​Introduction

Small fruit virology is one of the least studied areas of plant pathology. Unlike row crops or vegetables that are being grown around the world and are consumed in incalculable quantities, small fruits are primarily grown in temperate areas and account, in most cases, for a few thousand hectares, miniscule when compared to wheat, rice, maize or soybean. Therefore, there is a much greater emphasis on research on diseases of those crops than on small fruits. Also, small fruits are not conducive to applying modern, primarily molecular, techniques because they tend to have high levels of polysaccharides and other inhibitors of enzymatic reactions. At the same time, there is a renewed interest in production of small fruit as health foods because of their antioxidant and anti-inflammatory properties. This has led to a surge in the planted area of these crops across the globe. For example, China now grows more than 120,000 hectares of strawberries (2), whereas the blueberry and blackberry acreage in the southeastern United States has more than doubled in the past decade (1,13). With the expansion of crops into new production areas, it is common that new diseases emerge causing unforeseen problems and substantial yield losses. This has been the case for the three major small fruit crops: strawberry, Rubus (blackberry, red and black raspberry and their hybrids), and blueberry. In many cases with strawberry and Rubus, plants with single virus infections are symptomless, but symptoms appear when two or more viruses accumulate in the host (6,7). This is a major departure from the old paradigm where different symptomologies were attributed to mild and severe virus strains (3,5,10). In this communication, research performed on major small fruit virus diseases that emerged in the United States during the past decade will be presented.

Strawberry

The journey begins in 2002 along the west coast of North America when a severe decline with some mortality was observed in strawberry fruiting fields. Plants developed red discoloration in the older leaves and collapsed leading to major losses from British Columbia to southern California (Fig. 1). Losses were estimated in the $25 million range for each of the 2002 and 2003 seasons in the Watsonville, California area [(7) Mark Bolda, University of California Cooperative Extension]. Symptoms were strikingly similar between the highly diverse genotypes grown in California and the Pacific Northwest (PNW) and the different cultivation practices between the two areas, with the annual plasticulture in the south and the perennial matted row production in the PNW. Given that all commercial strawberries are propagated by a relative small number of nurseries and are sold as leafless, bare-rooted plants, it was assumed that there may have been contamination during the propagation process with one of the usual strawberry fungal pathogens causing the decline (Colletotricum acutatum, Phytophthora cactorum, P. fragariae, Verticillium dahliae). Once it was determined that the declining plants were free of those pathogens, the investigation turned to viruses. It was expected that viruses would be present in the perennial production systems of the PNW, but cultivars known to be tolerant to the common virus complex in the area were succumbing to decline. In the annual production systems used in California, virus diseases were rare in fruiting fields due to the short period of time that plants remained in the ground, usually less than one year.

Fortuitously, at the time of strawberry decline emergence, work was being conducted on strawberry pallidosis disease. At the time, pallidosis was considered a minor disease, since even in indicator plants it caused very subtle symptoms. Pallidosis was first described in 1957 (4) and it was not until 2001 that the pallidosis-associated viruses, Strawberry pallidosis associated virus (SPaV) and Beet pseudo-yellows virus (BPYV), were identified (16,23). Both viruses belong to the genus Crinivirus, family Closteroviridae and are solely transmitted by Trialeurodes vaporariorum, the greenhouse whitefly (GWF). What was unforeseen at the time was the great number of greenhouse whiteflies in decline-affected fields in California

Whiteflies were not reported as a major pest in strawberry fields until the GWF became the dominant genotype in California near the beginning of the 21st Century. The first samples tested showing decline symptoms were 90% and 40% infected with SPaV and BPYV, respectively. When grafted experiments were performed using both viruses on two virus susceptible cultivars, no symptoms were observed (Tzanetakis, unpublished), suggesting that there was more to decline than these two viruses. In addition, these two viruses were rare in declining plants in the Pacific Northwest. Thus, the research was directed to identify what other viruses might be contributing to this new disease in strawberry.

