyllids. Despite development of foliar symptoms that resemble those of ZC in plants exposed to liberibacter-free psyllids, no liberibacter was detected in these plants with psyllid yellows. Moreover, tubers from these plants with psyllid yellows did not exhibit any symptoms of ZC infection and tested negative for the bacterium. No liberibacter was detected in plants or tubers collected from the psyllid-infested potato field, suggesting that the observed symptoms were due to psyllid yellows. Furthermore, potato plants that were infected with liberibacter died sooner than plants that were infected with psyllid yellows. Although an association between liberibacter and ZC has been established, no pathogen is yet associated with potato psyllid yellows and mechanisms by which psyllid yellows symptoms are induced by the potato psyllid remain unclear.ResumenLas dos enfermedades de la papa, el amarillamiento de la papa por psílidos y zebra chip (ZC), están asociadas con el psílido de la papa Bactericera cockerelli (Sulc). Los síntomas aéreos de la planta por ambas enfermedades son similares, pero hay una diferencia en los síntomas del tubérculo. La ZC se ha asociado recientemente con una nueva especie de bacteria liberibacter, “Candidatus Liberibacter solanacearum” , también conocida como “Ca. Liberibacter psyllaurous”. No se han entendido los mecanismos por los cuales el psílido de la papa puede causar los síntomas, ya sea de la ZC o el amarillamiento de la papa por psílidos. Se condujeron estudios de la transmisión por insectos para demostrar la transmisión de ambas enfermedades por psílidos y para comparar los síntomas y el desarrollo de ambas enfermedades. Se expusieron plantas de papa a psílidos de la papa, tanto libres como con liberibacter, y se evaluaron posteriormente los síntomas de la planta y del tubérculo. Estas plantas y tubérculos se probaron después para liberibacter con la reacción en cadena de la polimerasa (PCR). Además, se colectaron plantas de papa que exhibieron síntomas severos de amarillamiento por psílidos/ZC, de un campo comercial de papa severamente infestado con el psílido de la papa, y se probaron para liberibacter. La PCR detectó “Ca. Liberibacter solanacearum” en plantas y tubérculos con síntomas de ZC, como resultado de su exposición a los psílidos con liberibacter. A pesar del desarrollo de síntomas foliares parecidos a los de ZC en plantas expuestas a los psílidos libres de liberibacter, no se detectó liberibacter en estas plantas con amarillamiento por psílidos. Aún mas, los tubérculos de estas plantas con amarillamiento por psílidos no exhibieron síntomas de infección por ZC y resultaron negativas a la bacteria. No se detectó liberibacter en plantas o tubérculos colectados del campo de papa infestado por psílidos, lo cual sugiere que los síntomas observados fueron debidos al amarillamiento por psílidos. Incluso, las plantas infectadas con liberibacter murieron más pronto que las infectadas con amarillamiento por psílidos. Aún cuando se ha establecido una asociación entre liberibacter y ZC, no se ha asociado a algún patógeno con el amarillamiento de la papa por psílidos y permanecen si aclararse los mecanismos por los cuales se inducen los síntomas del amarillamiento por el psílido de la papa.

Evidence that the Zebra Chip Disease and the Putative Causal Agent Can be Maintained in Potatoes by Grafting and In Vitro

