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The effect of hot-water treatment on fungi occurring in apparently healthy grapevine cuttings
Many grapevine nurseries are presently employing a hot-water treatment for propagation material as a prophylactic measure. The aim of this study was to determine the effect of this treatment on the endophyte population in grapevine cuttings. The number of fungal colonies isolated from the hot-water treated cuttings 6 months after planting were not significantly different (P = 0.05) from that of the untreated cuttings. This was in contrast to where isolations were made directly after treatment, when no known living fungal pathogens were found inside treated vine tissue. These results therefore indicate that the hot-water treatment is effective in eliminating the most well-known fungal pathogens and endophytes from grapevine tissue. Further research now needs to be focused on combining this treatment with biological control agents in an attempt to delay recolonisation of tissue by fungal pathogens.
Introduction
Young vine decline, which is associated with various pathogens, is a very serious problem in most of the grapevine producing countries of the world (Ferreira et al., 1994; Pascoe, 1999; Sidoti et al., 2000; Whitling et al., 2001). Recent observations have suggested that fungi such as Phaeomoniella chlamydospora (W. Gams et al.) Crous & W. Gams and Phaeoacremonium species may play a dominant role in this decline (Crous et al., 1996, Crous & Gams, 2000), though experimental proof has been slow in confirming pathogenicity (Ferreira et al., 1994; Scheck et al., 1998; Ferreira et al., 1999). Since researchers first became aware of these associations, however, there has been a frantic search for possible ways of eradication and control of the pathogens associated with young vine decline.
Von Broembsen and Marais (1978) reported that subjecting vine cuttings to a hot-water treatment gave effective control of Phytophthora cinnamomi, without any visual phytotoxicity. Other researchers have also reported effective control of phylloxera, nematodes and Pierce's disease using this technique (Moller and Fisher, 1961; Goheen et al., 1973). Using glass vials, spore suspensions and mycelial plugs, Whiting et al. (2001) found that a hot-water treatment may not be effective in reducing or eliminating P. chlamydospora and Phaeoacremonium inflatipes from vines. In contrast, however, this method is seen as effective against young vine decline by others (Waite, 1998). The aim of the present study, therefore, was to compare the fungal species occurring as endophytes within apparently healthy young vines and cuttings subjected to a 50øC hot-water treatment for 30 min, and in so doing determine the efficacy of this treatment for vine material.
Materials and methods
Hot water treatment
Rootstock and scion vines (20-25 cm long, 1-1.5 cm diam.) were treated by dipping them in a hot-water bath at 50øC for 30 min, immediately followed by a cold-water bath for a further 30 min. For the first experiment 45 hot-water treated and 45 untreated grapevine cuttings (Shiraz 99B grafted onto AA219A [101-14]) that were visually disease free, were planted in a nursery in the Paarl region of the Western Cape province for a period of 6 mo, after which they were uprooted and taken to the laboratory for isolations. For the second experiment, 100 shoots were cut from rootstocks (101-14) growing in a vineyard at Nietvoorbij, Stellenbosch in the Western Cape province. The lower 20-25 cm of each shoot was taken as representative of one cutting. Fifty of these were subsequently submitted to a hot-water treatment. Treated and untreated cuttings were immediately subjected to isolations.
Isolation and identification
For the first experiment, plant tissues were sampled from the roots, crown and scion area. Roots were surface-sterilised using triple sterilisation: 30 sec in 70% ethanol, 2 min in NaOCl (1%) and 15 sec in 70% ethanol. Ten root pieces were sampled per cutting. The cutting was then split open aseptically. Respectively 10 tissue pieces (2 mm x 2 mm) were sampled from the inside of the crown and scion area of each cutting. Tissue pieces were plated on Petri dishes containing potato dextrose agar (PDA, Biolab, Midrand, Johannesburg), amended with Streptomycin (1 ml/l), and incubated at 25øC. Fungal growth from plated tissue pieces was monitored every second day. Emerging colonies were hyphal-tipped over a period of 4 weeks, and transferred to Malt extract agar (MEA, Biolab) dishes for further identification. For the second experiment, the stems of the cuttings were split open aseptically and 10 tissue pieces (2 mm x 2 mm) were sampled from the inside of the basal end of the stem. The rest of the procedures followed were as explained for the first experiment.
Results and Discussion
From Table 1 it is apparent that the number of fungal colonies isolated from the hot-water treated cuttings was not significantly reduced (P = 0.05), compared to the untreated cuttings. Figures 1-5 indicate that the same was also true in the case of the more common vine pathogens occurring in the root, crown and scion areas of vine cuttings. These data represent treated and untreated cuttings that were growing in the field for a period of 6 months.
Very few Phaeomoniella strains were isolated, and these were all obtained from cuttings that underwent the hot-water treatment, suggesting that these cuttings were probably re-infected in the soil, or during the grafting process. No information is available about a possible soil-borne phase of P. chlamydospora, and this aspect is presently being investigated further. However, pycnidia of the fungus (alternate structures that occur in its life cycle) were recently found to be common on dead branches left in vineyards after pruning. The latter may possibly explain how healthy vines could be infected by means of splash dispersal via pruning wounds. Phaeomoniella appears, however, to be present in most vines (including healthy ones), which suggests that this slow dieback disease may be more complex than initially suspected, and may be largely due to incorrect viticultural practices, or incompatible rootstock/scion combinations that stress the vine. Currently it seems that Phaeomoniella occurs in most vines, and given its presence on discarded vine cuttings, and its ability to infect via pruning wounds, it may be close to impossible to keep out of uninfected vines. The presence of Phaeomoniella should thus no longer be seen as the sole reason for disease occurrence. Future research should thus focus on managing the disease, and reducing stress conditions, especially on young, 1-3-year-old vineyards.
