AN ABSTRACT OF THE DISSERTATION OF

Anne B. Haigren for the degree of Doctor of Philosophy in Botany and Plant Pathology, presented on January 24, 2006. Title: Characterization, Epidemiology, and Ecology of a Virus Associated with Black Raspberry Decline.

Abstract .aprud: Redacted for privacy

R. Martin

The objective of this study was to characterize an unknown agent associated with decline in black raspberry (Rubus occidentalis) in Oregon.A virus was found consistently associated with decline symptoms of black raspberries and was named Black raspberry decline associated virus (BRDaV). Double stranded RNA extraction from BRDaV-infected black raspberry revealed the presence of two bands of approximately 8.5 and 7 kilobase pairs, which were cloned and sequenced. The complete nucleotide sequences of RNA 1 and RNA 2 are 7581 nt and 6364 nt, respectively, excludingthe 3' poly(A) tails.The genome structure was identical to that of Strawberry mottle virus (SMoV), with the putative polyproteins being less than 50% identical to that of SMoV and other related sequenced viruses. The final 189 amino acids of the RNA-dependent- RNA-polymerase (RdRp) reveal an unusual indel with homology to A1kB-like protein domains, suggesting a role in repair of alkylation damage. This is the first report of a virus outside the Flexiviridae and ampeloviruses of the Closteroviridae to contain these domains. An RT-PCR test was designed for the detection of BRDaV from Rubus tissue. BRDaV is vectored non-persistently by the large raspberry aphid Amphorophora agathonica, the green peach aphid Mvzus persicae, and likely nonspecifically by other aphid species.Phylogenetic analysis of conserved motifs of the RdRp, helicase, and protease regions indicate that BRDaV belongs to the Sadwavirus genus. To assess the rate of spread BRDaV, four newly planted fields of black raspberries (Rubusoccidentalis)in Oregon were studied for three years. In an effort to characterize the suspected complexity of synergistic interactions between BRDaV and other Rubus-infecting viruses, the prevalence of ten additional Rubus viruses was also monitored in the study fields.The timing of BRDaV infection as it relates to aphid populations and flights was also determined. Testing of nearby vegetation identified several symptomless Rubus hosts of BRDaV, as well as detection in multiple cultivars of black raspberry and several non-Rubus weeds.It was determined that BRDaV spreads rapidly with a low aphid threshold and consistently is associated with decline of black raspberries in Oregon. © Copyright by Anne B. Haigren January 24, 2006 All Rights Reserved Characterization, Epidemiology, and Ecology of a Virus Associated with Black Raspberry Decline

by Anne B. Haigren

A DISSERTATION

submitted to

Oregon State University

in partial fulfillment of the requirements for the degree of

Doctor of Philosophy

Presented January 24, 2006 Commencement June 2006 Doctor of Philosophy dissertation of Anne B. Haigren presented on January 24. 2006.

APPROVED: Redacted for privacy

or Professor, representing Botany and Plant Pathology

Redacted for privacy

Chair of the Depftment çBotany and Plant Pathology

Redacted for privacy

Dean of tl* fIiadji'ate School

I understand that my dissertation will becomepartof the permanent collection of Oregon State University libraries. My signature below authorizes release of my dissertation to any reader upon request. Redacted for privacy

Anne B. Hahen. Author ACKNOWLEDGEMENTS

I extend my utmost appreciation to my major professor, Dr. Bob Martin, for his patience, support, and enthusiasm, and for teaching me the values of perseverance and independent thinking.

I thank my committee members Dr. Valerian Dolja, Dr. Tom Wolpert, Dr. Jennifer Parke, and Dr. Kate Field for their ideas, feedback, and genuine interest in my project.

The Oregon black raspberry growers made this research possible by allowing me to collect data in their fields. The financial support of the Northwest Center for Small Fruits Research and the Oregon Raspberry and Blackberry Commission also made this work possible. Financial support was also provided by the USDA-ARS, an NSF GK-12 Rural Science Education Program Fellowship, and two graduate teaching assistantships.

I thank the members of the Martin lab for supporting me so strongly. Special thanks go to Dr. loannis Tzanetakis, for sharing daily struggles and insights, and for challenging me to think more rigorously about the scope of my project, and Dr. Jennifer Kraus, who has extended to me her scientific wisdom and shown me the value of really listening.

I extend a big thank you to the whole crew at the USDA ARS Horticultural Crops Research Lab for their daily support and humor, and to the BPP office staff for keeping my program running smoothly.

I thank those who have made it possible for me to get to where I am: my professors at Bucknell University who encouraged my scientific development, my professors at Oregon State University for keeping the spark of excitement alive, my parents Nancy and Don for encouraging me to pursue higher education and supporting the decisions I have made, the BPP grad students and especially my good friend Rachael for being my life raft in the sea of grad school, my dogs for being unconditionally happy to see me every day, and my husband and greatest asset Jason McKerr for keeping me looking forward. TABLE OF CONTENTS

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Chapter 1. INTRODUCTION AND LITERATURE REVIEW...... 1

Raspberry history...... 1

Black raspberry botany...... 1 Black raspberry production systems in Oregon...... 2 Black raspberry viruses...... 3 Major aphid-transmitted viruses...... 4 Minor aphid-transmitted viruses...... 7 Nematode-transmitted viruses...... 8 European nematode-transmitted viruses...... 8 North American nematode-transmitted viruses...... 9 Pollen-borne viruses...... 10 Major pollen-borne viruses...... 11 Minor pollen-borne viruses...... 12 Viruses with unknown vectors...... 13 Insects as vectors...... 17

Biology of Amphorop ho ra...... 19 Strategies forRubusvirus control...... 20 Sadwavirus review...... 22 Overview of Dissertation ...... 24 References...... 24

Chapter 2. IDENTIFICATION, CHARACTERIZATION, AND DETECTION OF A VIRUS ASSOCIATED WITH DECLINE SYMPTOMS IN BLACK RASPBERRY...... 31 ABSTRACT...... 31 INTRODUCTION...... 32 MATERIALS AND METHODS...... 33 Test plants and virus isolates...... 33 Mechanical transmissions...... 34 TABLE OF CONTENTS (Continued)

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Aphid transmissions . 34 Virus purification...... 35 Isolation of dsRNA...... 36 cDNA synthesis and cloning...... 37 RT-PCR detection...... 38 PCR and RACE...... 40 Sequencing and genome analysis...... 40 Coat protein expression...... 42 RESULTS...... 43 Hostrange...... 43 Aphid transmissions...... 44 Properties of virions...... 45 Protein purification...... 46 Cloning from dsRNA...... 48 Nucleotide sequence and phylogenetic analysis...... 50 Non-coding regions...... 50 RNA1...... 50 RNA2...... 58 DISCUSSION...... 61 ACKNOWLEDGEMENTS...... 65 REFERENCES...... 65 Chapter 3. EPIDEMIOLOGY AND ECOLOGY OF BLACK RASPBERRY DECLINE ASSOCIATED VIRUS...... 70 ABSTRACT...... 70 INTRODUCTION...... 70 MATERIALS AND METHODS...... 75 Fielddesign...... 75 TABLE OF CONTENTS (Continued)

Monitoring of newly planted fields . 75 Time of transmission study...... 76 Cultivation practices...... 78 Detection...... 78 Alternate host and cultivar sampling...... 84 Statistical analyses...... 86 Reference isolates ofRubusviruses...... 86 RESULTS ...... 87 BRDaV distribution in newly planted fields...... 87 Timing of BRDaV infection...... 94 Infection with otherRubusviruses...... 101 Statistical analyses...... 105 Alternate host and cultivar sampling...... 105 Testing of reference isolates...... 106 DISCUSSION...... 106 ACKNOWLEDGEMENTS...... 110 REFERENCES...... 110 Chapter 4: GENERAL DISCUSSION...... 114 REFERENCES...... 118 BIBLIOGRAPHY...... 119 LIST OF FIGURES

Figure 2.1 Symptomatic leaf affected with Black raspberry decline associated virus, showing chiorosis, mottling, and puckering...... 32

2.2 Transmission electron microscope micrograph of a virus-like particle purified from systemically infected black raspberry...... 45

2.3 Detection RT-PCR on virion fractions from sucrose gradients ...... 46

2.4 Anti-His probed western blot of expressed CPs protein...... 47

2.5 DsRNA extracted from plants infected with Black raspberry decline associated virus (BRDaV)...... 49

2.6 Proposed genome map of BRDaV RNA 1 and RNA 2...... 51

2.7 Neighbor-joining analysis of the helicase of BRDaV and related viruses, based on the alignment of amino acids 577 through 673...... 54

2.8 Neighbor-joining analysis of the protease of BRDaV and related viruses, based on the alignment of amino acids 1214 through 1257...... 55

2.9 Neighbor-joining analysis of the RdRp motifs between amino acids 1338 and 1817 of BRDaV and homologous regions within related viruses ...... 56

2.10 ClustalW alignment of RNA-dependent RNA polymerase (RdRp) region encompassing motifs 1-VIlI (Koonin, 1991) between BRDaV and SMoV. . .57

2.11 Alignment of a 33 aa region (aa 182-2 14) of the movement protein of BRDaV with other closely related viruses...... 59

2.12 Alignment of a 47 aa region (aa 734-780) of the large coat protein of BRDaV with other closely related viruses...... 60 LIST OF FIGURES (Continued)

Figure 3.1 a Leaves infected with Black raspberry decline associated virus that show symptoms of mottling, chlorosis, and puckering ...... 71

3.lb Dieback of fruiting canes of black raspberry...... 71

3.2 Nymphs of the large raspberry aphid Amphorophora agathonica, wingless form...... 72

3.3 Schematic of experimental field set-up...... 77

3.4 Sticky trap for monitoring aphids ...... 78

3 .5a Average aphids caught per trap (plotted on the10Y axis) vs. daily maximum temperature, represented as 14-day moving average (plotted on the 2° Y axis) for each collecting trip for Fields 1, 2, and 3 in 2003 .....88

3.5bEnd of the growing-season (2003) summary-diagrams of each ofthe3fields...... 89

3 .6a Average aphids caught per trap (plotted on the10Y axis) vs. daily maximum temperature, represented as 14-day moving average (plotted on the 2° Y axis) for each collecting trip for Fields 1, 2, and 3 in 2004 ...... 90

3.6b End of the growing-season (2004) summary-diagrams of each ofthe3fields ...... 91

3 .7a Average aphids caught per trap (plotted on the10Y axis) vs. daily maximum temperature, represented as 14-day moving average (plotted on the 2° Y axis) for each collecting trip for Fields 1, 2, and 3 in 2005. ...92

3.7b End of the growing-season (2005) summary-diagrams of each ofthe 3 fields...... 93 LIST OF FIGURES (Continued)

Figure 3.8a2003 average aphids caught per trap (plotted on the10Y axis) vs. daily maximum temperature, represented as 14-day moving average (plotted on the20 Y axis) for Field 4...... 95

3. 8b 2003 average aphids caught per trap (line graph, plotted on the10Y axis) vs. # of plants (out of 16) for each rotation testing positive for BRDaV (histogram, plotted on the 2° Y axis) for Field 4...... 96

3 .9a 2004 average aphids caught per trap (plotted on the10Y axis) vs. daily maximum temperature, represented as 14-day moving average (plotted on the 2° Y axis) for Field 4 ...... 97

3.9b 2004 average aphids caught per trap (line graph, plotted on the10Y axis) vs. # of plants (out of 16) for each rotation testing positive for BRDaV (histogram, plotted on the 2° Y axis) for Field 4...... 98

3.1 Oa 2005 average aphids caught per trap (plotted on the10Y axis) vs. daily maximum temperature, represented as 14-day moving average (plotted on the 2° Y axis) for Field 4 ...... 99

3.lOb 2005 average aphids caught per trap (line graph, plotted on thej0Y axis) vs. # of plants (out of 16) for each rotation testing positive for BRDaV (histogram, plotted on the 2° Y axis) for Field 4...... 100 LIST OF TABLES

Table ig 1.1 Mode of spread, distribution, and taxonomy of significant viruses found naturally infecting Rubus...... 16

2.1 Indicator plants used for mechanical transmission of Black raspberry decline associated virus (BRDaV)...... 35

2.2 Viruses referenced in sequence alignments, with taxonomic designation and accession numbers...... 42

2.3 Transmission of Black raspberry decline associated virus (BRDaV) by Amphorophora agathonica and Myzus persicae...... 44

2.4 Position on genome and size of proposed functional proteins of Black raspberry decline associated virus, isolate BRDaV- 1 ...... 52

3.laNumber of treatments applied to Fields 1, 2, and 3 during the growing seasons of 2003, 2004, and 2005 ...... 79

3.lbNumber of treatments applied to Field 4 during the growing seasons of 2003, 2004, and 2005...... 79

3.2 Rubus virus detection primers used for reverse-transcriptase polymerase chain reaction (RT-PCR)...... 81

3.3 Rubus viruses tested for by ELISA...... 83

3.4 Non-Rubus weeds tested for Black raspberry decline associated virus byRT-PCR...... 84

3.5 Testing of nine alternate black raspberry cultivars for 11 Rubus viruses ..... 85

3.6 Cumulative viruses acquired by the final sampling date in 2005 ...... 102 LIST OF TABLES (Continued)

Table 3.7 Viruses acquired in the 'Timing of Infection' experiment for each plant set rotated through Field 4 ...... 103 Characterization, Epidemiology, and Ecology of a Virus associated with Black Raspberry Decline

CHAPTER 1: INTRODUCTION AND LITERATURE REVIEW

Raspberry history The diverse genus Rubus, of family Rosaceae, contains hundreds of wild and cultivated species. Commonly known as "brambles" or "caneberries", the cultivated Rubus species include the polyploid blackberries (comprising many species of Rubus), and the diploid (2n=l4) red raspberry (R. idaeus L.) and black raspberry (R. occidentalis L.). Rubus occidentalis is indigenous to eastern North America and was first cultivated in the early19thcentury (Jennings, 1995). Domestication involved selection for such traits as stronger floricanes and larger fruits. Subsequent crossing of black and red raspberries produced the purple raspberry hybrid. Further breeding between European and American species of red raspberry improved upon flavor, disease resistance and growth habit. Eventually, degeneration of superior selections due to virus infection prompted breeders to begin selecting for resistance to the aphid vectors of these viruses.Major-genes controlling resistance to both the American and European aphid vectors were found in red raspberry. These genes contributed to the improvement of red raspberry, and led to a long period of relative crop stability (Jones et al., 2000).

Black raspberry botany Black raspberries, as well as other caneberries, have a perennial root system and biennial shoots. During the first year, shoots or primocanes remain vegetative and then after dormancy become reproductive (floricanes) the following year.The floricanes produce fruit-bearing laterals and then senesce at the end of the growing season. Thus, in any given year except the planting year, both primocanes and floricanes are present in a planting. Primocanes grow rapidly in length during their vegetative phase, but cease elongating during the reproductive phase. In the spring, primocanes grow rapidly from 2 the basal buds of the plant. As the new primocanes grow and arch over, their tips tend to form adventitious roots when in contact with soil, thus increasing the radius of the plant's domain. Internode length is dependent on variety, light exposure and vigor. Each node produces a compound leaf with 3 or 5 leaflets and a cluster of 1 to 4 axillary buds: a primary, a secondary, and one or more tertiary buds.Shortening days and lower temperatures trigger flower bud initiation. When terminal bud growth begins to slow in the fall, axillary buds begin to differentiate. Initiation of differentiation moves from the primary axillary buds at the tip of the primocane downwards. Initiation of the secondary buds follows the same pattern.The primary and secondary buds comprise the future lateral shoots and flower clusters. After the primocanes overwinter, the primary axillary buds begin to elongate, forming fruiting laterals (Crandall and Daubeny, 1990). Blackcaps are self-fertile but require insect pollination to increase the percentage of drupelet set (Crandall and Daubeny, 1990). Once fertilized, each ovary develops into a single fruit called a drupelet. Each drupelet contains one seed. Thus, the structure we call the "fruit" actually consists of many drupelets adhering together on a common receptacle, collectively composing an aggregate fruit. When harvested, blackcaps and other raspberries separate from the receptacle.

Black raspberry production systems in Oregon The cool summer and mild winter climate of western Oregon is ideally suited for the production of black raspberries.Consequently, the vast majority of national black raspberry production takes place in the northern region of Oregon's Willamette Valley, surrounding Portland.In 2005, an estimated 1000 acres were harvested, mostly destined for processing.The market for black raspberries tends to fluctuate, creating a constant challenge for growers.For instance, in 2002, processed berries sold for $0.40/lb compared to$2.25/lb in 2004 (Oregon Agricultural Statistics Service, 2005). In response to the current market value, growers constantly are getting into and out of fruit production. 3

Most of the black raspberry plants grown in Oregon originate either from self- propagated cuttings, or from nurseries in Washington. Nurseries coordinate acquisition of their stock material with virus certification programs.Plants originating in these programs are grown from virus-tested plants. Usually the first several cycles of propagation are in tissue culture, followed by propagation in an insect-free environment. Certified clean plants are then shipped out for planting in spring. Optimal production of black raspberries is a function of site selection, cultural practices, and cultivar. Under ideal conditions, yields of 4000 pounds (1814 kg) of fruit per acre (Funt et al., 2001) can be attained. Current black raspberry cultivars in production demonstrate very little diversity in fruit quality, size, and fruiting season (Weber, 2003). The cultivars 'Jewel' and 'Bristol' are reported to have superior berry size (Funt et al., 2001), but the major cultivar grown in the Pacific Northwest is 'Munger', selected for its superior flavor and amenability to mechanical harvest. Harvest occurs over the course of several weeks in early surmner. Some of the berries will be destined for use as a natural colorant in juice or ink. The vast majority, however, are earmarked for processing and the "individually quick frozen" market, or IQF, and are harvested via machine. Consequently, desired cultivar traits include firm fruit, fruit release from receptacle when ripe, and strong fruiting laterals.Although a healthy black raspberry planting could potentially last several decades if maintained properly, black raspberries are typically in production for three to four seasons before they succumb to disease and no longer remain profitable.

Black raspberry viruses Worldwide, over 30 viruses and virus-like diseases viruses have been reported to infectRubusspp. (Martin, 2002).Twenty-one of these have been reported in North America (Converse, 1991 and unpublished data).The diseases caused by viruses and phytoplasmas can all be transmitted via graffing or vegetative propagation, unless the pathogen is not distributed evenly in the propagation material. Until the last decade, 4 virus detection has been limited to grafting, mechanical inoculation, serological assays, or aphid transmission from test plants to various indicator plants.Recent advances in sequencing and molecular biology, however, as well as the acquisition and archiving of whole virus genomes, have enabled researchers to detect viruses, including symptomless viruses, using reverse transcription-polymerase chain reaction (RT-PCR). Consequently, viruses that were described previously based on symptomatology in indicator plants can now be confirmed with sequence data. A brief review of the more important viruses of Rubus spp. that are aphid-, nematode-, and pollen-borne will be given here, as well as those without a known vector. All viruses are summarized in Table 1.1.

Major aphid-transmitted viruses Raspberry Mosaic Disease Complex A complex of 2-4 viruses in Rubus causes raspberry mosaic disease (RMD), which has a poorly understood etiology.In North America, the large raspberry aphid Amphorophora agathonica (Hottes) transmits the two viruses involved: Black raspberry necrosis virus (BRNV) and Rubus yellow net virus (RYNV) (Stace-Smith, 1956).In Europe, where RMD is also known as raspberry veinbanding mosaic disease, Raspberry leaf spot virus (RLSV) (Cadman, 1952) and Raspberry leaf mottle virus (RLMV) (Cadman, 1951) consistently are associated with the disease (Jones, 1991), but are vectored, along with BRNV and RYNV, by A. idaei (Börner) (Cadman, 1951). All four viruses are vectored in a semi-persistent maimer.The vector loses the viruses after feeding on a non-infected plant for several hours to several days, or when it molts. The viruses neither multiply in the vector, nor are they transmitted congenitally to the progeny (Stace-Smith, 1 955a). Viruses associated with RMD can occur in all cultivated Rubus species in North America, but cause the most damage to black raspberry. New infections in black raspberry are characterized by necrotic tip dieback, leaf blistering, and mottling. Infected red raspberry may show mild leaf mottling and veinbanding, or it may remain symptomless (Stace-Smith, 1 955a).Cultivated and wild blackberries are tolerant, not showing symptoms of RMD. 5

Most red raspberry cultivars grafted with BRNV-infected material do not exhibit visible symptoms. However, Stace-Smith (1955a) found an exception in the cultivars 'Taylor', and 'Washington', which show chiorotic spots and mottling along leaf veins. Further studies found that in mixed infections with Raspberiy bushy dwarf virus (Murant, 1976) in the red raspberry cultivar 'Lloyd George', BRNV induces bushy dwarf disease (Jones, 1979). In the absence of genetic or serological tests, the four viruses of the RMD complex traditionally have been characterized and differentiated by the symptoms they cause on test plants in a protected environment.Graft transmission of BRNV to R. occidentalis and R. henryi produces the characteristic tip necrosis of RMD 16 to 20 days post-graft. After the shoot tip curls downward and dies, leaves beneath the tip wilt and become necrotic.Following this reaction, new leaves display chlorosis and mosaic pattern (Stace-Smith, 1955a).Shoot tip necrosis also occurs initially when black raspberry is infected with RLMV or RLSV (Converse, 1991). RLMV and RLSV can be distinguished based on symptom response in red raspberry indicators 'Malling Landmark' and 'Norfolk Giant', respectively (Jones, 1982). RYNV induces uneven growth of the two basal leaflets of the trifoliates when grafted onto black raspberry (Stace-Smith, l955b). However, as BRNV induces few symptoms in red raspberry, graft indexing onto red raspberry in addition to black raspberry is necessary to identify the cause of tip necrosis.Transfer of individual aphids from doubly infected plants to a series of healthy blackcap seedlings may separate two viruses, though since these viruses have similar vector transmission characteristics this can be laborious. As many raspberry plants undergoing diagnosis are infected naturally with several aphid-transmissible viruses, graft and aphid-transmission based diagnosis can be ineffective (Stace-Smith, 1956). Many attempts have been made to isolate and purify the recalcitrant BRNV. BRNV can be transmitted mechanically but with great difficulty by grinding infected leaves in alumina, diatomaceous product, and 2% nicotine solution, and rubbing leaves of Chenopodiuin quinoa (Jones and Murant 1972). In C. quinoa, BRNV induces small local 6 lesions, systemic chiorosis, and tip distortion beginning about a week after inoculation. Attempts to purify BRNV were thwarted by low virus yields (Jones and Murant, 1972). BRNV virions eventually were purified by Murant et al. (1976).The particles are isometric, rounded, not enveloped, and28nm in diameter.However, only minute amounts of virus were recovered, and antiserum production attempts were unsuccessful. Jones and Mitchell (1986) produced antiserum against BRNV in mixed infection with a coinfecting virus, but the supply has been exhausted. It is suspected that BRNV originated in red raspberry and subsequently the virus was distributed with symptomless plant material to most raspberry production areas in the world (Stace-Smith, 1955a). As black raspberry is often grown in close proximity to red raspberry, the virus likely was transmitted by aphids from the symptomless host. Jones et al. (1974) were the first to elucidate the small bacilliform particles associated with rubus yellow net disease, and later work confirmed this suggestion (Jones and Roberts, 1976). Subsequently, primers for the detection of RYNV by RT-PCR were developed based on conserved sequences of the genus Badnavirus.Amplicons were sequenced and RYNV specific primers developed (Jones et al., 2002). These primers can be used to detect RYNV in infected Rubus plants and in viruliferous A. idaei. RLMV and RLSV are uncharacterized in terms of virion morphology, but as of 1991 they had not been found in North America (Converse, 1991).Both viruses are widespread in raspberry in the United Kingdom. Neither virus is sap-transmissible, limiting characterization. It is difficult to assess the individual economic impact of either virus, as they likely often occur in mixed infections, but it is quite probable that latent infections with both RLMV and RLSV significantly decrease plant yield and growth (Jones, 1987). These viruses cause symptoms in many cultivars of red raspberry grown in the United Kingdom and likely are having a significant impact on production, since there are biotypes of A. idaei that overcome most of the aphid resistant genes used in the breeding program (Jones and McGavin, 1998). 7

Minor aphid-transmitted viruses Raspberry leaf curl virus (RpLCV) One of the first virus diseases of Rubus to be recognized in North America, raspberry leaf curl virus (RpLCV) istransmitted persistently by Aphis rubicola (Oestlund). This virus is found only in North America, predominantly in the northeastern U.S. and Rocky Mountain regions. The vector is not found in the Pacific Northwest, explaining the absence of RpLCV there. Blackcaps infected with RpLCV produce small, oval, upturned leaves and stunted, brittle, unbranching canes (Stace-Smith and Converse, I 987a).

Raspberry vein chiorosis virus (RVCV) RVCV, an unassigned species within the Rhabdoviridae, is transmitted by the small European raspberry aphid Aphis idaei (van der Goot), and likely is persistent within its vector. RVCV induces veinal chlorosis, epinasty, and leaf distortion. This disease is widespread in Europe and Russia and spread largely via infected plant material (Jones et al., 1987).

Cucumber mosaic virus (CMV) CMV has a worldwide distribution and vastly wide host range, but is rare and rather insignificant in Rubus. Its distribution in Rubus is limited to Scotland and Russia (Converse, 1991). CMV can be transmitted nonpersistently by many aphid species, including Amphorophora idaei (Börner), Myzus persicae (Sulz.), and Macrosiphum euphorbiae (Thos.) (Jones, 1976). CMV is the type species in genus Cucumovirus, family Bromoviridae.

Thimbleberry ringspot virus (ThRSV) Thimbleberry (R. parvflorus Nutt.) is the only natural host of ThRSV.This virus was investigated initially because wild Rubus hosts have the potential to serve as virus reservoirs. ThRSV can be transmitted experimentally to R. occidentalis, inducing a 8 diffuse chiorosis and tip necrosis.Four aphid species Illinoia maxima (Mason), I. davidsonii (Mason), Amphorophora parvflorii (Hill), and Myzus ornatus have been found colonizing thimbleberry in British Columbia (Forbes and Chan, 1989), and the first three can transmit ThRSV semipersistently, yet inefficiently (Converse, 1991).

Raspberry Leaf Spot Virus, North American virus (RLSV-NA) There is dsRNA evidence for a multi-partite RNA virus that is aphid transmitted in red raspberry in the Pacific Northwest (Jelkmann and Martin, 1988). This virus, which has no relation to the RLSV of the mosaic complex from Europe, remains uncharacterized in terms of its effects in the field and host range.

Nematode-transmitted viruses Nematodes belonging to two different families vector viruses of two different genera. All virus-transmitting nematodes are ectoparasitic, meaning they feed on the root epidermalcells. Several species of Xiphinema and Longidorus in the family Longidoridae vector the nepoviruses, and species of Paratrichodorus and Trichodorus in the family Trichodoridae vector the tobraviruses.The following compiled nematode- transmitted viruses will be categorized as either European or North American, in order to both highlight their current geographical importance, and the importance of potential quarantine regulations.Without tightly enforced quarantines and standards, human movement of planting stock, infected seeds, and weeds acting as reservoirs have the potential to move these viruses to new continents.

European nematode-transmitted viruses Arabis mosaic virus (AMV) and Strawberry latent ringspot virus (SLRSV) AMV and SLRSV are often described together, as they share a common vector and are often found in association with each other. Both are vectored by Xiphinema and can cause major yield losses in red raspberry crops. AMV and SLRSV have been found in North America, but in hosts other than Rubus. In red raspberry in the United Kingdom, AMV andSLRSVtogether cause a disease called raspberry yellow dwarf. Infected plants produce little or no fruit, and develop speckling, mottling, and yellowing on their leaves.In infected fields, circular expanding patches of weak plants are indicative of a nematode vectored virus infection (Converse, 1991). Although vectored by Xzphinema,SLRSVpresently is grouped in the unclassified genus Sadwavirus, rather than with the nepoviruses of the Comoviridae, based on genome structure and sequence information (Mayo, 2005).

