Leafroll transmission

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Leafroll transmission and characterisation of infected vines (Cabernet Sauvignon/Richter 99) at Welgevallen experimental farm - A case study*

PG Goussard & JP Underhay
Department of Viticulture and Oenology, Stellenbosch University
Private Bag X1, 7602 Matieland
* Based on a Winetech funded Honours project of JP Underhay (2002)
Key words: Leafroll, reinfection, transmission, visual infection categories, anatomy, morphology, physiology.
Introduction
Reinfection of grapevines with leafroll associated viruses (hereafter leafroll) after establishment, even though material may be free of harmful viruses, is a well-known and serious problem in the SA viticultural industry (Carstens, 2001). The transmission of leafroll after reinfection in certain blocks does not always follow a set pattern. In addition to the direct involvement of acknowledged vectors such as mealybug (Engelbrecht & Kasdorf, 1990; Cabaleiro & Segura, 1997) the transmission tempo is influenced by various factors.

Fig. 1: The first leafroll symptoms were noticed three years after establishment in the southernmost row (left), adjacent to the severely infected leafroll vineyard on the right.

The vineyard block in question, measuring 1,5 ha, was established in 1988 using certified material of Cabernet Sauvignon/Richter 99 on high potential soil that was previously planted to leafroll vines, and on its southern border (at a distance of approximately 7 m) was a visually severely infected block of Cabernet Sauvignon. In the preceding 18 months (January 1987 - August 1988) the soil in question was allowed to "rest", except for the cultivation of triticale as a cover crop. Vine spacing amounts to 2,7 m x 1,2 m, the vineyard is trellised (five strand hedge with moveable foliage wires) and was not subjected to any supplementary irrigation during the entire cultivation period (1988 - 2002). The row direction is east-west with a prevailing wind direction of southwest. Ever since its establishment the vineyard has been charted on an annual basis as far as visual leafroll symptoms is concerned. A record was kept of (i) when and in which vines the first visual indications of reinfection were observed, (ii) transmission tempo and pattern and (iii) which vines currently (2002) appeared the most seriously "ill" or "healthy", judging from their visual appearance.

Fig. 2: In a period of 12 years (2000), the total block (in the background) was subject to reinfection. In marked contrast with the vines in the foreground (Merlot noir) this reinfection is characterised by delayed leaf drop (as is generally the case with leafroll infected vines).

Fig. 3: Despite overall reinfection, the two categories (visually lightly and severely infected) are clearly visible on the picture, taken during the early autumn of 2002.

With the above as background, the aims of the project (duration one year) were to characterise vines in the particular block in two specific categories, viz. (i) visually severely and (ii) visually lightly infected. In addition to judging general performance, an investigation was undertaken into the anatomical, morphological and physiological changes/reactions brought about by leafroll at various degrees of visual symptom expression. Globally the intention was to formulate explanations for general vineyard performance as influenced by the above-mentioned changes/reactions.
Material and methods

Reinfection and transmission
Characterisation of vines with regard to reinfection and transmission was based on the visual occurrence of leafroll during the cultivation period 1988 - 2002 (see background scenario as described in the Introduction).

Fig. 4: Vine size and leaf characteristics in a visually lightly infected vine.

Anatomical, morphological and physiological reactions/changes
Altogether 30 vines in this block were subjected to applicable examinations. From this group 15 vines were classified as being representative of visually lightly infected and 15 vines as being severely infected. Morphological reactions/changes with regard to vine size, leaf characteristics, bunch size, berry colour, root system characteristics and growth reactions vis … vis leaf surfaces and shoot masses were studied in both categories. The anatomical study was based on changes/reactions that were induced in the petioles (directly above the bunches). Physiological changes/reactions, combined with leafroll infection, included the determination of sugar and acid concentrations, pH and anthocyanin concentrations of grapes. All examinations/analyses were done in accordance with standard procedures. As a result of the short duration of the project no statistical interpretation of applicable data could be undertaken.

Table 1: The influence of various categories of leafroll infection on growth reactions (leaf surface and shoot mass).

Growth reactions

Visually lightly infected

Visually severely infected

Leaf surface^*

3 891.65 cm²

2 305.91 cm²

Shoot mass^**

1 649 g

737 g

^* Average of leaves deriving from 15 shoots (1 shoot/vine/category).

^** Average of total shoot numbers of 15 vines/category.

