California Seed & Plant Lab., Inc Pleasant Grove Rd, Elverta CA Phone: (916) Fax: (916)

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1 Viruses testing RT-PCR for Virus diagnosis We have developed a PCR procedure to detect various strains of each virus. A multiprimer cocktail is used to detect all strains of the virus. For many viruses, we use real-time PCR that provides extra sensitivity and specificity. High quality RNA is isolated from the samples and subjected to RT-PCR method. In this method, virus RNA is first converted to complimentary DNA (cdna) and then amplified by PCR. The technique is highly sensitive when compared to ELISA. Best time of the year for virus testing Some viruses reach their maximum concentrations in spring (fan leaf, arabis mosaic) while others reach their maximum concentration in the fall (leaf roll, stem infecting viruses), therefore, a highly sensitive method should be used for year round testing. Our PCR protocol includes testing of undiluted RNA (free of inhibitors) and therefore can be used year round. Virus Vector Best time for testing Arabis mosaic Nematode Spring Grapevine fan Leaf virus Nematode Spring Grapevine leaf roll associated virus 1, 2, 3, 4, 5, 7, 9 Mealy bug Fall, winter Grapevine leaf rollassociated virus 2 (stem lesion) Mealy bug Fall, winter Grapevine fleck virus None Fall, winter Grapevine Vitivirus A (Kober stem grooving) None Fall, winter Grapevine Vitivirus B (Corky bark) Long tailed mealy bug Fall, winter Grapevine Vitivirus D None Fall, winter Rupestris stem pitting virus None Fall, winter Tomato ring spot virus Nematode Fall, winter Tobacco ring spot virus Nematode Fall, winter

2 How to collect samples Since viruses may not be uniformly distributed in the plant, two stem sections (6-inch long, pencil thick), one section from each cordon, should be collected. Stem sections should be taken from the base of a new shoot. The sample should be placed in a plastic bag and properly labeled. Plastic bags should not be exposed to sunlight in order to avoid heating. The sample can be placed in a cardboard box and shipped by express mail.

3 Descriptions of important viruses 17 viruses are known to infect grapevines in California. Viruses are obligate pathogens and require a host for their survival. In general the infected plants do not die but show reduced vigor and yield, and produce poor quality grapes. The stem and the leaves show various deformations. Sometimes virus infections may be latent in some varieties. Although latent infections do not cause any symptoms, they are still a risk for the susceptible varieties in the vineyard if a vector is present. Nematodes and insects such as mealy bugs can transmit infections from one vine to another. Arabis mosaic virus (ArMV) (Figure 1) Symptoms include fan-like distortions of leaves, ringspots, line patterns, vein banding, yellowish mottling, and yellow mosaic patterns. ArMV can be present in a mixed infection with GFLV. ArMV is widespread in Europe, there are few cases in Canada and the United States, however none in California. ArMV is transmitted by nematode. Grapevine fanleaf virus (GFLV) (Figure 2) Figure 1 GFLV causes fanleaf degeneration (often referred to as fanleaf) which is the oldest and best characterized disease affecting V. vinifera. Expression of fanleaf degeneration can be in the form of one of three distinct syndromes. Symptoms of the first syndrome, infectious malformations, include severe leaf malformations, asymmetry, acute denticulations on leaves, abnormal branching, double nodes, short internodes, and sometimes chlorotic mottling on leaves. Symptoms of the second syndrome, yellow mosaic, include bright chromatic yellowing of all parts of the vine, and poor berry set. Symptoms of the third syndrome, Figure 2 veinbanding, start with chromatic yellowing along the main veins which eventually spreads into the interveinal areas. All syndromes result in reduced yield and poor berry set. GFLV is widespread affecting vineyards worldwide and is major viticultural problem in California. GFLV is transmitted by nematode.

