Current understanding of this mysterious disease

Introduction
The table and wine grape industry in South Africa has boomed over the past few years, resulting in an increasing demand for new plant material from grapevine nurseries. General take-percentages of young vines are however unsatisfactorily low (below 50%). This can be attributed to several factors including fungal, bacterial and viral diseases, insect and nematode pests, abiotic factors, as well as nutritional deficiencies and toxicities. One particular disease associated with young grapevines that has grabbed the attention of researchers worldwide is “black goo decline” or “Phialophora” grapevine decline. Ferreira, van Wyk and Venter (1994) identified the causal organism as Phialophora parasitica. This fungus however differed from other species in the genus Phialophora, and consequently Crous et al. (1996) proposed a new hyphomycete genus, Phaeoacremonium, including six species of which P. chlamydosporum, P. aleophilum, P. inflatipes and P. angustius occurred on grapevines. Research undertaken by Groenewald and Crous (unpubl.) in South Africa agrees with reports from elsewhere (Mugnai, 1998; Bertelli, Mugnai & Surico, 1998; Pascoe, 1998) that P. chlamydosporum is the most frequently isolated Phaeoacremonium species from grapevines. To further add to the confusion associated with this disease, P. chlamydosporum differed from the other species in the genus Phaeoacremonium to such a degree (Dupont, Laloui & Roquebert, 1998; Groenewald, Bellstedt & Crous, 2000) that it had to be re-classified as a new genus, namely Phaeomoniella chlamydospora (Crous & Gams, 1999). The biological name for the grapevine decline fungus identified by Ferreira et al. (1994) as Phialophora parasitica therefore changed to Phaeoacremonium chlamydosporum and more recently to Phaeomoniella chlamydospora.

Wynboer - August 2000 - Black goo decline of grapevine Wynboer - August 2000 - Black goo decline of grapevine Wynboer - August 2000 - Black goo decline of grapevine

Disease cycle
Although Phaeomoniella chlamydospora appears to lack a sexual state, it has quite a complex life cycle and is able to form chlamydospores (survival structures) as well as a synanamorph (more than one asexual state). It produces conidia that infect the host tissue, invade the xylem, and the plant responds by producing tyloses (balloon-like structures that block the vessels) and phenolics (black discolouration). The tyloses block the xylem vessels resulting in vine decline, since the transport of water and minerals from the roots to the other metabolically active parts of the plant is impeded. When the plant dies or is cut, the fungus can produce conidia on the exposed surfaces, which could be dispersed to other potential infection sites. Phaeomoniella chlamydospora also produces chlamydospores, which in other pathosystems are able to survive for long periods in plant debris or soil. This aspect has however not been proven for P. chlamydospora.

Current understanding of this disease is limited and several infection pathways have been proposed. The possibility that P. chlamydospora is soil-borne, survives as chlamydospores and infects vines in nurseries or vineyards via the root system has been suggested (Bertelli et al., 1998). Based on extensive isolations made from apparently healthy rooted cuttings, these authors found P. chlamydospora to be one of the most frequently isolated fungi from nursery material in Italy and France, occurring mostly from isolation sites near the base of the stem. Isolations from the roots, which would have broadened the understanding of this disease, especially the hypothesis regarding its soil-borne status, were unfortunately not made. Contrary to this suggestion Larignon (1998) concluded from aerial trapping experiments in France that P. chlamydospora might be an airborne fungus that penetrates the plant via pruning wounds. A phoma-like pycnidial synanamorph for this fungus was recently reported by Crous and Gams (1999). The possible role of the synanamorph is unclear. Dr Doug Gubler from the USA (pers. comm.) believes that the pathogen may be introduced to the grape rootstock production system during the callusing stage in nurseries. Recent observations in the laboratories of ARC Infruitec-Nietvoorbij however indicate that P. chlamydospora is present in apparently healthy propagation material in a latent or endophytic form. This observation is supported by reports from elsewhere (Bertelli et al., 1998). Stress conditions resulting from planting, drought (Ferreira, 1998a; 1998b; Ferreira, van Wyk & Calitz, 1999), poor drainage, nutrition deficiencies, soil compaction and/or infection by other root or trunk pathogens result in the expression of the disease.

Phaeomoniella chlamydospora is also associated with esca-disease of grapevines (Mugnai, Graniti & Surico, 1999). The general understanding of this disease is that infection of mature grapevines with P. chlamydospora predisposes the vine to attack or infection by the wood-rotting fungus, Fomitiporia punctata. The black goo fungus preconditions the wood for growth of F. punctata by breaking down certain substances in the host that are inhibitory to this wood-rotting fungus (Mugnai et al., 1999; Pascoe, 1999). This emphasizes the importance of preventing infection of young grapevines and controlling black goo disease.