Raspberiy ringspot virus (RpRSV) and Tomato black ring virus (TBRV) The Longidorus-vectored nepovirusesRpRSVandTBRVtogether cause a disease of red raspberry in Scotland known as raspberry leaf curl (not to be confused with the previously described yet completely unrelated North American Rubus virus disease of the same name!).RpRSVcauses chlorotic ring spots on infected red raspberries, and occasional down-curling and brittle leaves.RpRSVhas caused considerable economic losses in eastern Scotland.TBRV issomewhat less damaging by comparison, but some red raspberry cultivars develop chiorotic rings when infected withTBRValone, becoming progressively more dwarfed and producing crumbly fruit (Murant, 1987).

North American nematode-transmitted viruses Tomato ringspot virus (T0RSV) ToRSV,a nepovirus, is the most economically important of the nematode- transmitted viruses of Rubus in North America.ToRSVis transmitted by several species of Xihineina, but predominantly X americanum (Cobb). Symptoms vary with season, cultivar, and timing of infection, but may include yellow rings and vein chlorosis in the spring, followed by reduction of fruit yield and quality. Infections spread via viruliferous nematodes often form oval patches of diseased plants within a field.ToRSVis seedborne in Rubus, as well as in common weeds, thus germinating seeds can serve as new foci for infections (Stace-Smith and Converse, 1 987b). 10

Tobacco ringspot virus (TRSV) TRSV has a wide natural host range, causing ringspot disease on many herbaceous and woody, as well as annual and perennial crops. TRSV has been reported to cause damage in blackberry but has not been reported in red or black raspberry. TRSV has a less significant impact on Rubus production than the other nepoviruses.Like ToRSV, TRSV is vectored by X americanum, and is not established outside of the Western Hemisphere (Stace-Smith, 1987).

Cherry rasp leaf virus (CRLV) Like ToRSV and TRSV, CRLV is transmissible by the nematode Xiphinema americanum.CRLV also is transmitted through seed of several natural hosts and contributes to North American raspberry decline. CRLV previously was grouped among the nepoviruses, but recent phylogenetic analysis has assigned CRLV as the type member of the unassigned genus Cheravirus (Mayo, 2005). CRLV was first found in Rubus in a symptomless red raspberry imported into Scotland from Quebec (Jones et al., 1985). Previously, CRLV had only been found in western North America in weeds and cherry trees, where it is an economically important disease (Hansen et al., 1974).

Pollen-borne viruses There are two types of pollen transmission of plant viruses. The first results in seed transmission when a virus is transmitted to the embryo but not to the mother plant. This occurs with many of the nepoviruses mentioned above as well as with viruses in several other genera of plant viruses. The second type of pollen transmission occurs from the pollen to the mother plant as well as to the embryo. The transmission to seed (vertical through generations) and to mother plant (horizontal within a generation) can be at greatly different rates. The latter type of transmission occurs with members of the ilarvirus and idaeovirus genera, and some nepoviruses. The epidemiological importance of pollen-borne viruses originates from their potential to be transmitted horizontally, which is very important for vegetatively propagated plants, especially perennial plants 11

that may be exposed to infection for many years. Pollen may be carried by thrips for short distances, by bees for miles, by wind for many miles and by humans anywhere in the world. Survival in seed could be an adaptive mechanism for viruses that infect annual plants as hosts, or for those that have slow-moving or inefficient vectors as hosts.

Major pollen-borne viruses Raspberry bushy dwarf virus (RBDV) RBDV (Murant, 1976), genus Idaeovirus, spreads via pollen not only to the pollinated plant but also to the seeds. It is likely that bees serve as carriers of RBDV- infected pollen, inoculating the virus onto the plants as an inadvertent result of their foraging (Bulger et al., 1990).Fortunately, symptom expression varies considerably among cultivars (Daubeny et al., 1978), and control can be achieved by the planting of resistant cultivars, such as the red raspberry 'Willamette', 'Haida', 'Nootka', 'Chilcotin' or 'Preussen' (Stace-Smith, 1984, Jennings and Jones, 1989).However, this solution is of little solace to the growers of the susceptible cultivar 'Meeker', the most commonly grown red raspberry cultivar in the Pacific Northwest (Martin and Mathews, 2001).In such susceptible cultivars, RBDV causes reduced yield, reduced fruit size, and crumbly fruit. Crumbly fruit arises from the abortion and uneven development of drupelets. This condition causes fruit to fall apart or crumble when picked, resulting in economically unviable and unmarketable fruit.Leaves of affected plants of many cultivars are symptomless or may show chiorosis from very mild to bright yellow (Jones et al., 1996). RBDV does not, on its own, cause the "bushy dwarf' symptom that its name implies.Rather, its name was derived from the symptoms induced from a mixed infection with BRNV (Jones, 1979). Murant and Jones (1976) described a serologically distinct strain of RBDV in black raspberry that is distinguishable from the two known strains in red raspberry.It is not known whether this strain is restricted to black raspberry, or whether different strains can cross-pollinate between species.However, RBDV is found frequently in black raspberry fields in the Pacific Northwest, especially in combination with other viruses (personal observation). RBDV is an increasing 12 problem in both red and black raspberry production areas around the world, including North America, South Africa, Australasia, and Europe (Jones et al, 1996).

Tobacco streak virus (TSV) or Strawberry necrotic shock virus (SNSV) SNSV, genus Ilarvirus, has been characterized recently at the molecular level, and reclassification has been suggested. Previously, symptoms caused by this pollen-borne virus were attributed to Tobacco streak virus (TSV), first documented in the 1960s (Frazier et al., 1962). Converse (1972) recognized that a distinct strain of TSV, "TSV-R" infected 'Munger' black raspberry in Oregon.Once genetic information was available, Tzanetakis et al. (2004) demonstrated that the Rubus and Fragaria isolates of TSV were distinct serologically and genetically and proposed the name Strawberry necrotic shock for this group of isolates.Thus, up until 2004, all literature referred to this particular virus in Rubus as TSV or as its former name, black raspberry latent virus. Despite it's re-naming, SNSV is believed to share the same transmission properties as TSV.Sdoodee and Teakie (1987) demonstrated that Thrips tabaci (Lindeman) could transmit TSV, most likely by inoculating the plants with pollen borne virus in feeding wounds.This supports work by Converse (1980) in which he demonstrated that SNSV moves rapidly from infected 'Munger' plants to adjacent healthy plants, with an annual infection rate of 25% when plants were allowed to bloom. Moreover, he found that infection still occurred, though at a much-reduced rate when plants were deflowered.

Minor pollen-borne viruses Apple mosaic virus (ApMV) ApMV is an ilarvirus that was first reported in Rubus in 1975 (Converse and Casper, 1975). Infected Rubus generally are asymptomatic, producing normal fruit crop, though a bright yellow mosaic has been observed in some cultivars of red raspberry (Baumann et al., 1982).The virus can be spread by root grafting and mechanical inoculation, and is speculated to be transmitted via pollen (Converse, 1991). 13

Viruses with unknown vectors Blackberry calico (BCV) and Wineberry latent virus (WLV) BCV, genus Carlavirus, and WLV, presumed genus Potexvirus, are both described as being causal agents of blackberry calico disease. BCV is limited in range to North America. WLV was initially found in a symptomless wineberry (R. phoenicolasius Maxim.) plant in Scotland. WLV was speculated to be involved in blackberry calico disease after a graft-inoculated blackberry with a mixture of WLV and RBDV developed calico symptoms.Experimentally, WLV can be transmitted via grafting to black raspberry, but it is not known whether black raspberry is a natural host (Converse, 1991). Failed attempts by Jones et al. (1990) to detect WLV using anti-potexvirus antibodies led them to re-examine its classification as a potexvirus.Upon closer examination, its flexuous filamentous particle was found to measure a length quite close to that reported for BCV. Based on these findings, BCV and WLV may be one and the same.

Cherry leaf roll virus (CLRV) CLRV, genus Nepovirus, was first reported in wild blackberry in England, and subsequently in red raspberry in New Zealand, where diseased plants exhibit severe chlorotic ringspot and yellow vein netting. CLRV has not been reported in Rubus in North America, but it has a wide natural host range and does occur in other woody species in North America and Europe.Despite its classification among nepoviruses, CLRV appears not to be vectored by nematodes.It may be seed-borne, as it is in other natural hosts (Jones et al., 1985), or pollen-borne, as it is in walnut (Cooper et al., 1984).

Blackberry yellow vein associated virus (BYVaV) BYVaV was first characterized in 2004 (Martin et al.)Symptoms of leaf yellowing, mottling, and vein-banding were appearing in blackberry in the southern and southeastern United States beginning around 2001, prompting investigation. The 14 sequence of a virus associated with the symptoms designated it as a new member of the Closteroviridae family, genus Crinjvjrus. BYVaV has been found in red raspberry, but has not yet been found in any sampled black raspberry in Oregon (Halgren, unpublished data). No transmission studies have yet been performed, but it is likely to be vectored by whitefly, which is the only known vector of criniviruses.

'Toti'-like Virus The sequence of a virus showing homology to the fungi-infecting totiviruses was derived from a red raspberry (Tzanetakis and Halgren, unpublished data). This raspberry was obtained from the National Clonal Germplasm Repository (NCGR) in Corvallis, OR, and was described as having raspberry mosaic disease (RMD) based on tip necrosis symptoms produced on 'Munger' upon grafting. Based on the lack of characterization of RLMV and RLSV, and the often-inconsistent symptoms associated with plants containing various viruses of the RMD complex, the virus is being investigated further to determine if it might represent one of the three aphid-borne viruses that cause tip necrosis in 'Munger' (see RMD section above).

Raspberry mottle associated virus (R1'IaV) and Glen Clova bisect-like virus RMaV, genus Closterovirus, was first described in 2005 (Martin and Tzanetakis, 2005). During a routine graft index, an asymptomatic 'Glen Clova' raspberry accession from Scotland produced mottling, epinasty, and necrosis on indicator plants.Shotgun cloning and sequence analysis revealed the presence of a novel closterovirus. Based on this sequence, detection primers were developed which revealed the presence of RMaV in red raspberry from Washington, and multiple 'Munger' black raspberries among Oregon fields. A second virus, bearing resemblance to viruses of insects, was also extracted from the same 'Glen Clova' raspberry accession. Detection primers designed based on partial sequence of this virus enabled the detection of this virus in R. occidentalis (unpublished data). 15

Blackberry flexivirus This unnamed, unclassified virus originally was derived from symptomatic Arkansas blackberry, and gives closest sequence homology to members of the Foveavirus genus of the family Flex iviridae.It has been found in several Oregon black raspberry fields in association with other Rubus viruses (unpublished data). 16 Table 1.1: Mode of spread, distribution, and taxonomy of significant viruses found naturally infecting Rubus.

Virus Vector Genus Family Geographic Distribution in Rubus Black raspberry Aphid Sadwavirus Unassigned Pacific Northwest, decline (proposed) Ohio associatedvirus Black raspberry Aphid Unassigned Unassigned North America and necrosisvirus Europe Rubus yellow netAphid Badnavirus CaulimoviridaeNorth America and virus Europe Raspberry leaf Aphid Unassigned Unassigned Europe mottlevirus Raspberry leaf Aphid Unassigned Unassigned Europe spotvirus Raspberry leaf Aphid Unassigned Unassigned Northeastern US curl virus and Rocky Mountain area Raspberry vein Aphid Unassigned Rhabdoviridae Europe, Russia chlorosisvirus Raspberry leaf Aphid Unassigned Unassigned Pacific Northwest spot virus- North Americanvirus Cucumber Aphid CucumovirusBromoviridae Scotland, Russia mosaic virus Thimbleberry Aphid Unassigned Unassigned British Columbia ringspotvirus Arabis mosaic NematodeNepovirus Comoviridae Europe virus Strawberry NematodeSadwavirus Unassigned Europe latent ringspot virus Raspberry NematodeNepovirus Comoviridae Scotland ringspot virus Tomato black NematodeNepovirus Comoviridae Scotland ring virus Tomato ringspotNematodeNepovirus Comoviridae Western virus Hemisphere, mostly North America Tobacco NematodeNepovirus Comoviridae North America ringspot virus Cherry rasp leafNematodeCheravirus Unassigned Quebec virus 17

Table 1.1 (Continued)

Virus Vector Genus Family Geographic Prevalence in Ru/ms Raspberry bushy Pollen Idaeovirus Unassigned Worldwide dwarf virus Strawberry Pollen, ilarvirus Bromoviridae North America, Necrotic Shock thrip Australia

Apple mosaic Pollen ilarvirus Bromoviridae North America, virus Germany Blackberry calico Unknown Carlavirus orFlexiviridae North America virus Potexvirus Wineberry latent UnknownPotexvirus Flexiviridae Scotland

Cherry leaf roll Unknown,Nepovirus Comoviridae England, New virus possibly Zealand seed or pollen Blackberry yellowUnknown,Crinivirus Closteroviridae Southeastern U.S., vein associated possibly Pacific Northwest virus whitefly Toti-like virus Unknown Unassigned Unassigned Pacific Northwest Raspberry mottle Unknown,ClosterovirusClosteroviridaePacific Northwest associated virus possibly aphid 'Glen Clova' Unknown Unassigned Unassigned Pacific Northwest insect-like virus Blackberry Unknown Unassigned Unassigned; Arkansas, Pacific flexivirus Flexiviridae Northwest proposed

Insects as vectors Knowledge of insect vector-virus relationships is critical to the fundamental understanding of viral ecology and disease epidemiology. Without an understanding of thisinteraction,adequate disease controlisnotfeasible.Often,controlling the invertebrate vector is the primary and most feasible virus control strategy. 18

Plant viruses need a means by which to penetrate the impermeable plant cell wall. This issue could be overcome by avoiding the need for penetration altogether (i.e. pollen or seed transmission). More often, though, viruses have solved the issue of penetration by evolving specific relationships with invertebrate vectors. Among invertebrates and particularly among the insects, aphids have become the most important virus vector group. There is a spectrum of association types between virus and vector.All of the interactions take into account the acquisition phase, latent period, and retention (transmission) period (Hull, 2002).The acquisition phase is the minimum period in which the aphid needs to feed on infected material in order to transmit it.The latent period is the length of time after acquisition before a vector can transmit the virus to another host.This period does not apply to externally borne viruses.The retention period is the length of time after acquisition during which a vector is capable of transferring virus to a healthy host without reacquiring virus. Taking these three periods into account, there are four main modes of transmission utilized by vectors. Non-persistent transmission involves only a temporary, stylet-borne association of the virus particles with the vector.In this system, the virus may be acquired in seconds to several minutes, can be immediately inoculated into healthy hosts, and has a retention half-life in the vector of minutes.Non-persistently transmitted viruses include the potyviruses and cucumoviruses. A slightly more complex form of transmission is semi-persistent, or foregut-borne transmission, used by groups such as the caulimoviruses and closteroviruses. In this mode, the virus acquisition takes longer, but the virus may be retained for hours.In both non- and semi-persistent transmission the virus is associated with the exoskeletal structure of the vector via a specific interaction with either the capsid or other helper component. The last two types of association involve entry of virus particles into vector cells, which requires a latent period, after which the virus is acquired but before it can be transmitted. Also, the virus is retained for long periods. In persistent circulative transmission, particles move through the alimentary canal as the vector feeds, passing 19 into the hemolymph and through the salivary glands. The virus must pass the barriers of both the gut wall and the wall of the salivary glands. The luteoviruses are among the viruses best characterized for this mode.Lastly, there ispersistent propagative transmission in which viruses that are internally borne replicate in their vector.Often there is a delay of at least a week after acquisition before the vector can transmit the virus. This mode is most often associated with leathopper, planthopper, and thrip transmission, though members of the Rhabdoviridae replicate in their aphid vectors.

Biology of Amphorophora The genus Amphorophora (Buckton) contains about 27 species, 13 of which live on Rubus (Blackman and Eastop, 2000). They are rather large compared to most aphids, and vary in color from greenish to pale.Those species that colonize Rubus are often found clustered on succulent developing shoot tips, or solitary on leaf undersides (personal observation). The large raspberry aphid Amphorophora agathonica (Hottes) is the principal vector of BRNV and RYNV in North America, as demonstrated by Stace- Smith (1956).In Europe, the large European raspberry aphid A. idaei (Börner) has a similar biology and is the primary vector of viruses involved in RMD. Several other species of Amphorophora are able to transmit one or more viruses associated with RMD, though their importance as vectors is unknown (Converse et al., 1987). Raspberry aphids overwinter as eggs on Rubus. Eggs hatch in the spring, giving rise to nymphs that undergo several instars prior to molting to the adult form. During the summer, the aphid population becomes all females, which give birth to live young without sexual reproduction in a process called parthenocarpy.Most of these adult females remain wingless. However, increasing population density may prompt some to develop wings and colonize different plants.In this regard, virus spread can become exponential, as a burgeoning aphid population rapidly expands.When climate and precipitation are permissible, aphids can cycle through as many as 10 generations in a summer. In the intense heat of the summer, the aphid populations decline naturally. 20

Males and the winged forms are produced again with the cool weather of autumn, resulting in egg production by fertilized females (Blackman and Eastop, 2000).

Strategies for Rubus virus control Most procedures aimed at stopping the spread of Rubus viruses are directed toward reducing the inoculum source both within and outside the crop, limiting the spread of vectors, and minimizing the impact of infection on yield. The most effective virus control is preventative, and relies on certified virus-free planting stock, immune or tolerant cultivars, aphid resistant cultivars, control of nearby alternative hosts serving as reservoirs, and vector control. It is the goal of Rubus breeding programs to incorporate virus resistance along with desirable horticultural traits in their selections.Several single dominant genes, incorporated into red raspberry for resistance to A. agathonica in North America and A. idaei in Europe have been effective at maintaining a lower level of RMD virus complex infection in resistant cultivars.In Europe, the genes 'A1' and 'A10' have been used to confer resistance to two biotypes and all four known biotypes of A. idaei, respectively (Birch and Jones, 1988). However, a new biotype of the European aphid vector has evolved that overcame the resistance provided by both genes, prompting a closer look at the mechanisms at work behind the aphid resistance. Recent experiments involving A. idaei-resistant and -susceptible parent cultivars demonstrate that there may be more complex polygenic control involved, explaining some of the varying levels of resistance (Jones et al., 2000). Unfortunately, no such resistance genes are known to exist in black raspberry cultivars, suggesting that future breeding efforts may integrate red raspberry germplasm for resistance. Attempts to control local outbreaks of Rubus viruses would need to take into account the presence of infected but symptomless hosts adjacent to the diseased field. Isolation of a new Rubus field from either an established field or wild Rubus will likely increase the productive life of the field. 21

Lastly, control of virus disease must take into account the vector.Control of nematode-borne viruses involves preplant site preparation, combined with certified virus- free planting material.Soil fumigation is a short-term control strategy.Crop rotation with non-nematode hosts, control of weeds, and rouging of infected plants are good alternative strategies. As many nematode-vectored viruses can also be moved via seed, cover crops to reduce weed establishment should improve control of these viruses. The best strategy for pollen-borne virus control is the use of immune cultivars. However, the recent emergence of the resistance-breaking strain of RBDV has reduced the effectiveness of this approach in several European countries. Red raspberry with engineered resistance to RBDV is being developed and looks promising in field trials (Martin and Mathews, 2001). However, pending the release and the public acceptance of this contentious modified crop, other means of pollen-control strategies must be undertaken. Thus, the next best strategy is beehive placement. Beehives in the center of fields (containing certified material) will limit bee visits to potentially infected plants outside the field. Another strategy is to isolate new fields from established, potentially infectedRubusfields. Most of our current aphid control methods rely heavily upon the use of foliar systemic and contact aphicides. Although success stories exist, the ongoing problem is the development of resistance of aphids to aphicides. In several studies, aphicides were demonstrated to actually promote the spread of non-persistently transmitted viruses (Lowery and Boiteau, 1988, Roberts et al., 1993). Aphids briefly probe possible hosts, become deterred by the aphicide, and disperse in search of other plants. The spectrum of chemicals in aphicides approved for use inRubusis on the decline, due to the high registration price for this minor crop. Furthermore, environmental and health concerns regarding the toxicity and persistence of aphicides necessitate a shift in thinking regarding our prevailing methods of crop protection. Any program that utilizes chemical application needs to be based on the principles of integrated pest management. Such a program would integrate the establishment of action thresholds, vector sampling at various times in a season to help predict the likelihood of infection, and cultural controls. 22

Sadwavirus review The Sadwavirus genus was created in 2004, with Satsuma dwarf virus (SDV) as the type species (Mayo, 2005). This genus is in a current state of flux, but as more viral sequences are obtained, its status will be solidified.Currently unassigned to a family, sadwaviruses are also known as "SD V-like viruses". SDV originally was placed as a tentative nepovirus in the family Comoviridae, as this was the family with the closest genomic organization.Like other members of the Comoviridae, SDV is a bipartite, positive-stranded RNA virus. Its particles are isometric, approximately 26 imi in diameter, and its RNAs are encapsidated separately (Iwanami et al., 1999).Striking differences, however, separate SDV from the nepoviruses. SDV contains two coat proteins, whereas nepoviruses have a single coat protein. Furthermore, the RNA dependent RNA polymerase (RdRp) domain of SDV was found to be phylogenetically remote from those of the Comoviridae (Iwanami et al., 1999). A group of serologically-related SDV-like viruses has been described in Japan, encompassing Citrus mosaic virus (CiMV), Navel orange infectious mottling virus (NIMV), and Natsudaidai dwarf virus (NDV) (Gamsey and Koizumu, 1988). Previously, the only difference between CiMV and SDV was the size of one of the two coat proteins, when estimated by protein gel (Iwanami et al., 1993). Although not considered distinct species, sequence analysis suggests they should be considered distinct (Iwanami and leki, 1996; Iwanami et al., 1998; Iwanami et al., 2001). A new SDV-related virus, Hyuganatsu virus (HV) (Ito et al., 2004) shares serological properties with SDV strains, but is genetically unique enough to be a separate species. These SDV-related viruses cause a spectrum of symptoms on infected mandarin (Citrus unshiu), including green blotches, ring-shaped spots, enations, stunting, dwarfing, and reduction in leaf and shoot size (Usugi and Saito, 1979). Although a vector has not been identified, it is suspected that these virus strains are vectored slowly from tree to tree through soil (Iwanami et al., 1999). Strawberry mottle virus (SMoV) recently was designated as a sadwavirus (Mayo, 2005). SMoV is transmitted semi-persistently by the strawberry aphid Chaetoszphon 23 spp., and has isometric particles of approximately 30 nm diameter (Leone et al., 1995). Phylogenetically, SMoV shares its closest sequence similarity with SDV, though SMoV has a more nepovirus-like organization than SDV, and a long, conserved 3' noncoding region that is absent in SDV (Thompson et al., 2002).Recent analysis of the 3' noncoding region of multiple isolates gives insight into the high degree of variation of this virus (Thompson et al., 2003). Strawberry latent ringspot virus (SLRSV) (Murant, 1974), also once a tentative member of the nepoviruses, is now designated as a sadwavirus (Mayo, 2005). SLRSV is transmitted by the nematode Xiphinema diversicaudatum, has 30 nm isometric particles, and has a wide host range. SLRSV, like other members of the sadwaviruses, is bipartite and has two coat proteins (Kreiah et al., 1994). However, RNA 2 shows little sequence similarity with the RNA 2 molecules of other sadwaviruses, making it a likely candidate for genus reassignment. Other tentative species (Mayo, 2005) include Lucerne Australian symptomless virus (Remah et al., 1986) and Rubus Chinese seed-borne virus (Barbara, 1985), though no sequence information exists for these viruses yet. Sequence analysis of the coat proteins encoded by SDV, CiMV and NIMV revealed a region similar to that of animal picornaviruses (Karasev et al., 2001). The diverse super-family of picornaviruses includes pathogens isolated from a wide range of organisms, including plants, insects, and vertebrates. All viruses within this group share essential core characteristics, including a similar translational strategy involving a polyprotein precursor and a replicative gene block. Picorna-like viruses known to infect plants include the families Comoviridae, Sequiviridae, and Poiyviridae (Dolja and Carrington, 1992; Koonin and Doija, 1993) though based on phylogenetic analyses, the unassigned genera Sadwavirus and Cheravirus are also likely members (Thompson et al., 2004). Viruses within the Sequiviridae have monopartite genomes, and are divided into two genera based on insect vector and on the presence of a poly(A) tail.It has been suggestedthatinsect monopartite picorna-likevirusesinvaded plants,and the Sequiviridae are a remnant of this stage (Thompson et al., 2004). The majority of these picorna-like viruses then could have split into bipartite genomes multiple times, giving 24

rise to the multiple lineages of plant picorna-like viruses (Karasev et al., 2001). Based on the picorna-like properties that sadwaviruses share with the Sequiviridae, it is likely that the genus eventually will be moved to this family.

Overview of Dissertation The major objective of this thesis was to identify and characterize the biological properties, ecology, and epidemiology of an unknown virus associated with decline of black raspberries. Since some of the earliest descriptions of decline in black raspberries (Stace-Smith, 1955a), several attempts have been made to understand the agents responsible. We found a virus associated with symptoms in declining black raspberries, and named it Black Raspberry Decline associated Virus (BRDaV). BRDaV may or may not be one of the previously described viruses of the RMD complex. The dissertation is divided into four chapters, the first being the review of Rubus viruses presented here. Chapter two is devoted to biological, phenotypic, and molecular characterization of BRDaV. Chapter three describes the ecology and epidemiology of the virus, and chapter four summarizes how this research fits in a larger context, and detection and control strategies.

REFERENCES

Barbara, D.J., Ashby, S.C. and McNamara, D.G.1985.Host range, purification and some properties of rubus Chinese seed-borne virus. Ann. Appi. Biol. 107:45-55.

Baumann, G., Casper, R. and Converse, R.H. 1982. The occurrence of apple mosaic virus in red and black raspberry and in blackberry cultivars. Acta Hort. 129:13-20.

Birch, A.N.E. and Jones, A.T. 1988. Levels and components of resistance to Amphorophora idaei in raspberry cultivars containing different resistance genes. Ann. App!. Biol. 113:567-578.

Blackman, R.L. and Eastop, V.R. 2000.Aphids on the World's Food Crops-an identification and information guide.2ndedition. John Wiley and Sons, Ltd. Chichester, Pp. 220-222.

Bu!ger, M.A., Stace-Smith, R. and Martin, R.R. 1990. Transmission and field spread of 25 raspberry bushy dwarf virus. Plant Dis. 74:514-517.

Cadman, C. H. 1951. Studies inRubusvirus diseases. I. A latent virus of Norfolk Giant raspberry. Ann. Appi. Biol. 38:801-8 11.

Cadman, C.H.1952.Studies inRubusvirus diseases. V. Experiments in analysis of Lloyd George decline. Ann. App!. Bio!. 39:501-508.

Converse, R.H. 1972. Tobacco streak virus in black raspberry.Phytopathology 62:1001-1004.

Converse, R.H.1980.Transmission of Tobacco streak virus in Rubus. Acta Hortic. 95:53-62.

Converse, RH. 1991. Diseases Caused by Viruses and Viruslike Agents, Pp. 42-52, and Viral Diseases with Unknown Methods of Natural Spread, Pp. 52-5 8.In: Compendium of Raspberry and Blackberry Diseases and Insects.Ellis, M.A., Converse, R.H., Williams, R.N. and Williamson, B. (eds). American Phytopathological Society Press, St. Paul, Minnesota.

Converse, R.H. and Casper, R. 1975. A member of the apple mosaic virus group occurring in Rubus. Acta Hortic. 44:63-64.

Converse, R.H., Stace-Smith, R. and Jones, A.T. 1987. Raspberry Mosaic. Pp. 168-178. In: Virus Diseases of Small Fruits. R. H. Converse, ed. US. Dept. Agric. Agric Handb. 631.

Cooper, J.I., Massalski, P.R. and Edwards, M.L.1984.Cherry leaf roll virus in the female gametophyte and seed of birch and its relevance to vertical virus transmission. Ann. App!. Biol. 105:55-64.

Crandall, P.C. and Daubeny, H.A. 1990. Raspberry Management.In Galletta, G.J., and Himeirick, D.G. (eds). Small Fruit Crop Management. Prentice Hall Career and Technology, Englewood Cliffs, New Jersey.

