A systems-based approach to manage strawberry virus diseases

Abstract

Since the turn of the 21st century, there has been a significant increase in the number of viruses known to infect strawberry. This is due in part to advances in detection technologies, which allow for the identification of agents associated with graft-transmissible diseases, and the expansion of both crop production and virus vectors into new geographic areas. This paper identifies the virus diseases affecting strawberry in North America and summarizes steps and measures that can be taken to minimize their impact on strawberry yield, starting from cultivar selection to nursery propagation and field production. The implementation of these measures can minimize virus impact on strawberry yield and fruit quality.

Résumé

Depuis le début du 21^e siècle, nous avons noté une forte augmentation du nombre de virus qui s’attaquent à la fraise. Cela est dû principalement aux avancées dans les technologies de détection, qui permettent l’identification d’agents associés aux maladies transmissibles par greffage, ainsi qu’à l’accroissement des surfaces en production et à l’augmentation des vecteurs de virus dans de nouvelles régions géographiques. Cet article décrit les maladies virales qui s’attaquent à la fraise en Amérique du Nord et résume les étapes qui peuvent être suivies et les mesures qui peuvent être appliquées pour minimiser leurs répercussions sur la production de fraises, allant de la sélection des cultivars en passant par la propagation en pépinière, et ce, jusqu’à la production en champ. La mise en œuvre de ces mesures peut minimiser les effets des virus sur la production de fraises et la qualité des fruits.

Keywords:

• advanced detection technologies
• breeding
• disease detection
• Fragaria × ananassa
• large scale sequencing
• nursery production

Mots clés:

• détection des maladies
• Fragaria × ananassa
• production en pépinière
• sélection séquençage à grande échelle
• technologies avancées de détection

Introduction

Strawberry Fragaria × ananassa Duchesne is the most popular berry crop and is grown around the world, from the subarctic to subantarctic areas (Hummer Hancock 2009). The world production in 2011 was estimated at over 4.5 million tons, with the USA the leading producer with 29% of total production, followed by Spain and Turkey with 11 and 6.5% of production, respectively (Anonymous 2016a). Strawberry production has increased by an estimated 80% over the last 20 years (Anonymous 2016a), primarily because of the global movement to an annual plasticulture system. In the USA alone, strawberry production increased by 130% from 0.57 to 1.31 million tons between 1990 and 2011 (Anonymous 2016a). The increase in production is attributed to improved germplasm, to adequate fertilization and enhanced pest/disease control under plastic (Hancock 1999). The increased quality of the planting material is due to strict pest and disease management practices. Despite this, disease outbreaks can occur at the nursery or the field level, leading to disease epidemics. Problems encountered during propagation or production, where a management component is misused or mishandled, can lead to pest outbreaks. The occurrence of a new pest or pathogen in a new area can result in breakdown of plant resistance, leading to significant economic losses, as seen in California fields in 2002–2004 and propagation nurseries in Eastern Canada in 2012 (Martin & Tzanetakis 2006, 2013; Xiang et al. 2015).

Any model applied for the control of virus diseases needs to consider variables such as the type of virus and associated vectors, plant genotype, and time and location of planting. The viruses that infect strawberry and associated vectors have been well studied (Table 1). In general terms, as production moves from temperate/humid to drier/arid conditions, there is a decrease in aphid- and pollen-borne viruses, with an increase in viruses transmitted by whiteflies (Table 1; Martin & Tzanetakis 2006; 2013). Nematode-transmitted viruses have always been of concern in non-fumigated soil but they do not pose a threat to the strawberry industry at the present time (Martin & Tzanetakis 2006). This may change in the future when new, more environmentally friendly nematicidal compounds are developed which have lower potency against nematodes, resulting in the reemergence of nematode-borne viruses. In many cases, location and timing of strawberry planting are determined by plant genotype, climatic conditions, soil fertility and the targeted market for the fruit, which all determine the strawberry production system that will be used.

Table 1. Nomenclature, transmission method, detection method and geographic distribution of strawberry viruses.