In the last several years, a disorder of chipping potatoes that causes internal browning of raw tubers and very dark chip color when the tubers are fried has been described from the southwestern United States. The discoloration often shows as rays or stripes and the common name “zebra chip” (ZC) has been used to describe the disorder. Foliar symptoms include chlorosis, purpling of shoot tips, and leaf scorch. Aerial tubers are sometimes produced on diseased plants. The disease has been associated with the potato psyllid, Bactericera cockerelli, but a suspected pathogen that may be associated with the ZC disorder has only very recently been described. In our efforts to identify the causal agent we have been maintaining the disease in the greenhouse by tip grafting symptomatic potato shoots onto healthy plants of several commercially important cultivars of potatoes, including Atlantic, Russet Burbank, and Russet Norkotah. Most of the grafted plants subsequently developed ZC symptoms. Many of the grafted plants produced tubers with symptoms typical of the disease. In cultivar Atlantic, 104 of 138 grafted plants developed foliar symptoms of ZC and 72 of the grafted plants produced ZC-symptomatic daughter tubers. Grafted plants without foliar symptoms did not produce symptomatic tubers. Herein we report the results of these grafting experiments and describe the symptoms produced in grafted plants and daughter tubers. We have also maintained the disease by placing surface-sterilized symptomatic shoots onto growth medium in vitro. In addition, the putative ZC pathogen, a new Candidatus Liberibacter spp., has been detected in symptomatic grafted plants, symptomatic in vitro plants, and potato psyllids by PCR.ResumenEn los últimos años se ha descrito en el sudoeste de EE.UU. un desorden en papas para fritura que causa el oscurecimiento interno de los tubérculos crudos y un color completamente oscuro de las hojuelas. La decoloración se muestra a menudo como rayas o franjas y se ha usado el nombre común de “hojuelas zebra (ZC)” para describirla. Los síntomas foliares incluyen clorosis, enrojecimiento de la punta de los brotes y chamuscado de las hojas. A veces se producen tubérculos aéreos en las plantas enfermas. La enfermedad ha sido asociada con el psílido de la papa Bactericera cockerelli, pero recientemente se ha descrito un patógeno que se sospecha esté asociado con el ZC. En el esfuerzo de identificar el agente causal hemos mantenido la enfermedad en el invernadero injertando los retoños de plantas sintomáticas a plantas sanas de varios cultivares de papa comercialmente importantes, incluyendo Atlantic, Russet Burbank y Russet Norkotah. La mayoría de las plantas injertadas desarrollaron síntomas de ZC. Muchas de las plantas injertadas produjeron tubérculos con síntomas típicos de la enfermedad. En el cultivar Atlantic, 104 de 138 de plantas injertadas desarrollaron síntomas foliares de ZC y 72 de las plantas injertadas produjeron tubérculos hijos sintomáticos. Plantas injertadas sin síntomas foliares no produjeron tubérculos sintomáticos. Por lo tanto informamos los resultados de estos experimentos y describimos los síntomas producidos en las plantas injertadas y los tubérculos hijos. Hemos mantenido también la enfermedad poniendo brotes sintomáticos esterilizados superficialmente en medio de crecimiento in vitro. Además, el supuesto patógeno de ZC, un nuevo Candidatus Liberibacter spp., ha sido detectado por PCR en las plantas sintomáticas injertadas, plantas sintomáticas in vitro y los psílidos de papa.

A new haplotype of "Candidatus Liberibacter solanacearum" identified in the Mediterranean region

Abstract“Candidatus Liberibacter solanacearum”, a phloem-limited and Gram-negative bacterium that is spread from infected to healthy plants by psyllid insect vectors, is an economically important pathogen of solanaceous and carrot crops in the Americas, New Zealand and Europe. Three haplotypes of “Ca. L. solanacearum” have previously been described, two (LsoA and LsoB) in relation to solanaceous crops in the Americas and New Zealand and the third (LsoC) to carrots in Finland. Herein, we describe a fourth haplotype of this ‘Candidatus Liberibacter’ species (LsoD), also associated with carrots, but from Spain and the Canary Islands and vectored by the psyllid Bactericera trigonica. In addition, LsoC was confirmed in carrot and psyllid samples recently collected from Sweden and Norway. Phylogenetic analysis of the 16S rRNA gene suggests that two of the haplotypes, one in the Americas and the other in northern Europe are closer to each other in spite of a large geographic separation and host differences. Furthermore, during this study, potatoes with symptoms of zebra chip disease recently observed in potato crops in Idaho, Oregon and Washington states were analyzed for haplotype and were found to be positive for LsoA. This liberibacter haplotype was found in psyllids associated with the diseased potato crops as well. This finding contrasts with an earlier report of LsoB from psyllids in Washington which came from a laboratory colony originally collected in Texas.

Seasonal Occurrence and Abundance of the Potato Psyllid, Bactericera cockerelli, in South Central Washington

