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D.A. Golino
CAUSAL AGENT
Xylella fastidiosa, a gram-negative aerobic bacterium found only in the xylem tissue of infected plants (Wells et a/., 1987; Davis, Whitcomb and Gillaspie, 1981), has recently been named as the causal agent of Pierce's disease (PD).
GEOGRAPHICAL DISTRIBUTION
PD was first described in 1892 in southern California by N.B. Pierce, after whom it was named. PD has been held responsible for the destruction of the once-extensive grape plantings in that area. It is widespread throughout areas of the Western Hemisphere with mild winter temperatures, including all of the southern United States from California to Florida. It limits the cultivation of Vitis vinifera in many of these areas. Since the development of techniques for cultivation and serological detection of the pathogen have facilitated identification of the causal agents Chile, Costa Rica Mexico and Venezuela have been added to the list of countries in which PD occurs. Reports of infection outside the Americas have recently been made (Boubals, 1989).
ALTERNATE HOSTS
Other Vitis species are hosts of X. fastidiosa; native Vitis species are believed to serve as reservoirs of infection in much of the southeastern United States (Hopkins, 1988). In addition, the pathogen has a wide natural host range that includes both annual and perennial plants of many genera (Raju, Goheen and Frazier, 1983). Many plant hosts show either no symptoms or mild symptoms. Xylella fastidiosa causes several diseases, including almond leaf scorch (Figure 132), alfalfa dwarf and a disease of macadamia in Costa Rica. Other Xilella species are known to cause phony peach disease and a scorching disease of numerous tree species. The taxonomic relationship of X. fastidiosa to the strains that cause these diseases is as yet undetermined (Hopkins, 1988).
FIELD SYMPTOMS
Obvious PD symptoms are scorched or dry leaves on one or a few canes. Symptoms are usually spread asymmetrically in the canopy until late in the development of the disease. Infected vines often exhibit delayed bud break in the spring.
Early season foliage may show interveinal chlorosis (Figure 133) and develops more slowly than foliage on surrounding healthy vines. In midsummer, as water stress from vascular plugging begins to affect the plant, asymmetrical yellowing of the leaf margins is seen. These discoloured regions become progressively more necrotic until autumn (Figures 134 to 136). Yields are reduced, and clusters are often shrivelled (Figure 137). Leaves fall prematurely, leaving the petiole intact on the vine (Figure 138). This symptom is considered diagnostic for PD. Canes mature unevenly in the autumns leaving patches of immature green wood (Figures 139 and 140).
Vines deteriorate rapidly after appearance of symptoms. Infected plants grow progressively weaker as symptoms become more pronounced (Figure 141). PD is normally fatal to infected vines. The life expectancy of diseased vines is reported to vary from one to a few years.
The symptoms of PD can be easily confused with other phenomena that cause water stress. In addition, some other diseases such as Eutypa dieback (Eutypa armeniaca), oakroot fungus (Armillariella mellea) and measles may cause similar symptoms. Nutrient imbalances can also cause chlorosis and scorching.
NATURAL SPREAD
PD is spread by xylem-feeding insects which insert their mouthparts directly into those tissues in which the bacteria are found (Figure 142). All species of sharpshooters (Cicadellidae) and spittle bugs (Cercopidae) that have been tested under experimental conditions are capable of transmitting the bacterium, whereas phloem-feeding leafhoppers, which occasionally probe xylem tissues, do not transmit the disease. This suggests that feeding behaviour is an important component of the relationship between the vectors and X. fastidiosa (Purcell, 1989). The bacteria can be readily observed in large numbers with the scanning electron microscope on the surfaces of the feeding apparatus of vector species (Figure 143).
In the western United States spread into vineyards is thought to occur from surrounding vegetation. Vine-to-vine spread is not believed to contribute significantly to diseases in these areas (Purcell, 1974). In contrasts in the southeastern United States diseased grapes may (Goheenserve as sources of inoculum (Goheen and Hopkins, 1988).
DETECTION
PD is normally detected by the observation of symptoms in the late summer and autumn. Detection is also possible in the dormant season or early spring by experienced workers.
IDENTIFICATION
Symptomatology
PD can be diagnosed with some confidence in European grapes on the basis of symptoms during the late summer and autumn (Figures 134 to 140). Spring symptoms (Figure 133) are more easily confused with other conditions. The pathogen is non-symptomatic in many of its host plants. Absolute identification relies on cultivation of the bacterium on selective media and/or the use of serological techniques.