Detection tests for all the viruses known to infect strawberry were developed and massive testing was performed. The most important strawberry viruses, those transmitted by aphids, were present in California at low numbers and there were several plants showing severe symptoms but were only found infected with SPaV and BPYV. Consequently, it was decided to characterize all graft-transmissible agents known to infect strawberry to determine whether those agents were the missing link in the development of strawberry decline. This quest yielded several new viruses along with known viruses that had never been reported in strawberry in the US and/or elsewhere. We discovered two new criniviruses, closely related to SPaV and BPYV, and both found in a large subset of diseased samples (Tzanetakis and Martin, unpublished). We also discovered Strawberry latent ringspot virus (8), a quarantine virus for the US, Strawberry necrotic shock virus, a virus species mislabeled as Tobacco streak virus for more than 30 years (18), Apple mosaic virus, the probable causal agent of strawberry leafroll (19), Fragaria chiloensis latent virus, a virus thought only to occur in the Southern hemisphere (20) and Strawberry chlorotic fleck associated virus, a new closterovirus (21). The myriad of new viruses gave new insight into strawberry decline. The whitefly-transmitted viruses and some of the old, but newly identified as strawberry-infecting viruses were detected in declining plants in California, whereas the whitefly- transmitted viruses where virtually absent from the northern latitudes where the aphid-borne viruses dominated. In the PNW, especially in northern Washington and British Columbia, Strawberry crinkle virus, which had not been reported from that area previously, was one of the most common viruses in declining plants. A surprise at the time was the fact that the decline symptoms in strawberry were a reaction to mixed virus infections, the identity of which was of minimal importance for symptom development. The other important part of the story was the strawberry nursery industry.

In clonally propagated crops, all plants of a particular cultivar come from a single cross that may have occurred seven to ten years before commercial release. This provides time for viruses to accumulate in plants. Even when all appropriate measures are taken before a cultivar is released, including heat and chemical therapy, meristem culture, and rigorous testing, there is always the possibility that one or more unknown viruses may not be detected or eliminated during the process. As viruses accrue in plants with latent infections, symptoms develop and major epidemics, such as the one described for strawberry decline, may occur. For this reason, a systems-based approach was implemented by the strawberry nursery industry where G1 plants (first level or first generation of plants in the propagation scheme) were tested for the new viruses, propagation fields were heavily scouted, and chemical control was implemented targeting all potential aerial vectors, including whiteflies. Previously, since there were no known whitefly transmitted viruses in strawberry, nurseries did not attempt to control them. Once these strategies were implemented, the incidence of strawberry viruses in nursery plants dropped dramatically. As a result, in the annual production system in California, decline symptoms have not been observed since 2005 and it has not been necessary to control vectors in fruiting fields despite a significant number of plants that become infected with one or more viruses over the 11-12 months the plants are in the fruiting fields. The number of viruses per plant does not reach a critical level to result in decline symptoms. In the perennial matted row system, vector control is still important since there is high disease pressure on new fields from 2-4 year old neighboring fields. Commercially grown strawberry plants are the major source of inoculum in the region. Control measures that were developed were targeted to the weakest link, the vector that moves slowest and is the easiest to control. In the PNW, especially northern Washington and British Columbia, the dominant viruses are aphid-borne and their mode of transmission is persistent or semi-persistent, providing sufficient time for pesticides to act between virus acquisition and virus transmission, thus minimizing virus movement. At the present time, growers that implement aphid control are able to produce strawberries economically for three years in a cropping cycle. The ideal scenario for the PNW would be a coordinated vector control program over a large area, such as the Fraser Valley or the Skagit Valley. This would entail aphid control by all growers for four years to reduce inoculum levels in the area.

Rubus

At the time strawberry decline emerged along the west coast, a new disorder named blackberry yellow vein disease (BYVD) was documented in blackberry in the southeastern US. Plants developed bright oak-leaf chlorosis along main leaf veins and ringspots whereas malformed leaves were also observed (Fig. 2). The major impact of the disease was rapid decline, which significantly reduced yield and plant longevity. Normally, blackberry fields should remain productive with profitable yields for 20 years in the southeastern US. Today, because of BYVD many fields are in a 5-year rotation, increasing grower input costs and reducing profitability.