Daubeny, H.A., Stace-Smith, R. and Freeman, J.A.1978. The occurrence and some effects of raspberry bushy dwarf virus in red raspberry.J. Am. Soc. Hortic. Sci. 103:519-522.

Dolja, V.V. and Carrington, J.C. 1992.Evolution of positive-strand RNA viruses. Semin. Virol., 3:315-326.

Forbes, A.R. and Chan, C.K. 1989. Aphids of British Columbia. Ag. Canada research Branch Technical Bulletin 1989-1E,p. 205. 26

Frazier, N.W., Jorgensen, P.S., Thomas, H.E. and Johnson, H.A., Jr.1962.Necrotic shock: a virus disease of strawberries. Plant Dis. Rep. 46:547-550.

Funt, R.C., Ellis, M.A., Williams, R.N., Doohan, D., Scheerens, J.C. and Welty, C. 2001. BramblesProduction, Management, and Marketing. Ohio State University Extension Bulletin 782. Media Distribution, The Ohio State University, 385 Kottman Hall, 2021 Coffey Road, Columbus OH 432 10-1044. http://ohioline.osu.edu/b782/index.html

Garnsey, S.M. and Koizumi, M. 2000. Satsuma dwarf virus group. In: Timmer, L. W., Garnsey, S.M. and Grahan, J.H. (eds).Compendium of citrus diseases,2nded. APS Press, The American Phytopathological Society, St. Paul, p 59.

Hansen, A.J., Nyland, G., McElroy, R.D. and Stace-Smith, R. 1974. Origin, cause, host range, and spread of cherry rasp leaf disease in North America. Phytopathology 64:72 1- 727.

Hull, R. 2002. Transmission 1: By Invertebrates, Nematodes, and Fungi. In: Matthews' Plant Virology. Academic Press, San Diego, Pp 485-532.

Ito, T., Iwanami, T., leki, H., Shimomura, K., Shimizu, S. and Ito, T. 2004. A new virus related to Satsuma dwarf virus:the nucleotide sequence of the 3 '-terminal regions of Hyuganatsu virus RNAs 1 and 2.Arch. Virol. 149:1459-1465.

Iwanami, T. and leki, H. 1996. Nucleotide sequence of the coat protein genes of citrus mosaic virus. Virus Res. 42:18 1-186.

Iwanami, T., Koizumi, M. and leki, H.1993. Diversity of properties among Satsuma dwarf virus and related viruses. Ann. Phytopathol. Soc. Jpn 59: 642-6560.

Iwanami, T., Kondo, Y. and Karasev, A.V. 1999. Nucleotide sequences and taxonomy of satsuma dwarf virus. J. Gen. Virol. 80:793-797.

Iwanami, T., Kondo, Y., Kobayashi, M., Han, S.S. and Karasev, A.V. 2001. Sequence diversity and interrelationships among isolates of Satsuma dwarf-related viruses. Arch. Virol. 146:807-813.

Iwanami, T., Kondo, Y., Makita, Y., Azeyanagi, C. and leki, H. 1998. The nucleotide sequence of the coat protein genes of satsuma dwarf virus and navel orange infectious mottling virus. Arch. Virol. 143:405-412.

Jelkmann, W. and Martin, R.R. 1988.Complementary DNA probes generated from double-stranded RNAs of a Rubus virus provide the potential for rapid in vitro detection. Acta Hortic. 236:103-109. 27

Jennings, D.L. 1995. Raspberries and blackberries: Rubus (Rosaceae). Pp. 429-434. In: J. Smartt and N.W. Simmonds (eds.), Evolution of crop plants. 2nd ed. Longman Scientific & Technical. Essex, England.

Jennings, D.L. and Jones, A.T. 1989. Further studies on the occurrence and inheritance of resistance in red raspberry to a resistance-breaking strain of raspberry bushy dwarf virus. Ann. Appi. Biol. 114:317-323.

Jones, A.T., 1976. An isolate of cucumber mosaic virus from Rubus phoenicolasius Maxim. Plant Pathol. 25:137-140.

Jones, A. T., 1979. The effects of black raspberry necrosis and raspberry bushy dwarf viruses in Lloyd George raspberry and their involvement in raspberry bushy dwarf disease. J. Hortic. Sci. 54:267-272.

Jones, A.T.1982.Distinctions between three aphid-borne latent viruses of raspberry. Acta Hortic. 129:41-48.

Jones, A.T. 1987.Raspberry leaf mottle and Raspberry leaf spot.Pp. 183-187.In: Virus Diseases of Small Fruits. R. H. Converse, ed. US. Dept. Agric. Agric Handb. 631.

Jones, A.T. 1991. Rubus host range of rubus yellow net virus and its involvement with other aphid-borne latent viruses in inducing raspberry veinbanding mosaic disease. Ann. App!. Biol. 118:331-338.

Jones, A.T. and McGavin, W.J. 1998.Infectibility and sensitivity of UK raspberry, blackberry and hybrid berry cultivars to Rubus viruses. Ann. Appl. Biol. 132:239-251.

Jones, A.T. and Mitchell, M.J.1986.Propagation of black raspberry necrosis virus (BRNV) in mixed culture with solanum nodiflorum mottle virus, and the production and use of BRNV antiserum. Ann. App!. Biol. 109:323-336.

Jones, A.T. and Murant, A.F. 1972. Some properties of a mechanically transmissible virus widespread in raspberry (Rubus idaeus) in Scotland. Plant Patho!. 21:166-170.

Jones, A.T. and Roberts, I.M.1976.Ultrastructural changes and small bacifliform particles associated with infection by rubus yellow net virus. Ann. Appi. Biol. 84:305- 310.

Jones, A.T., Mayo, M.A. and Henderson, S.J. 1985. Biological and biochemical properties of an isolate of cherry rasp leaf virus from red raspberry. Ann. Appi. Biol. 106:101-110. 28

Jones, A.T., Mayo, M.A. and Murant, A.F.1996. Raspberry Bushy Dwarf Idaeovirus. The Plant Viruses, Volume 5:Polyhedral Virions and Bipartite RNA Genomes. B. D. Harrison and A. F. Murant, eds. Plenum Press, New York. Pp. 283-301.

Jones, A.T., McGavin, W.J. and Birch, A.N.E. 2000.Effectiveness of resistance genes to the large raspberry aphid, Amphorophora idaei Bömer, in different raspberry (Rubus idaeus L.) genotypes and under different environmental conditions.Ann. Appl. Biol. 136:107-113.

Jones, A.T., McGavin, W.J., Geering, A.D.W and Lockhart, B.E.L. 2002. Identification of Rubus yellow net virus as a distinct badnavirus and its detection by PCR in Rubus species and in aphids. Ann. Appl. Biol. 141:1-10.

Jones, A.T., Mitchell, M.J., McGavin, W.J. and Roberts, I.M.1990. Further properties of wineberry latent virus and evidence for its possible involvement in calico disease. Ann. AppI. Biol. 117: 571-581.

Jones, A.T., Murant, A.F. and Stace-Smith, R.1987. Raspberry Vein Chiorosis.Pp. 194-197.In:Virus Diseases of Small Fruits.R. H. Converse, ed. US. Dept. Agric. AgricHandb. 631.

Jones, A.T., Roberts, I.M. and Murant, A.F. 1974.Association of different kinds of bacilliform particle with vein chlorosis and mosaic diseases of raspberry (Rubus idaeus). Ann. App. Biol. 77:283-288.

Karasev, A.V., Han, S.S. and Iwanami, T.2001.Satsuma Dwarf and related viruses belong to a new lineage of plant Picorna-like viruses. Virus Genes 23:1, 45-52.

Koonin, E.V. and Dolja, V.V. 1993. Evolution and taxonomy of positive-strand RNA viruses: implications of comparative analysis of amino acid sequences. Critical Reviews in Biochemistry and Molecular Biology 28:375-43 0.

Kreiah, S., Strunk, G. and Cooper, J.I.1994. Sequence analysis and location of capsid proteins within RNA2 of strawberry latent ringspot virus. J. Gen. Virol. 75:2527-2532.

Leone, G., Lindner, J.L. and Schoen, C.D. 1995. Unstable infectivity and abundant viral RNAs associated with strawberry mottle virus. Acta Hortic. 3 85:76-85.

Lowery, D.T. and Boiteau, G. 1988. Effects of five insecticides on the probing, walking, and settling behavior of the green each aphid and the buckthorn aphid (Homoptera: Aphididae) on potato. J. Econ. Entomol. 81:208-214.

Martin, R.R.2002.Virus Diseases of Rubus and Strategies for Their Control. Acta Hortic. 585:265-270 29

Martin, R.R. and Mathews, H. 2001. Engineering resistance to Raspberry bushy dwarf virus. ActaHortic. 551:33-37.

Martin, R. and Tzanetakis, I.2005.Characterization of three novel viruses infecting raspberry. Phytopathology 95:S66.

Martin, R.R., Tzanetakis, I.E., Gergerich, R., Fernandez, G., and Pesic, Z. 2004. Blackberry yellow vein associated virus: a new crinivirus found in blackbeny. Acta Hortic. 656:137-142.

Mayo, M.A. 2005. Changes to virus taxonomy 2004. Arch Virol. 150:189-198.

Murant, A.F.1974. Strawberry latent ringspot virus. CMIIAAB Descriptions of Plant Viruses, no. 126.

Murant, A.F., 1976.Raspberry bushy dwarf virus. CMTIAAB Descriptions of Plant Viruses, No. 165.

Murant, A.F.1987. Raspberry Ringspot and Associated diseases ofRubuscaused by Raspberry ringspot and Tomato black ring viruses. Pp. 211-220. In: Virus Diseases of Small Fruits. R. H. Converse, ed. US. Dept. Agric. Agric. Handb. 631.

Murant, A.F. and Jones, A.T. 1976. Comparison of isolates of raspberry bushy dwarf virus from red and black raspberries. Acta Hortic. 66:47-52.

Murant, A.F., Jones, A.T. and Roberts, I.M. 1976.Recent research on 52V virus of raspberry. Acta Hortic. 66:39-46.

Oregon Agricultural Statistics Service, January 27, 2005 Press Release. http://www.nass.usda.gov/or/fruit/berrieso 105 .pdf

Remah, A., Jones, A.T. and Mitchell, M.J. 1986. Purification and properties of lucerne Australian symptomless virus, a new virus infecting lucerne in Australia.Ann. Appl. Biol. 109:307-315.

Roberts, J.M.F., Hodgson, C.J., Jackai, L.E.N., Thottappilly, G. and Singh, S.R.1993. Interaction between two synthetic pyrethroids and the spread of two non-persistent viruses in cowpea. Ann. Appl. Biol. 122:57-67.

Sdoodee, R. and Teakle, D.S.1987. Transmission of tobacco streak virus by Thrips tabaci:A new method of plant virus transmission. Plant Pathol. 36:377.

Stace-Smith, R. 1955a. Studies on Rubus virus diseases in British Columbia. II. Black raspberry necrosis. Can. J. Bot. 33:314-322. 30

Stace-Smith, R. 1955b. Studies on Rubus virus diseases in British Columbia. I. Rubus yellow-net. Can. J. Bot. 33:267-274.

Stace-Smith, R. 1956.Studies on Rubus virus disease in British Columbia.III. Separation of components of raspberry mosaic. Can. J. Bot. 34:435-442.

Stace-Smith, R.1984.Red raspberry virus diseases in North America.Plant Dis. 68:274-279.

Stace-Smith, R.1987.Tobacco Ringspot Virus inRubus.Pp. 227-228.In:Virus Diseases of Small Fruits. R. H. Converse, ed. US. Dept. Agric. Agric Handb. 631.

Stace-Smith, R, and Converse, R.H. 1987a. Rubus Leaf Curl. Pp.194-197. In:Virus Diseases of Small Fruits. R. H. Converse, ed. US. Dept. Agric. Agric Handb. 631.

Stace-Smith, R. and Converse, R.H. l987b. Tomato ringspot virus inRubus.Pp. 223- 227 In: Virus Diseases of Small Fruits.R. H. Converse, ed. US. Dept. Agric. Agric. Handb. 631.

Thompson, J.R. and Jelkmann, W. 2003. The detection and variation of Strawberry mottle virus. Plant Dis. 87:385-390.

Thompson, J.R., Perry, K.L. and De Jong, W. 2004. A new potato virus in a new lineage of picorna-like viruses. Arch. Virol. 149:2141-2154.

Thompson, R.R., Leone, G., Lindner, J.L, Jelkmann, W. and Schoen, C.D.2002. Characterization and complete nucleotide sequence of Strawberry mottle virus: a tentative member of a new family of bipartite plant picorna-like viruses.J. Gen. Virol. 83:229-239.

Tzanetakis, I.E., Mackey, I.C. and Martin, R.R. 2004. Strawberry necrotic shock virus: a new virus previously thought to be tobacco streak virus. Acta Hortic. 656: 27-31.

Usugi, T. and Saito, Y.1979. Satsuma dwarf virus. CMLAAB Descriptions of Plant Viruses, no. 208.

Weber, C.A.2003.Genetic Diversity in Black Raspberry(Rubus occidentalis L.) Detected by RAPD Markers. HortScience 38(2):269-272. 31

CHAPTER 2: IDENTIFICATION, CHARACTERIZATION, AND DETECTION OF A VIRUS ASSOCIATED WITH DECLINE SYMPTOMS IN BLACK RASPBERRY

*The nucleotide sequence data reported in this chapter have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers DQ344639 and DQ344640 for RNA 1 and 2, respectively

ABSTRACT

The objective of this study was to characterize an unknown agent associated with decline in black raspberry (Rubus occidentalis) in Oregon. Using standard methods a virus was found consistently associated with decline symptoms of black raspberries and was named Black raspberry decline associated virus (BRDaV). Grafting and aphid transmissions do not induce the characteristic tip necrosis of Black raspberry necrosis virus (BRNV), a previously described but poorly characterized virus of black raspberry. Double stranded RNA extraction from BRDaV-infected black raspberry revealed the presence of two bands of approximately 8.5 and 7 kilobase pairs, which were cloned and sequenced. The complete nucleotide sequences of RNA 1and RNA 2 are 7581 nt and 6364 nt, respectively, excluding the 3' poly(A) tails. The genome structure was identical to that of Strawberry mottle virus (SMoV), with the putative polyproteins being less than 50% identical to that of SMoV and other related sequenced viruses. The final 189 amino acids of the RNA-dependent-RNA-polymerase (RdRp) reveal an unusual indel with homology to AIkB-like protein domains, suggesting a role in repair of alkylation damage. These domains, though widespread among eukaryotes and bacteria, are found in plant viruses mainly within a subgroup of the Flexiviridae and this is the first report of a virus outside the Flexiviridae and ampeloviruses of the Closteroviridae to contain these domains. An RT-PCR test was designed for the detection of BRDaV from Rubus tissue.BRDaV is vectored non-persistently by the large raspberry aphid Amphorophora agathonica and by 32

the green peach aphid Myzus persicae, and likely nonspecifically by other aphid species. Phylogenetic analysis of conserved motifs of the RdRp, helicase, and protease regions indicate that BRDaV belongs to the Sadwavirus genus.

INTRODUCTION

Black raspberry decline (BRD) is a disease of major concern to black raspberry (Rubus occidentalis L.) growers in the United States. In Oregon, where nearly 100% of the black raspberry production in the USA occurs (Oregon Agricultural Statistics Service, 2004), growers in recent years have observed a decline in production. Black raspberries, or "blackcaps" affected with the disease initially display leaf chlorosis and puckering (Fig. 2.1).Eventually, the fruiting canes of this biennial plant prematurely die back, resulting in a rapid and severe reduction in yield. Although a healthy black raspberry planting could last several decades if maintained properly, those affected with BRD are typically in production for three to four growing seasons before they succumb to disease and no longer remain profitable. A single cultivar, 'Munger', is grown predominantly in Oregon.Several other black raspberry cultivars in production demonstrate very little genetic diversity (Weber, 2003), and also succumb to BRD (see chapter 3).

Fig. 2.1: Symptomatic leaf affected with Black raspberry decline associated virus, showing chlorosis, mottling, and puckering. 33

The BRD syndrome may be caused by a component(s) of raspberry mosaic disease (Stace-Smith, 1956), which consists of Rubus yellow net virus (RYNV) (Stace- Smith, 1 955a), Black raspberry necrosis virus (BRNV) (Stace-Smith, 1 955b), Raspberry leaf mottle (Cadman, 1951) or Raspberry leaf spot (Cadman, 1952) viruses, or by a previously unidentified virus(es) infecting Rubus.The etiology of raspberry mosaic disease is complex and poorly understood. All identified members of the complex can be detected by petiole leaflet grafting into different cultivars of Rubus species (Jones and Jennings, 1980).It was speculated (Martin, personal observation) that BRD may be caused by BRNV, but the characteristic tip necrosis of black raspberries infected with BRNV did not develop when grafted onto indicators. Interestingly, most red raspberry cultivars are tolerant of BRNV, thus detection requires indexing on suitable indicator plants (Stace-Smith, 1955b). Early attempts to purify BRNV were thwarted by low virus yields (Jones and Murant, 1972, Murant et al.,1976).Jones and Mitchell (1986) produced antiserum against BRNV in mixed infection with a coinfecting virus, Solanum nodiflorum mottle (SNMV), but the supply has been exhausted. This report identifies a virus consistently associated with declining black raspberries, which was named Black raspberry decline associated virus (BRDaV). Despite the absence of necrotic tip dieback, BRDaV shares many biological attributes with BRNV. The biological and molecular properties of BRDaV are described and data suggest BRDaV is a new member of the genus Sadwavirus.

MATERIALS AND METHODS

Test plants and virus isolates A collection of BRDaV isolates was maintained in nursery-grown Rubus occidentalis plants. The isolates were obtained by aphid transmissions using Amphorophora agathonica (Hottes) collected from an infected field near Sandy, Oregon. One isolate, BRDaV- 1, was chosen as the reference isolate for obtaining the nucleotide sequence of BRDaV. BRDaV-1 also was used as a source for aphid transmissionstudies. Healthy plants for rearing aphids, tested previously for all major Rubus viruses, were 34 derived from cuttings of virus-free nursery grown 'Munger', and were tested frequently to ensure absence of virus.Healthy and infected plants were kept in separate greenhouses. Plants ('Munger') for aphid transmission studies were grown from seed of healthy tissue culture-derived plants.Black raspberry plants were maintained in a greenhouse between 20-26°C with supplemental lighting to maintain at least 14 hours daylight.

Mechanical transmissions BRDaV- 1 was used as inoculum for mechanical transmission to 20 herbaceous hosts of seven different families, including a transgenic tobacco line containing a viral RNA silencing suppressor (Carrington et al., 1990) (Table 2.1). At least two sets of 16 plants of each species were inoculated.Inoculum was prepared by grinding infected black raspberry leaf tissue at about a 1:50 dilution in PBS, pH 7.4, containing 2% nicotine.Test plants were dusted with Carborundum powder (600 mesh) prior to inoculations.Inoculum was rubbed lightly onto recipient leaves with a foam pad and rinsed off with tap water after an hour.

Aphid transmissions Clonal colonies of aviruliferous aphids were maintained in aphid cages in a separate greenhouse under aforementioned light and temperature settings. Colonies of A. agathonica and Myzus persicae (Suizer) were established by transferring nymphs as they were born to healthy virus-free black raspberry and Chenopodium quinoa (Wilid.), respectively. In order to determine aphid acquisition and transmission times, aphids were fed on detached 'Munger' leaves from plants infected with virus isolate BRDaV-1 for 20 sec-i mm, 1h, 5 h or 24 h.After specified feeding times, 10 aphids were transferred with a fine bristle paintbrush to each of 5 plants to feed for 20 sec- 1 mm, 1 h, 5 h, 24 h, or 7 days for inoculation access. All plants were at the same age and stage of growth. Plants were evaluated for virus infection via reverse-transcriptase polymerase chain reaction (RT-PCR) at 5 weeks post-inoculation. 35

Table 2.1: Indicator plants used for mechanical transmission of Black raspberry decline associated virus (BRDaV).

Plant name Family Antirrhinum majus Scrophulariaceae Beta vulgaris var. cicla* Chenopodiaceae Brassica junceaica Brassicaceae Capsicum annum Solanaceae Chenopodium amaranticolor Chenopodiaceae Chenopodium quinoa Chenopodiaceae Cucumis sativus var. sativus Cucurbitaceae Datura stramonium Solanaceae Glycine max Fabaceae Lactuca sativa Asteraceae Nicotiana benthaniana* Solanaceae Nicotiana clevelandii Solanaceae Nicotiana occidentalis* Solanaceae Nicotjana rustica* Solanaceae Nicotiana tabacum* So!anaceae Nicotiana tabacum-HC Pro mutant* Solanaceae Phaseolus vulgaris Fabaceae Pisum sativum Fabaceae Spinacia oleracea Chenopodiaceae Vigna unguiculata subsp. Unguiculata* Fabaceae______*Indicates species tested positive for BRDaV

Virus purification Virus was purified from infected 'Munger' leaf tissue using a modified protocol of Murant et al. (1976) used for BRNV. Extraction buffer contained 0.05 M citrate buffer, pH 6, with 1% Triton X-100 and 0.2% thioglycolic acid.Tissue was homogenized in a blender with the buffer, using 2 rnL buffer/g tissue. Sap was expressed through cheesecloth and stirred for 2 hours at 4°C, then centrifuged at 10,000 g for 20 mm. The supernatant was collected, polyethylene glycol (MW 8000) (8% w/v) and NaC1 to 0.1 M were added, and the mixture was stirred at 4°C for 2 hours.The precipitated virus was collected by centrifugation for 20 mmat 10,000 g, and the pellet resuspended in 20 mL of 0.05 M citrate buffer, pH 6.0,containing 0.1% 13- mercaptoethanol. The sample was then subjected to differential centrifugation at 10,000 36 g for 20 mm followed by 110,000 g for 2.5 h in a Beckman 50.2 Ti rotor topellet virus. The pellet was resuspended in citrate buffer (0.05 M) and subjected to sucrose density gradient centrifugation in a SW 40 Ti rotor at 180,000 g for 90 mm. One mL fractions were collected from the gradient and diluted, and finally the virus was pelleted at252,000 g for 60 mm in a Beckman Type 80 Ti rotor. The pellets wereresuspended in 200 tL citrate buffer (0.05 M). Virus was also purified from Nicotiana benthamiana L. and N. occidentalis (Wheeler) adapting a protocol for Tomato ringspot virus (T0RSV) (Stace- Smith, 1984). This protocol was similar to that described above, with two exceptions: the extraction buffer contained 0.03 M dibasic sodium phosphate, pH 9.0, with 3.52 gIL ascorbic acid and 1.56 mLIL 3-mercaptoethano1, and prior to the second centrifugation step, the pH was adjusted to 5.0 with HC1. Each fraction was tested by RT-PCR for the presence of BRDaV. The RT-PCR procedure was performed as describedbelow, except that 1tL of the virus preparation served as template for the RT reaction, and the virus, water and primer components were all incubated at 70°C prior to combination with remaining components of the first-strand eDNA synthesis reaction.Fractions testing positive were sent to the electron microscope facility for analysis, where they were stained with 2% ammonium molybdate and viewed by a Philips CM- 12 scanning transmission electron microscope.

Isolation of dsRNA Fresh and frozen black raspberry tissue collected in the spring and fall from a severely infected field originally was used as source material for dsRNA. Total and dsRNA from BRDaV- 1 was later used to confirm the entire sequence as a single isolate. For dsRNA isolation a modified Yoshikawa and Converse (1990) method was used. Briefly, 20 g of leaf tissue was homogenized in liquid nitrogen with a mortar and pestle. The resulting powder was mixed with 4 volumes (80 mL) of 2x STE (100 mM Tris Base, 0.2 M NaCI, and 2mM EDTA) pH 7.0, 1 mL of 3-mercaptoethanol, 0.5 volumes of 10% SDS (10 mL) and 1.5 volumes STE-saturated phenol (30 mL). Afler shaking for I h at room temperature, the mixture was centrifuged for 20 mm at 10,000 g at 4°C in aSorvall 37

SLC 1500 rotor. The aqueous phase was collected and adjusted to 16.5% EtOH, 2.5 g coarse textured, CF- 11 fibrous cellulose (Whatman) was added, and the mixture was shaken at room temperature for 20 minutes. The mixture was washed by centrifuging at 10,000 g for 10 minutes in a Sorvall SLC 1500 rotor at 4°C, the supernatant was decanted and the pellet resuspended in 16.5% EtOH in STE. The washing step was repeated, and the resulting slurry was poured into a 2.5-cm diameter Bio-Rad chromatographic column with a fitted glass filter, where the liquid was allowed to drain through by gravity. The cellulose pellet was washed with an additional 80 ml of STE-ethanol. Bound nucleic acid was eluted off the column using 25 mL of STE. Single-stranded RNA was digested with 1 pL/mL of Ti RNase (Sigma) at 37°C for 30 minutes, followed by addition of 1/60 volume 2 MMgC12and DNase (0.5 Kunitz units/j.tL) (Sigma). After a further 30 mm incubation, 0.5 M EDTA (pH 8.0) was added. Addition of 0.3 g coarse textured cellulose followed and the mixture was adjusted to 20% EtOH.The mixture was shaken 30 minutes at room temperature, poured into a 0.5 cm diameter chromatographic column, and washed with 50 mL 18% EtOH in STE. The resulting bound dsRNA was eluted with 3.5 mL STE, precipitated in 1/10 vol of 3 M, pH 5.2 sodium acetate and 2 volumes of 100% EtOH, aliquoted into microfuge tubes, and stored at -80°C.For analysis, a microfuge tube was centrifuged for 45 mmat 16,000 g, and after decanting, the pellet was dried, resuspended in loading buffer (diluted 6x Bromophenol blue/Xylene cyanol FF), loaded onto either 0.8% agarose or 12% polyacrylamide gels, separated by gel electrophoresis, stained with ethidium bromide, and visualized under UV light. For use in cloning, dsRNA from several microfuge tubes was washed twice in cold 70% EtOH, dried under vacuum, resuspended in RNasefree H20 and combined. eDNA synthesis and clonin Synthesis of eDNA was performed using dsRNA as a template.DsRNA combined with 0.3 jig of random hexanucleotides (Invitrogen), was denatured in the presence of 20 mM methylmercury (II) hydroxide, as described (Jelkmann et al., 1989). The reverse transcriptase mixture was added to the denatured dsRNA to create a total volume of 50 tL, which contained 10 tL of 5X cDNA synthesis buffer (250 mM Tris acetate (pH 8.4 at 25°C), 375 mM potassium acetate, 40 mM magnesium acetate), 5 tL of 10 mM dNTPs, 5 tL of 100 mM DTT, 1 jiL (40U) of RNase OUT (Invitrogen), 1 jL (15U) of Thermoscript (Invitrogen), and 19.5 tL H20. The mixture was incubated for 5 mm at room temperature, then 1 h at 60°C. First-strand cDNA was precipitated with 1/10 volume of 3 M NaAcetate and 2.5 volumes (v/v) 100% EtOH. The precipitated and dried cDNA pellet was redissolved in 100 L of second-strand buffer, containing 82 jtL of

H20, 10 j.iL oflOx E. coilligase buffer (300 mM Tris-HC1, 40 mM MgC12, 10 mM DTT,

260 iM NAD, and 500 tg/ml BSA, pH 8.0 @ 25 °C) 1 j.iL (lOU) ofE. coilligase, 5 iL of 10mM dNTPs, 1.5 .tL (15U) E. coil DNA polymerase I, and 0.5 tL (1U) of RNase H. All enzymes were from New England Biolabs. The reaction was incubated for 1 h at 12°C and 1 h at 22°C. eDNA was then adenylated at the 3' ends using 1 unit Taq polymerase and 0.2 mM dATP for 20 mmat 72°C. cDNA was then purified using the rapid PCR purification system (Novagen), cloned into the pCR4 TOPO vector (Invitrogen), and transformed into DH5a E. co/i cells (Invitrogen).Plasmids were purified using the Eppendorf miniprep kit, digested withEcoRI (New England Biolabs) and analyzed via agarose gel electrophoresis.