4 Grapevine fleck virus (GFkV) (Figure 3) GFkV causes fleck disease on V. rupestris, other vitis species can be infected but do not show any symptoms. Young V. rupestris leaves infected with GFkV exhibit clearing flecks in vienlets of young leaves. On older leaves the flecks diffuse to create a mosaic pattern and the leaves curl upward. Symptoms disappear during hot weather, however, the infection remains intact. GFkV is grafttransmissible. Figure 3 Grapevine vitivirus A (GVA) (Kober Stem Grooving) (Figure 4) The grapevine vitiviruses are associated with the rugose wood complex. Diseases in the rugose wood complex are associated with the pitting and grooving of the trunk and stem, and possible foliar symptoms similar to leafroll. GVA is associated with Kober Stem Grooving. Infected vines may exhibit swelling at the graft union and may fail to thrive. Ungrafted vines may be infected, but usually do not show symptoms. GVA is graft-transmissible. Grapevine vitivirus B (GVB) (Corky Bark) (Figure 5) Figure 4 GVB is associated with corky bark disease and only affects grafted vines. Expression of corky bark is more severe in vines infected with other rugose complex diseases. The symptoms include grooving and pitting on the entire surface of grafted cultivars. In LN 33, corky bark disorder also causes severe stunting of grafted plants with internodal swelling, leaf rolling, and reddening. These disorders remain latent or symptomless in ownrooted or ungrafted wine grape cultivars, American Vitis species, and rootstock hybrids. Depending on the rootstock-scion combination, woody grapevine cylinder alterations occur on rootstocks, scions, or both. Any type of swelling above the graft union, marked difference between the relative diameter of the scion and rootstock, poor bud take, graft incompatibility or Figure 5 decline, slow growth, or delayed bud opening can be manifestations. The virus can be spread by long tailed mealy bug (Pseudococcus longispinus).

5 Grapevine vitivirus D (GVD) GVD is not known to cause disease, however, it matches the structure of the other vitiviruses. Grapevine leaf roll associated viruses (GLRaV) (Figure 6) There are several viruses in this group. GLRaV 1, 2, 3, 4, 5, 7 and 9 are found in California. These viruses are known to cause leafroll. Affected grapevines are slightly smaller than normal in their entirety, the leaves start turning yellowish or reddish and eventually roll downward later in the season with the interveinal areas of the leaves bright yellow or red. Fruit ripening is Figure 6 delayed and at harvest time fruit is small, low in sugar, and pale. Symptoms resemble mechanical damage and in most cases the infected plants do not die, thus, it is sometimes difficult to make a field diagnosis. Mixed infections can result in death. Leafroll is responsible for a reduced yield of 20%. Leafroll is widespread and can be found worldwide. Grapevine leaf roll 2 stem lesion associated virus This variant of leaf roll 2 appears to have symptoms of incompatibility. This virus was first found on Red Globe and then on other varieties. Rupestris stem pitting-associated virus (RSPaV) (Figure 7) RSPaV is associated with rupestris stem pitting. Infected vines exhibit basipetal stem pitting symptoms extending downward below the graft unions in the V. rupestris cultivar St. George, cultivars LN 33 and Kober 5BB remain symptomless. Infected Figure 7 plants slowly decline in terms of growth. The size difference between infected and healthy plants becomes apparent after several years. Fruit ripening is slightly delayed and at harvest time fruit is slightly smaller than normal and slightly lower in sugar than normal.

6 Tomato ring spot virus (ToRSV) (Figure 8) ToRSV is associated with a number of symptoms and diseases. Differences in disease expression seem to be correlated with differences in temperature. In colder regions, like the Northeast or Canada, the symptoms are short internodes, small and distorted leaves, and stunted plants. In colder areas and highly susceptible varieties infected plants are severely affected and uneconomical by the third year of infection. In California, where temperatures are higher, ToRSV causes grape yellow vein. Grape yellow vein is characterized by slow decline, yellow flecks on leaves along the veins and other parts of the lamina. The most consistent Figure 8 symptom across all temperatures is a reduced yield due to a poor berry set. ToRSV is transmitted by nematode.