Symptoms
Symptoms of this disease are well recorded (Ferreira et al., 1994; Ferreira, 1998a; 1998b; Scheck et al., 1998; Pascoe, 1999). This pathogen has been associated with decline of young and very old vines (Pascoe, 1998). Typical symptoms include stunted growth, mildly chlorotic foliage and a general decline of young vines (Fig 1) resulting in plant death. Cross-sections of diseased vines reveal typical “black goo”-symptoms.
The blackened xylem tissue appears as black dots in the cross-section with a shiny, tarry substance exuding from the severed vessels (Fig 2). Diagnosis should however never be made on visual symptoms alone, since several other diseases and physiological factors may cause similar vascular streaking in grapevines.

Electron microscopy studies indicated that the “black goo” in the xylem is parenchymatous, indicating that it might be the effect of a resistance reaction of the plant (Ferreira et al., 1994). Although it may be infected, symptoms are generally not visible in young one-year-old wood.

Control measures
Researchers of the Disease Management Division at ARC Infruitec-Nietvoorbij and the Department of Plant Pathology at the University of Stellenbosch are currently investigating various methods of controlling this disease. Plant pathologists from other countries across the world have only recently started investigating black goo decline and very little has been recorded on possible methods of disease control. At present recommendations to nurseries and producers are therefore based on limited available information:

Ensure good quality, disease-free plant material. Grapevine plant material from nurseries in South Africa must be certified as free from most viruses (fanleaf, Shiraz-disease, stem grooving, fleck, corky bark, leafroll, yellow speckle viroid, vein necrosis, vein mosaic), crown gall and blight bacteria, as well as Pythium and Phytophthora fungal infections. Diagnostic tools for the large-scale detection of the causal organisms of trunk diseases in grapevines do not exist and plant material can therefore not be certified as “trunk disease free” at present. Groenewald et al. (2000) have recently made a significant breakthrough in this regard and have developed primers that successfully detect P. chlamydospora in asymptotic grapevine material using polymerase chain reaction as a diagnostic tool. These primers bind to certain unique sections in the DNA structure of P. chlamydospora and thus allow diagnosticians to determine the presence of the pathogen in apparently healthy plant material. Further tests are however necessary before this technique can be used in a commercial certification scheme. Nonetheless, producers should refrain from buying uncertified, sub-standard plant material, since past experiences have shown that these grafted vines are more likely to have the black goo fungus, as well as other trunk and root disease pathogens.

Hot water treatment. Dormant grapevine cuttings can be treated in hot water to minimize the possibility of transferring diseases and pests from nurseries to vineyards. The temperature and duration of this treatment is of the utmost importance. A drench for 30 min in 50C water is recommended (Delaine, 1998). Temperature above 51C would cause damage to the cutting and reduce viability, while temperatures below 49C would allow survival of the pathogens. Normal practice in South Africa does not involve hot water treatment and producers should insist on this treatment and the correct application thereof. It is essential that cuttings be planted immediately after the cooling down phase of the hot water treatment in order to maintain viability and prevent the fine rootlets from drying out (Ferreira, 1998a; 1998b). Delaine (1998) gave an excellent account of the procedure for hot water treatment of grapevine cuttings used by the Australasian Vine Improvement Association. Dr Gubler (USA) recently presented arguments at a grapevine trunk disease workshop in Siena, Italy, stressing that this technique does not kill P. chlamydospora. The controversy surrounding the use of this technique must therefore still be resolved.