RT-PCR detection Total RNA was extracted from plant samples for use in RT-PCR detection using a modified Spiegel and Martin (1993) method. 100 mg of leaf tissue representative of all leaves in the sample was homogenized in one mL of extraction buffer (200 mM Tris base, pH 8.5, 300 mM lithium chloride, 1.5% lithium dodecyl sulfate, 10 mM EDTA, 1% deoxycholic acid, 2% PVP, 1% NP 40 "Tergitol" and 3-mercaptoethanol was added to result in a 1% solution (v/v) just before use). An equal volume of potassium acetate (2.8 M with respect to potassium and 6 M with respect to acetate, pH 6.5) was added to the extract and chilled at 20°C for at least 30 mm.After thawing, the mixture was centrifuged at 16,000 g for 10 mmin a microfuge and 0.7 ml of the supernatant transferred to a new tube. An equal amount of isopropanol was added and mixed via 39 inversion and centrifuged for 20 mmat 16,000 g.Finally, the pellet was washed twice with cold 70% EtOH and dried briefly under vacuum. The dried pellet was resuspended in 40 tl of RNase-free water. RNA constituted 1- 5% of the first-strand DNA synthesis reaction volume. Typically, a reaction would consist of 1tL of total RNA, 0.5 tL of 300 ng/tL random primers, 4 jiL of 5X eDNA Synthesis Buffer (250 mM Tris acetate, pH 8.4 at 25°C, 375 mM potassium acetate, 40 mM magnesium acetate), 2 tL of 10 mM dNTPs, 2 .iL of 100 mM DTT, 0.4 jtL (l6U) RNase OUT (Invitrogen), and 0.3 tL (60 U) SuperScript III RT (Invitrogen) in a final volume of 20 xL. The reaction was incubated for one hour at 5 0°C. The RT product constituted no greater than 4% of the total PCR reaction volume to prevent inhibition from plant secondary metabolites carried over from the RNA. PCR reactions consisted of 1 jtL of RT reaction, 0.8 tL of 10 mM dNTPs,loxGenscript Buffer (500 mM KC1, 100 mM Tris-HCI (pH 9.0 at 25°C), 15 mM MgC12, 1% Triton X- 100), 0.25L each of forward and reverse primers (40 tM each), and 0.25 jiL (1.25 U) Taq Polymerase (Genscript) in a total volume of 25tL. Two sets of primers were designed and used in tandem for detection. Primer set 1 of BRDaV amplified a 417 nt fragment of the RNA dependent RNA polymerase (RdRp), and consisted of forward primer 5 'ATGCTGAGCCACTTGTGA3' and reverse primer 5'ATCTGGTGTGTTCCGCAT3'. Primerset2of BRDaV,forwardprimer 5'CAATGTCTTGGAAGCCAC3' and reverse primer 5'AGCATGGTTCGTCATCTG3', amplified a 350 nt fragment further downstream at the very 3' end of the RdRp. The PCR program for detection consisted of initial denaturation for 5 mm at 94°C followed by 40 cycles with denaturation for 30 see at 94°C, annealing for 45 sec at 55°C and extension for 30 sec at 72°C, with a final 10-minute extension step at 72°C. The samples were subjected to electrophoresis through agarose gels containing 10 jtg/mL of ethidium bromide and amplicons visualized by exposure to a UV light.Amplification of the highly conserved plant gene NADH dehydrogenase ND2 subunit(ndhBgene) was used as an internal control to verify the quality of the RNA extraction and effectiveness of the

RT-PCR (Martin et al.,in press). To verify the amplification of the viral genes, numerous PCR products were sequenced. All PCR reactions were carried out using a Robocycler (Stratagene) thermocycler.

PCR and RACE Total RNA was extracted from BRDaV- 1 infected plants for use in RT-PCR to fill in the sequence gaps between clones. RT reactions were performed with random primers, as described above, for use in multiple specific PCR reactions. PCR primers were developed based on the sequence adjacent to gaps obtained in the random cloning described above. The PCR program consisted of an initial denaturation at 94°C for 5 mm, followed by 40 cycles of 94°C for 30s, varying temperatures for annealing (often on a gradient and dependent on the primer melting temperatures) for 30s, and extension of varying times (dependent on the estimated length of the amplicon) at either 68°C if using Takara LaTaq(Takara Shuzo Co.) or 72°C using GenscriptTaqpolymerase. The sequences of the 5' and 3' termini of RNA 1 and RNA 2 were determined using 'Rapid Amplification of cDNA Ends' (RACE) reactions.This involved poly- adenylating the dsRNA template (Sambrook and Russell, 2001), first-strand synthesis with methylmercury (II) hydroxide (Jelkmann et al., 1989) using a universal anchored primer(5'GACTCGAGTCGACATCGA(T)173'), and nested PCR using a sequence specific primer and a poly(dT) primer as described in Thompson et al. (2002). The 5' end of the RNA 1 and RNA 2 was confirmed via poly(C) tailing using the 5' RACE System kit, version 2.0 (Invitrogen) with total nucleic acid as a template. This system uses terminal deoxynucleotidyl transferase to add homopolymer poly(C) tails to the reverse transcriptase products.Amplification of all termini using both protocols was performed twice.

Sequencing and genome analysis All clones and PCR products were sequenced with an ABI 3730XL DNA sequencer at Macrogen, Inc. facilities (Seoul, Korea), identified using BLAST (Altschul et al., 1997) and analyzed using CAP3 software (Huang, 1996). ORFs were verified using the NCBI ORF finder (http://www.ncbi.nlm.nih. gov/gorf/gorf.html). Conserved 41 domains were identified using CD-Search (Marchler-Bauer, 2004). The amino acid (aa) identity of BRDaV proteins with orthologous proteins of related viruses was calculated using MatGAT (Campanella et al., 2003). PCR products and clones generated from each reaction were sequenced at least twice in both directions. GenBank accessions of related viruses were obtained using BLAST. Virus names, abbreviations, and taxonomic families of all referenced viruses are shown in Table 2.2.Multiple alignments and phylogenies were constructed with the CLUSTALW program (Thompson et al., 1994) with its default parameters, after bootstrapping in 1000 pseudoreplicates. Cluster algorithm phylogenetic trees in Phylip tree format of conserved helicase, protease, and polymerase motifs were visualized with TreeView (Page, 1996). Branches with bootstrap values below 70% are not shown, as they were considered statistically unreliable. 42

Table 2.2: Viruses referenced in sequence alignments, with taxonomic designation and accession numbers.

Virus Full Name GenBank accessionVirus family/genus BRDaVBlack Raspberry DQ3 44639 Unassigned/Unassigned Decline associated VirusDQ344640 SMoV Strawberry mottle virus NP 599086.1 Unassigned/Sadwavirus NP 599087.1 SDV Satsumadwarfvirus NP_620566 Unassigned /Sadwavirus NP_620567 NIMV Navel orange infectious BAA74537 Unassigned /Sadwavirus* mottling virus CiMV Citrus mosaic virus BAA92935 Unassigned /Sadwavirus* MCDVMaizechloroticdwarf NP6 19716.1 Sequiviridae/Waikavirus virus RTSV Rice tungro spherical Q91PP5 Sequiviridae/Waikavirus virus PYFV Parsnzp yellow fleck NP_6 19734.1 Sequiviridae/Sequivirus virus BBWVBroad bean wilt virus 2 NP_i 49012 Comoviridae/Fabavirus 2 CAB63085 CPMV Cowpea mosaic virus NP_6 13283.1 Comoviridae/Comovirus P03599 ToRSVTomato ringspot virus NP_620765. 1 Comoviridae/Nepovirus NP_62 0762.1 SLRSVStrawberry latent YP227367. 1 Unassigned /Sadwavirus ringspot virus YP_227368. 1 ALSV Apple latent spherical NP 620568 Unassigned /Cheravirus virus NP 620569 CRLV Gieriy rasp leaf virus AAW92 113 Unassigned /cheravirus CAF2 1714 PVY Potato Virus Y p18247 Potyviridae/Potyvirus *Not yet officially accepted into Sadwavirus genus

Coat protein expression In an attempt to overcome the difficulties associated with large-scale virus purification from plant tissue, an effort was made to express the small coat protein (CPs) of BRDaV in E. coli. RT-PCR products of the complete CPs gene were produced using as a template RNA from isolate BRDaV- 1. The forward PCR primer contained 4 extra 43 bases on the 5' end, enabling directional cloning into the pET1O1/D-TOPO vector (Invitrogen). The cassette contained a C-terminally positioned hexahistidine tag (His- tag) upstream of a stop codon, providing the means for detection and purification of the recombinant fusion protein. The construct was transformed into TOP 10 competent E. coil (Invitrogen) for characterization, propagation, and maintenance, and the insert was sequenced three times in both directions from a single colony to ensure the gene was in frame with the 5' and 3' regulatory sequence and His-tag. Once correct positioning was confirmed, the plasmid was transformed into BL2 1star (DE3) expression cells (Invitrogen). Cultures of BL2 1transformation reactions were induced with 1PTG during logarithmic growth phase, following the method supplied (Invitrogen).Aliquots from each culture were removed at several timepoints, pelleted, and frozen.Similarly, an uninduced control was also grown and sampled in parallel. Total cell extracts from each timepoint were examined by SDS-polyacrylamide gel electrophoresis (PAGE) via both western blot analysis and Coomassie Brilliant Blue staining (Sambrook and Russell, 2001). Western blot analysis was performed using monoclonal anti-histidine and goat anti-mouse antibodies conjugated with alkaline phosphatase (Sigma) for detection with NBT/BCIP substrate (Sigma).

RESULTS

Host range Seven of the 20 mechanically inoculated indicator species tested positive for BRDaV though none was infected reliably upon large-scale inoculation, and all plants that tested positive were asymptomatic.BRDaV systemically infected Nicotiana benthamiana, N. occidentalis, N. rustica, N. tobacco, N. tobacco HC-Pro mutant, Vigna unguicuiata, and Beta vuigaris (Table 2.1). ririi

Aphid transmissions Symptoms onMpersicae and A. agathonica-inoculated black raspberry plants developed approximately one month post-inoculation. The results of the transmission experiments are shown in Table 2.3. Based on these results, it is evident that BRDaV is transmitted in a non-persistent, stylet-borne mode byMpersicae.Although the percentage of infected plants increased as the feeding times were extended, 1/5 plants inoculated using A. agathonica that had been given 20 sec 1 mm feeds for acquisition and inoculation transmission times tested positive.

Table 2.3: Transmission of Black raspberry decline associated virus (BRDaV) by Amphorophora agathonica and Myzuspersicae.

Treatment # Acquisition Inoculation Numberofinfected access time*access time* plants/plants fed on A. agathonicaMpersicae

1 20 sec-1 20 sec-i mm 1/5 2/5 mm 2 1 hour 1 hour 4/5 3/5

3 5 hours 24 hours 2/5 0/5

4 24 hours 24 hours 5/5 0/5

5 24 hours 7 days 3/5 0/5

* Aphids fedon a detached leaf from a 'Munger' plant infected with isolate BRDaV-1 for indicated times. After feeding, 10 aphids were transferred to each of 5 plants to feed for the same amount of time. Plants were tested for virus by RT-PCR at 5 weeks post- inoculation. 45

Properties of virions Electron microscopy of purified virus preparations revealed isometric particles of approximately 35 nm in diameter (Fig. 2.2). The particles were hexagonal, and appeared as empty or full.Purification from N. occidentalis and N. benthamiana was successful, but electron microscope analysis revealed low numbers of virions. Purification attempts from Vigna unguiculata (cowpea) were unsuccessful. Though recalcitrant and low yielding, black raspberry was the most reliable host for purification. Not enough virus was present to visualize discrete bands of coat protein on a silver-stained polyacrylamide gel in individual fractions. However, RT-PCRon individual fractions served as a useful indicator for those that contained viral RNA (Fig. 2.3).

Fig. 2.2: Transmission electron microscope micrograph ofa virus-like particle purified from systemically infected black raspberry. Bar represents 50nm. - Fraction #

rM J - r Ite N - - - Z Z

Fig. 2.3:Detection RT-PCR on virion fractions from sucrose gradients.Twelve fractions were collected from the sucrose gradient of a BRDaV purification from black raspberry. Fractions were resuspended in 200 tL citrate buffer. One tL of each fraction was used per RT reaction. PCR primer set 2 was used, yielding a 350 bp amplicon.

Protein purification The expected size of CPs, including the His-tag, is 58,873 Da.However, immunoblotting with anti-His antibody revealed that expressed protein appeared to have migrated as three separate species (Fig. 2.4).The largest protein appeared to be the anticipated size, but an intermediate, much brighter protein band, and smaller protein band were present.Proteolytic degradation was suspected as the cause, so pilot expression was repeated, and samples were treated with protease inhibitors at time of harvest.This treatment had no effect, indicating that either proteolysis was occurring during the induced expression phase of cell growth, or another mechanism was at work. Coomassie staining on replicate samples demonstrated a similar outcome; three bands of the same MW as those observed in the western blot were overexpressed in comparison to the uninduced control, and relative expression levels were consistent between the two analyses (not shown). The anti-His blot demonstrated that all three proteins contain the C-terminal His tag, and the same-sized bright bands on the Coomassie stained gel reinforced the idea that these bands were not the product of cleavage. 47

1.5h 3h 45h Likely product Da - C..) - C..) - C size (Da)

124,000

80,000

-59,000 Da 49,100

34,800 -_&. . -- -31,000 Da

28,900

20, 600

-14,000 Da

Fig. 2.4: Anti-His probed western blot of expressed CPs protein. 1= induced reaction, C= uninduced control. Cells were harvested at 1 .5h, 3h, and 4.5h timepoints. Cloning from dsRNA DsRNA extraction from declining black raspberry revealed two major bands of about 8.5 and 7 Kbp based on agarose gel electrophoresis (Fig. 2.5 a, b, c).Cloning of dsRNA produced several clones that provided enough genomic coverage to develop PCR primers and obtain the missing sequence between non-overlapping clones. The presumed regions of the genome to which these clones belonged were derived based on comparison toStrawberry mottle virus (SMoV)(Thompson et al., 2002). bp - (1 . iie N -

8454 5686

3675

2323

1929

Fig. 2.Sa Fig. 2.5b Fig. 2.5c

Fig. 2.5: DsRNA extracted from plants infected with Black raspberry decline associated virus (BRDaV).Double-sided arrows indicate the position of the dsRNA doublet of BRDaV, about 8.5 and 7 Kbp. Fig. 2.5a. Lane I: ?.BstE II DNA marker (NEB); Lane 2: dsRNA extracted from field-collected tissue with mixed virus infections, demonstrating the presence of high MW dsRNA in disproportionate amount relative to BRDaV (1/6 the mass of plant tissue used in this lane as compared to all others); Fig. 2.Sb. Lane 3: Plant infected with BRDaV and Raspberry bushy dwa,f virus purified from greenhousegrown tissue; Lanes 4, 5, and 6: dsRNA from field-collected tissue.Lane 5 contains other viruses; Lane 7: dsRNA from N. tabacum testing positive via RT-PCR for BRDaV, demonstrating the increased sensitivity of RT-PCR over dsRNA; Lane 8: dsRNA from mutant N. tabacum, containing potyviral HC-Pro gene, which acts as a suppressor of post transcriptional gene silencing; plants tested positive via RT-PCR for BRDaV; Lane 9: dsRNA extracted from a healthy black raspberry plant; Lane 10: dsRNA extracted from fruiting canes only (contains other viruses in addition to BRDaV); Fig. 2.5c. Lane 11: dsRNA extracted from Western brackenfern (Pteridium aquilinumvar. pubescens), an alternate host for BRDaV. 50

Nucleotide sequence and phylogenetic analysis Non-coding regions The 5' noncoding regions of RNA 1 and RNA 2 of BRDaV are 146 and 223 bp, respectively, with the first 42 nucleotides (nt) being identical. This identity is similar to that of SMoV (Thompson et al., 2002), which contains an initial 36 nt consensus sequence. Also characteristic of SMoV and theComoviridae,the 3 '-noncoding regions of RNA 1 and RNA 2 share much greater sequence identity than the 5'-noncoding regions, with 94% identity.

RNA1 RNA 1 is 7581 nt long, excluding the 3' poly(A) tail. RNA 1 encodes a single open reading frame (ORF) that presumably is cleaved proteolytically into its respective functional proteins. ORF 1 is predicted to begin at the AUG nt 147-149 and terminate at UAA (nt 6636-6638), yielding a putative polypeptide with MW of 242 kDa. The first AUG initiation codon that is in good translational context (GAAGAUGUCGU) (Kozak, 2002) would produce a 6492 nt ORF. The polyprotein encoded by RNA 1 probably is cleaved into five functional proteins involved in replication: a putative protease cofactor (Pro-C), helicase (Hel), viral genome-linked protein (VPg), protease (Pro), and an RNA-dependent RNA polymerase (RdRp). Alignment of the entire 242 kDa protein with the corresponding polyprotein of other closely related viruses suggested the most likely locations for cleavage sites. The resulting most likely dipeptide proteolytic cleavage sites are Q/G, E/G, Q/N, and Q/Q at amino acids 515/516, 1020/1021, 1046/1047, and 1280/1281 (Fig. 2.6). This is partially in line with the cleavage specificity of 3C-like proteases, which cleave primarily between Q (or E), and G (or S) dipeptides (Palmenberg, 1987).The predicted polyprotein cleavage would result in proteins of the following MW: Pro-C, 56 KDa; Hel 57 KDa; VPg, 3 KDa; Pro 25 KDa; and RdRp 101 KDa (Table 2.4). The N-terminus region of the polyprotein has the Pro-C (aa 1-515), which is 0 1 2 3 4 5 6 7 8Kb

RNA1

Pro-C Hel VPgPro RdRp U

Q/G E/G Q/N Q/Q

RNA 2

MP CP1 CPs

QIG EIG

Fig. 2.6: Proposed genome map of BRDaV RNA 1 and RNA 2. Scale at top is marked in kilobases (Kb). The lines indicate non- coding regions, the boxes represent putative polyprotein encoded by ORFs, and vertical lines within the boxes indicate the most probable cleavage sites, based on conserved cysteine protease cleavage sites and comparison with other sadwaviruses. Below probable cleavage sites are the peptide sequence at each site. Coding regions are protease cofactor (Pro-C), helicase (He!), viral genome-linked protein (VPg), protease (Pro), RNA-dependent RNA polymerase (RdRp), movement protein (MP), large coat protein (CP1), and small coat protein (CPs). Hatched region at 3' end of RNA 1 ORF represents A!kB-homo!ogous domain. 52

Table 2.4: Position on genome and size of proposed functional proteins of Black raspberry decline associated virus, isolate BRDaV- 1.

Gene Nucleotide No. of No. of amino Molecular Positions Nucleotides acids mass (kDa) Pro-C 147-1691 1545 515 56.22 Hel 1692-3206 1515 505 56.95 VPg 3207-3284 78 26 2.82 Pro 3285-3986 702 234 25.43 RdRp 3987-6638 2652 883 100.70 MP 224-1195 972 324 36.14 CP1 1196-3958 2763 921 102.71 CPs 3959-5425 1467 488 55.25 related most closely with the Strawberry mottle virus (SMoV) Pro-C, having 48% amino acid sequence identity. The conserved amino acid motifFx27Wx11Lx21LxE (Ritzenthaler et al., 1991) identified within the Pro-C region of some Comoviridae was not detected within the corresponding region of BRDaV. Following Pro-C, the helicase (aa 516-1020) aligns most closely with SMoV with 72% sequence identity in a conserved 200 aa overlap, and more distantly with Satsuma Dwarf Virus (SDV), with 42% sequence identity in the same orthologous overlapping region.The highly conserved helicase domains A (GKS), B (DE), and C (N) (Gorbalenya et al., 1990) are found at amino acid positions 577-9, 623-4, and 673.Alignment and phylogenetic analysis of these conserved motifs (aa 577-673) groups BRDaV most closely with SMoV and SDV among the sadwaviruses (Fig. 2.7).Note that Strawberry latent ringspot virus (SLRSV) currently is classified with the sadwaviruses (Mayo, 2005) yet does not share significant sequence identity in this region. The putative VPg (aa 1021-1046) only shows sequence similarity with SMoV, with 50% identity in the entire protein overlap. The consensus sequence E/D-x(13)-Y-x(3)-N-x(45)-R found among some comoviruses and nepoviruses, proposed by Mayo and Fritsch (1994) was found partially in the putative VpG of BRDaV. E-x3-Y is present, but the latter half of the consensus is missing, and does not fit the modified scheme proposed by Thompson et al. (2002).The closest sequence identities in a 234 aa overlap within the putative protease protein (aa 1047-1336) are with 53

SMoV (26%), SDV (22%), and Navel orange infectious mottling virus (NIMV), a virus serologically-related to SDV, with 21%. Alignment of the BRDaV putative protease with proteases of related viruses revealed the presence of conserved cysteine protease motifs (Gorbalenya et al., 1989). The amino acids H, D and C of the 3C protease catalytic triad are located at residues 1100, 1164, and 1230. Phylogenetic analysis of BRDaV amino acids 1214-1257 with corresponding sequence in related viruses revealed a clustering of BRDaV with SMoV and SDV of the sadwaviruses (Fig. 2.8). The final region within the 242 kDa ORF is the putative RdRp (aa 1337-2 163), that has aa sequence identities in a 268 aa conserved overlap of 77%, 48%, and 47% with SMoV, SDV, and NIMV, respectively. The BRDaV RdRp clusters with supergroup I of positive-strand RNA viruses, and contains the conserved RdRp motifs 1-Vill (Koonin and Dolja, 1993, Koonin, 1991). A conserved domain between aa 1338 and 1817 detected by the Conserved Domain Database (Marchler-Bauer et al., 2004) encompassing the 8 motifs was aligned with corresponding sequences in related viruses to generate a phylogenetic tree (Fig. 2.9). Two distinct clades emerged, one containing branches for the Comoviridae and proposed cheraviruses, and the other containing the sadwaviruses. A very unique feature of the BRDaV RdRp, compared to other related viruses, is an unusual indel at the C terminus. There is a one aa overlap where the alignment of SMoV ends, and a 189 aa mdcl begins. The mdcl region contains a motif homologous to the polymerase fragments of Flexiviridae and Closteroviridae members (Fig. 2.10).Most closely related are Little cherry virus 2, with 43% sequence identity in a 59 aa overlap, Cherry green ring mottle virus, with 42% identity in a 67 aa overlap, and Grapevine virus A, with 41% identity in a 56 aa overlap. Specifically, the conserved domains within this region correspond to A1kB, an alkylated DNA and RNA repair protein recently shown to be a member of the 20G-Fe(II) oxygenase superfamily (Aravind and Koonin, 2001). 54

cadwa virus

cequiviridae

Corn oviridae

Cheravirus

0.1

Fig. 2.7: Neighbor-joining analysis of the helicase of BRDaV and related viruses, based on the alignment of amino acids 577 through 673.Abbreviations of virus names and accession numbers are as in Table 2.2. Numbers by each node are bootstrap values for 1000 replicates. Bootstrap values under 70% are not shown. The scale bar represents the average number of residue substitutions per site. 55

Sequiviridae

Corn oviridae

Sadwa virus

Cheravirus

PvY

0.1

Fig. 2.8: Neighbor-joining analysis of the protease of BRDaV and related viruses, based on the alignment of amino acids 1214 through 1257. Abbreviationsof virus names and accession numbers are as in Table 2.2. Numbers by each node are the bootstrap values for 1000 replicates. Bootstrap values under 70% are not shown. The scale bar represents the average number of residue substitutions per site. The phylogram is rooted with the outgroup PVY. 56

Ch eravirus

SLRSV

CPMV 988 BBWV2 Corn oviridae

SDV 000 NIMV Sadwa virus 99 SMoV 1000 BRDaV

MCDV 998 RTSV Sequ iviridae

PYFV

PVY 0.1

Fig 2.9: Neighbor-joining analysis of the RdRp motifs between amino acids 1338 and 1817 of BRDaV and homologous regions within related viruses. Abbreviations of virus names and accession numbers are as in Table 2.2. The numbers by each node arethe bootstrap values for 1000 replicates.Bootstrap values under 70% are not shown. The scale bar represents the average number of residue substitutions per site. The phylogram is rooted with the outgroup PVY. 57 BRDaV PAKELDELEQSVAREDFDGKI ITI ACAKDE KTKLIKVYEKPKTRI FEI LPYHYN ILVRKY SM0V VLDDLKELEEAI KSDDFKGGI ITI ACAKDE KTI.1KRVREVPKTRI FEI LPFHYN ILVRKY .:*.***::: :**.* *************. * **********:*********

BRDaV YLFFMQ FIMRMHNVLPCKVGLNPFSQDWDEMJjAEHTRFEHHFNGDYSG FDTGTPRQLLLK SM0V YLFFMQ FIMSLHDFLPCKVGLNVY SKS WDTMHAEHNRFAYHFNGDYTG FDTATPRVLMMK ********* *:** ******* :******:****.*** *::*

BRDaV FADLIS ELAADGRRNKVI RRNLMQLAVDRR ILVLAELYHVRGGTPSGFALTVI I NSMVNQ SMoV IADMVSNLAGDGRENAIVRRNLMKMAVERR ILVLRDLYQVKGGTPSGFALTVI I NSVVNQ :**: :* :**.***.*: :*****: :**:****** :**:*:***************:***

BP.DaV FYLMWAWRKIMS RISPSM VTYRVMRTHCTFSVYGDDNWSFSLAVRDMYNLCTI ?JDEL SM0V FYLMWAWRKIMS RIDPGLVPYRVMRSHCTFSVYGDDNWSFSLQVKDMYNLVTI AAELKT ***************.******:***************** *:***** *** ****

BPJJaV IGVTLS DGKXTGVLIKWTGFKDLD FLKRRWELEPGHGFKCPLDKMAI E ERLFWVRKSEDN SMoV IGVNLS DGKKTGNLVKWMEFSELD FLKRRWVLYSGQGFLCPLHKSAIEERLFWVRSSEDS ***.******** *:** :******** * *:** **** *************

BRDaV MESLDDNCYSALMEAFHHGPEYFHDLREKI LDGYQSAGLAAP VLLHYQEARS IWFEQHKV SM0V VETLDDNCYSALMEAFHHGRDYFN FLRARI QDAYDKAGLYQPHLLHFNEAQAIWLEQHSI :*:**************** :**: **:* **:*** * ***::**::**:***.

BRDaV GAESDF FAGVQNHLLPS IVGKDVI RTISVDWSSI RRYNEAFASGNYNRTKVFLDPLP.K SMoV APPNDYLDGIKKEVLPLTAGRDVMRTITTr IDATSVKGYNKAHKEQSYHRTKVFLNPLT *::: .:** *:**:***:**:* :*: **:* *:******:***

BRDaV EILWLKSKSGPHLSIPASLRKETFQSIIEG INRVVPG-EICVVDGRGDKSGLVVALALAM SM0V EITWLRSGQAAJLDVPAKL}jRR1qFEGIAIKvpj.LLKGENCCI VEGTCG INSFALALP.IGF ** **.* ** .** * :

BRDaV DREEIS QAQGHNMLLALVTTNKEVEYGLQL FNVLEATSRPSVSLSPKFT]PPAELLKTIV SM0V LRELTGVGCNLLSYSSNSYYTGLAMLTTVVG ** ::..