Symptoms, especially ringspots and malformed leaves had long been attributed to the nematode-transmitted Tobacco ringspot virus (TRSV), which was widespread in areas where the disease was prevalent. However, the overwhelming majority of symptomatic samples were not infected by this virus, nor were they infected with other Rubus viruses known to occur in the US at the time. As the strawberry decline story was unfolding, the idea of new viruses in blackberry became the dominant hypothesis behind this emerging blackberry disease. Several plants were assayed for the presence of new viruses and all were found infected with a new crinivirus, the most closely related virus to SPaV and BPYV known to date (22). The virus was named Blackberry yellow vein associated virus (BYVaV) and was thought to be the causal agent of BYVD as all symptomatic samples tested were infected with it. However, grafting experiments showed that it is asymptomatic in indicators and some cultivars, and it was also detected in symptomless nursery material (14). The experience with strawberry decline led to the hypothesis that BYVD was also caused by virus complexes.

BYVaV was discovered in 2002 (9). The number of characterized Rubus viruses has more than doubled in the past decade, primarily because of a myriad of new viruses found in BYVD material. The distribution and incidence of the new viruses identified in BYVD affected plants varies by location. There are species within the newly identified viruses that can be transmitted horizontally and vertically through pollen and seed, as well as viruses that can be transmitted by thrips, mites, hoppers, aphids and whiteflies (6). Vectors of some of these new virus species are yet to be identified, as they cannot be deduced based on genomic data. As in the case of strawberry decline, we now understand that the identity of the viruses found in the plants are not as important as the numbers of viruses present in affected material. That being said, certain combinations of viruses can have a major impact on plant productivity and as shown in the BYVaV/Blackberry virus Y (BVY). BVY is one of the new viruses mentioned above, and in combination with BYVaV, can even lead to plant death (Fig. 3) (15).

Although there are obvious similarities between strawberry decline and BYVD, there are major differences between the two crops, including cultivation practices and growing environment. In strawberry, the vast majority of production is based on an annual system that eliminates most of the inoculum reservoirs, since there is usually at least a short strawberry-free period between cropping cycles. Blackberries, on the other hand are perennial and fields are in production for many years providing constant inoculum to new fields, somewhat similar to the strawberry virus situation in the Pacific Northwest. In addition, and unlike strawberry, there are blackberry growers that propagate their own material from plants that may have been in the field for several years but appear healthy, which exacerbates the situation. In addition, most strawberry production fields are isolated from major populations of native Fragaria species, whereas wild blackberries are very often found in close proximity to production fields. It is likely that vectors prefer feeding on vigorously growing plants, and thus move from wild to cultivated material as the season progresses, vectoring viruses in the process. The microenvironments for blackberry production are very diverse and several viruses that affect the crop are found in limited areas. Additionally, with the diversity of viruses of blackberry and multiple types of vectors, there is not a single strategy that can be used to manage BYVD, making the management of this virus complex much more challenging than strawberry decline. In addition to the very diverse array of vectors, the mode of transmission of the new blackberry viruses spans from non-persistent to persistent propagative. Given the situation, it is obvious that BYVD cannot be eradicated using cost effective approaches. For this reason, a systems-based approach has also been used in the management of BYVD focusing on the virus/vector/environment/ locality combinations; we identify the components that can be controlled in a particular area or microenvironment. This includes elimination of wild blackberry populations, targeted vector control or even the enhancement of natural predatory populations that can minimize vector movement in and out of the field. Given the complexity of the issue, the 20-year blackberry production span in the southeastern US is likely a thing of the past. Realistically, research will provide information that will result in developing strategies that would extend the present 5-year rotation by several years to enhance grower profitability.