Chemical control. Based on glasshouse trials, Ferreira (1998a) proposed the application of metalaxyl (40 g/m2 Ridomil granules), phosphorous acid (400 ml/100 l Phytex) or fosetyl-Al (400 g/l Aliette) as drenches around young vines in the vineyard. Groenewald et al. (submitted) also tested several other systemic and contact fungicides for in vitro mycelial inhibition of P. chlamydospora. Systemic fungicides like benomyl, kresoxim-methyl, fenarimol, prochloraz manganese chloride and tebuconazole gave effective inhibition of P. chlamydospora at low concentrations (EC50-values 0.01-0.5 g/ml). The contact fungicides, thiram and chlorothalonil, and to a lesser extent iprodione, also proved to be effective inhibitors of mycelial growth with EC50-values ranging from 1.38 to 5.15 g/ml. Contrary to results from glasshouse experiments by Ferreira (1998a), Groenewald et al. (submitted) observed no mycelial inhibition of P. chlamydospora by metalaxyl. This was attributed to the fact that metalaxyl is a specific fungicide targeting the oomycete group of fungi (Bruin, 1980; Kerkernaar & Kaars Sijpesteijn, 1981). The inexplicable efficacy of metalaxyl against a non-oomycete fungus reported by Ferreira (1998a) was partly explained by the author at a later stage. The formulated product used in these glasshouse trials was Ridomil MZ, which contains metalaxyl and mancozeb. It is therefore possible that the mancozeb partner fungicide controlled the black goo decline in his experiments. Groenewald et al. (submitted) however found that mancozeb failed to inhibit in vitro mycelial growth of P. chlamydospora. Inhibition of spore germination was not tested in this study. All the fungicides with the potential to control P. chlamydospora will be tested for inhibition of spore germination in future trials at ARC Infruitec-Nietvoorbij. Subsequent to these trials, several potential systemic and contact fungicides will be evaluated in glasshouse and nursery experiments. Producers applying metalaxyl as a soil drench for the control of black goo decline must take note of the fact that this compound is not effective against non-oomycete fungi. Repeated use of metalaxyl can also lead to the buildup of resistance in the oomycete population (Phytophthora and Pythium sp.) in the soil. The current recommendation is two to three foliar applications of fosetyl-Al (Aliette) or phosphorous acid (Rootmaster) to the affected vines at a spray interval of 3 weeks. These compounds are not fungicidal or fungistatic (Groenewald et al., submitted), but rather stimulate host resistance (Schwinn & Staub, 1995). It would therefore inhibit P. chlamydospora (Ferreira, 1998a) and other foliar, root and trunk pathogens by making the plant more resistant to the pathogen.

Biological control. Although research on the biological control of P. chlamydospora has not been done, several reports on the beneficial use of Trichoderma products in grapevine propagation as well as in established vineyards have been published (Anonymous, 1998; Hunt, 1999; Messina, 1999). The use of Trichoderma products containing a mixture of T. harzianum and T. viride in callusing boxes resulted in a stronger graft union and root system in a shorter callusing period. Uncovered scions were also protected from disease by soaking in Trichoderma solutions (Messina, 1999). Hunt (1999) reported symptom improvement of vines suffering from certain trunk diseases (Eutypa lata and Botryosphaeria stevensii) after two seasons’ treatment with Trichoderma products. These products are administered to the mature grapevine trunk by injecting a Trichoderma solution or placing a wood dowel that has been impregnated with the biocontrol agent into a 6 mm hole drilled into the trunk of the vine. The efficacy of Trichoderma and other biocontrol agents against P. chlamydospora in mature grapevines, young graftlings and vines are presently being studied at ARC Infruitec-Nietvoorbij.

Optimize vine growth. Cultural practices should aim at minimizing stress and keeping the vines as vigorous and healthy as possible. According to the general understanding of the black goo disease, latent or endophytic infections of P. chlamydospora are triggered to express when the plant experiences a stress situation. Normal viticultural practices like grafting, hot water treatment, soil preparation, planting, irrigation, nutrition, disease and pest management, pruning, etc. should be done according to the best recommendations in order to ensure optimum growth of vines. These plants will naturally be more resistant to diseases than vines subjected to a stress situation.

Wound management. Several researchers regard wound sites, such as pruning or grafting wounds, as entry portals for P. chlamydospora (Gubler, pers. comm.; Larignon, 1998). Larignon (1998) demonstrated that pruning wounds stay susceptible to infection by P. chlamydospora for up to 14 weeks, depending on the pruning date. Pruning wounds made later during the dormant season were susceptible to infection for a shorter period than wounds made earlier in the dormant season. Infections furthermore correlated with rainfall. These findings emphasize the importance of pruning later in the winter and also applying wound sealant after pruning. The use of registered wound sealant products is also recommended. Hunt (1999) discussed Trichoderma wound sealant products that provide fresh wounds with a living protective layer. Grafting should also be done in a sterile environment and equipment must be sterilized as frequently as possible to prevent contamination of the grafting wounds.
Current research
Researchers at ARC Infruitec-Nietvoorbij and the Department of Plant Pathology, University of Stellenbosch, are currently involved in research on P. chlamydospora, focusing on various means of control, as well as aspects concerning the disease cycle of this fungus. Results from these studies will hopefully contribute to the understanding of the disease and allow nurseries and producers to grow healthy vines.