BRDaV GVELYEVTEAVKKMPFGSVGPSLRGRMALFYSDGAFDYAHDKYHYTSQGWPREVDDLAKK SM0V

BRDaV LGGYNS CLVQKYDKGAYI PFHADD EPCYDDNDSVITVNLNGRATFIVRNKTrGAETRREL SM0V

BRDaV HHGS ILEMLPSCQKLCKHSVNVRDQGRVSLTFRRQRRTMNGTPI SMoV

Fig. 2.10:ClustaiW alignment of RNA-dependent RNA polymerase (RdRp) region encompassing motifs I-Vu! (Koonin, 1991) between BRDaV and SMoV. Alignment consists of aa 181-883 of BRDaV, and 179-694 of SMoV. The end of the overlap represents the C-terminus of SMoV RdRp. Red region represents indel at C terminus of BRDaV RdRp. Asterisk: residues strictly conserved; colon: strong conservation; single dot: weak conservation. RNA2 RNA 2 consists of 6364 nt, while its predicted single ORF encodes a 1734 aa polyprotein. The predicted initiation codon, AUG224, like that of RNA 1, has a purine in the -3 position and is in good but not excellent translational context (Kozak, 2002). The predicted ORF2 of BRDaV potentially begins at AUG (nt positions 224-226) of RNA 2 and terminates at UAG (nt 5200-5202), yielding a 194 kDa polypeptide. The polyprotein encoded by RNA 2 likely is cleaved to give rise to three proteins: a movement protein (MP), large coat protein (CP1) and small coat protein (CPs). The most probable cleavage sites in RNA 2, based on alignments with SMoV, are Q/G and E/G at residues 324/325 and 1245/1246, respectively (Fig. 2.6). One EQG, a conserved motif within SMoV (Thompson et al., 2002) was found at residues 907/908, as well as three other Q/G dipeptides and eight E/G dipeptides. The predicted MW of the mature proteins are: MP 36 KDa; CP1 103 KDa, and CPs 55 KDa (Table 2.4). When the first putative protein, MP (aa 1-324), was used as the query in a BLAST search only the MP of SMoV, with 42% sequence identity in a 279 aa overlap was identified. A 33 aa region from aa 182-214 which encompasses a conserved sequence motif in the 30K superfamily of plant virus movement proteins (Mushegian and Koonin, 1993) was located and aligned with MPs of closely related viruses (Fig. 2.11). There was conservation of the 30K superfamily LxD motif in BRDaV, as seen in Fig. 2.11, and the first common motif, LxP (aa 24-26) is present (Melcher, 2000). Consensus between BRDaV and the Sequiviridae was marginal, but slightly greater with the Comoviridae. The lack of consensus between alignment of the BRDaV MP and that of the most closely related viruses underlies the previously noted poor sequence conservation among the 30K superfamily (Melcher, 2000), and reinforces the idea that the MPs of the Satsuma dwarf- related viruses may have been acquired from the Comoviridae (Karasev et al., 2001). The next protein, CP1 (aa 325-1245), aligns most closely with SMoV with 42% identity in a 921 aa overlap, but very little with other Satsuma dwarf related viruses. Though better known for their conservation of secondary structure than amino acid motifs (Doija and Koonin, 1991), a 47 aa region of BRDaV CPI was aligned with a previously identified coat protein region conserved among several calici-, picorna- and sequiviruses 59

(Karasev Ct al., 2001) (Fig. 2.12).This alignment revealed a universally conserved G motif among all viruses compared, whereas several other motifs were conserved specifically among sadwaviruses,Sequiviridae,or comoviruses. The last protein, CPs (aa 1246-1733), only showed similarity with SMoV, with 24% sequence identity in a 264 aa overlap.

S DV C IMV Q VLSVT A C PMV Q FASPES L- G BBWV-2 DGFASVNS T G- Y BRDaV AVSTKES PT SNVk SMoV AVNTTNSKQPS SPIP P T0RSV DPVGMP DT fT cNSDD PY FV TLMHQ DSRECI-Y CRiD consensus lhvg leivvs gd maimlsd hk

Fig. 2.11: Alignment of a 33 aa region (aa 182-214) of the movement protein of BRDaV with other closely related viruses.Similar residues are highlighted in gray.Identical residues are highlighted in black. The conserved motif LXD of the 30K MP superfamily (Mushegian and Koonin, 1993; Melcher, 2000) is present in BRDaV. Abbreviations of virus names are in Table 2.2. 60

CIMV ---YLGLTAS FS N YI K LS--GSC NIMV ---YLGLTAS FS N Yl K LS--GSC SDV ---YLGLGAS FSL ------Y K ------D LS--GSC PYFV ---LLS S T ------FE C--SAST SM0V ---KGST SS CSH ------S G S SA--SSV BRDaV ---YGS S CAN ------N C S N TA-- NV MCDV ---TQT SI H R ------KFT ------TFGA--SMFT RTSV ---YQT S H K ------S KYT ------FVFGA--SMFD

ALSV ---THN I-I STT ------HYQ ------TY--DRN CRLV ---THN HY SCT ------TYE TY--NSRV SLRSV ---YPSCSSS LENH ------E ------IFNL--PAMG BBWV2 --ILPK T DHAP ------KEL FKQ-GERVAYSFE NFG-PQT CPMV ---CEPT YH DCQN LPLNR TFPQGVTSEVRIP SIG-GGAG QAF T0RSV FSNSFG P TLPHI RLDIGY PTEJDLTSTPAPNAYLFGLSTAI consensus 1 1 yaf wr Gtl 1 v v k ag i

CIMV YVP P TSD- NIMV YVP P TSD- SDV YIP P SSD- PYFV SVT 1L SMoV YT T TFVH- BRDaV YTL P YNTR- MCDV I KRKD- RTSV SA QFRNS- ALSV AFYTTNNES- CRLV AFYTSQVED- SLRSV FANDSCSR- BBWV2 TSTFASSYCLS CPMV NMINSWISM- ToRSV TLNANQ LRFFISNZT- consensus ± p g a

Fig. 2.12: Alignment of a 47 aa region (aa 734-780) of the large coat protein of BRDaV with other closely related viruses. Similar residues are highlighted in gray. Identical residues are highlighted in black. Abbreviations of virus names are in Table 2.2. 61

DISCUSSION

BRDaV reveals a genomic sequence and structure closest to that of Strawberry mottle virus (SMoV) (Thompson et al., 2002), a new member of the genus Sadwavirus (Mayo, 2005). Both viruses share long 3' noncoding regions, a characteristic that ties them to some nepoviruses (Rott et al., 1991) but sets them apart from the Sadwavirus type member, SDV (Iwanami et al., 1998; Iwanami et al., 1999). Until recently, this unclassified genus had been termed "SDV-like viruses", consisting of members with genomic similarity to SDV yet with no consistency of vector.Like the nepoviruses, sadwaviruseshaveasingle-stranded,positive-senseRNA,bipartitegenome. Furthermore, the genomes of both genera are encapsidated in icosahedral particles, and each RNA produces a proteolytically cleaved polyprotein. RNA 1 encodes replication- related proteins, whereas RNA 2 encodes the cell-to-cell movement protein and coat proteins.Unlike the nepoviruses however, sadwaviruses produce two distinct coat protein subunits of differentsizes,a feature found among definitivefaba- and comoviruses. While the BRDaV genome organization closely resembles viruses of the family Comoviridae, the RdRp is related to viruses both among and outside of the sadwaviruses, Sequiviridae, and Comoviridae. BLAST searches reveal relationships with Cherry rasp leaf virus of the newly-established unassigned Cheravirus genus (Mayo, 2005) and more remote viruses such as Acute bee paralysis virus (Govan et al., 2000), Kashmir bee virus (De Miranda et al., 2004) of the unassigned Dicistroviridae, and Varroa destructor virus 1 (Ongus et al., 2004), a virus of mites in the unassigned genus Iflavirus. This relatedness among seemingly diverse viruses supports the growing concept of thepicorna-like virus "superfamily". Genomes within this grouping share the same replicative core proteins, in the order: Hel-VPg-Pro-RdRp, having a Type III helicase domain and a typeI polymerase domain (Koonin and Dolja, 1993). Further defining characteristics include similarities in RNA termini, involving a 3' poly(A) tail (with the exception of the Sequivirus genus) and a covalently-linked VPg at the 5' end, and a 3C-like cysteine 62 protease (Gorbalenya etal.,1989). Based on these affinities, the International Committee on Taxonomy of Viruses (ICTV) recently proposed the creation of an order, termed Picornavirales which would include those viruses of vertebrates, plants, and insects sharing similar replicative gene blocks and expression mechanisms, and encompassing the sadwaviruses (Christian et al., 2005). Karasev et al. (2001) suggest an SD V-like lineage likely occurred by the splitting of an ancestral, monopartite picorna-like insect virus genome, generating independent evolutionary lineages with bipartite genomes. The most atypical aspect of the BRDaV genome is the 189 aa indel at the C- terminus of the RdRp with homology to the AIkB domain of viruses of the Fiexiviridae and ampeloviruses of the Closteroviridae. The source of this unusual sequence is unknown, but it likely arose from a recombination event between two co-infecting viruses. It is possible that this sequence was integrated from a previously required helper virus, one of which is documented for at least one member of the Sequiviridae, Parsnip yellow fleck virus (Harrison and Murant, 1984). Because BRDaV has been detected in multiple species of Rubus (see Chapter 3), it is likely that the recombination event could have occurred in a different host before moving to black raspberry. This transfer of genetic material could have conferred an adaptive advantage of the virus to the aphid vector or plant host. Genes for AIIkB homologues are prevalent in nature, occurringin eukaryotes, bacteria, and a narrow subset of plant viruses (Aravind and Koonin, 2001; Bratlie and Drabløs, 2005.).Conservation of catalytic residues suggested that these homologues, traditionally thought to protect only DNA against damage from methylating agents, might also modify RNA in a similar way. Enzymatic RNA modifications are often associated with control of gene expression, so it was postulated that if A1kB could use RNA as a substrate, it could be implicated in defense ofpost-transcriptional gene silencing (PTGS) (Aravind and Koonin, 2001). Indeed, Aas et al. (2003) demonstrated that AIkB can repair alkylation damage in RNA. Thus, perhaps the acquisition and retention of this functional domain supplied BRDaV with an adaptive mechanism of counterdefense. This same region has been detected in isolates of BRDaV obtained from 63

Ohio and California, which reveals that this is not an event unique to Oregon isolates. This insert bears no homology to the newly discovered blackberry flexivirus (see Chapter 3), which also occasionally appears in declining black raspberries. The goal of CPs protein expression was to use this recombinant protein for the production of antibodies, which could then be employed in a BRDaV-specific ELISA. The generation of truncated gene products associated with heterologous gene expression in E. coli has been recorded previously.It has been suggested that this phenomenon is attributable to sequence within the RNA encoding the protein resembling sequence utilized by prokaryotic ribosomes for initiation of protein synthesis (Halling and Smith, 1985). Such sequence is the Shine-Dalgarno sequence, a purine rich sequence (AAGGAGG) within the ribosome binding site (RBS) and located approximately 5-13 nt upstream of the initiation codon (Shine and Dalgarno, 1974).Preibisch et al. (1988) found that the removal of such sequences abolished truncated protein synthesis when internal start codons fell 4-20 nt downstream. Halling and Smith (1985) also experienced truncated expression products, and demonstrated via introduction of ineffective upstream stop codons that the smaller products result from independent internal translations. Closer examination of the sequence of the CPs reveals a series of 11 internal, in-frame start codons. The sixth such codon, at aa 246, corresponds to sequence that occurs 13-19 nt downstream from the exact Shine-Dalgarno sequence within the RBS (nt 7 17-738, 5' AAGGAGGCGGCCGGAACCUA IJG 3'). If translation were to initiate at this internal methionine, a product of 31.5 kDa would result, which could correspond to the bright, middle band on the anti-His western blot (Fig. 2.4). Further toward the C terminus at aa 397 resides the 2u to last internal in-frame methionine. The AUG corresponding to this position lies just downstream of purine-rich sequence resembling the Shine-Dalgarno sequence (nt 1177-1191 5' GGGAGAAAGGAAAUG 3').If the ribosome recognized this site and translated from this start codon, the translation product would be 13.7 kDa, which corresponds to the lowest protein on the anti-His western blot (Fig. 2.4). Perhaps ribosomes recognize this site less frequently than its upstream counterpart, which could explain the weakness of the band. Why the ribosome would recognize the internal ribosome-binding site more strongly than that which is built into the cloning cassette upstream from the cloning site is not known, but could be related to interference from secondary structure. To verify that BRDaV is the causal agent of black raspberry decline, the steps of Koch'spostulateswere undertaken,withmodificationsforobligateparasites. Greenhouse grown clonal black raspberry plants containing isolate BRDaV-1 that showed mottling and chiorosis symptoms typical of declining black raspberry in fields were used. Plants with such symptoms consistently tested positive for BRDaV via RT- PCR. The characteristic high MW dsRNA doublet and -35 nm virus-like particles were purified from these plants. No other knownRubusviruses were detected in these plants, and no other dsRNA bands were present that would indicate mixed infections.After feeding on these infected plants, the large raspberry aphid was used to transmit BRDaV to healthy black raspberry.These recipient hosts developed the same mottling and chiorosis, tested positive for BRDaV via RT-PCR (which was confirmed by sequencing), and had the same dsRNA banding pattern.In the absence of a full-length clone of BRDaV, Koch's rules are not completely carried out. However, given the overwhelming circumstantial evidence, BRDaV appears to be the causal agent of BRD. Several characteristics of BRDaV point to its likeness to BRNV. Both viruses have a limited experimental host range, are found in low titers in herbaceous plants, and are recalcitrant to purification (Jones and Murant, 1972; Murant et al., 1976). Both infect red raspberry with indistinct or no symptoms (Stace-Smith, 1955b), and both are transmitted by the large raspberry aphid, though with different transmission modes. Both have similar particle structure consisting of both empty and filled particles, though BRNV, at30 nm diameter, was reportedly smaller than BRDaV (Jones and Murant, 1972).Cross-referencing of molecular and biological characteristics of BRDaV with European plant accessions containing original BRNV isolates will likely reveal whether BRDaV and BRNV are different strains of the same virus, and will furthermore provide insight into the evolutionary dynamics of the A1kB domain indel. 65

ACKNOWLEDGEMENTS

The authors are grateful to Dr. Mike Nesson at the Oregon State University Electron Microscope Facility for his technical expertise. Thank you to the Carrington lab for providing seeds for the mutant tobacco. Thank you also to Dr. loannis Tzanetakis and Dr. Jennifer Kraus for many helpful discussions.

REFERENCES

Aas, P.A., Otterlei, M., Flanes, P.O., Vagbo, C.B., Skorpen, F., Akbari, M., Sundheim, 0., Bjoras, M., Slupphaug, G., Seeberg, E. and Korkan, H.E. 2003. Human and bacterial oxidative demethylases repair alkylation damage in both RNA and DNA. Nature 421:859-863.

Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D.J.1997. Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res. 25:33 89-3402.

Aravind, L. and Koonin, E.V.2001.The DNA-repair protein AIkB, EGL-9, and leprecan define new families of 2-oxoglutarate- and iron-dependent dioxygenases. Genome Biology 2(3):research0007. 1-0007.8

Bratlie M.S. and Drabløs F. 2005. Bioinformatic mapping of A1kB homology domains in viruses. BMC Genomics 6:1.

Cadman, C. H. 1951. Studies inRubusvirus diseases. I. A latent virus of Norfolk Giant raspberry. Ann. Appi. Biol. 38:801-811.

Cadman, C.H.1952.Studies inRubusvirus diseases. V. Experiments in analysis of Lloyd George decline. Ann. Appl. Biol. 39:50 1-508.

Campanella, J.J., Bitincka, L. and Smalley, J.2003. MatGAT: an application that generatessimilarity/identity matrices using proteinor DNA sequences. BMC Bioinformatics 4:29.

Can-ington, J.C., Freed, D.D. and Oh, C.S. 1990. Expression of potyviral polyproteins in transgenic plants reveals three proteolytic activities required for complete processing. EMBO J. 9:1347-1353. Christian, P., Fauquet, C.M., Gorbalenya, A.E., King, A.M.G., Knowles, N., LeGal!, 0. and Stanway, G. 2005. Picornavirales: A proposed order of positive sense RNA viruses. ICTV Poster session at the International Congress of Virology, San Francisco, Moscone Convention Center, July 23-28, 2005. http://www.danforthcenter.org/iltab/ictvnet/sanfran.htm de Miranda, J., Shen, M., Cameron, C.E., Stoitz, D.B. and Camazine, S.M.2004. Complete nucleotide sequence of Kashmir bee virus and comparison with acute bee paralysis virus. J. Gen. Virol. 85:2263-2270.

Doija, V.V. and Koonin, E.V. 1991. Phylogeny of capsid proteins of small icosahedral RNA plant viruses. J. Gen. Virol. 72:1481-1486.

Gorbalenya, A.E., Donchenko, A.P., Blinov, V.M. and Koonin, E.V.1989.Cysteine proteases of positive strand RNA viruses and chymotrypsin-like serine proteases. FEBS Letters 243:103-114.

Gorbalenya, A.E., Koonin, E.V. and Wolf, Y.I.1990. A new superfamily of putative NTP-binding domains encoded by genomes of small DNA and RNA viruses.FEBS Letters 262:145-148.

Govan, V.A., Leat, N., Allsopp, M. and Davison, S.2000. Analysis of the complete genome sequence of acute bee paralysis virus shows that it belongs to the novel groupof insect-infecting RNA viruses. Virology 277:457-463.

Hailing, S. M. and Smith, S.1985. Expression in Escherichia coli of Multiple Products from a Chimaeric Gene Fusion: Evidence for the Presence of Procaryotic Translational Control Regions within Eucaryotic Genes. Bio/Technology 3:715-720.

Harrison, B.D. and Murant, A.F. 1984. Involvement of virus-coded proteins in transmission of plant viruses by vectors.In:Mayo, M.A., and Harrap, K.A. (eds), Vectors in Virus Biology, Academic Press, London, pp. 13-14.

Huang, X. and Madan, A. 1999. CAP3: A DNA Sequence Assembly Program. Genome Research 9:868-877.

Iwanami, T., Kondo, Y., Makita, Y., Azeyanagi, C. and leki, H.1998. The nucleotide sequence of the coat protein genes of satsuma dwarf virus and navel infectiousmottling virus. Arch. Virol. 143:405-412.

Iwanami, T., Kondo, Y. and Karasev, A.V. 1999. Nucleotide sequences and taxonomy of satsuma dwarf virus. J. Gen. Virol. 80:793-797. 67

Jelkmann, W., Martin, R.R. and Maiss, E.1989.Cloning of four plant viruses from small quantities of double-stranded RNA. Phytopathology 79:1250-1253.

Jones, A.T. and Murant, A.F.1972. Some properties of a mechanically transmissible virus widespread in raspberry(Rubus idaeus)in Scotland. Plant Pathol. 21:166-170.

Jones, A.T. and Jennings, D.L.1980. Genetic control of the reactions of raspberry to black raspberry necrosis, raspberry leaf mottle and raspberry leaf spot viruses.Ann. Appl. Biol. 96:119-123.

Jones, A.T. and Mitchell, M.J.1986.Propagation of black raspberry necrosis virus (BRNV) in mixed culture with solanum nodiflorum mottle virus, and the production and use of BRNV antiserum. Ann. App!. Biol. 109:323-336.

Karasev, A.V., Han, S.S. and Iwanami, T. 2001. Satsuma Dwarf and Related Viruses Belong to a New Lineage of Plant Picorna-like Viruses. Virus Genes 23 :45-52.

Koonin, E.V.1991. The phylogeny of RNA-dependent RNA polymerases of positive- strand RNA viruses. J. Gen. Virol. 72:2197-2206.

Koonin, E.V. and Doija, V.V.1993. Evolution and taxonomy of positive-strand RNA viruses: implications of comparative analysis of amino acid sequences. Critical Reviews in Biochemistry and Molecular Biology 28:375-430.

Koonin, E.V., Mushegian, R.R., Ryabov, E.V. and Doija, V.V. 1991. Diverse groups of plant RNA and DNA viruses share related movement proteins that may possess chaperone-like activity. J. Gen. Virol. 72:2895-2903.

Kozak, M.2002.Pushing the limits of the scanning mechanism for initiation of translation. Gene 299:1-34.

Marchler-Bauer A. and Bryant, S.H. 2004. CD-Search: protein domain annotations on the fly. Nucleic Acids Res. 32:W327-33l.

Martin R.R., Tzanetakis, I.E., Sweeney, M. and Wegener, L. A virus associated with blueberry fruit drop disease. Acta Hortic,in press.

Mayo, M.A. 2005. Changes to virus taxonomy 2004. Arch. Virol 150:189-198.

Mayo, M.A. and Fritsch, C. 1994. A possible consensus sequence for VPg of viruses in the family Comoviridae. FEBS Lett. 354:129-130.

Meicher, U. 2000. The '30K superfamily of viral movement proteins.J. Gen. Virol. 81:257-266. 1Sr]

Murant, A.F., Jones, A.T. and Roberts, I.M.1976.Recent research on 52V virus of raspberry. Acta Hortic. 66:39-46.

Mushegian, A.R. and Koonin, E.V.1993.Cell-to-cell movement of plant viruses. Insights from amino acid sequence comparisons of movement proteins and from analogies with cellular transport systems. Arch. Virol. 133:239-257.

Ongus, J.R., Peters, D., Bonmating, J.M., Bengsch, E., Viak, J.M. and van Oers, M.M. 2004. Complete sequence of a picorna-like virus of the genus Iflavirus replicating in the mite Varroa destructor. J. Gen. Virol. 85:3747-3755.

Oregon Agricultural Statistics Service. 2004. p://www.nass.usda. gov/or/fruit.htm

Page, R.D.M. 1996.TREEVIEW: An application to display phylogenetic trees on personal computers. Comp. Appi. Bio. 12: 357-358.

Palmenberg, A. C.1987. Picornaviral processing: Some new ideas. J. Cell. Biochem. 33, 191-198.

Preibisch, G., Ishihara, H., Tripier, D. and Leineweber, M. 1988. Unexpected translation initiation within the coding region of eukaryotic genes expressed in Escherichia coli. Gene 72:179-186.

Ritzenthaler, C., Viry, M., Pinck, M., Margis, R., Fuchs, M. and Pinck, L.1991. Complete nucleotide sequence and genetic organization of grapevine fanleaf nepovirus RNA1. J. Gen. Virol. 72:2357-2365.

Rott, M.E., Tremaine, J.H. and Rochon, D.M.1991.Comparison of the 5' and 3' termini of tomato ringspot virus RNA1 and RNA2: evidence for RNA recombination. Virology 185:468-472.

Sambrook, J. and Russell, D.W. 2001. Molecular Cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, NY: Cold Spring Harbor Laboratory.

Shine, J. and Dalgarno, L.1974. The 3'-Terminal Sequence of Escherichia coli 16S Ribosomal RNA: Complementarity to Nonsense Triplets and Ribosome Binding Sites. Proc. Natl. Acad. Sci. U.S.A. 71:1342-1346.

Spiegel, S. and Martin, R.R.1993.Improved detection of potato leafroll virus in dormant potato tubers and microtubers by the polymerase chain reaction and ELISA. Ann. App!. Biol. 122:493-500.

Stace-Smith, R.1 955a. Studies on Rubus virus diseases in British Columbia. I. Rubus yellow-net. Can. J. Bot. 33:267-274. Stace-Smith, R. 1955b. Studies on Rubus virus diseases in British Columbia. II. Black raspberry necrosis. Can. J. Bot. 33:314-322.

Stace-Smith, R. 1956.Studies onRubusvirus disease in British Columbia. III. Separation of components of raspberry mosaic. Can. J. Bot. 34:435-442.

Stace-Smith, R. 1984. CMIIAAB Descr. P1. Viruses No. 290.

Thompson J., Higgins D., Gibson T., Thompson J.D., Higgins D.G. and Gibson, T.J. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22 :4673-4680.

Thompson, J.R., Leone, G., Lindner, J.L., Jelkmann, W. and Schoen, C.D. 2002 Characterization and complete nucleotide sequence of Strawberry mottle virus:A tentative member of a new family of bipartite plant picorna-like viruses.J. Gen. Virol. 83:229-239.

Weber, C.A.2003.Genetic Diversity in Black Raspberry(Rubus occidentalisL.) Detected by RAPD Markers. HortScience 38:269-272.

Yoshikawa, N. and Converse, R.H. 1990. Strawberry pallidosis disease: distinctive dsRNA species associated with latent infections in indicators and in diseased strawberry cultivars. Phytopathology 80:543-548. 70

CHAPTER 3: EPIDEMIOLOGY AND ECOLOGY OF BLACK RASPBERRY DECLINE ASSOCIATED VIRUS

ABSTRACT

Four newly planted fields of black raspberries(Rubus occidentalis)in Oregon were studied for three years to assess the rate of spread of Black raspberry decline associated virus (BRDaV). In an effort to characterize the suspected complexity of synergistic interactions between BRDaV and other Rzibus-infecting viruses, the prevalence of ten additionalRubusviruses was also monitored in the study fields. The timing of BRDaV infection as it relates to aphid populations and flights was also determined. Testing of nearby vegetation identified several symptomlessRubushosts of BRDaV, as well as detection in multiple cultivars of black raspberry and several non-Rubus weeds.It was determined that BRDaV spreads rapidly with a low aphid threshold and was associated with symptoms of decline in black raspberries in Oregon.

INTRODUCTION

Oregon is the largest producer of black raspberries in the United States (Oregon Agricultural Statistics Service, 2005).Despite the region's success in growing black raspberries, or "blackcaps", however, Black raspberry decline (BRD) is an increasing problem for growers. The serious decline problem in black raspberry in Oregon was brought to our attention in 1996 when the USDA-ARS small fruit virology program was reestablished in Corvallis, OR. BRD has a significant impact on the yield and vigor of black raspberry plants. Leaves may become puckered with foliar chlorosis and mosaic in the cooler spring and fall (Fig. 3. la), followed by cane dieback (Fig. 3.lb).As the disease progresses, there is yield decline due to increased death of fruiting canes. A healthy black raspberry planting can last several decades, yet most growers in Oregon are replacing affected fields every 3-4 years due to BRD. 71

Fig. 3.la: Leaves infected with Black raspberry decline associated virus that show symptoms of mottling, chiorosis, and puckering.

Fig. 3.lb: Dieback of fruiting canes of black raspberry. 72

One virus consistently was associated with declining black raspberries, and was named Black raspberry decline associated virus (BRDaV; see chapter 2). The large raspberry aphid Amphorophora agathonica (Fig. 3.2) vectors this virus, along with several other known viruses associated with raspberry mosaic disease. Black raspberry decline may be a component of raspberry mosaic disease described previously (Stace-Smith, 1956) and caused by a complex of Rubus yellow net virus (RYNV) (Stace-Smith, 1955a) and Black raspberry necrosis virus (BRNV) in North America (Stace-Smith, 1955b), and Raspberry leaf mottle (Cadman, 1951) and Raspberry leaf spot viruses in Europe (Cadman, 1952).Other than RYNV (Jones et al., 2002), viruses of the mosaic disease complex remain uncharacterized in terms of particle morphology, serology and nucleotide sequence.