Virus Name	Acronym	Genus	Transmission	Detection	North America	South America	Europe	Africa	Asia	Australia/New Zealand
Apple mosaic virus	ApMV	Ilarvirus	Pollen/seed*	ELISA, RT-PCR	Yes	Yes	Yes	Yes	Yes	Yes
Arabis mosaic virus	ArMV	Nepovirus	Nematode/persistent#*	ELISA, RT-PCR	Yes	Yes	Yes	Yes	Yes	Yes
Beet pseudo yellows virus	BPYV	Crinivirus	Whiteflies/semi-persistent	ELISA, RT-PCR	Yes	Yes	Yes	Yes	Yes	Yes
Cucumber mosaic virus	CMV	Cucumovirus	Aphids/non-persistent#	ELISA, RT-PCR	Yes	Yes	Yes	Yes	Yes	Yes
Fragaria chiloensis cryptic virus	FCCV	Deltapartitivirus	Pollen/seed#*	RT-PCR	No	Yes	N/A1	N/A	N/A	N/a
Fragaria chiloensis latent virus	FClLV	Ilarvirus	Pollen/seed#*	ELISA, RT-PCR	Yes	Yes	N/A	N/A	N/A	N/A
Raspberry ringspot virus	RpRSV	Nepovirus	Nematodes/persistent#*	ELISA, RT-PCR	No	No	Yes	N/A	Yes	No
Strawberry chlorotic fleck virus	SCFaV	Closterovirus	Aphids/semi-persistent	RT-PCR	Yes	N/A	N/A	N/A	N/A	N/A
Strawberry crinkle virus	SCV	Cytorhabdovirus	Aphids/persistent	RT-PCR	Yes	Yes	Yes	N/A	Yes	Yes
Strawberry latent C virus	SLCV	Nucleorhabdovirus	Aphids/persistent?	N/A	Yes	N/A	N/A	N/A	N/A	N/A
Strawberry latent ringspot virus	SLRSV	Unassigned/Secoviridae	Nematodes/persistent#*	ELISA, RT-PCR	Yes	No	Yes	Yes	Yes	Yes
Strawberry leaf curl virus	StLCV	Begomovirus	Whiteflies/semi-persistent	ELISA, PCR	N/A	N/A	N/A	Yes	N/A	N/A
Strawberry mild yellow edge virus	SMYEV	Potexvirus	Aphids/persistent	ELISA, RT-PCR	Yes	Yes	Yes	Yes	Yes	Yes
Strawberry mottle virus	SMoV	Sadwavirus	Aphids/semi-persistent	RT-PCR	Yes	Yes	Yes	N/A	Yes	Yes
Strawberry necrotic shock virus	SNSV	Ilarvirus	Pollen/seed#*	ELISA, RT-PCR	Yes	N/A	Yes	N/A	Yes	Yes
Strawberry pallidosis associated virus	SPaV	Crinivirus	Whiteflies/semi-persistent	RT-PCR	Yes	Yes	N/A	N/A	N/A	Yes
Strawberry polerovirus-1	SPV-1	Polerovirus	Aphids/persistent	RT-PCR	Yes	Yes	N/A	N/A	N/A	N/A
Strawberry pseudo mild yellow edge virus	SPMYEV	Carlavirus	Aphids/non-persistent	ELISA?	Yes	N/A	N/A	N/A	Yes	N/A
Strawberry vein banding virus	SVBV	Caulimovirus	Aphids/semi-persistent	PCR	Yes	Yes	Yes	Yes	Yes	Yes
Tobacco necrosis virus D	TNVD	Necrovirus	Olpidium?#*	ELISA, RT-PCR	Yes	Yes	Yes	Yes	Yes	Yes
Tobacco streak virus	TSV	Ilarvirus	Pollen/seed#*	ELISA, RT-PCR	Yes	Yes	Yes	Yes	Yes	Yes
Tomato black ring virus	TBRV	Nepovirus	Nematodes/persistent#*	ELISA, RT-PCR	No	N/A	Yes	N/A	Yes	N/A
Tomato ringspot virus	ToRSV	Nepovirus	Nematodes/persistent#*	ELISA, RT-PCR	Yes	Yes	Yes	Yes	Yes	Yes

^aData are as of December 2016.

