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R. Carstens
ARC Infruitec-Nietvoorbij, Private Bag X5026, Stellenbosch, 7599
E-mail: roleen@infruit.agric.za

Introduction

Leafroll is the most significant virus disease found in vineyards all over the world (9). A complex of viruses (grapevine leafroll-associated viruses 1 - 8) that belong to the closterovirus group, are being associated with leafroll. Leafroll-associated viruses are restricted to the phloem of the grapevine and are easily transmitted by grafting.

History

For years leafroll has been known under the following names: rougeau, flavescence and brunisure in France (1853); Rollkrankheit in Germany and red-leaf in California. Scheu (1936) was the first in Europe to prove that leafroll is caused by a virus. Leafroll was identified for the first time in California in 1958 on the red table grape cultivar Emperor. Due to leafroll the grapes of this cultivar did not colour and the disease was forthwith called White Emperor Disease (22). Since then considerable research has been done on leafroll in order to combat the virus, but in spite of that it continues to be a serious problem in all vine-growing countries.

Symptoms

Leafroll symptoms vary depending on environmental conditions as well as the cultivars concerned. Symptoms are best observed in the period between harvesting and shedding of leaves (late summer and early autumn). If vines are subjected to conditions of stress, the symptoms tend to appear earlier. The leaf margins roll downwards and the leaf veins stay green. In red wine cultivars the areas between the veins turn red, whereas yellowing of the same leaf areas occur in white wine cultivars. In older leaves symptoms first appear near the base of the shoot. The extent to which the leaves roll differ from cultivar to cultivar. In cultivars such as Chardonnay and Sauvignon blanc, the leaf margins roll down severely, whereas cultivars such as Sultanina and Chenin Blanc display only minor rolling of the leaves. It is therefore extremely difficult to visually identify leafroll in these cultivars. Rootstocks never display leafroll symptoms and therefore the virus can be transmitted unknowingly.

Leafroll causes a degeneration of phloem tissue in young shoots, leaves, petioles and rachis. Symptoms similar to that of leafroll can be brought about by actions that damaged the phloem, for example mechanical damage to the trunk, breaking of shoots, poor graft unions, girdling and the pinching effect of ropes. Low temperatures, as well as leafhoppers, may cause discoloration, which can be confused with leafroll. All red discoloration of vineyards should therefore not inadvertently be attributed to leafroll.

Detection of the virus

The standard procedure for the detection of leafroll viruses is indexing. It entails the grafting of one or two buds from a vine with unknown virus status on an indicator vine (Cabernet franc) and symptom expression is then evaluated over a 3-year period. The green-grafting technique, where young green test plant material is grafted onto an actively growing indicator plant, presents symptoms at an earlier stage compared to standard indexing (21). Due to erratic results, this method is not much in use. Commercially manufactured antibodies against leafroll-associated viruses 1, 2 and 3 are currently available and can be used in ELISA tests for quick detection (2-3 days) of leafroll viruses. A more sensitive and strain specific method targeted at the nucleic acid of the virus, namely the polymerase chain reaction or PCR technique, can be used as an alternative detection method. This is, however, an expensive method and cannot be used for routine testing of large numbers of vines.

Situation then and now

Commercial wine and rootstock cultivars in the Western Cape had been tested in 1970 for a variety of viruses. An extremely high percentage (99,9%) of vines had been infected with viruses (17). Leafroll virus represented 68,4% of these virus infections. A much lower rate of infection was detected in the rooted (tame) scion cultivars. The high percentage virus infection can be attributed to the use of rootstocks since the turn of the century.

Rootstocks serve as symptom less carriers of leafroll virus. According to earlier research the use of infected rootstocks will result in an increase in the incidence of the virus that again depends on the original percentage infection, as well as the number of consecutive propagations that are performed (6). An initial infection rate of 10% of the rootstocks will within a period of 10 years (10 propagations) lead to an infection of 65% of scion material, which initially had been free from major harmful viruses. Similarly, an initial 40% infection rate of rootstocks within the same period will result in an infection of 99% of scion material, which initially had been virus-free.

Since 1970 no survey has been undertaken on the incidence of leafroll in the vineyards of the Western Cape and therefore no statistics in this regard are available. The perception that there is an increase in leafroll infection in South African vineyards can be attributed to the fact that more red wine cultivars, that display more visible symptoms, are cultivated.