-

Fig. 3.2: Nymphs of the large raspberry aphid Amphorophora agathonica, wingless form. 73

There are more than 30 known viruses and virus-like diseases that infect Rubus (Martin, 2002), some of which have been characterized for many years, others that have been discovered only recently, and several that still are characterized only in terms of vectors and symptoms. The two main black raspberry viruses in Oregon (Martin, 1999), are the pollen-borne Raspberiy bushy dwarf virus (RB DV) (Murant,1976), genus Idaeovirus, and Strawberry necrotic shock virus (SNSV) (Frazier et al., 1962), formerly known as Tobacco streak virus (Tzanetakis et al., 2004), genus Ilarvirus. The incidence of these viruses in black raspberry fields in Oregon exceeds 50% (Martin, 1999). Tomato ringspot virus (ToRSV) and Tobacco ringspot virus (TRSV) are nepoviruses transmitted by the nematode Xiphinema americanum. ToRSV is the most economically important of the nematode-transmitted viruses of Rubus in North America (Stace-Smith and Converse, 1987). TRSV has been reported in blackberry (Rush and Gooding, 1970) but not in red or black raspberry. Several other viruses of Rubus have been characterized recently at the molecular level, but their effect on black raspberry and other Rubus species has not been studied. One such virus, Blackberry yellow vein associated virus (BYVaV) has been associated with vein yellowing and mosaic in blackberry in the southeastern United States (Martin et al., 2004), but is likely part of a virus complex that causes the symptoms in the field (Susaimuthu et a!, 2005). The effects of several other black raspberry viruses in the field remain unknown, including that of Raspberry leaf spot virus-North America (RLS V-NA), a multipartite aphid-transmitted virus (with no relation to the RLSV of the mosaic complex from Europe) (Jelkmann and Martin, 1988); a closterovirus tentatively named Raspberry mottle associated virus (RMaV) and an insect-like virus from 'Glen Clova' red raspberry (Martin and Tzanetakis, in press, a); a newly identified flexivirus from blackberry (Martin and Tzanetakis, in press, a); and a novel virus originally isolated from black raspberry from the USDA-ARS National Clonal Germplasm Repository, in Corvallis, Oregon, with homology to the fungi-infecting Totiviruses (Halgren et al., 2005). Although there are varying levels of information on each of the above-described viruses, no studies have examined their incidence in black raspberry fields. Development and implementation of strategies for control of viruses relies on an 74 understanding of virus vectors and virus reservoirs.Symptomless hosts can serve as sources of inoculum. These alternate hosts could be either weeds or cropplants. Thus, it is important to identify and eliminate such hosts in the case of weeds, or if possible isolate the growing site from alternate crop hosts. Most often, the use of chemical insecticides is the first method used to control insect-vectored plant viruses. However, the application of insecticides to manage the spread of aphid-borne viruses can be expensive and ineffective if sprays are not well timed. Raspberry aphids may be winged or wingless, the former type emerging in spring and re-appearing in late summer, and the latter predominating through most of the summer.Infestations of the large raspberry aphid can vary from year to year or geographically illustrating the unpredictability of this insect pest and the need for aphid monitoring. Although ultimate virus control resides in the use of virus- and/or vector- resistant cultivars, the predominant black raspberry cultivar 'Munger' grown in the Pacific Northwest is susceptible to BRD and the large raspberry aphid, the major vector of BRDaV. Thus, until replacement cultivars with resistance or tolerance are developed that are equally superior in taste and amenable to mechanical harvest, control of virus spread will continue to rely on pest management and elimination of virus reservoirs. The primary objective of the research presented here was to assess the distribution and incidence of BRDaV, identify viruses that are associated consistently with symptoms, and determine the timing of infection as it relates to aphid populations and flights. 75

MATERIALS AND METHODS

Field design Monitoring of newly planted fields Monitoring aphid populations and virus incidence in three black raspberry fields located southwest of Portland, Ore. began in 2003. These fields had been planted in late spring and early summer of 2003, in a north-south row orientation, using nursery-grown, tissue culture-derived plants as source material.Plants were virus-free when planted, but disease pressure was high, as each field was planted either adjacent to or within close proximity to mature, symptomatic, BRDaV-positive black raspberries.Field 1 was planted east of a red raspberry field with low incidence of BRDaV and was buffered by a blueberry field from a black raspberry field further east with nearly 100% incidence of BRDaV. Field 2 was planted to the east of a 2-year-old 'Munger' field, with over 50% incidence of BRDaV.Field 3 was planted west of a 2-year-old BRDaV infected 'Munger' field with over 50% incidence of BRDaV. Immediately following planting, 16 plants in a 4x4-grid pattern were tagged and labeled in each field (Fig. 3.3). All plants tested negative in all virus assays at the beginning of the trial and were chosen solely on their representative position in the field.Additionally, yellow ScoutTM sticky traps (OlympicHorticultural Products) were placed at canopy level in an"x"formation across each field, in order to monitor the population of potential vectors (Fig. 3.3 and Fig. 3.4). On a bi-weekly basis in the spring and summer of 2003 and 2004, and on a monthly basis in the spring, summer, and fall of 2005, several leaves, representative of both the primocanes and the fruiting canes (when present) were collected from each of the tagged plants. Furthermore, leaves were collected from several positions along the length of the cane, in order to ensure that if virus were present it would be represented in the sample. Tissue for analysis was stored at 4°C until testing.Sticky traps were collected and replaced at the time of leaf collection. All aphids, regardless of species, were counted, and an average was taken for each field.This average would represent the average number of winged aphids caught since the previous collection, and would be considered 76 alongside the number of plants testing positive since the last collection. Sticky cards that had fallen or were damaged during harvest were not included. Average daily temperature for these fields was compiled from Oregon Climate Service for the city of Forest Grove, Ore.

Time of transmission study A second series of field experiments was carried out in a fourth field, "Field 4" near Sandy, Ore., southeast of Portland. This 12-acre field was plantedwith cuttings of BRDaV-infected plants.Thus, disease pressure within the field was intense.In the spring and summer of 2003, 2004, and spring, summer, and fall of 2005, 16 virus-tested, tissue-culture-derived, potted black raspberry plants were placed in this field and replaced with new plants every two weeks. The test plants were placed within the rows of the permanent plants, in a grid-like formation similar to that of the previously described fields.Aphid populations in the field were monitored using 12 sticky traps that were placed within the field and collected with each set of plants. After two weeks in the field, plants were returned to the lab, where they were treated with either granular or foliar MarathonTM insecticide, and stored outside. Seven sets of plants from the 2003 season, including one set which overwintered in the field, and 12 sets of plants from 2004, including one field overwintering set, were tested in May and October 2005. Fourteen sets of plants from 2005 were tested in November 2005.All plants were tested for 11 Rubusviruses, as described below. Average daily temperature for Field 4 was compiled from Weather Underground, Inc., for the city of Boring, Ore. P L H D

0 K G C

N J F B

M I E A

Fig 3.3: Schematic of experimental field set-up. Each of the 16 plants sampled is represented bya letter "A" though "P". Yellow sticky traps, represented by a yellow box above, form a field-wide "X". Only in Field 1were sampled plants set up in border rows, as this particular field was limited to four rows. 78

Fig. 3.4: Sticky trap for monitoring aphids.

Cultivation practices Growers treated fields as though no experiments were taking place. Tables 3.1 a and 3. lb list the chemical treatments and target organisms of insecticides.Thus, our resulting models of virus transmission are based on fields undergoing several typical management schemes.

Detection The timing and extent of BRDaV infection and the testing for 10 otherRubus viruses was conducted via reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). RT-PCR was used for detection of BRDaV and six other newly described viruses for which no antibodies exist, and ELISA was used for the remaining four viruses. Only plants from the 'Time of transmission' experiment (Field 4) and the samples from 2005 in the 'Monitoring of newly planted fields' experiment were tested for the additional 10 viruses. All plants from 2003 and 2004 Field 4 were tested twice after overwintering, and the 2005 samples from Field 4 were tested once. 79

Table 3.la. Number of treatments applied to Fields 1, 2, and 3 during the growing seasons of 2003, 2004, and 2005.

Field 1 Field 2 Field 3 Application typea Field season Field season Field season 2003 20042005200320042005 200320042005 Fungicide 1 2 4 4 8 4 4 5 Herbicide 2 2 2 3 4 6 3 3 2 Insecticideb 1 5 2 4 2 2 3

1 aGrowers applied treatmentsas they normally would, without regard to theexperiment. blnsecticidesprays and their target pests, in parentheses, were as follows: Field 1- Malathion (moth, aphid) applied 6/03. Field 2- Azinphos-Methyl (broad-spectrum) applied 7/17/03, 7/31/03, 9/4/03; Diazinon (broad-spectrum) applied 8/12/03, 9/4/03; Bacillus Thuringiensis (leafroller) applied 5/11/04, Diazinon applied 3/15/04, Fenamiphos Nematicide applied 1/5/04; Esfenvalerate (broad-spectrum) applied 6/11/05, Bacillus Thuringiensis applied 5/22/05, Diazinon applied 3/3/05, and 0,0-Dimethyl S- [(4-oxo 1 ,2,3-benzotriazin-3(4H)-yl)methyl]phosphorodithioate (Guthion Solupak) (broad-spectrum) applied 7/15/05. Field 3- Bacillus Thuringiensis applied 5/15/03, 6/10/03; Bacillus Thuringiensis applied 5/13/04, 6/1/04; Bacillus Thuringiensis applied 5/6/05, Imidacloprid (broad-spectrum) and Spinosad (broad-spectrum) applied 6/4/05.

Table 3.lb. Number of treatments applied to Field 4 during the growing seasons of 2003, 2004, and 2005.

Field 4 Application typea Field season 2003 2004 2005 Fungicide 6 3 4 Herbicide 5 3 4 Insecticideb 0 0 1 aGrowers applied treatmentsas they normally would, without regard to theexperiment. blnsecticidesprays and their target pests, in parentheses, were as follows: Diazinon (broad-spectrum) applied 2/28/05. Total RNA was extracted from plant samples for use in RT-PCR detection tests using a modified Spiegel and Martin (1993) protocol.100 mg of combined leaf tissue (representative of all points of collection from a sample plant) was homongenized in one mL extraction buffer (200 mM Tris base, pH 8.5, 300 mM lithium chloride, 1.5% lithium dodecyl sulfate, 10 mM EDTA, 1% deoxycholic acid, 2% PVP, and 1% NP 40 "Tergitol"). fE5-mercaptoethanol was added to create a I % final concentration just before use. An equal amount of potassium acetate (2.8 M with respect to potassiumand 6 M with respect to acetate, pH 6.5) was added to the extract and chilled at 20°C for at least 30 mm.After thawing, the mixture was centrifuged at 16,000 g for 10 mmin a microfuge and 0.7 ml of the supernatant transferred to a new tube. An equal amount of isopropanol was added and mixed via inversion and centrifuged for 20 mmat 16,000 g. Finally, the pellet was washed twice with cold 70% EtOH and dried briefly under vacuum. The dried pellet was resuspended in 40 jtl of RNase-free water. For first-strand DNA synthesis, total RNA constituted 1- 5% of the reaction volume. Typically, a reaction would consist of 1tL of total RNA, 0.5 iL of 300 ng!.iL random primers, 4 tL of 5X eDNA Synthesis Buffer (250 mM Tris acetate, pH 8.4 at 25°C, 375 mM potassium acetate, 40 mM magnesium acetate), 2 iL of 10 mM dNTPs, 2 tL of 100 mM DTT, 0.4tL (16U) RNase OUT (Invitrogen), and 0.3j.iL (60 U) SuperScript III RT (Invitrogen) in a final volume of 20L. The reaction was incubated for one hour at 50°C. The RT product constituted no greater than 4% of the total PCR reaction volume to prevent inhibition from plant secondary metabolites carried over from the RNA. PCR reactions consisted of 1tL of RT reaction, 0.8 tL of 10 mM dNTPs, loxGenscript Buffer (500 mM KCI, 100 mM Tris HC1 (pH 9.0 at 25°C), 15 mMMgC12, 1% Triton X-100), 0.25 jiL each of forward and reverse primers (40 tM each), and 0.25 tL (1.25 U) Taq Polymerase (Genscript) in a total volume of 25 !IL. Based on BRDaV sequence obtained previously (Chapter 2), two sets of primers were designedand used in tandem for detection (Table 3.2) to minimize false positives due to sequence variation. Primers based on other newly described viruses of unknown etiology, encompassing RLSV-NA (Jelkmann and Martin, 1988), RMaV, 'Glen Clova' insect-like ('GC-I'), Table 3.2:Rubusvirus detection primers used for reverse-transcriptase polymerase chain reaction (RT-PCR).

Virus AbbreviationPrimersa Expected Amplicon Size Black raspberry BRDaV lF: ATG CTG AGC CAC TTG TGA 417bp decline associated 1R: ATC TGG TGT GTT CCG CAT virus 2F: CAA TGT CU GGA AGC CAC 350bp 2R: AGC ATG GTT CGT CAT CTG

Raspberry leaf spot RLSV-NA iF: CCA CGC CCA ACC TCC AAA TAA 465bp virus-North 1R: ACC CCG TCT CCC CAT CTG C Americanvirus Raspberry mottle RMaV GC-HSPF: CGA AAC TTY TAC GGG GAA C 452bp associated virus GC-HSPR: CCT TTG AAY TCT TTA ACA TCG T

GC-P6OF: TTT GCA CTG CTC GAA G 380bp GC-P6OR: GTG AGC GGG AGT GGA

aprimersequences are listed 5' -3'. Some viruses have several primer detection pairs asnoted. All primers anneal at 5 5°C. Table 3.2: (Continued)

Virus AbbreviationPrimersa Expected Amplicon Size 'Glen Clova' GC-I GC-I IF: GCC ATC ACT AAC AGG CA 450bp insect-like virus GC-I 1R: CTC GCT GAC GGG TCA TA

GC-I 2F: GGA TCG ACG AAG GAA T 450bp GC-I 2R: GCT ACA CCT CGG TAA CT

Blackberry yellow BYVaV BYVaV-CP F: AAT CAA CGG GAG AAT GTT AT 458bp vein associated BYVaV-CPR: GGA TTG GCA ACG TCC G virus BYVaV-polF: CTT CAG CAG AGA GAT TGG TG 318bp BYVaV-polR: CCA AGA ACA TCG GCG AGT

Totivirus-like Toti iF: CAC GCA TTA CAC GCC C 573bp 1R: CTG TGC CGG TAG TCT T

2F: AAG ACT ACC GGC ACA G 514bp 2R: GTT AAT CGC CAT CCC GAA A

Blackberry BB Flexi 2F: CAC CTA GCA GCC TTG A 400bp flexivirus 2R: TGG TTT GAC CAG CGA T sequences are listed 5' -3'. Some viruses have severalprimer detection pairs as noted. All primers anneal at 55° blackberry flexivirus ('BB-Flexi') (Martin and Tzanetakis, in press, a), BYVaV (Martin et at., 2004), black raspberry Totivirus ('Toti') (Haigren et al., 2005) are alsolisted in Table 3.2. All detection programs consisted of initial denaturation for 5 mmat 94°C followed by 40 cycles with denaturation for 30 sec at 94°C, annealing for 45 sec at55°C and extension for 30 sec at 72°C, with a final 10-minute extension step at72°C. The samples were subjected to electrophoresis through agarose gels containing 10 tg/ml of ethidium bromide and amplicons visualized by exposure to a UV light. Amplification of the highly conserved plant gene NADH dehydrogenase ND2 subunit (ndhB gene) was used as an internal control to verify the quality of the RNA extraction and effectiveness of the RT-PCR (Martin et al., in press, b). To verify the amplification of the viral genes, over 20 PCR products were sequenced. All PCR reactions werecarried out using a Robocycler (Stratagene) thermocycler. Samples were tested for four viruses by ELISA (Table 3.3).Triple antibody sandwich tests were performed for RBDV and SNSV using antibodies and protocols as described by Martin (1999). Double antibody sandwich tests were used for detection of ToRSV and TRSV as described previously (Clark and Adams, 1977).

Table 3.3: Rubus viruses tested for by ELISA.

Virus Abbreviation Type of ELISAa Raspberry Bushy Dwaif RBDV TAS Virus Virus______Strawberry Necrotic Shock SNSV TAS Tomato Ringspot Virus ToRSV DAS

Tobacco ringspot virus TRSV DAS

DAS = Double antibody sandwich; TAS= Triple antibody sandwich. Alternate host and cultivar sampling Samples were collected from plants growing near fields of BRDaV-infected black raspberry.Three plant categories were considered: commercial Rubus fields (i.e. red raspberry, blackberry or hybrid berries) adjacent to infected black raspberry, volunteer and native Rubus growing in woody areas and in hedges surrounding infected fields, and a range of weed species common within the fields (Table 3.4). Samples werecollected at several times throughout the growing season, and were processed as described above. To determine if resistance or tolerance to BRDaV exists in black raspberry, nine additional cultivars grown in a test block along the eastern border of Field 2 were sampled (Table 3.5).There were between 5-10 plants representing each cultivar, and some plants of each cultivar exhibited varying degrees of symptoms.Leaves were collected from several points on both the primocanes and fruiting canes on the most symptomatic plant of each cultivar. Samples were collected on June 6 and October 6, 2005, and tested for BRDaV as well as the 10 other Rubus viruses described above.

Table 3.4: Non-Rubus weeds tested for Black raspberry decline associated virus by RT- PCR.

pecies Family Common Name Sonc/zus arvensis Asteraceae Perennial sowthistle* Sonchüs asper Spiny sowthistle Taraxacum officinale Dandelion Cirsium vulgare " Bull thistle Pteridium aquilinum var. Dennstaedtiaceae Western brackenfern* pubescens Trfolium repens Fabaceae White clover Malva neglecta Malvaceae Common mallow Ruinexcrispus Polygonaceae Curly dock Galium aparine Rubiaceae Catchweed bedstraw * Indicates at least one plant of this species tested positive for BRDaV. Table 3.5: Testing of nine alternate black raspberry cultivars for 11Rubusviruses.

, I111 fli'ii' I 1 1t'AFt'IT 1A 1 :1 P1' 'p

Ik.T Ike Ikei Tfl11 T fl1T Ii Ik.i Iksi

I IITrT . lks Ii lks Ikei Iks

Iks Iki

Iksi

IrJ,r,JiI Iks Ik. *'pos' indicates at least one of the two samples tested from each cultivar tested positive for specified virus. Statistical analyses Regression analysis was used to test the following relationships: number of plants testing BRDaV-positive versus aphid numbers, and maximum daily temperature versus aphid numbers. Tukey-Kramer family-wise multiple comparison analysis of variance, P<0.05, was used to compare between mean numbers of aphids collected in each field in each season. For all analyses, S-Plus version 7.0 (Insightful Corporation) was used.

Reference isolates ofRubusviruses The Scottish Crop Research Institute (SCRI) (Invergowrie, Scotland) contains a collection ofRubusvirus isolates. Many of these viruses have been described solely on the basis of symptom expression, experimental host range and vector relations. The sets of primers described above were used to test desiccatedRubusleaf tissue from plants infected with various viruses obtained from Dr. H. Barker at 5CR!, in an attempt to correlate the recently cloned virus sequences with previously described virus diseases. Desiccated plant tissue was reconstituted in RNA extraction buffer at a 1:50 tissue to buffer ratio and tested via RT-PCR for a range ofRubusviruses as described above. RESULTS

BRDaV distribution in newly planted fields In 2003 plants were sampled June 6, June 20, July 11, and August 5, though two weeks prior to the initial sampling, sticky traps were set out, and the plants underwent an initial test to ensure all were BRDaV-free at the beginning of the trials.The alate (winged) aphid population, as represented by those caught on traps, declined afterthe first sampling date in Fields I and 2, and after the second sampling date in Field 3 (Fig. 3.5a). Apterous (non-winged) aphids were only observed on plants during the June20 sampling. Only one plant total, in field 1, tested positive for BRDaV during the 2003 field season (Fig. 3.5b). During the 2004 growing season plants were sampled eight times: May 4, May 19, June 2, June 17, July 6, July 20, August 3, and August 18. Aphid numbers on traps increased until mid June, and then declined (Fig. 3 .6a). Apterous aphids wereobserved on all but the first and last sampling dates. In fields 1, 2and 3, respectively 14/16, 13/16, and 11/16 plants tested positive (Fig. 3.6b). In 2005 plants were sampled 6 times on a monthly basis, on May 4, June 6, June 30, August 5, September 1, and October 6. Aphid traps were set out in earlyMarch to monitor the emergence of alates, which peaked between May 4 and June 6 (Fig. 3.7a). Field 1 had 100% infection by the third sampling date (June 30) (Fig. 3.7b). Soon after this date, this field was plowed under and thus no more samples could be obtained. However, several traps from the field border were recovered, and therefore a datapoint exists for the following collection date. In Field 2 the incidence of infection with BRDaV reached 100% by the end of the field season. In Field 3, which had the lowest aphid numbers throughout the study, the incidence of infection with BRDaV at the end of the 3rdseason was 81%. Average Aphids/Trap: 2003

16 100 14

90 LI. 12 0 10

8 4 0) 6

(5 1 4 C) ) 60 4 2 0 50 >1 Dl (5 S S S ---Fieldl I I I I I N 0 1(1 Field2 'I. N 1I N C N N Field 3 Date Max Daily Temp

Fig. 3.5a:Average aphids caught per trap (plotted on the10Y axis) vs. daily maximum temperature, represented as 14-day moving average (plotted on the 2° Y axis) for each collecting trip for Fields 1, 2, and 3 in 2003. Average aphid # corresponds to the average # of aphids collected in a particular field since the previous collecting trip. For the first data point, sticky traps had been set out 2 weeks earlier. Field 1 Field 2 Field 3

Fig. 3.5b: End of the growing-season (2003) summary-diagrams of each of the 3 fields. Lines representrows. Circles represent the 16 test plants, in the order described in Fig. 3.3. Green circles represent plants that tested BRDaV-negative throughout theyear. Red circles cumulatively represent those plants that tested positive for BRDaV atsome point during the year. Average Aphids/Trap: 2004

14 LW) 12 (5 90 I- U. i_10 U) 80 E a, .c I- 46 70 = a, (5

a, 60

0 50 >. >. > C C C C (5 (5 (5 jj 5 5 I-) I-) I- I) I-) P , i 4 4 4 I I I N Cl ( 0 1 '.4 '--Fieldi '.4 '.4 N m 'o m 0 N N N '.4 NUField2 Field 3 Date Max Daily Temp

Fig. 3.6a:Average aphids caught per trap (plotted on the 1° Y axis) vs. daily maximum temperature, represented as 14-day moving average (plotted on the 2° Y axis) for each collecting trip for Fields 1, 2, and 3 in 2004. Average aphid # corresponds to the average # of aphids collected in a particular field since the previous collecting trip. For the first data point, sticky traps had been set out 2 weeks earlier. Fig. 3.6b: End of the growing-season (2004) summary-diagrams of each of the 3 fields. Lines represent rows. Circles represent the 16 test plants, in the order described in Fig. 3.3. Green circles represent plants that tested BRDaV-negative throughout the year. Red circles cumulatively represent those plants that tested positive for BRDaV at some point during the year. Average Aphids/Trap: 2005

100

L.5(5 I- 90

(1

<3 a) rs2 I- a) <1> 60

[I] (5(5(5I- I- I- I- I- - I- . (5(5(5(5 a) a)a)a) --FieIdl I I I i u u u u i i u Co Ifl .D m 0 i-I Co II II 0 -4NNCo i-i N NNN C Nl-lNQ -4,INm' ---Field 2 Field 3 Date Max Daily Temp

Fig. 3.7a: Average aphids caught per trap (plotted on the10Y axis) vs. daily maximum temperature, represented as 14-day moving average (plotted on the 2° Y axis) for each collecting trip for Fields 1, 2, and 3 in 2005. Average aphid # corresponds to the average # of aphids collected in a particular field since the previous collecting trip. For the first data point, sticky traps had been set out 2 weeks earlier.

'C Field 1 Field 2

Fig. 3.7b: End of the growing-season (2005) summary-diagrams of each of the 3 fields. Lines representrows. Circles represent the 16 test plants, in the order described in Fig. 3.3. Green circles represent plants that tested BRDaV-negative throughout theyear. Red circles cumulatively represent those plants that tested positive for BRDaV at some point during theyear. Timin2 of BRDaV infection During the summer of 2003, aphid numbers on traps peaked in late-May, then steadily declined (Fig. 3.8a). Regardless of the decline, infection of experimental plants continued through the season.Alate aphids were present on the traps throughout the testing season but apterous aphids were observed on the plants only on June 25. Collection dates of each rotation and number of plants (out of 16) testing positive were as follows: May 16: 6, May 30: 8, June 13: 6, June 25: 11, July 11: 5, and July 28: 1 (Fig. 3.8b). Similarly, in 2004 aphid numbers on traps peaked in early to mid June, but infection occurred throughout the test period (Fig. 3.9a).Collection dates of each rotation and number of plants (out of 16) testing positive are as follows: June 2: 14, June 17: 11, July 6:9, July 20:6, August 3:2, August 18:2, September 2:6, September 16:2, September 30: 4, October 14: 1, and October 28: 1 (Fig. 3.9b). Apterous aphids were observed in the field on only the first 4 rotation collection dates. In 2005, traps were set up just once per month in early March and early April, but then were collected on a biweekly basis throughout the rest of the season with each plant rotation. The earliest traps revealed that aphid flights began in early to mid April, though the apterous Amphorophora agathonica was not observed on black raspberry leaves until late June (Fig. 3.1 Oa).Many fewer plants tested positive this year compared to the previous 2 years (Fig. 3.lOb). Plant rotation collection dates and corresponding number of positives are as follows: April 20: 0, May 4: 0, May 18: 1, June 6: 0, June 20: 0, June 30: 0, July 14: 0, August 5: 0, August 18: 0, September 1: 0, September 16: 12, October 6: 5, October 20: 0, and November 8: 1. Average Aphids/Trap: 2003

4 100

D. 3.5 (5 I.. 90 ii. I 3

! 2.5

C, o 1.5

1 E I 0.5 0 50 >1 >' >" >. >. C C C C 5 5 (5 (5 (5 (5 (5 i- i-

' '.4 I I I I I m In N Ci D ,1 In N '-I N i-I N m -I N N iI Average Aphids: 2003 Date -Max Daily Terrp

Fig. 3.8a: 2003 average aphids caught per trap (plotted on the 1° Y axis) vs. daily maximum temperature, represented as 14-day moving average (plotted on the 2° Y axis) for Field 4. Average Aphids/Trap vs. Positives: 2003

4 16 # of BRDaV positi.es 3.5 Aerage Aphids: 2003 14

0. U) 3 ------25Jon 12 4) >

2.5 10 U) 0 30-May Q. 8 > U) 16-May 1 3-Jun 1.5 6 I >a, < 1 4

0.5 2 I I I I I

A 0 >1 >. >. >. c c c c

? -? -? ? 4 in 0 F.. 1 C1 ' C4 C1

Date

Fig. 3.8b: 2003 average aphids caught per trap (line graph, plotted on the 1° Y axis) vs. # of plants (out of 16) for eachrotation testing positive for BRDaV (histogram, plotted on the 2° Y axis) for Field 4. Average Aphids/Trap: 2004

VA 100

6 I- I U.

. 80 I- >. ) 3 70 (5 2 > x 60 1

0 " ccc U U U U (5(5(5(5 Q) WQ) w 00 I I I I ifl N C .D . I I I I I A . , i1 in ON'I rINN NNCDmODmON"4N m ,l NN '4 'l N Average Aphids: 2004 Date Max Daily Terrp

Fig. 3.9a:2004 average aphids caught per trap (plotted on the 1° Y axis) vs. daily maximum temperature, represented as 14-day moving average (plotted on the 2° Y axis) for Field 4.

-1 Average Aphids/Trap vs. Positives:2004

7 16 #of posith.es 2-Jun Arage Aphids:2004 6 14

U) 17-Jun 12 ø 5

:- . 6-Jul 10 a. a- 0

c. 3 20-Jul ------2-Sep ------6 2 4 3-Aug 18-Aug 16-Sep 1 ;oect 2 0

Date

Fig. 3.9b: 2004 average aphids caught per trap (line graph, plotted on the10Y axis) vs. # of plants (out of 16) for each rotation testing positive for BRDaV (histogram, plotted on the 2° Y axis) for Field 4. Average Aphids/Trap: 2005 100 a.6 U-

I- 1-5 a. U,, E 4 80 0) I- a. >. 70 '-2 x > 60 1

ri 50 c > a.a.a.a. 55 0)0) 0

I I

I I I nis9999z i u i I I i i i -INm Date Average Aphids: 2005 Max Daily Temp

Fig. 3.lOa: 2005 average aphids caught per trap (plotted on the 1° Y axis) vs. daily maximum temperature, represented as 14-day moving average (plotted on the 2° Y axis) for Field 4. Average Aphids/Trap vs. Positives: 2005

7 16 # of posiths 2005 Average Aphids 14 16-Sep 1' I >

1---6r------

1

riu 0 L L - - l - - I. CC C555 > > > c c c c a)a) 000 o11 rc0.ZZZ i-C'1Cs1 iC\1 -C1C) TC\1C1 -C\1C) -

Date

Fig. 3.lOb: 2005 average aphids caught per trap (line graph, plotted on the 1° Y axis) vs. # of plants (out of 16) for each rotation testing positive for BRDaV (histogram, plotted on the 2° Y axis) for Field 4. C 101

Infection with otherRubusviruses RBDV and SNSV reached very high rates of infection in the newly planted fields, which is consistent with their reported rapid rate of spread (Bulger et al., 1990; Converse, 1980). Field 1 had 14/16 and 7/16 positives for RBDV and SNSV, respectively, before it was plowed under. This field showed the greatest overall decline. Fields 2 and 3 both were 100% infected with RBDV by the end of testing in 2005, and had 13/16 and 16/16 positive plants, respectively, for SNSV (Table 3.6). However, Field 3 showed the least overall field-wide symptoms of decline.Whether these two viruses play a role in enhancing symptoms of BRDaV is unclear. RLSV-NA was found in 2, 2, and 6 of the 16 plants in Fields 1, 2, and 3 respectively, and in two of the trap plants from Field 4 (Table 3.7). 'Toti' was detected in 1, 2, and 2 of the 16 plants in Fields 1, 2, and 3 respectively, and three trap plants in Field 4 in 2004. GC-I was not detected in any of the newly planted fields, and RMaV was detected in one plant. In the time of infection study, Field 4, GC-I was detected in one plant, and RMaV was detected in four plants; both of these viruses were detected in the 2004 trap plants. 'BB-flexi' was found in moderate numbers in all fields, but only in 2004. BYVaV, TRSV, and ToRSV were not detected in any of the fields. Table 3.6: Cumulative viruses acquired by the final sampling date in 2005.