Of the other pathogens isolated Fusarium and Cylindrocarpon spp. proved to be dominant. Although the devastating effect of Cylindrocarpon black foot rot is well known, not much is known about Fusarium wilt, and the importance of this disease complex will thus also have to be elucidated. Species of Botryosphaeria that cause black dead arm were also found, though in low numbers. Although it has recently been demonstrated that Botryosphaeria spp. can survive as endophytes in apparently healthy woody material (Smith et al., 1996; Mostert et al., 2000), not much is known about the virulence and pathogenicity of these endophytic strains, and further research is now also being focused on these aspects. Other root pathogens that are common in soils in the Western Cape province, namely Macrophomina and Rhizoctonia spp. were also recovered, though their importance to grapevine propagation remains to be determined.
In the case of the cuttings where isolations were made directly after treatment (Fig. 6), a drastic reduction in the pathogen population occurring in the stems of the hot-water treated cuttings, compared to the untreated cuttings was evident. These results therefore lead us to conclude that the hot-water treatment is effective in eliminating endophytes in the stems of grapevine cuttings, as well as most of the common pathogens. As can be seen in Table 1, however, it is evident that the treated cuttings get re-infected in the field once planted out, and that the advantage of being "fungal free" may be short lived. This does, however, raise interesting possibilities of combining the hot-water treatment with a biological control agent such as Trichoderma, which could ensure that plants remain pathogen free for a longer period. Although the data obtained in the present study cannot adequately address all the questions pertaining to the young vine decline disease complex, it did indicate that the hot-water treatment was very effective against most of the pathogens associated with the decline of young vines.
Acknowledgements
We are grateful to Tobie Oosthuizen from the Plant Improvement Section of KWV for the donation of the plants used in this study. Francois Halleen (ARC-Nietvoorbij) is also thanked for contributing the material used in the second study. This work represents a practical project done by the final year plant pathology students (Plant Pathology 414). Their hard work and dedication is acknowledged, as well as financial support from the Department of Plant Pathology, University of Stellenbosch.
Literature cited
Crous, P.W. and W. Gams, 2000. Phaeomoniella chlamydospora gen. et comb. nov., a causal organism of Petri grapevine decline and esca. Phytopathologia Mediterranea, 39, 112-118.
Crous, P.W., W. Gams, M.J. Wingfield and P.S. van Wyk, 1996. Phaeoacremonium gen. nov. associated with wilt and decline diseases of woody hosts and human infections. Mycologia, 88, 786-796.
Ferreira, J.H.S., P.S. van Wyk and E. Venter, 1994. Slow dieback of grapevine: association of Phialophora parasitica with slow dieback of grapevines. South African Journal of Enology and Viticulture, 15, 9-11.
Ferreira, J.H.S., P.S. Van Wyk, and F.J. Calitz, 1999. Slow dieback of grapevine in South Africa: stress-related predisposition of young vines for infection by Phaeoacremonium chlamydosporum. South African Journal of Enology and Viticulture, 20, 43-46.
Goheen, A.C., G. Nyland and S.K. Lowe, 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.
Moller, W.J. and J.M. Fisher, 1961. Hot water treatment effective for nematode infested grapevine rootlings. Australian Journal of Agricultural Research, 12, 38.
Mostert, L., P.W. Crous and O. Petrini, 2000. Endophytic fungi associated with shoots and leaves of Vitis vinifera, with specific reference to the Phomopsis viticola complex. Sydowia, 52, 46-58.
Pascoe, I., 1999. Grapevine trunk diseases - black goo decline, esca, Eutypa dieback and others. Australian Grape Grower Winemaker, 429, 24-28.
Scheck, H., S. Vasquez, D. Fogle and W.D. Gubler, 1998. Three Phaeoacremonium spp. cause young grapevine decline in California. Plant Disease, 82, 590.
Sidoti, A., E. Buonocore, T. Serges and L. Mugnai, 2000. Decline of young grapevines associated with Phaeoacremonium chlamydosporum in Sicily (Italy). Phytopathologia Mediterranea, 39, 87-91.
Smith, H., M.J. Wingfield, P.W. Crous and T.A. Coutinho, 1996. Sphaeropsis sapinea and Botryosphaeria dothidea endophytic in Pinus spp. and Eucalyptus spp. in South Africa. South African Journal of Botany, 62, 86-88.
Von Broembsen, S. and P.G. Marais, 1978. Eradication of Phytophthora cinnamomi from grapevine by hot water treatment. Phytophylactica, 10, 25-27.
Waite, H. 1998. Hot-water treatment of vinifera and rootstock cuttings. Current status and issues. Interim report of the University of Melbourne, Dookie College, Australia.
Whiting, E.C., A. Khan and W.D. Gubler, 2001. Effect of temperature and water potential on survival and mycelial growth of Phaeomoniella chlamydospora and Phaeoacremonium spp. Plant Disease, 85, 195-201.
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