Literature cited
Anonymous. 1998. Trichopel- improves grapevine roots. Trichoderma News 8.
Bertelli, E., Mugnai, L. & Surico, G. 1998. Presence of Phaeoacremonium chlamydosporum in apparently healthy rooted grapevine cuttings. Phytopath. Medit. 37: 79-82.
Bruin, G.C.A. 1980. Resistance in Peronosporales to acylalanine-type fungicides. In: A.P.J. Trinci and J.F. Ryley (eds.)
Mode of action of antifungal agents. The Pitman Press, Bath. p. 240.
Crous, P.W., Gams, W., Wingfield, M.J. & van Wyk, P.S. 1996. Phaeoacremonium gen. nov. associated with wilt and decline diseases of woody host and human infections. Mycologia 88: 786-796.
Crous, P.W. & Gams, W. 1999. Phaeomoniella chlamydospora gen. et. comb. nov., the causal organism of Petri grapevine decline. Phytopath. Medit. (In press).
Delaine, A. 1998. Addendum 2: Hydration and Disinfestation. Seminar and Workshop on Black Goo Symptoms and Occurrence of Grape Declines – IAS/ICGTD Proceedings 1998: 107-110.
Dupont, J., Laloui, W. & Roquebert, M.F. 1998. Partial ribosomal DNA sequences show an important divergence between Phaeoacremonium species isolated from Vitis vinifera. Mycol. Res. 102: 631-637.
Ferreira, J.H.S. 1998a. Phialophora terugsterwing – ‘n Algemene probleem by jong wingerde. Wynboer Tegnies March: T6-T8.
Ferreira, J.H.S. 1998b. Researching P. chlamydosporum in rootstocks: summary, highlights, & excerpts. Seminar and Workshop on Black Goo Symptoms and Occurrence of Grape Declines – IAS/ICGTD Proceedings 1998: 94-97.
Ferreira, J.H.S., van Wyk, P.S. & Calitz, F.J. 1999. Slow dieback of grapevine in South Africa: stress-related predisposition of young vines for infection by Phaeacremonium chlamydosporum. S. Afr. J. Enol. Vitic. 20: 43-46.
Ferreira, J.H.S., van Wyk, P.S. & Venter, E. 1994. Slow dieback of grapevine: association of Phialophora parasitica with slow dieback of grapevines. S. Afr. J. Enol. Vitic. 15: 9-11.
Groenewald, M., Bellstedt, D.U. & Crous, P.W. 2000. A PCR-based method for the detection of Phaeomoniella chlamydospora in grapevines. S. Afr. J. Sci. 96: 43-46.
Groenewald, M., Denman, S. & Crous, P.W. Fungicide sensitivity of Phaeomoniella chlamydospora, the causal organism of black goo decline, to twelve fungicides. S. Afr. J. Enol. Vitic. (submitted).
Hunt, J.S. 1999. Wood-invading fungi: protective management in the vineyard. Australian Grapegrower & Winemaker Annual Technical Issue: 125-126.
Kerkernaar, A. & Kaars Sijpesteijn, A. 1981. Antifungal activity of metalaxyl and furalaxyl. Pestic. Biochem. Physiol. 15: 71-78.
Larignon, P. 1998. Esca disease from a European perspective. Seminar and Workshop on Black Goo Symptoms and Occurrence of Grape Declines – IAS/ICGTD Proceedings 1998: 43-55.
Messina, J. 1999. The use of beneficial Trichoderma in grapevine propagation. Proceedings of the 1999 Conference of the Australian Region of the International Plant Propagators’ Society, Launceston, Tasmania.
Mugnai, L. 1998. A threat to young vineyards: Phaeoacremonium chlamydosporum in Italy. Seminar and Workshop on Black Goo Symptoms and Occurrence of Grape Declines – IAS/ICGTD Proceedings 1998: 35-42.
Mugnai, L., Graniti, A. & Surico, G. 1999. Esca (black measles) and brown wood-streaking: two old and elusive diseases of grapevines. Plant Disease 83: 404-414.
Pascoe, I. 1998. Grapevine trunk diseases in Australia: Diagnostics and taxonomy. Seminar and Workshop on Black Goo Symptoms and Occurrence of Grape Declines – IAS/ICGTD Proceedings 1998: 56-77.
Pascoe, I. 1999. Grapevine trunk diseases – black goo decline, esca, Eutypa dieback and others. Australian Grapegrower & Winemaker, September: 24-28.
Scheck, H., Vasquez, S., Fogle, D. & Gubler, W.D. 1998. Grape growers report losses to black foot and grapevine decline. California Agriculture I, July – August: 19-23.
Schwinn, F. & Staub, T. 1995. Oomycete fungicides. In: H. Lyr (Ed.). Modern Selective Fungicides – Properties, Applications, Mechanisms of Action. Gustav Fischer Verlag, Jena. pp. 323-346.

The Authors:

Paul Fourie (pictured), Francois Halleen and Michelle Groenewald: Disease Management Division, ARC Infruitec-Nietvoorbij, Stellenbosch
Pedro Crous: Department of Plant Pathology, University of Stellenbosch, Stellenbosch

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