BRDaV SNSVTotiRLSVRMaVGC-IBYVaVRBDVBB TRSVToRSV -NA Flexi Field 16 7 1 2 14 6 1*

Field 16 13 2 2 1 16 8 2* Field 13 16 2 6 16 4 3* *Fields were only tested for viruses in addition to BRDaV in 2005. Numbers represent how many plants (out of 16 for each field) acquired a virus. Table 3.7: Viruses acquired in the 'Timing of Jnfection' experiment for each plant set rotated through Field 4.

Set 3RDaVSNSV Foti RLSV- MaVGC-I BYVaVRBDV B Flexi1'RSV foRSV NA 2003* 1 6 8 1 6

1 11 5 5 6 1 W04* 1 14 2 2 11 3 9 2 1 6 5 6 2 7 6 1 1 1 8 2 1 7 1 1 4

10 1 1 3 11 1 1 3 *Sets from 2003 and 2004 were tested twice for all viruses in May and October of 2005, while sets from 2005 were tested only once, in November 2005. Table 3.7: (Continued)

Set BRDaVSNSV [oti ELSV-EMaVGC-I BYVaV BDV 3B FlexiRSV roRsv

005* 1

3

1 1 5 6 7 8 ) 10 11 12 12 5 13 14 1 * Sets from 2003 and 2004 were tested twice for all viruses in May and October of 2005, while sets from 2005 were tested only once, in November 2005. 105

Statistical analyses There was no evidence of a linear correlation between maximum daily temperature and aphid number (R2=0.008, y=0.0289x + 0.542, p-value = 0.3926 on 82 D. F.) When testing the relationship between aphid number and the number of BRDaV- positive plants, only the 2003 and 2004 field seasons of Field 4 were included. These plants overwintered prior to testing, unlike those from the 2005 field season.Over- wintering may be important for virus titers to reach levels for reliable detection. For this reason the 2005 field season was not included in the analysis. Similarly, in Fields 1-3, there was no precise method to determine when a plant became infected, and thus no valid statistical correlation can be made between aphid numbers on the traps and rates of virus infection. Given these parameters, there was a statistically significant relationship between aphid number and number of BRDaV-positive plants for the 2003 and 2004 plants of Field 4 (R2= 0.4338, y=l.7587x + 2.6198, p-value = 0.004 on 15 D. F.). There was no evidence that any one field's mean aphid number over the course of a growing season differed from any other field's mean aphid number.

Alternate host and cultivar sampling BRDaV was found to be symptomless in several commercial Rubus species commonly grown alongside black raspberry, including red raspberry (R.idaeus), Evergreen blackberry (R. laciniatus), and 'Marion' blackberry (a complex hybrid with R. idaeus, R. armeniacus, and R. ursinus in its background). BRDaV was also found in Himalaya blackberry (R. armeniacus), a weedy Rubus that commonly grows in the borders of fields, and Pacific blackberry (R. ursinus) found growing in field perimeters and hedgerows. Two of the nine non-Rubus in-field weeds tested (Table 3.4), perennial sowthistle (Sonchus arvensis) and western brackenfern (Pteridium aquilinum var. pubescens) tested positive for BRDaV. As western brackenfern seemed such an unusual host, being from such a disparate plant family (Dennstaedtiaceae), it was sampled multiple times.Multiple tests from fern collected in two different fields from several collecting dates confirmed fern in fact to be an alternate host for BRDaV. 106

'John Robertson' was the only cultivar of black raspberry tested that did not test positive for BRDaV on either of the two sampling dates.However, since 'John Robertson' was symptomatic, it is likely that either plant inhibitors interfered with detection, or sampling was insufficient. SNSV and RBDV were found in high incidence among the alternate cultivars.Whereas the Toti virus, RLSV-NA, RMaV, and 'BB- Flexi' were all detected within the adjacent 'Munger', only RLSV-NA and 'BB-Flexi' appeared in the alternate cultivars only in 'Dundee' (Table 3.5).

Testing of reference isolates Four samples were received from SCRI, described as having Raspberry leaf mottle virus (RLMV), Raspberry leaf spot virus (RLSV), Raspberry vein chlorosis virus (RVCV), and Hawaiian rubus leaf curl virus (HRLCV). The sample containing HRLCV tested negative for sevenRubusviruses in RT-PCR tests.The RLMV, RLSV, and RVCV samples all tested positive for RMaV using the GC-P60 primers.Testing of reference isolates of previously described raspberry mosaic disease complex viruses from for BRDaV is underway.

DISCUSSION

Considering the disparity between plants testing positive for BRDaV at the end of 2003 and the beginning of the 2004 field season in Fields 1, 2, and 3, it is likely that inoculation of plants occurred in 2003, but BRDaV titer did not reach detectable levels until 2004. In these experiments, the results were cumulative, and thus the inoculation date of plants could not be determined. It is likely there is a lag time between inoculation and detection time that is dependent on an array of factors that affect plant physiological status, including temperature, precipitation, nutrition, and pre-existing disease.Perhaps there is also an overwintering component to the equation, such that a virus is capable of reaching higher levels or is more evenly distributed after a plant has been through 107 dormancy. Such a phenomenon has been observed with Bluebmy shock virus (MacDonald et al., 1991). This could explain the reason for the few positives detected in Field 4 in the 2005 'Timing of BRDaV Infection' experiment. These plants weretested before they had a chance to overwinter, possibly before BRDaV could reach a detectable titer or spread systemically throughout the plant. Another factor that could impact the ability to detect any one virus is the presence of other viruses. In mixed infections, some viruses interact to cause an increase inviral concentration and symptom severity (Karyeija et al., 2000, Scheets, 1998). It may be that viral counterdefense of RNA silencing plays a role in increasing the synergistic interactions in mixed infections, increasing the titer of each of the viruses involved (Pruss et al., 1997).Often, greater rates of BRDaV transmission were found in greenhouse mechanical transmission trials (data not shown) using coinfected plants as inocula,which may support an important role for viral suppressors of RNAsilencing. There was a correlation between BRDaV incidence and peak numberof colonizing aphids (R2= 0.433 8) in the 'Timing of Transmission assay', though the highest disease incidence did not consistently occur when aphid numbers were highest. This suggests two scenarios.Either aphid threshold for infection is extremely low, or other variables relating to plant physiology or environmental conditions play a role in disease development. It is possible that date of aphid emergence as related to levels of predators plays a great role, rather than strict aphid number alone. Thackray et al. (2000) suggest that earlier arrival of colonizing aphids is correlated with greatest spread of Cucumber mosaic virus, and greater rainfall influences earlier arrival. Although there was nodirect correlation between aphid number and temperature in this study, it is possible that there could be a role between aphid predators and temperature (Elliott et al., 1998),with predator populations being triggered to increase earlier with warmer temperatures. Furthermore, because a minimum prey density is often required to support a population of predators, there is likely interplay between predator and prey levels that in turnaffects virus incidence. The numbers of aphids caught in traps may not correspond to colonizing aphids found on the plant, as demonstrated by Tatchell et al. (1988). However, transient winged aphids represented on the traps likely contribute to stylet-borne virus spread considerably more than colonizing, non-winged aphids, which tend to settle onto one plant(Berlandier et at., 1997).Acquisition and inoculation of virus is fleeting but efficient with non colonizing aphids, which involve brief sampling probes and saliva injection as they sample sap (Powell, 2005).Considering the extensive number of both weedy and cultivated alternate hosts that harbor BRDaV in proximity to the observed black raspberry plantings, non-colonizing aphids may be important vectors. As species other than the large raspberry aphid were found occasionally on the sticky traps, these non- colonizers likely shared responsibility for the rapid spread of virus through Fields 1-3.

The green peach aphidMyzus persicaealready has been demonstrated as a vector of BRDaV in the laboratory. Furthermore, a population of aphids of unidentified species was found colonizing the western brackenfern leaf undersides. As thisplant was found to be a host for BRDaV, these aphids may have been responsible for contributing to transient transmission. Most current aphid control methods rely heavily upon the use of foliar systemic and contact aphicides. Although synthetic chemical pesticides have enabled enormous boosts in productivity, many problems exist. One such problem is the development of resistance of aphids to aphicides. As the spectrum of chemicals in aphicides approved for use inRubusis declining due to the high registration price for this minor crop, this problem will only continue. Another problem is the elimination of natural enemies by broad-spectrum pesticide sprays.It is the destruction of the relationship between the aphids and the naturally occurring biocontrol agents that can result in pest outbreaks (Thacker, 2002). In several studies, aphicides were demonstrated to actually promote the spread of non-persistently transmitted viruses (Lowery and Boiteau, 1988; Roberts et at., 1993).Aphids briefly probe potential hosts, become deterred by the aphicide, and disperse in search of other plants. Finally, environmental and health concerns regarding the toxicity and persistence of aphicides are too costly to ignore. Although organo- 109 phosphates have been the most successful group of synthetic chemical insecticides, they are neurotoxic to nontarget organisms, including humans. Diazinon hasespecially high toxicity to bees, as well as to birds and fish (Extension Toxicology Network, 1996). There are chemical alternatives to organophosphates for aphid control, but the same problems with aphid dispersal exist with some of these newer chemicals (Roberts et al., 1993). These problems necessitate a shift in thinking regarding our prevailing methods of crop protection. Ultimate control of BRDaV and other viruses relies largely on breeding programs to incorporate new sources of resistance into black raspberry. The few existing alternate cultivars of black raspberry in production do not demonstrate tolerance to BRDaV. However, cultivars that demonstrate some level of tolerance may make useful lines for further breeding.It is likely that red raspberry will provide a source for resistance to BRDaV, as it is a tolerant host for the virus.Widening the genetic base ofRubus breeding programs will enable cultivar selection based on the presence of aphid and/or virus resistance, both of which recently have been shown to share overlapping signaling pathways (Stout et al., 1999). However, it must be considered that while a virus control strategy dependent on aphid rejection of plant tissue may reduce overall colony levels, the brief sampling behavior of noncolonizing, nonpersistent transmitters such as M persicaewould not be affected. Based on the data presented in this communication, it appears that BRDaV spreads rapidly and is consistently associated with BRD. Six additional viruses tested for were found to coinfect black raspberries but only BRDaV wasassociated consistently with decline symptoms of the crop. Field tests showed that RBDV and SNSV quite often occur in mixed infections with BRDaV, but they were not associatedwith declining symptoms in the absence of BRDaV. The blackberry flexivirus, RLSV-NA, the two viruses isolated from 'Glen Clova' raspberry, and the Toti-like virus were found less frequently in association with declining black raspberries. Possibly, these viruses are less important in causing severe symptoms in black raspberry. The presence of the newly characterized viruses in commercial fields underlies the importance of quarantines for 110 limiting long-distance dispersal of viruses. The use of tissue culture for the maintenance and transport of virus-free lines of vegetatively propagated crops such as blackraspberry is also critical for limiting the spread of these viruses.

ACKNOWLEDGEMENTS:

The authors express their gratitude to the Oregon black raspberry grower cooperatorsfor enabling the field experiments. Thank you to the Northwest Center forSmall Fruits Research and the Oregon Raspberry and Blackberry Commission for funding.Thank you also to Janelle Barnes for helping with the processing of field tissue.

REFERENCES:

Berlandier, F.A., Thackray, D.J., Jones, R.A.C., Latham, L.J. and Cartwright, L.1997. Determining the relative roles of different aphid species as vectors of cucumbermosaic and bean yellow mosaic viruses in lupins. Ann. Appl. Biol. 131:297-314.

Bulger, M.A, Stace-Smith, R. and Martin, R.R. 1990. Transmission andfield spread of raspberry bushy dwarf virus. Plant Dis. 74:514-517.

Cadman, C.H. 1951. Studies inRubusvirus diseases. I. A latent virus of Norfolk Giant raspberry. Ann. AppI. Biol. 38:801-811.

Cadman, C.H.1952.Studies inRubusvirus diseases. V. Experiments in analysis of Lloyd George decline. Ann. Appl. Biol. 39:50 1-508.

Clark, M.F. and Adams, A.N. 1977.Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses.J. Gen. Virol. 34:475-483.

Converse, R.H.1980.Transmission of tobacco streak virus in Rubus. ActaHortic. 95:53-6 1.

Elliott, N.C., Kieckhefer, R.W., Lee, J.H. and French, B.W. 1998. Influenceof within- field and landscape factors on aphid predator populations in wheat. Land. Ecol.14:239- 252. 111

Extension Toxicology Network. 1996: http://extoxnet.orst.edu/pips/diazinon.htm

Frazier, N.W., Jorgensen, P.S., Thomas, H.E. and Johnson, H.A. Jr.1962.Necrotic shock: a virus disease of strawberries. Plant Dis. Rep. 46:547-550.

Haigren, A., Tzanetakis, I. and Martin, R.2005.Raspberry mosaic disease:An expanding virus universe. Phytopathology 95:S39.

Jelkmann, W. and Martin, R.R.1988.Complementary DNA probes generated from double-stranded RNAs of a Rubus virus provide the potential for rapid in vitro detection. Acta Hortic. 236:103-109.

Jones, A.T., McGavin, W.J., Geering, A.D.W. and Lockhart, B.E.L. 2002. Identification of Rubus yellow net virus as a distinct badnavirus and its detection by PCR inRubus species and in aphids. Ann. App!. Biol. 141:1-10.

Karyeija, R.F., Kreuze, J.F., Gibson, R.W. and Valkonen, J.P.T.2000.Synergistic interactions of a potyvirus and a phloem-limited crinivirus in sweet potato plants. Virology 269:26-3 6.

Lowery, D.T. and Boiteau, G. 1988. Effects of five insecticides on the probing, walking, and settling behavior of the green each aphid and the buckthorn aphid (Homoptera: Aphididae) on potato. J. Econ. Entomol. 81:208-214.

MacDonald, S.G., Martin, R.R. and Bristow, P.R. 1991. Characterization of an ilarvirus associated with a necrotic shock reaction in blueberry. Phytopathology 81: 210-214.

Martin, R.R. 1999.Raspberry viruses in Oregon, Washington, and British Columbia. Acta Hortic. 505:259-262.

Martin, R.R.2002.Virus Diseases ofRubusand Strategies for Their Control. Acta Hortic. 585:265-270

Martin, R.R., Tzanetakis, I.E., Gergerich, R., Fernandez, G. and Pesic, Z. 2004. Blackberry yellow vein associated virus: a new crinivirus found in blackberry. Acta Hortic. 656:137-142.

Martin, R.R. and Tzanetakis, I.E.Characterization of three novel viruses infecting raspberry. Acta Hortic,in press, a.

Martin R.R., Tzanetakis, I.E., Sweeney, M. and Wegener, L. A virus associatedwith blueberry fruit drop disease. Acta Hortic,in press, b.

Murant, A.F., i976. Raspberry bushy dwarf virus. CML/AAB Descriptions of Plant 112

Viruses, No. 165.

Oregon Agricultural Statistics Service, January 27, 2005 Press Release. http://www.nass.usda.gov/or/fruit.htm

Oregon Climate Service.2005. http://www.ocs.oregonstate.edu

Powell, G. 2005. Intracellular salivation is the aphid activity associated with inoculation of non-persistently transmitted viruses. J. Gen. Virol. 86:469-472.

Pruss, G., Ge, X., Shi, M.X., Carrington, J.C. and Vance, V.B. 1997.Plant viral synergism:The poyviral genome encodes a broad range pathogenicity enhancer that transactivates replication of the heterologous viruses. Plant Cell 9:859-868.

Roberts, J.M.F., Hodgson, C.J., Jackai, L.E.N., Thottappilly, G. and Singh, S.R.1993. Interaction between two synthetic pyrethroids and the spread of two non-persistent viruses in cowpea. Ann. Appi. Biol. 122:57-67.

Rush, M.C. and Gooding, G.V., Jr.1970. The occurrence of tobacco ringspot virus strains and tomato ringspot virus in hosts indigenous to North Carolina. Phytopathology 60:1756-1760.

Scheets, K. 1998.Maize chiorotic mottle machiovirus and wheat streak mosaic rymovirus concentrations increase in the synergistic disease corn lethal necrosis. Virology 242:28-3 8.

Spiegel, S. and Martin, R.R.1993.Improved detection of potato leafroll virus in dormant potato tubers and microtubers by the polymerase chain reaction and ELISA. Ann. Appl. Biol. 122:493-500.

Stace-Smith, R. 1955a. Studies on Rubus virus diseases in British Columbia. I. Rubus yellow-net. Can. J. Bot. 33:267-274.

Stace-Smith, R. 1955b. Studies on Rubus virus diseases in British Columbia. II. Black raspberry necrosis. Can. J. Bot. 33:314-322.

Stace-Smith, R. 1956.Studies on Rubus virus disease in British Columbia. Ill. Separation of components of raspberry mosaic. Can. J. Bot. 34:435-442.

Stace-Smith, R. and Converse, R.H. 1987. Tomato Ringspot Virus inRubus. Pp.223- 227. In:Virus Diseases of Small Fruits. R. H. Converse, ed. US. Dept. Agric. Agric Handb. 631. 113

Stout M.J., FidantsefA.L., Duffey S.S. and Bostock, R.M. 1999. Signal interactions in pathogen and insect attack: systemic plant-mediated interactions between pathogens and herbivores of the tomato, Lycopersicon esculentum. Physiol. Mo!. P!ant Patho!. 54: 115- 130

Susaimuthu, J., Tzanetakis, I., Gergerich, R. and Martin, R. 2005. Evidence for mixed infections by two or more viruses causing severe symptoms and decline of blackberry. Phytopathology 95: Si 01.

Tatchell, G.M., Plumb, R.T. and Carter, N. 1988. Migration of alate morphs of the bird cherry aphid (Rhopalosiphum padi) and implications for the epidemiology of barley yellow dwarf virus. Ann. App!. Biol. 112:1-11.

Thacker, J.R.M.2002. An Introduction to Artbropod Pest Control. Cambridge University Press, Cambridge, United Kingdom, pp 112-134.

Thackray, D.J., Jones, R.A.C, Bwye, A.M. and Coutts, B.A. 2000. Further studies on the effects of insecticides on aphid vector numbers and spread of cucumber mosaic virusin narrow-leafed lupins (Lupinus angustfolius). Crop Protection 19:121-139.

Tzanetakis, I.E., Mackey, I.C. and Martin, R.R. 2004. Strawberry necrotic shock virus: a new virus previously thought to be tobacco streak virus. Arch.Virol. 149: 2001-2011.

Weather Underground, Inc. 2005. http://www.wunderground.com 114

CHAPTER 4: GENERAL DISCUSSION

Virus transmission by graft inoculation and aphid vectors enabled distinctionof Rubus diseases as early as 1927 (Bennett) and by the mid-1950's, many major virus diseases of Rubus had been identified and described.An important advance in the detection and eradication of viruses was the finding that prolonged exposure of a plant to high temperatures decreases the rate of virus replication and movement (Chambers, 1954). This enables removal of actively growing shoot tips to generate virus-free clones. Not only was this milestone critical for the production of virus-free breedingmaterial, but it also enabled the production of virus-free indicator plants.Clean indicator plants provided one of the most fundamental tools for routine viral diagnosis. Cadman'sfinding in 1954 that nicotine averts the inhibitory action of raspberry tannins facilitated efforts to transfer Rubus viruses into herbaceous hosts. A further development, electron microscopy, opened up a new field of knowledge regarding the size, shape,and cytological localization of virus particles. Our present efforts in Rubus virus research are in the development of identification techniques that provide the tools forepidemiological studies. For large-scale field surveys, studies of viral host range, or vectortransmission studies in the field, diagnostics are needed that are capable of processing large numbers of samples both accurately and quickly. Two widely used diagnostic methods based on viral protein and nucleic acid properties that have become integral to modem diagnostics are RT-PCR andserologicalprocedures,most notablytheEnzyme Linked Immunosorbent Assay (ELISA) (Clark and Adams, 1977). RT-PCR is a powerful tool for virus detection. A large part of this research project involved the design and use of RT-PCR detection tests to detect and monitorthe spread of BRDaV and other Rubus viruses in the field. Primer choice allows for designed specificity, and can distinguish between viral strains or generically detect whole families of virus. The drawback, however, is that primers annealing to homologous regionsof a contaminating gene promote the amplification of non-viral material. Thus, it is important to optimize RT-PCR conditions to ensure fidelity of diagnosis. RT-PCR isalso extremely sensitive, which enables detection in samples with very low virus titer. However, the 115 potential exists for false negatives, if viral titer is too low, or if a part of theplant is sampled where virus is not distributed.To complicate detection further,RubusRNA templates come with the inherent risk of reaction interference due to the large amountsof tannins in the leaf tissue.These tannins can interfere with downstream enzymatic reactions, underlying the importance of using an internal PCR control toverify the quality of the RNA extraction and effectiveness of the RT-PCR. ELISA procedures rely on the interaction between an antigen in the pathogenwith antibodies raised against it in a vertebrate. Antibodies for use in ELISA werehistorically produced against whole virions. This method ensures that antibodies generatedagainst the virus will be against subunits of a viral protein coat.One potential drawback, however, is the presence of contaminating plant material in the immunogen preparation. The resulting contaminating antibodies raised against plant proteins will interferewith the specificity of the viral detection.Such contamination proved to be a pitfall of antibody production against BRDaV in this project.Expression of individual coat proteins is a useful alternative technique to whole vinon purification. Purifiedantibody raised against the coat protein is free of plant contaminants that might otherwise cause background in an ELISA, and protein expression systems can producesubstantial amounts of target protein. However, some proteins may be made ininsufficient amounts. Target proteins may interfere with cellular gene expression, possibly acting as acellular toxin. Some proteins may decrease in synthesis as the target protein accumulates. One further disadvantage to generating antibodies against expressed coat proteins isthe possibility that the expressed protein will not adopt its native tertiary structureafter expression in an in vitro system, thereby reducing the effectiveness of theantibody in recognizing virions. ELISA is quite fast, economical and easily adapted toquantitative measurement. However, depending on the antigen against whichthe antibodies were generated, this assay may either fail to discriminate between strains of avirus, or overlook detection of a particular strain The major objective of this research study was to identify andcharacterize the physical properties, ecology, and epidemiology of an unknown virus associatedwith 116 decline of black raspberries.The underlying goal was to gain insight from our understanding of the vector, rate of spread, timing of transmission, and host range, to recommend a control strategy. Towards that goal, we have examined many biological and molecular properties of the virus. Unexpectedly, we came across several interesting facets of the virus that will not only lend information toward a control strategy, but also perhaps shed some light on viral evolution. Of foremost interest was the finding of a 189 amino acid mdcl at the C terminus of the RNA-dependent RNA polymerase. To the best of our knowledge, this is the first published finding of an AIkB domain outside the Flexiviridae and the ampeloviruses of the Closteroviridae.This finding opens up a set of questions regarding the adaptive advantages conferred by this domain, and whether this domain has played a role in the successful spread of this virus within the field. This work also serves to highlight trends invirusevolution. Comparativeanalysisof individualgenesunderliesthe recombinatorial shuffling concept typical of RNA viruses, yet reinforces the concept of universal building block conservation. The provided analysis of the BRDaV genome illustrates its phylogenetic relationship to a taxonomically uncertain group of picoma-like plant viruses, which is changing the paradigm with which we view the history of RNA virus evolution.It is likely that recombination and reassortment were the mechanisms responsible for the modular nature of BRDaV, as they are with many RNA viruses (Lai, 1992).However, an additional suggested evolutionary scenario proposes that insect monopartite viruses of a picorna-like nature invaded plants, resulting in a monopartite lineage of Sequiviridae and two independent bipartite lineages, the Comoviridae and the Satsuma dwarf virus-like group (Karasev et al., 2001; Thompson et al., 2004). The experimental host range was examined with the goal of finding a host more likely to generate a high titer of BRDaV, though none provided higher concentrations of dsRNA or virions than black raspberry. The natural host range of BRDaV was examined in order to understand potential sources of infection within or adjacent to fields.Of particular interest was the finding that two pervasive species of weed, perennial sowthistle (Sonchus arvensis) and Western brackenfern (Pteridium aquilinum var. 117 pubescens) were hosts. This finding, combined with the finding that at least five different Rubus species often found growing near black raspberries could host BRDaV asymptomatically, makes eradication of infection sources a very difficult task. Understanding vector transmission is key to mapping a viral control strategy. It was demonstrated that the large raspberry aphidAmphorophora agathonica transmits BRDaV in a non-persistent maimer. BRDaV can spread rapidly throughout ablackcap field, even with reduced populations of winged aphids. However, the green peach aphid Myzus persicae has also been demonstrated to transmit BRDaV, which mayindicate BRDaV can spread by transient aphids.The reason for attempting to determine the transmission modes of both A. agathonica and M persicae is related to the desire to understand rate of virus spread in the field.Non-persistently transmitted viruses are acquired within a matter of seconds from a plant. These viruses are transmitted during the initial host-sampling behavior of the vector, when the aphid is sampling the epidermal cell sap.Vectors of viruses transmitted in such a mode are often non-colonizers of a particular crop species. Though transient, these vectors may do more damage than the colonizers, such as A. agathonica, which are tightly associated with the crop plant (Berlandier et al., 1997). We have determined that BRDaV spreads rapidly with an extremely low aphid threshold and is consistently associated with symptoms of decline in black raspberries in Oregon.Although six other viruses were found to coinfect black raspberries, only BRDaV was associated consistently with decline symptoms of the crop.Given the positive correlation found between average winged-aphid number/field and numberof plants testing positive for BRDaV, it is evident that there is a need for vector control. However, considering that aphicides can encourage greater movement of transient aphids (Lowery and Boiteau, 1988; Roberts et al., 1993), compounded with the very lowaphid threshold required to initiate infection, alternative control strategies are needed. The presiding commercially grown alternate black raspberry cultivars demonstrate little resistance or tolerance to BRDaV. Thus, it is likely that a concerted effort to incorporate germplasm from more diverse sources, potentially from Rubus idaeus, will provide the 118 black raspberry industry with the resistance that is required to control this disease.

REFERENCES:

Bennett, C. W.1927. Virus diseases of raspberries. Mich. Agr. Expt. Sta. Tech. Bull. 80.

Berlandier, F.A., Thackray, D.J., Jones, R.A.C., Latham, L.J. and Cartwright, L. 1997. Determining the relative roles of different aphid species as vectors of cucumber mosaic and bean yellow mosaic viruses in lupins. Ann. Appl. Biol. 131:297-314.

Cadman, C.H. 1954. Dept. of Plant Pathol., Scot. Hort. Res. Inst.1stAnn. Rept. Pp. 17- 20.

Chambers, J. 1954. Heat therapy of virus-infected raspberries. Nature 173:595-596.

Clark, M.F. and Adams, A.N. 1977.Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses.J. Gen. Virol. 34:475-483.

Karasev, A.V., Han, S.S. and Iwanami, T. 2001. Satsuma Dwarf and Related Viruses Belong to a New Lineage of Plant Picorna-like Viruses. Virus Genes 23 :45-52.

Lai, M.M.C.1992. RNA Recombination in Animal and Plant Viruses.Micro. Rev. 56:61-79.