In the virus research program the distribution patterns of leafroll are currently researched in mother blocks. This project should enhance tendencies regarding the tempo of infection as well as the incidence of leafroll. This project will at a later stage be extended to include spreading of the disease in commercial vineyards.

Pseudococcus longispinus

Excellent progress has been made with the development of techniques regarding the successful elimination of all known harmful viruses. This improved plant material is being propagated under the directive of the SA Plant Certification Scheme for Wine Grapes and is cultivated in mother blocks. Due to the problem of re-infection of the sources in the field, it, alas, is impossible to guarantee that the plant material in the mother blocks would stay virus-free. Sources in the Scheme are inspected regularly and, as in the case of scion cultivars, only plant material with no visible virus symptoms is issued as certified plant material to nurserymen.

International Situation

Leafroll occurs worldwide in all grape-growing areas. According to calculations, Germany suffered an 80% leafroll infection in 1936 (22). In Nuriootpa, Southern Australia, natural spreading of leafroll symptoms in Pinot noir clone material was observed. In 1993, 6% of vines displayed leafroll symptoms, which escalated to 21 and 36% in 1994 and 1995 respectively (14). The incidence of leafroll infection over a period of 5 years in New Zealand had increased from 11 to 100% (15). In France some vineyards are reported to be 80% infected with leafroll (Sforza, R. - personal communication).

The incidence of leafroll and the problem of re-infection of healthy vineyards are therefore not unique to South Africa.

Effect on vineyards and wine

Leafroll does not destroy the grapevine, but causes a decline in fruit yield and quality of grapes. In some cases crop losses of 40 to 60% have been recorded (23). Bunches of leafroll-infected vines are usually less and smaller compared to those of healthy vines (10). Leafroll lowers the quality of the grapes by delaying the accumulation of sugar and lowering the production of anthocyanin in red wine cultivars. This result in the grapes being low in sugar concentration, discolour insufficiently and harvesting is mostly delayed by several weeks. Spectrographical analyses of wine made from healthy, as well as leafroll-infected Mission grapes, indicated that leafroll is responsible for a 50% loss in pigment concentration (18).

Planococcus ficus

The most prominent effect of a serious degree of leafroll infection in Ruby Cabernet is a delay in the ripening of the grapes, as well as a crop loss. Wine made from these grapes is lower in alcohol, colour and tannin compared to Ruby Cabernet that displays less drastic symptoms of leafroll (1).

Vine material infected with leafroll-associated viruses 1 and 3, as well as a similar clone from which the viruses have been eliminated by means of meristem tip culture in combination with heat therapy, were compared, as was the wine that have been made from these vines (16). It was found that vineyards free from leafroll-associated viruses have higher chlorophyll content and higher photosynthetic activity compared to infected vines of the same clone. An accompanying earlier ripening, indicated by higher accumulation of anthocyanin in the grapes, had also been observed among these healthy grapevines. The quality of the grapes improved and according to a wine-tasting panel, the quality of wine from the healthy grapevines also improved, although not in the first years of harvesting. It is probable that the negative effect of the virus infection could increase with ageing of the vines. By removing leafroll-associated virus 1 from the vines, the colour of the grapes had improved.

South African research regarding the elimination of viruses by means of heat treatment of clone material indicated that this plant material had shown better results than similar clones that had not received heat treatment. A higher sugar concentration and lower total acid were recorded (4).

Impact on the health of humans

Plant viruses pose no health hazard to human beings. The consumption of wine made from grapes harvested from leafroll-infected grapevines may undoubtedly be declared as totally safe. According to a recent article, leafroll virus is not found in grapes (5).

Re- infection

Internationally the leafroll virus is being transmitted mainly by infected propagation material. It has been found worldwide in vine-growing areas that the disease could also be transmitted by mealybugs (3, 7, 11, 12, 19) and scale insects (2). A variety of mealybug species that transmit leafroll virus has been identified worldwide. The vine mealybug, Planococcus ficus, that is commonly found, and the long-tailed mealybug, Pseudococcus longispinus, that is found to a lesser extent in South African vineyards, proves to be the most important factors in the transmission of the disease in South Africa. The fact that mealybug had recently been found on grapevine roots up to 60cm below the surface, affects the current strategy of control (Refer: Article Wingerdwitluis: Biologie en beheerstrategie in Wynland, March 2001 p. 75-78).