Raspberry production in the PNW has experienced a major problem with Raspberry bushy dwarf virus (RBDV) since the early 1990s with the transition from the RBDV resistant cultivar Willamette to more productive and ‘appealing’ cultivars. RBDV, a pollen-transmitted idaeovirus, affects several major cultivars and causes abortion of fruit drupelets causing what is known as crumbly fruit (Fig. 4) (12). The raspberry receptacle separates from the fruit and when drupelets have not developed properly the fruit falls apart during harvest. The crumbly fruit is only suitable for the low value juice, jam or puree markets and not for the high value fresh or individually quick frozen (IQF) markets. It is now thought that crumbly fruit is another case of a disease caused by a virus complex causing disease. In addition to RBDV, two aphid-borne viruses, Raspberry leaf mottle virus (RLMV), a closterovirus (17) and Raspberry latent virus (RpLV), a reovirus (11) have been found in very high incidence in areas where severe crumbly fruit was observed. RLMV infection rates reach 100% in 3-year old fields in northern Washington state, the primary raspberry production area in the United States, whereas RpLV is less prevalent with approximately 50% infection in 5-year old fields (Quito-Avila et al., unpublished). Triple-infected plants (RBDV, RLMV, and RpLV) showed reduced growth and vigor compared to virus-free or single-infected plants (Quito-Avila et al., unpublished). It is hypothesized that fruit from triple-infected plants will exhibit a severe crumbly fruit phenotype. Work is underway on the epidemiology of the two new viruses that would assist in the development of control strategies in the area.

Blueberry

Blueberry is the small fruit crop that appears to have the least number of problems with virus infections, although a generalized decline of yet unknown etiology has been observed in the US midsouth. Blueberry is a plant adapted to acid soils that has an unusual vascular architecture that leads to slow lateral movement resulting in uneven virus distribution. Blueberry tissue is also very acidic, which leads to problems with virus and RNA extractions for ELISA or RT-PCR assays, respectively. In most cases single virus infections in blueberry can cause significant losses as has been shown for Blueberry shoestring, Blueberry leaf mottle, Tomato ringspot (ToRSV), Blueberry scorch and Blueberry shock (3); however, symptoms are enhanced in mixed infections. In blueberry, most viruses are regional in their distribution, with the exception of ToRSV and TRSV. Thus, it is critical that only virus-tested material be moved from area to area to avoid increasing the distribution of these viruses.

In the last four years a new disease named blueberry necrotic ring blotch has emerged in several areas in North and South Carolina, Florida and Mississippi. The presence of this disease is manifested by concentric rings that appear on both sides of leaves of affected plants (Fig. 5). This can lead to severe defoliation of affected bushes, resulting in losses due to lack of fruit photosynthates. A new virus has been identified in all plants with typical symptoms. The positive-strand RNA virus will probably be the first member of a new genus with distant relationships to members of the Virgaviridae (Martin et al., unpublished). The rapid progression of disease symptoms in the field indicates an aerial vector and work is under way to identify and characterize the vector, based on genomic sequences the vector is speculated to be an eriophyid mite.

The Future

As outlined above, all major small fruit crops have suffered substantial losses in the past decade because of the emergence of new virus diseases. Similar scenarios have been observed for most vegetatively propagated crops. For this reason, combined with experience on the value of plants free of known viruses through establishment and productivity of clonally propagated fruit and nut crops, the United States Department of Agriculture has developed a new initiative, the National Clean Plant Network (NCPN). Berries are a major component of NCPN, which aims to develop and maintain high quality fully virus-tested G1 propagation material that will feed into National Certification Programs which are presently being formulated. As new viruses are found in the crops, the G1 plants will be tested for these viruses to continually improve the health status of the G1 blocks, eliminating infected material from propagation. The primary strategy for virus control in vegetatively propagated crops is to use plants free of known viruses, something that would allow for field longevity and grower profitability.

Acknowledgments

The work presented here would not have been possible without the moral and scientific support of Dr. R. R. Martin (USDA-ARS, Corvallis, OR). The help of Dr. W.M. Wintermantel, Dr. S. Sabanadzovic, the Tzanetakis and Martin labs is also greatly appreciated. The work presented here was made possible with funds from the North American Strawberry Growers Association, the California Strawberry Commission, the North American Raspberry and Blackberry Association, the Northwest Center for Small Fruits Research, USDA-NIFA-SCRI, USDA-APHIS-NCPN, the Southern Region Small Fruit Consortium, and the Arkansas Agricultural Experimental Station.

Literature Cited

1. Anonymous. 2010. USDA Economics, Statistics and Market Information System: U.S. Blueberry Industry. Online. USDA-ERS, Washington, DC.