*Also transmitted by pollen-feeding arthropods.

#Pollen and seed transmitted.

¹Not tested.

Germplasm plays a significant role in the prevalence of virus diseases and generally, current strawberry cultivars show tolerance to many different viruses. Most cultivars grown are asymptomatic with single or even double virus infections, excluding those vectored by nematodes, and symptoms only develop when multiple viruses infect the plants (Martin & Tzanetakis 2006). Given the complexity of the production system (plant genotype, timing of infection and virus isolates/strains), determining the symptomatology and disease onset with different virus combinations is challenging.

In the current production systems, there is minimal ability to change any of the components required for disease control. For this reason and at least for the near and intermediate future, control strategies will be systems-based and will focus on best management practices from the time of genotype selection to nursery multiplication to production fields.

This paper will focus on the application of new knowledge in the development of best management practices for strawberry virus diseases, from the time a breeding accession is selected for release as a new cultivar to the commercial production in the field.

Breeding for virus resistance

Breeding materials require assessment for seven or more years in the field for the selection and evaluation of desired traits before they are released as a named cultivar. Over this time period, there is a high probability that the plants will be infected by one or more viruses. Since breeders normally eliminate visibly weak plants, most advanced selections are tolerant to single infections and in areas with high vector and virus pressure selections are often tolerant to mixed infections. These selections, if infected by one or more viruses and symptomless, could allow for a pathway for virus dispersal if virus(es) were not detected and eliminated prior to cultivar release. The normal procedure is to have a selection indexed using laboratory tests such as ELISA and PCR for known viruses prior to release, but most importantly also testing for unknown viruses by graft indexing (Gergerich et al. 2015). Grafting has been an invaluable tool for virus identification since the introduction of Fragaria vesca L. and F. virginiana Duchesne clones UC 4–6 and UC10-12, respectively, which are used as indicators for virus infection (Converse 1987). Present guidelines require that candidate G1 plants (advanced selections being considered for cultivar release) be indexed for viruses by grafting onto F. vesca and F. virginiana indicators. If symptoms are observed, the selection undergoes therapy combined with meristem-tip culture to eliminate the agent(s); if plants remain asymptomatic, then the selection is released for propagation (Converse 1987). However, this approach has a blind spot: what if an unknown agent(s) which does not cause symptoms on these standard indicator hosts enters the plant production system? Such a case has occurred recently, where a plant indexed negative on F. virginiana and F. vesca (UC-5), both accepted indicators for strawberry viruses, but when the plants were reindexed upon introduction to the USA using F. vesca ‘Alpine’ as an indicator, symptoms were observed (R.R. Martin, personal observations). After the test cultivar was subjected to thermal therapy and meristem-tip culture, regenerated plants indexed negative on F. vesca ‘Alpine’ in repeated tests. Similar problems have occurred with biological indexing in Rubus (Susaimuthu et al. 2007, 2008) and grapes (Al Rwahnih et al. 2015).

Disease development on indicator host species is a well-tested approach for virus indexing and is considered the ‘gold standard’ for screening plant genotypes before they enter a plant certification system. However, the results are subjective, requiring specialized expertise for grafting and recognizing symptoms, and as mentioned above, is not always effective for mild strains or unknown viruses. New technologies including large scale sequencing (LSS) combined with the development of bioinformatic pipelines have also changed the way detection and discovery of plant viruses is conducted (Barba et al. 2014; Ho & Tzanetakis 2014; Martin et al. 2016). LSS and bioinformatics analysis present a neutral approach, even though the human involvement and the depth of knowledge in the databases can affect the accuracy and effectiveness of the analysis. As part of the National Clean Plant Network (http://nationalcleanplantnetwork.org/), the effectiveness of LSS combined with bioinformatics is being evaluated for detection of major strawberry viruses as well as viruses of other crops included in the network. We are evaluating depth of sequencing and multiplexing samples to reduce costs, and also comparing the sensitivity of this technology with grafting. The goal is to provide recommendations based on the sensitivity and specificity of LSS. This could lead to a shift from grafting to LSS as the method of choice in evaluating virus infections in candidate G1 plants before they enter certification schemes. We hypothesize that the unbiased nature of LSS will outperform grafting as has been shown for grapevine (Al Rwahnih et al. 2015). However, we need to be cognizant of the importance of the quality and depth of the sequence databases in the annotation of the results. If all new strawberry viruses are found within the established virus families, then it is almost certain that LSS combined with bioinformatics will be successful in identifying new viruses in strawberry, as has been done recently with Strawberry polerovirus-1 (Xiang et al. 2015). On the other hand, if a virus is ‘exotic’ and does not present any significant homology to annotated genes and proteins, then there is the risk of an ‘unknown’ diagnosis which can escape detection, as was recently reported for Tilapia lake virus (Bacharach et al. 2016). In such a case, grafting may represent a better choice. However, since decisions are usually based on risk assessment, if the implementation of both approaches cannot be done, then the best method under current conditions should be adopted.