Danger of top grafting existing grapevines to other cultivars

Due to changing wine consumption trends worldwide, a need for alternative cultivars has developed. A tendency could then develop to top-work existing grapevines to other cultivars in order to save time and costs to establish new vineyards. Wine producers should realise that leafroll is transmitted by grafting. They should also be aware of the danger attached to top-working existing vineyards to other cultivars, except if tests done on the existing vineyards prove them to be free of leafroll and other harmful viruses (20). Apart from leafroll virus, other diseases could also occur in existing vineyards, e.g. Shiraz disease for which no instant detection method exists at the moment. The disease can be latent in existing grapevines and could then be transmitted to the cultivar onto which it is grafted.

Control

Existing vineyards infected with viruses cannot be restored to a healthy state. The only way in which leafroll can be controlled, is to use certified virus-free rootstock and scion material to establish new vineyards and to control vectors (Protokol vir die bekamping van witluisverspreiding in 'n wingerdblok - V. Walton). The use of uncertified plant material holds a risk for the wine producer and is not recommended.

Propagation material that is free from harmful viruses

The first link in the chain of the availability of propagation material of a high phytosanitary quality is based on technological expertise to successfully eliminate damaging viruses. Research in South Africa contributed to the optimising of protocols for successful plant regeneration (scion and rootstock cultivars) by means of tissue culture techniques (meristem tip culture and spontaneous somatic embryogenesis). In addition, it is scientifically proven that these techniques separate and/or in combination with heat therapy, are extremely effective and reliable regarding the elimination of all known harmful grapevine viruses (8, 13). It is a given fact that propagation material that is free from leafroll-associated viruses could be supplied to wine producers.

S.A. Plant Certification Scheme for Wine Grapes

The SA Plant Certification Scheme for Wine Grapes, in accordance with the Plant Improvement Act (Act no 53 of 1976), was announced in the Government Gazette. According to this the National Minister of Agriculture authorised the Executive Board of the Vine Improvement Association to implement the Scheme.

The Scheme makes provision for continuous upgrading of plant material and certification of clones that meet the minimum requirements set by the Scheme. This ensures that only the best available plant material within each cultivar will be made available to the wine industry.

Plant material that qualifies for certification has to, according to the Scheme, meet the minimum phytosanitary requirements set for scion and rootstocks. Rootstocks are symptom less carriers of virus diseases and as a result they have to be tested free of all prescribed virus diseases. Scion cultivars should only test free from fanleaf and be visually free from the following virus diseases: fleck, leafroll, corky bark, stem grooving and Shiraz disease. The plants should also be visually free of the prescribed fungal and bacterial diseases and insects.

Furthermore, all plant material should adhere to the minimum physical requirements as prescribed by the Scheme for each category of plant material, including minimum length and width. According to the Scheme only plant material that is true to cultivar, descended from a registered clone and cultivated in a registered unit, may be certified. It is further prescribed that plant material be developed according to a specific order, starting at nucleus unit, then foundation unit, mother unit and nursery unit. These units should meet the prescribed isolation requirements and the soil should be raw soil or test free from the vector for fanleaf. Certification entails that the prescribed processes, according to which the plant material had been developed, had been controlled and that the grapevines had been inspected.

Certified nursery vines receive the blue Scheme label which serves as guarantee to wine producers that the plant material had been controlled and that all the requirements had been met. Certification, however, does not mean that the nursery vines are free of viruses, but certifies that nursery vines tested clean with regards to fanleaf, and that they are visually free of all prescribed virus diseases, as determined by the Scheme.

Furthermore, the Scheme offers the advantage that certified vines could be traced back to a specific clone and source whenever a complaint regarding the plant material is received.

Management practices for leafroll infected vineyards

Implementation of the following practices could result in the production of higher quality grapes from leafroll infected vines or blocks:

• Prune severely for a lower yield. When only isolated cases of leafroll virus are reported in a block, these vines could be marked and then pruned so as to ensure a lower yield, or these vines could be harvested at a later stage.
• Reduce stress to the vine from pea-size stage for more active growth later in the season.
• Irrigate and fertilise for more active late season growth.
• Cut back shoots in late November. This will result in side-shoots of 30-40 cm growth in December.
• Remove old leaves from certain red wine cultivars, e.g. Shiraz.