Table 2: The influence of various categories of leafroll infection of grape composition during the harvest.^*

Grape composition

Visually lightly infected (Average of
15 vines)

Visually severely infected (Average of 15 vines)

Sugar concentration

22.3°B

21.4°B

Concentration titratable acid

6.79 g/l

6.43 g/l

PH

3.61

3.67

Anthocyanin concentration

92 mg/l

74 mg/l

^* All values are based on 2 representative bunches that were gathered during the harvest from each vine/category and then mixed to determine averages/category.
Results

Reinfection and transmission
The first visual leafroll symptoms were observed in 1991 (three years after establishment) in the southernmost row (adjacent to the existing leafroll infected, mealybug infected Cabernet Sauvignon block) (Fig. 1). At this stage the reinfection occurred in vines that were not necessarily adjacent to each other. In the same year the presence of mealybug was also noted in the three-year-old block. Despite regular and thorough input to control mealybug effectively in the new block, this goal could not be achieved in the course of the total cultivation period (1988 - 2002). After 1991 leafroll spread systematically throughout the block (based on visual observations), at a tempo of approximately 0,5% - 1% per annum until 1996. With the exception of the first three southernmost rows, it was noticeable that transmission in the northerly direction (across rows) occurred more rapidly than inside the rows (east-west). After 1996 the tempo of transmission increased considerably (> 20% per annum). Within 12 years (2000) the total vineyard was reinfected (Fig. 2) with typical spots where vines could be seen to be either sligthly or severely infected. The same situation applied in 2002 when the project was in progress (Fig. 3).

Fig. 5: Vine size and leaf characteristics in a visually severely infected vine.

Morphological reactions

Vine size and leaf characteristics
The morphological appearance of visually severely infected as opposed to visually lightly infected vines is represented in figures 4 and 5. Although vines in the former category appeared smaller than in lightly infected vines, no drastic differences could be observed. By contrast obvious differences could be noted in leaf characteristics in the two categories. Leaf discolouration with the accompanying curling of leaf edges was more prominent and occurred earlier in severely infected than in lightly infected vines. During ripening leaves of lightly infected vines also started to discolour, but the curling of leaf edges was delayed until the harvest. The occurrence of higher numbers of green leaves (more chlorophyll) was characteristic of visualy lightly infected vines.
Bunch size and berry colour
Smaller and looser bunches were characteristic of visually heavily infected vines (Fig. 6) in contrast with bigger and fuller bunches (Fig. 7) on lightly infected vines. In both categories bunches were subject, however, to suboptimal discolouration.
Root systems
Root distribution and density in the two categories are represented in Figures 8 and 9. Although there is no direct correlation between leafroll infection and root distribution and density and it should be borne in mind that growth balance above and below the surface is usually maintained, smaller root systems with fewer active root tips were noticeable in visually severely infected vines.
Leaf surface and shoot mass
The two categories of infection displayed drastic differences in leaf surface (as determined at the time of the harvest) and shoot mass (as determined before winter pruning) (Table 1), despite the fact that no drastic differences in vine size could be observed visually.

Fig. 6: Smaller and looser bunches were characteristic of visually severely infected vines.

Fig. 7: Bigger and fuller bunches in visually lightly infected vines.

Anatomical reactions
Phloem degeneration (petioles) was observed in both categories (Figure 10). Anatomical changes were characterised particularly by a wavy occurrence of the cambium and obliteration (compression), deformation and necrosis of sieve-cells. This situation was more intense in the petioles of visually severely infected vines.

Physiological reactions
Physiological reactions with regard to grape composition are indicated in Table 2. As far as average sugar and acid concentrations, pH and in particular anthocyanin concentrations are concerned, the differences between the two categories were obvious.
Discussion and conclusions
Optimal grape quality and quantity are inextricably bound to the phytosanitary quality of the vine material. At the same time it is of cardinal importance that vineyard management practices be specifically directed at the creation of conditions under which vines can operate physiologically at optimum levels to achieve the desired vine performance. This investigation once again highlighted the importance of the phytosanitary quality of the material. Initially the block performed optimally, but in reaction to reinfection it started to deteriorate gradually, despite correct and applicable industry management practices. Of particular importance was the rapid reinfection and the subsequent high transmission tempo, which means that the particular block will have to be uprooted within a time frame of just 14 years as a result of poor performance. In this instance mealybug, deriving from a neighbouring leafroll vineyard, was almost certainly the main cause of the above situation, despite the fact that the particular soil had a rest period of 18 months.

Fig. 8: Root systems in visually lightly infected vines.

Fig. 9: Root systems in visually severely infected vines.