Nursery propagation of germplasm

Assuming that a germplasm accession is found to be free of viruses of importance as tested by grafting or LSS, it is then released to nurseries for propagation. In almost all cases, nurseries follow the four propagation cycle system, referred to as generations G1–G4. G1 represents the top tier plant that has been found free of all viruses of importance and is maintained under protected culture, and G4 is the material which is shipped to producers for field establishment (Gergerich et al. 2015). Because of the importance of the material and the likelihood that undetected virus infection at the nursery level could result in an epidemic in the production fields, nurseries have to deploy systems-based protocols to minimize the potential for infection. In nurseries, the focus is on virus control, which includes many of the symptomless viruses. This is especially important since single virus infections are symptomless in most if not all currently grown commercial strawberry cultivars. Thus, virus infections in the nursery most often go unnoticed. For this reason, there are best management practices that are based primarily on vector biology in place that when followed should minimize the possibility of significant virus infection during the propagation process. As plants move through the propagation pipelines (G1 → G2 → G3 → G4), testing and isolation guidelines become less stringent, since an infection late in the propagation pipeline will affect fewer plants. This is based on the need to have a balance between safeguarding the material and allowing for the economic sustainability of nursery operations. For example, during the first propagation step (G2), the buffer (isolation) zone between the plant material and native strawberries or weeds that may harbour strawberry viruses may reach 200 m or more (Anonymous 2016b). Often G2 material is grown in protected culture, which greatly reduces isolation distance requirements. In addition, these plants are monitored regularly for all strawberry viruses of importance that occur in the area of propagation, since virus introduction at this level will lead to the propagation of infected plants during the following propagation cycles. As plants are further propagated and grown in the open, it is not economical to test all plants for all viruses of interest. At this point, it is more suitable to perform testing based on infection risk parameters. Limited testing is conducted for the one or two viruses that are most likely to be present in the area where the nursery is located. This approach can identify potential problems in the system with sampling intensity based on a hypergeometric table, thereby providing the desired balance of safeguarding the crop and addressing the economics of nursery production. During the final propagation step (G4), and depending on the local conditions and presence of viruses, plant material may be tested once more, but at a threshold that aims to eliminate the possibility of epidemics. This testing may be done at a frequency to detect a 5% infection level with 95% confidence limits. Based on the ISMP 31 (Anonymous 2008) this requires 59 samples (single or composite) per block – this may be 59 samples in millions of plants. The ISMP 31 also gives numbers for other levels of confidence and detecting other levels of infection. If the samples can be tested as composites of 5, then it is 12 tests per block. In addition to the obvious strategies to minimize virus movement (vector control, deflowering, etc.), nurseries need to have contingency plans on how to address problems such as the presence of a new virus in the area or the emergence of a vector – aerial, soil- or water-borne (Anonymous 2016b). If this procedure is followed, there will be minimal risk for virus infection in the nursery.