Cultivation
Xylella fastidiosa cannot grow on commonly used bacteriological media. Specialized media have thus been developed (Hopkins 1988). A method for isolating and growing the PD bacterium is outlined in Part III. Although the PD3 medium is quite satisfactory other media may be required for some pathovars. Petioles from symptomatic leaves are ideal for isolation purposes, although other grapevine parts may be acceptable. Xylella fastidiosa is slow growing, and colonies may require one to three weeks to develop. Colonies are white and smooth, with complete margins (Davis Purcell and Thompson, 1978).
Serology
Antisera to X. fastidiosa are readily prepared by the use of either whole cells (Davis, Purcell and Thompson, 1978) or sonicated cultured cells (Hopkins, 1988) as antigen. Antisera and type culture of the bacterium are both available from the American Type Culture Collection (ATCC, 12301 Parklawn Drive, Rockville, MD 20852, USA). Purified antibodies can be used either in ELISA or in Outcherlony gel diffusion tests as described in Part III. No serological differences have been observed between the strains of X. fastidiosa that cause PD and those that cause elm leaf scorch, phony peach, plum leaf scald and periwinkle wilt (Hopkins, 1988).
Electron microscopy
When fixed and prepared for electron microscopy, X. fastidiosa can be seen as rodshaped bacterial elements 0.25 to 0.5 mm in diameter and 1.0 to 4.0 mm in length (Mollenhauer and Hopkins, 1974). A rippled cell wall is characteristic of the species.
In cross-sections through the xylem elements of infected grapes, bacteria can be seen blocking some vessels, while other vessels remain clear (Figure 144).
Pathogenicity
Inoculation of grapes and other plants is readily accomplished by needle inoculation with the cultured bacterium. A drop of turbid suspension of the bacterium in phosphate-buffered saline is placed on a leaf or in the angle of a petiole, and a sterile needle is inserted through the droplet into the plant tissues. Xylem tension will draw the inoculum into the plant. A syringe filled with the inoculum may also be used. On grapes, PD symptoms develop rapidly. Pathogenicity of the cultured organism is easily lost in culture, so early passages of new isolates should be preserved by storing at -20°C if the pathogenicity of an isolate needs to be determined or maintained.
It is also possible to transmit PD by grafting from infected to healthy vines. The graft must contain the active xylem tissues which harbour the PD agent. This technique can provide a useful diagnostic check if culture media, antisera or electron microscope facilities are not available. In pathogenicity testing one should always keep in mind that avirulent and mild strains of the bacterium are known to occur (Hopkins, 1 984).
SANITATION
PD is normally lethal for individual vines. Although treatment with antibiotics will result in a remission of symptoms the expense of this treatment, the recurrence of symptoms when the treatment is discontinued and environmental concerns about field applications of antibiotics make chemical therapy infeasible. Control strategies have been based largely on eliminating vector species or host-plant inoculum. Knowledge of the identity of the natural vector species and alternative plant hosts in a given region is an essential component of the development of sanitary procedures.
Given the wide host range of the PD pathogen and the large number of sharpshooter species that are vectors, control of the disease in areas where X. fastidiosa is established in native vegetation is extremely difficult. In the Central Valley of California, PD is transmitted largely by the green- and red-headed sharpshooters Draeculacephala minerva and Carneocephala fulgida. Field studies have established that the alternate hosts of these sharpshooters include weeds growing in alfalfa fields and some of the perennial grasses that grow at the margins of the fields. Elimination of the weed species and application of pesticides to neighbouring alfalfa fields have been effective in controlling the disease.
The north coast valleys of California have a high incidence of PD foci. Control of alternate hosts of the pathogen and elimination of vector populations are difficult since they originate in riparian areas which are protected as wildlife refuges. Only limited control of vector species is possible with pesticides, since applications to woodlands are both impractical and illegal. Often a border effect is observed, with a high mortality of vines adjacent to native vegetation. Vine-to-vine spread is not believed to be of importance in these locations.
In the Gulf Coastal Plains of the United States, eastern coastal Mexico and the tropical Americas, X. fastidiosa is well established in native Vitis species and other vegetation. Neither V. vinifera nor Vitis labrusca survives more than a few years in these areas before becoming infected. Control strategies are limited to planting of PD-resistant varieties of native American genera such as Muscadinia or Euvitis and the development of resistant hybrids between those species and their more susceptible relatives.