Anatomical, morphological and physiological changes/reactions as induced by leafroll in vines, correspond with the available literature (Esau, 1948; Goheen & Cook, 1959; Goheen, 1965, 1988; Over de Linden & Chamberlain, 1970; Weber et al., 1993). Information forthcoming from the investigation made it possible to draw a parallel between the above responses and the eventual unacceptable grape and wine quality. In global terms the beginning of the problems should probably be sought in anatomical reactions/changes, which in turn cause the necessary physiological processes to be negatively influenced and to find expression in abnormal morphological characteristics of infected vines. In a nutshell, these reactions can be explained as follows:

Vine size
Phloem degeneration limits/impedes the translocation of photosynthetic products such as carbohydrates to important storage organs such as roots, trunks and cordons. These reserves play a cardinal role in the initial growth in the following year. Additionally the photosynthetic capacity of leaves is impeded (loss of chlorophyll) which results in a similar situation. Smaller vines are furthermore associated with smaller root systems which are characterised by lower root tip numbers in particular.

Leaf characteristics
Due to phloem degeneration in petioles, products of photosynthesis such as sucrose cannot make sufficient progress to the bunches, resulting in lower sugar concentrations in particular. Precursors of colourants in grapes are also present in leaves and are similarly exposed to hampered translocation. These and other products of photosynthesis accumulate in leaf blades, petioles, bunch stems and berry stems and are the cause, inter alia, of brittleness and the curling of leaves and suboptimal discolouration of bunches.

Bunches
As is known, cytokinins, gibberellins and auxins in particular are closely involved in berry set and the subsequent growth and development. These hormones do not act separately; there is a very delicate balance and interaction in order to eventually ensure optimal grape quality and quantity. It may be argued that as a result of the smaller number of root tips in leafroll vines, fewer cytokinins are produced, thus causing the balance to be disturbed. This, together with insufficient reserves, may result in smaller and looser bunches.

Leaf surface and shoot mass
It is well known that leafroll results in smaller vines and shorter shoots with fewer and smaller leaves. Vines are less vigorous, which in turn may be ascribed to lower reserves, less cytokinin and lower photosynthetic capacity.

Fig. 10: Phloem degeneration (petioles) in visually lightly infected vines. A = dead sieve-cells; B = obliterated sieve-cells.

Conclusion
In the light of the above background, phloem degeneration may be singled out as an important, if not the most important cause of insufficient and disturbed physiological activities, which in turn give rise to abnormal morphological characteristics. In order to manage a leafroll infected vineyard, some of the most important proposed practices are to subject vines to the least amount of stress and to retain green leaves on the vines for as long as possible. On the whole one of the most important messages forthcoming from this investigation is the extremely short period within which clean, certified plant material may be reinfected - a situation which cannot be allowed, both from an economic point of view and otherwise.
References Cabaleiro, C. & Segura, A., 1997. Field transmission of grapevine leafroll associated virus 3 (GLRaV-3) by the mealybug Planococcus citri. Plant Dis. 81, 283-287.
Carstens, R., 2001. Rolblaar oorsig. LNR Infruitec-Nietvoorbij/Winetech publication. 8 p.
Engelbrecht, D.J. & Kasdorf, G.G.F., 1990. Transmission of grapevine leafroll diseases and associated closteroviruses by the vine mealybug, Planococcus ficus. Phytophylactica 22, 341-346.
Esau. K., 1948. Some anatomical aspects of plant virus disease problems, II. Bot. Rev. 14, 413-449.
Goheen, A.C., 1965. Reactions of grapevine to leafroll virus. In: Proc. Int. Conf. On virus and vector on perennial hosts, with special reference to Vitis, 6-10 September 1965, California. pp. 135-138.
Goheen, A.C., 1988. Leafroll disease. In: Pearson, R.C. & Goheen, A.C. (eds). Compendium of grape diseases, APC Press, Saint Paul. p 52.
Goheen, A.C. & Cook, J.A., 1959. Leafroll (red-leaf or rougeau) and its effects on vine growth, fruit quality and yields. Am. J. Enol. Vitic. 10, 173-181.
Over de Linden, A.J. & Chamberlain, E.E., 1970. Effect of grapevine leafroll virus on vine growth and fruit yield and quality. NZ J. Agric. Res. 13, 689-698.
Weber, E., Golino, D.A. & Rowhani, A., 1993. Leafroll disease of grapevines. Practical winery and vineyard (March/April). pp. 21-25.
SUMMARY
Investigations were carried out on anatomical, morphological and physiological responses of grapevines (Cabernet Sauvignon/Richter 99) as induced by long-term exposure to potential reinfection with leafroll-associated viruses. Although the entire vineyard (1,5 ha) appeared visually reinfected after only 14 years, a clear distinction could be drawn between (i) lightly and (ii) severely infected vines regarding visual appearance and general performance. Anatomical investigations confirmed the existence of phloem degeneration to a greater extent within lightly infected compared to severely infected vines. In both categories important physiological processes are hampered as a result of these anatomical changes/responses, leading eventually to abnormal morphological characteristics of visually infected vines.

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