2. Beckman, C., and Lei, Z. 2009. GAIN Report Number CH6056.

3. Converse, R. H. (ed.) 1987. Virus Diseases of Small Fruits. Agriculture Handbook No. 631, USDA ARS, Washington, DC.

4. Frazier, N. W., and Stubbs, L. L. 1969. Pallidosis: A new virus disease of strawberry. Plant Dis. Rept. 53:524-526.

5. 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. 96:59-65.

6. Μartin, R. R., MacFarlane, S., Sabanadzovic, S., Quito-Avila, D. F., Poudel, B., and Tzanetakis, I. E. Viruses and Virus Diseases of Rubus. In preparation.

7. Μartin, R. R., and Tzanetakis, I. E. 2006. Characterization, detection and management of strawberry viruses. Plant Dis. 90:384-396.

8. Martin, R. R., Tzanetakis, I. E., Barnes, J. E., and Elmhirst, J. F. 2004. First report of Strawberry latent ringspot virus in strawberry in USA and Canada. Plant Dis. 88:575.

9. 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.

10. McGavin, W. J., and MacFarlane, S. A. 2010. Sequence similarities between Raspberry leaf mottle virus, Raspberry leaf spot virus and the closterovirus Raspberry mottle virus. Ann. Appl. Biol. 156:439-448.

11. Quito-Avila, D. F., Jelkmann, W., Tzanetakis, I. E., Keller, K., and Martin, R. R. 2011. Complete sequence and genetic characterization of Raspberry latent virus, a novel member of the family Reoviridae. Virus Res. 155:397-405.

12. Strik, B., and Martin, R. R. 2003. Impact of Raspberry bushy dwarf virus on ‘Marion’ blackberry. Plant Dis. 87:294-296.

13. Strik, B. C., Clark, J. R., Finn, C. E., and Bañados, M. P. 2007. Worldwide production of blackberries, 1995 to 2005 and predictions for growth. HortTech. 17:205-213.

14. Susaimuthu, J., Gergerich, R. C., Bray, M. M., Clay, K. A., Clack, J. R., Tzanetakis, I. E., and Martin, R. R. 2007. The incidence and ecology of Blackberry yellow vein associated virus. Plant Dis.91:809-813.

15. Susaimuthu, J., Tzanetakis, I. E., Gergerich, R. C., Kim, K. S., and Martin, R. R. 2008. Viral interactions lead to decline of blackberry plants. Plant Dis. 92:1288-1292.

16. Tzanetakis, I. E., Halgren, A. B., Keller, K. E., Hokanson, S. C., Maas, J. L., McCarthy, P. L., and Martin, R. R. 2004. Identification and detection of a virus associated with strawberry pallidosis disease. Plant Dis.88:383-390.

17. Tzanetakis, I. E., Halgren, A. B., Mosier, N., and Martin, R. R. 2007. Identification and characterization of Raspberry mottle virus, a novel member of the Closteroviridae. Virus Res.127:26-33

18. Tzanetakis, I. E., Mackey, I. C., and Martin, R. R. 2004. Strawberry necrotic shock virus is a distinct virus and not a strain of Tobacco streak virus. Arch. Virol. 149:2001-2011.

19. Tzanetakis, I. E., and Martin, R. R. 2005. First report of strawberry as a natural host of Apple mosaic virus. Plant Dis.89:431.

20. Tzanetakis, I. E., and Martin, R. R. 2005. New features in the genus Ilarvirus revealed by the nucleotide sequence of Fragaria chiloensis latent virus. Virus Res. 112:32-37.

21. Tzanetakis, I. E., and Martin, R. R. 2007. Strawberry chlorotic fleck: Identification and characterization of a novel Closterovirus associated with the disease. Virus Res.124:88-94.

22. Tzanetakis, I. E., Susaimuthu, J., Gergerich, R. C., and Martin, R. R. 2006. Nucleotide sequence of blackberry yellow vein associated virus, a novel member of the Closteroviridae. Virus Res.116:196-200.

23. Tzanetakis, I. E., Wintermantel, W. M., and Martin, R. R. 2003. First report of Beet pseudo yellows virus in strawberry in the United States: A second crinivirus able to cause pallidosis disease. Plant Dis. 87:1398.