Quarantine regulations that limit the movement of Vitis species from the Americas are aimed at preventing the introduction of X. fastidiosa from the warm grape-growing regions where PD is common into other regions. Fortunately. dormant cuttings harbouring the pathogen are normally short-lived. In addition, hot water treatment of dormant cuttings (immersion at 45°C for three hours or at 50°C for 20 minutes) will destroy X. fastidiosa (Goheen, Nyland and Lowe, 1973).
REFERENCES
Boubals, D. 1989. La maladie de Pierce arrive dans les
vignobles d'Europe. Prog. Agric. Vitic.,
106: 85-87.
Davis, M.J., Purcell, H.A. & Thompson, S.V. 1978. Pierce's disease of grapevines: isolation of the causal bacterium. Science, 199: 75-77.
Davis, M.J., Whitcomb, R.F. & Gillaspie, A.G.M. 1981. Fastidious bacteria of plants and insects (including so-called rickettsia-like bacteria). In M.P. Starr, H.O. Stolp, H.G. Truper, A. Balows & H.G. Schelegel, eds, The prokaryotes: a handbook on habitats, isolation and identification of bacteria, 2: 2171-2188. Berlin, Springer-Verlag.
Goheen, A.C. & Hopkins, D.L. 1988. Pierce's disease. In R.C. Pearson and A.C. Goheen, eds, Compendium of grape diseases, p. 44-45. St Paul, MN, USA, Am. Phytopathol. Soc.
Goheen, A.C., Nyland, G. & Lowe, K.S. 1973. Association of a rickettsialike organism with Pierce's disease of grapevines and alfalfa dwarf and heat therapy of the disease in grapevines. Phytopathology, 63: 341-345.
Hewitt, W.B., Frazier, N.W., lacob, H.E. & Freitag, H.J. 1942. Pierce's disease of grapevines. Calif. Agric. Exp. Sta. Circ. No.353. 32 pp.
Hopkins, D.L. 1984. Physiological and pathological characteristics of virulent and avirulent strains of the bacterium that causes Pierce's disease of grapevine. Phytopathology, 75: 713-717.
Hopkins, D.L. 1988. Xylella fastidiosa and other bacteria of uncertain affiliation. In N.W. Schaad, ea., Laboratory guide for the identification of plant pathogenic bacteria, 2nd ea., p. 95-103. St Paul, MN, USA, Am. Phytopathol. Soc.
Mollenhauer, H.H. & Hopkins, D.L. 1974. Ultrastructural study of Pierce's disease bacterium in grape xylem thisue. J. Bacteriol., 119: 612618.
Purcell, A.H. 1974. Spatial patterns of Pierce's disease in the Napa Valley. Am. J. Enol. Vitic., 25: 162-167.
Purcell, A.H. 1989. Homopteran transmission of xylem-inhabiting bacteria. Adl'. Dis. VectorRes., 6: 243-266.
Raju, B.C., Goheen, A.C. & Frazier, N.W. 1983. Occurrence of Pierce's disease bacterium in plants and vectors in California. Phytapathology, 73: 1309- 1313.
Wells, J.M.,Raju, B.C., Hung, H.Y., Weisburg, W.G., Mandelco-Paul, L. & Brenner, D.G. 1987. Xylella fastidiosa gen. nov., sp. nov.: gram negative, xylemlimited, fastidious plant bacteria related to Xanthomonas spp. Int. J. Syst. Bacteriol., 37: 136143.
Summary: Pierce's disease detection
GRAFT TRANSMISSION
Indicators
Several Vitis vinifera cultivars (Chardonnay, Merlot)
No. plants/test
3-5 rooted cuttings
Inoculum
Wood chips, single buds, bud sticks
Temperature
Field conditions
Symptoms
Scorching, yellowing or reddening of the leaves within the first
year after inoculation
OTHER TESTS
Serology (immunodiffusion, ELISA)
FIGURE 134 Marginal scorching of cv. Merlot leaves caused by Pierce's disease in autumn
FIGURE 136 Close up of autumn symptoms in cv. Chardonnay
FIGURE 141 Final stage of a vine infected by Pierce's disease