Field production

Unlike the nursery environment where virus control is the objective and control of all virus vectors is required, the producers only need to identify and control factors that lead to losses in fruit yield and/or quality. Thus, fruit growers are concerned with disease control and preventing virus complexes that can cause crop losses. If the propagation material is free of viruses of importance to start and the producer is using an annual production system, with fumigation treatment before planting, there may not be a need for vector control. While viruses may be present and accumulate in plants over the course of the production cycle, infection rates will be under the threshold to cause economic damage (Martin & Tzanetakis 2013). Thus, the producer can benefit economically since vector control costs should be reduced. For producers using the matted-row perennial system, where the plants are maintained for production over multiple years (usually 2–4), vector control is critical if they are known to occur in the area of production. Even if virus infections are not obvious in the first season, if they accumulate over time, yield and fruit quality can decline rapidly (Martin & Tzanetakis 2013).

There are always instances when the two growing regimes described above are not applicable. For example, in a growing area which is 100% in annual plasticulture production, if vectors for the viruses are not prevalent in the area, producers may opt not to control them and may observe little or no effect on production that season. But, if the plants are retained for a second year by the producers to save on planting costs, the virus dynamics will have changed drastically and virus diseases will probably develop. This practice means that there is not a strawberry-free period between crops and the viruses can persevere in the production area and be spread from crop to crop, causing yield and quality losses in subsequent plantings. This is especially true for the aphid-transmitted viruses. For example, the common strawberry aphid (Chaetosiphon fragaefolii Cockerell) feeds primarily on strawberry (Converse 1987) and a strawberry-free period serves to greatly reduce the vector population without the need for insecticides.

Management of weeds is important within the production field as well as in outlying areas, since they could serve as reservoirs for strawberry viruses present in the area. One example are the strawberry whitefly-transmitted viruses: their host range includes several of the weed species that are commonly found in and around strawberry fields (Wintermantel 2004; Tzanetakis et al. 2006). In such a case, it is important that producers control weeds within the fields and also around them to minimize movement of vectors and viruses to the crop. If this is not done, the whiteflies, which are strong fliers, would enter the field when the weed hosts senesce, since strawberry plants remain green and attractive to insects during production. In such a scenario, the virus influx may result in plant collapse at the peak of production, resulting in significant yield losses (Martin & Tzanetakis 2006, 2013). This type of scenario has occurred along the south-central coast of California, where the whitefly transmitted viruses, Strawberry pallidosis associated and Beet pseudo yellows viruses are prevalent in some of the fruit production areas because of the alternate hosts (crops or weeds) for the viruses and whitefly vector. In the summer of 2013, growers were impacted by a large influx of whiteflies from adjacent vegetation and subsequent development of virus disease in strawberry caused by Strawberry pallidosis associated virus and Strawberry mottle virus. The Strawberry mottle virus was present in the strawberries but not causing disease until whitefly transmitted viruses were introduced into the fields. This situation was also compounded by overlapping strawberry production cycles and thus a lack of a strawberry-free time during the production year.

Conclusions

The strawberry industry in North America is vibrant and fruit production and quality is continuously improving annually, with dramatic increases in yield having occurred in the last 20 years (Anonymous 2016a). A major issue that could affect this growth is the quality of propagation material. Infection by viruses during nursery production may lead to epidemics in the field that could cause tens of millions of dollars of losses as observed on the west coast of North America from 2002–2004 (Strawberry mottle virus, Strawberry crinkle virus, Strawberry vein banding virus, Strawberry mild yellow edge virus, Strawberry pallidosis associated virus and Beet pseudo yellows virus) and in the south-eastern USA in 2012–2013 (Strawberry mottle virus and Strawberry mild yellow edge virus). With warming climates, higher abundances of insect vectors and increased range of viruses, there can be increased disease pressure in some areas (Martin & Tzanetakis 2013). On the other hand, improved detection technologies for strawberry viruses, such as LSS, can aid in rapid diagnosis and provision of G1 plants that are clean for propagation within the nursery system. Of importance is the implementation of best management practices that are tailor-made for the locality of the propagation nursery (Gergerich et al. 2015; Anonymous 2016b). Knowledge of the viruses affecting strawberries in North America can assist producers in making appropriate and timely decisions, to ensure a sustainable and profitable production scheme.

Additional information

Funding

This work was supported by the North American Strawberry Growers Association; Hatch Project [1002361]; USDA-APHIS [Farm Bill 10007 4.0104]; USDA [Interagency Agreement 6-2072-5-002]