The potato psyllid, Bactericera cockerelli Sulc, has recently been identified as a vector of Candidatus Liberibacter solanacearum, the putative causal agent of zebra chip potato disease. Zebra chip is causing millions of dollars in losses to the potato industry in the United States, Mexico, Central America, and New Zealand. Currently, the most effective strategy to manage this potato disease is to target the potato psyllid with insecticides. Effective management of this insect pest requires knowledge of its biology, ecology, geographic distribution, and population dynamics. Although it is well documented that the potato psyllid is common throughout the western United States, several reports have indicated that this insect pest does not occur in Washington and Oregon. However, this insect has recently been observed and collected in this region. Studies were conducted from 2005 to 2008 to document and determine the seasonal occurrence of the potato psyllid in this important potato growing region of the United States. The potato psyllid was monitored in untreated experimental potato plots at Moxee and Prosser in south central Washington. Contrary to previous reports, the potato psyllid was found to occur in Washington and appears to migrate into the region late in the growing season. Upon arrival in south central Washington in late July, this insect readily reproduces in potatoes and appears to have at least one generation a year. The origin of potato psyllids migrating to Washington has not yet been determined. Information from this study will help potato growers in Washington manage the potato psyllid to better prevent potential zebra chip outbreaks.ResumenEl psílido de la papa, Bactericera cockerelli Sulc, se ha identificado recientemente como un vector de Candidatus Liberibacter solanacearum, el presunto agente causal de la enfermedad de la papa Zebra chip. Esta enfermedad esta causando millones de dólares en pérdidas en la industria de la papa en Estados Unidos, México, Centroamérica y Nueva Zelanda. Actualmente, la estrategia más efectiva para manejar esta enfermedad de la papa es enfocarse al psílido de la papa con insecticidas. El manejo efectivo de este insecto plaga requiere del conocimiento de su biología, ecología, distribución geográfica y dinámica poblacional. Aún cuando está bien documentado que el psílido de la papa es común a lo largo del oeste de los Estados Unidos, varios reportes indican que este insecto no se presenta en Washington y Oregon. No obstante, se le ha observado y colectado a este insecto en esta región. Se han hecho estudios de 2005 a 2008 para documentar y determinar la ocurrencia estacional del psílido de la papa en esta región tan importante de cultivo de papa en los Estados Unidos. El psílido de la papa se ha monitoreado en lotes experimentales de papa no tratados en Moxee y Prosser en la parte centro-sur de Washington. Contrario a reportes previos, se ha visto que el psílido de la papa se presenta en Washington y parece migrar al interior de la región al final del ciclo de cultivo. Al llegar al centro sur de Washington a finales de julio, el insecto se reproduce rápidamente en papa y parece tener por lo menos una generación al año. Aun no ha sido determinado el origen de los psílidos de papa que emigran a Washington. La información de este estudio ayudará a los productores de papa en Washington a manejar el psílido para una mejor prevención de establecimientos potenciales de zebra chip.

First Report of the Necrotic Strain of Potato virus Y (PVYN) on Potatoes in the Northwestern United States

More than 50 isolates of Potato virus Y (PVY) with characteristics of strains that cause tobacco veinal necrosis (PVYN) were obtained from potatoes (Solanum tuberosum L.) grown in the northwestern United States. These isolates are being characterized at the biological and molecular levels. Isolate RR1 was obtained from leaves of potato cv. Ranger Russet showing distinct mottling and leaf deformity, which is in contrast to the leaf-drop and necrosis usually observed with ordinary strains of PVY (PVYO) in this variety. Isolate AL1 was obtained from tubers of potato cv. Alturas showing distinct internal light brown rings and blotches. When RR1 and AL1 were transmitted to tobacco (Nicotiana tabacum L. cvs. Samsun NN and 423), they caused systemic veinal necrosis, including stem and petiole lesions typical of PVYN strains (2). Symptoms induced by RR1 and AL1 on tobacco appeared 9 to 11 days after inoculation, whereas some other isolates caused delayed veinal necrosis. All isolates that produced veinal necrosis on tobacco were detectable with PVY polyclonal antisera. Potato virus X was not detected by enzyme-linked immunosorbent assay in tobacco plants showing veinal necrosis. Some isolates, including AL1, failed to react in serological tests using PVYN-specific monoclonal antibodies obtained from three commercial sources. Other isolates, including RR1, were detectable with these monoclonal antibodies. Reverse transcription-polymerase chain reaction (RT-PCR) products obtained with primers specific for the coat protein (CP) open reading frame (ORF) were cloned and sequenced. AL1 possesses a CP more closely related to PVYO type isolates, which would account for its failure to react with PVYN monoclonal antibodies. In this regard, AL1 is similar to the PVYN-Wilga isolate (1). Other isolates that are detectable with the PVYN monoclonal antibodies possess a CP more consistent with N strains of the virus. Results of RT-PCR tests using primers derived from the P1 ORF sequence (3), and the restriction enzyme analysis and sequencing of the RT-PCR products, all suggest that AL1 and RR1 are related to European-type members of PVY tuber necrotic (NTN) or N strains. However, other isolates under investigation appear to be more closely related to previously reported North American NTN types (3). The symptomatology of these viruses on tobacco and potato, and the serological and molecular data clearly show that at least two distinct variants of PVYN have been found for the first time in a major potato production area of the United States, and pose a potential threat to the potato industry. References: (1) B. Blanco-Urgoiti et al. Eur. J. Plant Pathol. 104:811, 1998. (2) J. A. de Bokx and H. Huttinga. Potato virus Y. Descriptions of Plant Viruses. No. 242, CMI/AAB, Surrey, England, 1981. (3) R. P. Singh et al. Can J. Plant Pathol. 20:227, 1998.