Lowery, D.T. and Boiteau, G. 1988. Effects of five insecticides on the probing, walking, and settling behavior of the green each aphid and the buckthorn aphid (Homoptera: Aphididae) on potato. J. Econ. Entomol. 81:208-214.

Roberts, J.M.F., Hodgson, C.J., Jackai, L.E.N., Thottappilly, G. and Singh, S.R.1993. Interaction between two synthetic pyrethroids and the spread of two non-persistent viruses in cowpea. Ann. Appl. Biol. 122:57-67.

Thompson, J.R., Peny, K.L. and Dc Jong, W. 2004. A new potato virus in a new lineage of picorna-like viruses. Arch. Virol. 149:2141-2154. 119

BIBLIOGRAPHY:

Aas, P.A., Otterlei, M., Flanes, P.O., Vagbo, C.B., Skorpen, F., Akbari, M., Sundheim, 0., Bjoras, M., Slupphaug, G., Seeberg, E. and Korkan, H.E. 2003. Human and bacterial oxidative demethylases repair alkylation damage in both RNA and DNA. Nature 421:859-863.

Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D.J.1997. Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res. 25:33 89-3402.

Aravind, L. and Koonin, E.V.2001.The DNA-repair protein A1kB, EGL-9, and leprecan define new families of 2-oxoglutarate- and iron-dependent dioxygenases. Genome Biology 2(3):research0007. 1-0007.8

Barbara, D.J., Ashby, S.C. and McNamara, D.G.1985.Host range, purification and some properties of rubus Chinese seed-borne virus. Ann. Appl. Biol. 107:45-55.

Baumann, G., Casper, R. and Converse, R.H. 1982. The occurrence of apple mosaic virus in red and black raspberry and in blackberry cultivars. Acta Hort. 129:13-20.

Bennett, C. W.1927. Virus diseases of raspberries. Mich. Agr. Expt. Sta. Tech. Bull. 80.

Berlandier, F.A., Thackray, D.J., Jones, R.A.C., Latham, L.J. and Cartwright, L.1997. Determining the relative roles of different aphid species as vectors of cucumber mosaic and bean yellow mosaic viruses in lupins. Ann. Appl. Biol. 131:297-314.

Birch, A.N.E. and Jones, A.T. 1988. Levels and components of resistance to Amphorophora idaei in raspberry cultivars containing different resistance genes. Ann. AppI. Biol. 113:567-578.

Blackman, R.L. and Eastop, V.R.2000. Aphids on the World's Food Crops-an identification and information guide. 2' edition. John Wiley and Sons, Ltd. Chichester, Pp. 220-222.

Bratlie M.S. and Drabløs F. 2005. Bioinformatic mapping of A1kB homology domains in viruses. BMC Genomics 6:1

Bulger, M.A., Stace-Smith, R. and Martin, R.R. 1990. Transmission and field spread of raspberry bushy dwarf virus. Plant Dis. 74:514-517. 120

Cadman, C.H. 1951. Studies inRubusvirus diseases. I. A latent virus of Norfolk Giant raspberry. Ann. Appl. Biol. 38:801-811.

Cadman, C.H.1952.Studies inRubusvirus diseases. V. Experiments in analysis of Lloyd George decline. Ann. Appi. Biol. 39:501-508. Cadman, C.H. 1954. Dept. of Plant Pathol., Scot. Hort. Res. Inst.1stAnn. Rept. Pp. 17- 20.

Campanella, J.J., Bitincka, L. and Smalley, J.2003. MatGAT: an application that generatessimilarity/identity matrices using protein or DNA sequences. BMC Bioinformatics 4:29.

Carrington, J.C., Freed, D.D. and Oh, C.S. 1990. Expression of potyviral polyproteins in transgenic plants reveals three proteolytic activities required for complete processing. EMBO J. 9:1347-1353.

Chambers, J. 1954. Heat therapy of virus-infected raspberries. Nature 173:595-596.

Christian, P., Fauquet, C.M., Gorbalenya, A.E., King, A.M.G., Knowles, N., LeGall, 0. and Stanway, G. 2005. Picornavirales: A proposed order of positive sense RNA viruses. ICTV Poster session at the International Congress of Virology, San Francisco, Moscone Convention Center, July 23-28, 2005. http://www.danforthcenter.org/iltab/ictvnetlsanfran.htm

Clark, M.F. and Adams, A.N. 1977.Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses.J. Gen. Virol. 34:475-483.

Converse, R.H. 1972.Tobacco streak virus in black raspberry.Phytopathology 62:1001-1004.

Converse, R.H.1980.Transmission of tobacco streak virus in Rubus. Acta Hortic. 95:53-61.

Converse, R.H. 1991. Diseases Caused by Viruses and Viruslike Agents, Pp. 42-52, and Viral Diseases with Unknown Methods of Natural Spread, Pp. 52-5 8.In: Compendium of Raspberry and Blackberry Diseases and Insects.Ellis, M.A., Converse, R.H., Williams, R.N. and Williamson, B. (eds). American Phytopathological Society Press, St. Paul, Minnesota.

Converse, R.H. and Casper, R. 1975. A member of the apple mosaic virus group occurring in Rubus. Acta Hortie. 44:63-64. 121

Converse, R.H., Stace-Smith, R. and Jones, A.T. 1987. Raspberry Mosaic. Pp. 168-178. In: Virus Diseases of Small Fruits. R. H. Converse, ed. US. Dept. Agric. AgricHandb. 631.

Cooper, J.I., Massaiski, P.R. and Edwards, M.L.1984.Cherry leaf roll virus in the female gametophyte and seed of birch and its relevance to vertical virus transmission. Ann. Appi. Biol. 105:55-64.

Crandall, P.C. and Daubeny, H.A. 1990. Raspberry Management.In Galletta, G.J., and Himeirick, D.G. (eds). Small Fruit Crop Management.Prentice Hall Career and Technology, Englewood Cliffs, New Jersey.

Daubeny, H.A., Stace-Smith, R. and Freeman, J.A.1978. The occurrence and some effects of raspberry bushy dwarf virus in red raspberry.J. Am. Soc. Hortic. Sci. 103 :5 19-522. de Miranda, J., Shen, M., Cameron, C.E., Stoltz, D.B. and Camazine, S.M.2004. Complete nucleotide sequence of Kashmir bee virus and comparison with acute bee paralysis virus. J. Gen. Virol. 85:2263-2270.

Doija, V.V. and Carrington, J.C.1992.Evolution of positive-strand RNA viruses. Semin. Virol., 3:315-326.

Doija, V.V. and Koonin, E.V. 1991. Phylogeny of capsid proteins of small icosahedral RNA plant viruses. J. Gen. Virol. 72:1481-1486.

Elliott, N.C., Kieckhefer, R.W., Lee, J.H. and French, B.W. 1998. Influence of within- field and landscape factors on aphid predator populations in wheat. Land. Ecol. 14:239- 252.

Extension Toxicology Network. 1996: http://extoxnet.orst.edulpips/diazinon.htm

Forbes, A.R. and Chan, C.K. 1989. Aphids of British Columbia. Ag. Canada research Branch Technical Bulletin 1989-1E, p. 205.

Frazier, N.W., Jorgensen, P.S., Thomas, H.E. and Johnson, H.A. Jr.1962.Necrotic shock: a virus disease of strawberries. Plant Dis. Rep. 46:547-550.

Funt, R.C., Ellis, M.A., Williams, R.N., Doohan, D., Scheerens, J.C. and Welty, C. 2001. BramblesProduction, Management, and Marketing. Ohio State University Extension Bulletin 782. Media Distribution, The Ohio State University, 385 Kottman Hall, 2021 Coffey Road, Columbus OH 43210-1044. http://ohioline.osu.edu/b782/index.html 122

Gamsey, S.M. and Koizumi, M. 2000. Satsuma dwarf virus group. In: Timmer, L. W., Garnsey, S.M. and Grahan, J.H. (eds).Compendium of citrus diseases,2nded. APS Press, The American Phytopathological Society, St. Paul, p 59.

Gorbalenya, A.E., Donchenko, A.P., Blinov, V.M. and Koonin, E.V.1989.Cysteine proteases of positive strand RNA viruses and chymotrypsin-like serine proteases. FEBS Letters 243:103-114.

Gorbalenya, A.E., Koonin, E.V. and Wolf, Y.I.1990. A new superfamily of putative NTP-binding domains encoded by genomes of small DNA and RNA viruses.FEBS Letters 262:145-148.

Govan, V.A., Leat, N., Allsopp, M. and Davison, 5.2000. Analysis of the complete genome sequence of acute bee paralysis virus shows that it belongs to the novel group of insect-infecting RNA viruses. Virol. 277:457-463.

Halgren, A., Tzanetakis, I. and Martin, R.2005.Raspberry mosaic disease:An expanding virus universe. Phytopathology 95:S39.

Halling, S. M. and Smith, 5.1985. Expression in Escherichia coli of Multiple Products from a Chimaeric Gene Fusion: Evidence for the Presence of Procaryotic Translational Control Regions within Eucaryotic Genes. Bio/Technology 3:715-720.

Hansen, A.J., Nyland, G., McElroy, R.D. and Stace-Smith, R. 1974. Origin, cause, host range, and spread of cherry rasp leaf disease in North America. Phytopathology 64:721 - 727.

Harrison, B.D. and Murant, A.F. 1984. Involvement of virus-coded proteins in transmission of plant viruses by vectors.In:Mayo, M.A., and Harrap, K.A. (eds), Vectors in Virus Biology, Academic Press, London, pp. 13-14. http://www.nass.usda.gov/or/fruit/berrieso 105 .pdf

Huang, X. and Madan, A. 1999. CAP3: A DNA Sequence Assembly Program. Genome Research 9:868-877.

Hull, R. 2002. Transmission 1: By Invertebrates, Nematodes, and Fungi. In: Matthews' Plant Virology. Academic Press, San Diego, Pp 485-532.

Ito, T., Iwanami, T., leki, H., Shimomura, K., Shimizu, S. and Ito, T. 2004. A new virus related to Satsuma dwarf virus:the nucleotide sequence of the 3 '-terminal regions of Hyuganatsu virus RNAs 1 and 2.Arch. Virol. 149:1459-1465.

Iwanami, T. and leki, H. 1996. Nucleotide sequence of the coat protein genes of citrus mosaic virus. Virus Res. 42:18 1-186. 123

Iwanami, T., Koizumi, M. and leki, H.1993. Diversity of properties among Satsuma dwarf virus and related viruses. Ann. Phytopathol. Soc. Jpn 59: 642-6560.

Iwanami, T., Kondo, Y. and Karasev, A.V. 1999. Nucleotide sequences and taxonomy of satsuma dwarf virus. J. Gen. Virol. 80:793-797.

Iwanami, T., Kondo, Y., Kobayashi, M., Han, S.S. and Karasev, A.V. 2001. Sequence diversity and interrelationships among isolates of Satsuma dwarf-related viruses. Arch. Virol. 146:807-813.

Iwanami, T., Kondo, Y., Makita, Y., Azeyanagi, C. and leki, H.1998. The nucleotide sequence of the coat protein genes of satsuma dwarf virus andnavel orange infectious mottling virus. Arch. Virol. 143:405-4 12.

Jelkmann, W. and Martin, R.R.1988.Complementary DNA probes generated from double-stranded RNAs of a Rubus virus provide the potential for rapid in vitro detection. Acta Hortic. 236:103-109.

Jelkmann, W., Martin, R.R. and Maiss, E.1989.Cloning of four plant viruses from small quantities of double-stranded RNA. Phytopathology 79:1250-1253.

Jennings, D.L. 1995. Raspberries and blackberries:Rubus(Rosaceae). Pp. 429-434. In: J. Smartt and N.W. Simmonds (eds.), Evolution of crop plants. 2nd ed. Longman Scientific & Technical. Essex, England.

Jennings, D.L. and Jones, A.T. 1989. Further studies on the occurrence and inheritance of resistance in red raspberry to a resistance-breaking strain of raspberry bushy dwarf virus. Ann. Appl. Biol. 114:317-323.

Jones, A.T., 1976. An isolate of cucumber mosaic virus fromRubus phoenicolasius Maxim. Plant Pathol. 25:137-140.

Jones, A.T., 1979. The effects of black raspberry necrosis and raspberry bushy dwarf viruses in Lloyd George raspberry and their involvement in raspberry bushy dwarf disease. J. Hortic. Sci. 54:267-272.

Jones, A.T.1982.Distinctions between three aphid-borne latent viruses of raspberry. Acta Hortic. 129:41-48.

Jones, A.T. 1987.Raspberry leaf mottle and Raspberry leaf spot.Pp. 183-187.In: Virus Diseases of Small Fruits. R. H. Converse, ed. US. Dept. Agric. Agric Handb. 631. 124

Jones, A.T. 1991. Rubus host range of rubus yellow net virus and its involvement with other aphid-borne latent viruses in inducing raspberry veinbanding mosaic disease. Ann. Appl. Biol. 118:331-338.

Jones, A.T. and Jennings, D.L.1980. Genetic control of the reactions of raspberry to black raspberry necrosis, raspberry leaf mottle and raspberry leaf spot viruses.Ann. Appl. Biol. 96:119-123.

Jones, A.T. and McGavin, W.J. 1998.Infectibility and sensitivity of UK raspberry, blackberry and hybrid berry cultivars to Rubus viruses. Ann. Appl. Biol. 132:239-251.

Jones, A.T. and Mitchell, M.J.1986.Propagation of black raspberry necrosis virus (BRNV) in mixed culture with solanum nodiflorum mottle virus, and the production and use of BRNV antiserum. Ann. Appl. Biol. 109:323-336.

Jones, A.T. and Murant, A.F.1972. Some properties of a mechanically transmissible virus widespread in raspberry (Rubus idaeus) in Scotland. Plant Pathol. 21:166-170.

Jones, A.T. and Roberts, I.M.1976.Ultrastructural changes and small bacilliform particles associated with infection by rubus yellow net virus. Ann. App!. Biol. 84:305- 310.

Jones, A.T., Mayo, M.A. and Henderson, S.J. 1985.Biological and biochemical properties of an isolate of cherry rasp leaf virus from red raspberry. Ann. App!. Biol. 106:101-110.

Jones, A.T., Mayo, M.A. and Murant, A.F.1996. Raspberry Bushy Dwarf Idaeovirus. The Plant Viruses, Volume 5:Polyhedral Virions and Bipartite RNA Genomes. B. D. Harrison and A. F. Murant, eds. Plenum Press, New York. pp. 283-301.

Jones, A.T., McGavin, W.J. and Birch, A.N.E. 2000.Effectiveness of resistance genes to the large raspberry aphid, Amphorophora idaei Börner, in different raspberry (Rubus idaeus L.) genotypes and under different environmental conditions.Ann. Appl. Biol. 136:107-113.

Jones, A.T., McGavin, W.J., Geering, A.D.W. and Lockhart, B.E.L. 2002. Identification of Rubus yellow net virus as a distinct badnavirus and its detection by PCR inRubus species and in aphids. Ann. Appi. Biol. 141:1-10.

Jones, A.T., Mitchell, M.J., McGavin, W.J. and Roberts, I.M.1990. Further properties of wineberry latent virus and evidence for its possible involvement in calico disease. Ann. Appl. Biol. 117: 571-581. 125

Jones, A.T., Murant, A.F. and Stace-Smith, R.1987. Raspberry Vein Chiorosis.Pp. 194-197.In:Virus Diseases of Small Fruits.R. H. Converse, ed. US. Dept. Agric. Agric Handb. 631.

Jones, A.T., Roberts, I.M. and Murant, A.F. 1974.Association of different kinds of bacilliform particle with vein chiorosis and mosaic diseases of raspberry(Rubus idaeus). Ann. App. Biol. 77 :283-288.

Karasev, A.V., Han, S.S. and Iwanami, T.2001. Satsuma Dwarf and related viruses belong to a new lineage of plant Picorna-like viruses. Virus Genes 23:1, 45-52.

Karyeija, R.F., Kreuze, J.F., Gibson, R.W. and Valkonen, J.P.T.2000.Synergistic interactions of a potyvirus and a phloem-limited crinivirus in sweet potato plants. Virology 269:26-36.

Koonin, E.V. 1991. The phylogeny of RNA-dependent RNA polymerases of positive- strand RNA viruses. J. Gen. Virol. 72:2197-2206.

Koonin, E.V. and Doija, V.V. 1993. Evolution and taxonomy of positive-strand RNA viruses: implications of comparative analysis of amino acid sequences. Critical Reviews in Biochemistry and Molecular Biology 28:375-430.

Koonin, E.V., Mushegian, R.R., Ryabov, E.V. and Dolja, V.V. 1991. Diverse groups of plant RNA and DNA viruses share related movement proteins that may possess chaperone-like activity. J. Gen. Virol. 72 :2895-2903.

Kozak, M.2002.Pushing the limits of the scanning mechanism for initiation of translation. Gene 299:1-34.

Kreiah, S., Strunk, G. and Cooper, J.I.1994. Sequence analysis and location of capsid proteins within RNA2 of strawberry latent ringspot virus. J. Gen. Virol. 75:2527-2532.

Lai, M.M.C.1992. RNA Recombination in Animal and Plant Viruses.Micro. Rev. 56:61-79.

Leone, G., Lindner, J.L. and Schoen, C.D. 1995. Unstable infectivity and abundant viral RNAs associated with strawberry mottle virus. Acta Hortic. 385:76-85.

Lowery, D.T. and Boiteau, G. 1988. Effects of five insecticides on the probing, walking, and settling behavior of the green each aphid and the buckthorn aphid (Homoptera: Aphididae) on potato. J. Econ. Entomol. 81:208-214.

MacDonald, S.G., Martin, R.R. and Bristow, P.R. 1991. Characterization of an ilarvirus associated with a necrotic shock reaction in blueberry. Phytopathology 81: 210-214. 126

Marchler-Bauer A. and Bryant, S.H. 2004. CD-Search: protein domain annotations on the fly. Nucleic Acids Res. 32:W327-331.

Martin, R.R. 1999. Raspberry viruses in Oregon, Washington, and British Columbia. Acta Hortic. 505:259-262.

Martin, R.R.2002.Virus Diseases ofRubusand Strategies for Their Control. Acta Hortic. 585:265-270

Martin, R.R. and Mathews, H. 2001. Engineering resistance to Raspberry bushy dwarf virus. ActaHortic. 551:33-37.

Martin, R. and Tzanetakis, I.2005.Characterization of three novel viruses infecting raspberry. Phytopathology 95:S66.

Martin, R.R. and Tzanetakis, I.E.Characterization of three novel viruses infecting raspberry. Acta Hortic,in press.

Martin, R.R., Tzanetakis, I.E., Gergerich, R., Fernandez, G. and Pesic, Z. 2004. Blackberry yellow vein associated virus: a new crinivirus found in blackberry. Acta Hortic. 656:137-142.

Martin R.R., Tzanetakis, I.E., Sweeney, M. and Wegener, L. A virus associated with blueberry fruit drop disease. Acta Hortic,in press.

Mayo, M.A. 2005. Changes to virus taxonomy 2004. Arch Virol. 150:189-198.

Mayo, M.A. and Fritsch, C. 1994. A possible consensus sequence for VPg of viruses in the family Comoviridae. FEBS Lett. 354:129-130.

Melcher, U. 2000. The '30K superfamily of viral movement proteins.J. Gen. Virol. 81:257-266.

Murant, A.F. 1974. Strawberry latent ringspot virus. CMI!AAB Descriptions of Plant Viruses, no. 126.

Murant, A.F., 1976.Raspberry bushy dwarf virus. CML'AAB Descriptions of Plant Viruses, No. 165.

Murant, A.F.1987. Raspberry Ringspot and Associated diseases ofRubuscaused by Raspberry ringspot and Tomato black ring viruses. Pp. 2 11-220. In: Virus Diseases of Small Fruits. R. H. Converse, ed. US. Dept. Agric. Agric. Handb. 631. 127

Murant, A.F. and Jones, A.T.1976. Comparison of isolates of raspberry bushy dwarf virus from red and black raspberries. Acta Hortic. 66:47-52.

Murant, A.F., Jones, A.T. and Roberts, I.M.1976. Recent research on 52V virus of raspberry. Acta Hortic. 66:39-46.

Mushegian, A.R. and Koonin, E.V.1993.Cell-to-cell movement of plant viruses. Insights from amino acid sequence comparisons of movement proteins and from analogies with cellular transport systems. Arch. Virol. 133:239-257.

Ongus, J.R., Peters, D., Bonmating, J.M., Bengsch, E., Vlak, J.M. and van Oers, M.M. 2004. Complete sequence of a picorna-like virus of the genus Iflavirus replicating in the mite Varroa destructor. J. Gen. Virol. 85:3747-3755.

OregonAgriculturalStatistics Service,January27,2005 PressRelease. http://www.nass.usda.gov/or/fruit.htm

Oregon Climate Service.2005. http://www.ocs.oregonstate.edu

Page, R.D.M.1996.TREEVIEW: An application to display phylogenetic trees on personal computers. Comp. Appl. Bio. 12: 357-358.

Palmenderg, A. C.1987. Picornaviral processing: Some new ideas. J. Cell. Biochem. 33, 191-198.

Powell, G. 2005. Intracellular salivation is the aphid activity associated with inoculation of non-persistently transmitted viruses. J. Gen. Virol. 86:469-472.

Preibisch, G., Ishihara, H., Tripier, D. and Leineweber, M. 1988. Unexpected translation initiation within the coding region of eukaryotic genes expressed in Escherichia coli. Gene 72:179-186.

Pruss, G., Ge, X., Shi, M.X., Carrington, J.C. and Vance, V.B. 1997.Plant viral synergism:The poyviral genome encodes a broad range pathogenicity enhancer that transactivates replication of the heterologous viruses. Plant Cell 9:859-868.

Remah, A., Jones, A.T. and Mitchell, M.J. 1986. Purification and properties of lucerne Australian symptomless virus, a new virus infecting lucerne in Australia.Ann. Appl. Biol. 109:307-315.

Ritzenthaler, C., Viry, M., Pinck, M., Margis, R., Fuchs, M. and Pinck, L.1991. Complete nucleotide sequence and genetic organization of grapevine fanleaf nepovirus RNA1. J. Gen. Virol. 72:2357-2365. 128

Roberts, J.M.F., Hodgson, C.J., Jackai, L.E.N., Thottappilly, G. and Singh, S.R.1993. Interaction between two synthetic pyrethroids and the spread of two non-persistent viruses in cowpea. Ann. Appi. Biol. 122:57-67.

Rott, M.E., Tremaine, J.H. and Rochon, D.M.1991.Comparison of the 5' and 3' termini of tomato ringspot virus RNA1 and RNA2: evidence for RNA recombination. Virology 185:468-472.

Rush, M.C. and Gooding, G.V., Jr.1970. The occurrence of tobacco ringspot virus strains and tomato nngspot virus in hosts indigenous to North Carolina. Phytopathology 60:1756-1760.

Sambrook, J. and Russell, D.W. 2001. Molecular Cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, NY: Cold Spring Harbor Laboratory.

Scheets, K. 1998.Maize chlorotic mottle machiovirus and wheat streak mosaic rymovirus concentrations increase in the synergistic disease corn lethal necrosis. Virology 242:28-38.

Sdoodee, R. and Teakle, D.S.1987.Transmission of tobacco streak virus by Thrips tabaci: A new method of plant virus transmission. Plant Pathol. 36:377.

Shine, J. and Dalgarno, L.1974. The 3'-Terminal Sequence of Escherichia coli 16S Ribosomal RNA: Complementarity to Nonsense Triplets and Ribosome Binding Sites. Proc. Nati. Acad. Sci. U.S.A. 7 1:1342-1346.

Spiegel, S. and Martin, R.R.1993.Improved detection of potato leafroll virus in dormant potato tubers and microtubers by the polymerase chain reaction and ELISA. Ann. Appi. Biol. 122:493-500.

Stace-Smith, R, and Converse, R.H. 1987a. Rubus Leaf Curl. Pp.194-197. In:Virus Diseases of Small Fruits. R. H. Converse, ed. US. Dept. Agric. Agric Handb. 631.

Stace-Smith, R. 1955. Studies on Rubus virus diseases in British Columbia.I. Rubus yellow-net. Can. J. Bot. 33:267-274.

Stace-Smith, R. 1955. Studies on Rubus virus diseases in British Columbia. II. Black raspberry necrosis. Can. J. Bot. 33:314-322.

Stace-Smith, R. 1956.Studies on Rubus virus disease in British Columbia.III. Separation of components of raspberry mosaic. Can. J. Bot. 34:435-442.

Stace-Smith, R. 1984. CMIIAAB Deser. P1. Viruses No. 290. 129

Stace-Smith, R.1984. Red raspberry virus diseases in North America. PlantDisease 68:274-279.

Stace-Smith, R.1987.Tobacco Ringspot Virus in Rubus. Pp. 227-228.In:Virus Diseases of Small Fruits. R. H. Converse, ed. US. Dept. Agric. Agric Handb. 631.

Stace-Smith, R. and Converse, R.H. 1987. Tomato ringspot virus in Rubus. Pp.223-227 In: Virus Diseases of Small Fruits. R. H. Converse, ed. US. Dept. Agric.Agric. Handb. 631.

Stout M.J., Fidantsef A.L., Duffey S.S. and Bostock, R.M. 1999. Signalinteractions in pathogen and insect attack: systemic plant-mediated interactions betweenpathogens and herbivores of the tomato, Lycopersicon esculentum. Physiol. Mo!. Plant Pathol. 54:115- 130.

Susaimuthu, J., Tzanetakis, I., Gergerich, R. and Martin, R. 2005. Evidencefor mixed infections by two or more viruses causing severe symptoms and decline ofblackberry. Phytopathology 95:S 101.

Tatchell, G.M., Plumb, R.T. and Carter, N. 1988. Migration of alate morphsof the bird cherry aphid (Rhopalosiphum padi) and implications for the epidemiologyof barley yellow dwarfvirus. Ann. App!. Biol. 112:1-11.

Thacker, J.R.M.2002.An Introduction to Arthropod Pest Control. Cambridge University Press, Cambridge, United Kingdom, pp 112-134.

Thackray, D.J., Jones, R.A.C, Bwye, A.M. and Coutts, B.A. 2000. Furtherstudies on the effects of insecticides on aphid vector numbers and spread of cucumber mosaicvirus in narrow-leafed lupins (Lupinus angustifolius). Crop Protection 19:121-139.

Thompson J., Higgins D., Gibson T., Thompson J.D., Higgins D.G. andGibson, T.J. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties andweight matrix choice. Nucleic Acids Res. 22:4673-4680.

Thompson, J.R. and Jelkmann, W. 2003. The detection and variation ofStrawberry mottle virus. Plant Dis. 87:385-390.

Thompson, J.R., Leone, G., Lindner, J.L., Jelkmann, W. and Schoen, C.D.2002 Characterization and complete nucleotide sequence of Strawberry mottle virus: A tentative member of a new family of bipartite plant picorna-like viruses.J. Gen. Virol. 83:229-239. 130

Thompson, J.R., Perry, K.L. and De Jong, W. 2004. A new potato virus in a newlineage of picorna-like viruses. Arch. Virol. 149:2141-2154.

Thompson, R.R., Leone, G., Lindner, J.L, Jelkmann, W. and Schoen, C.D. 2002. Characterization and complete nucleotide sequence of Strawberry mottle virus: a tentative member of a new family of bipartite plant picorna-like viruses.J. Gen. Virol. 83:229-239.

Tzanetakis, I.E., Mackey, I.C. and Martin, R.R. 2004. Strawberry necroticshock virus: a new virus previously thought to be tobacco streakvirus. Acta Hortic. 656: 27-31.

Usugi, T. and Saito, Y. 1979. Satsuma dwarf virus. CMI/AAB Descriptionsof Plant Viruses, no. 208.

Weather Underground, Inc. 2005. p://www.wunderground.com

Weber, C.A.2003.Genetic Diversity in Black Raspberry(Rubus occidentalisL.) Detected by RAPD Markers. HortScience 3 8:269-272.

Yoshikawa, N. and Converse, R.H. 1990. Strawberry pallidosis disease:distinctive dsRNA species associated with latent infections in indicators and in diseasedstrawberry cultivars. Phytopathology 80:543-548.