Eutypa Dieback Appearance in Some
Grapevine Growing Regions in Serbia
Recently the fungus Eutypa lata (Pers.: Fr.) Tul. i C. Tul. is also numbered among dangerous disease causal agents of grapevine. It causes disease called Eutypa dieback of grapevine. It was discovered and identified for the first time in the grapevine growing region of Australia (Carter, 1973), and at this moment is considered to be the most dangerous fungal pathogen of grapevine. Several years later it was also identified in the grapevine growing regions of Europe. This fungus was previously known as Eutypa armeniacae (Honsf. & Carter) because of discovery on apricot, and now is known as Eutypa lata. On apricot it causes Eutypa canker (or Eutypa dieback) formerly Gummosis. Important citation: ''The pathogen on apricot was originally described as E. armeniacae. However, in many recent publications this species has been considered conspecific with E. lata, a species with a very broad host range. In contrast, some researchers maintain that only the strains pathogenic to cultivated fruit trees and grapevine belong to E. armeniacae and that isolates from other woody dicots are E. lata. Molecular data support the existence of two distinct species, both occurring on cankered grapevines. For descriptions of the genus Eutypa, see Glawe and Rodgers and Rappaz'' (Munkvold, 2001).
In Europe this disease was reported in Greece for the first time (Kouyeas et al., 1976), and then in Switzerland (Bolay and Moller, 1977), Czechoslovakia (Venek, 1978), Hungary (Lehoczky and Moller, 1979), France (Bolay, unpubl., Mur, 1979; Dubos et al., 1980; 1981), Austria (Nieder, 1980) and Italy (Carter and Bolay, unpubl.) (loc. cit. Delibašić et al., 2001). In South Africa the existence of this disease was reported by Ferreira (2000), and Halleen et al. (2000).
In France rapid and successful identification of Eutypa lata is reported by Lecomte et al. (2000) using PCR. In the USA E. lata is identified by more specific method - PCR-RFLP (Rolshausen, Trouillas & Gubler, 2004).
In the past this pathogen was ''hidden'' by disease called Phomopsis cane blight and leaf spot (excoriose) of grapevine which is also caused by the fungus - Phomopsis viticola. As the matter of fact P. viticola often inhabits diseased and cankered wood surfaces made of so called "Eutypa cells". Those cells cause necrosis of armlets, trunks and even the whole shoots of diseased vines (Dye and Carter, 1976; Rumbos, 1989, loc. cit. Delibašić et al., 2001). Particulary susceptible vine cultivar is "Kardinal" (Delibašić et al., 2001).
Eutypa lata has a wide range of hosts. It attacks over 90 plant species from 28 botanical families including ornamental and silvan plants (Berberis darwinii L., Viburnum opulus L.) (Bolay and Carter, 1985; Carter, 1957, 1960, 1975, 1982; Carter and Bolay, 1972; Carter and Moller, 1971; Carter and Talbot, 1974; Kouyeas, 1978; Rumbos, 1984, loc. cit. Delibašić et al., 2001). Among other cultivated fruit trees besides grapevine, hosts of this pathogen are: apricot, plum tree, blackcurrant, cherry, almond, pistachio, olive, lemon, apple, quince, pear, walnut etc. This fungus was described for the first time on apricot in Australia (Samuel, 1933, loc. cit. Carter et al., 1983), then in USA (English et al., 1962, loc. cit. Carter et al., 1983), and after that in Europe (Carter et al., 1964, loc. cit. Carter et al. 1983) (loc. cit. Delibašić et al., 2001).
It is established that this pathogen occurs more often in areas where the rainfall is higher than 600 mm per annum, while in areas where the rainfall is lower than 250 mm pathogen is not present (Ivanović, 2001).
Today Eutypa dieback of grapevine, previously known as "Dead hand" or "Dying hand", is one of the most significant diseases of wood tissue in commercial grapevine plantations. It’s becoming the main barrier world wide for longevity of vineyards. In Australia Eutypa lata causes the most destructive disease of apricot known as Gummosis (Ivanović, 2001).
Delibašić et al. (2001) state that causal agent of Eutypa dieback, fungus Eutypa lata is a serious problem in grapevine growing which was obtruded during the former years. Authors further state that in Serbia and Montenegro E. lata is not yet officially discovered. However, bearing in mind it’s way of spreading (by the wind or by raindrops) and the fact that our country is great fruit and grape producer as well, they warn that it is necessary to pay attention especially on this dangerous, polyfagous pathogen. Same authors conclude that there are certain indications that E. lata is present in our grapevine growing regions (personal communication, 2002).
During the last ten years in some grapevine growing regions of Vojvodina, it is noticed that grapevines are dying off as a result of one dangerous disease which is determined as Eutypa dieback (Cindrić et al., 2002).
Stojanović et al. (2002) state that in some vineyard plantations of Serbia, vines with massive appearance of symptoms such as stunted shoots with small, chlorotic, distorted leaves and without or poorly developed bunches of grapes, have been noticed. Later on, these vines slowly die off. Spurs or cordons die first, then trunk and eventually the entire vine is killed. That is accompanied with wood cracks along the dead trunk or cordons. Authors state that this symptoms are typical for disease called "Arm dieback" (Eutypa dieback) of grapevine.
Based on detected and photographed symptoms, it is concluded that Eutypa dieback of grapevine is the most probably present in Serbia (Delibašić & Ivanović, personal communication, 2004). Based on visual detection of vines with specific Eutypa dieback symptoms, wood samples were collected in the vineyards from grapevine growing regions of Vršac, Fruška gora and Kruševac. The first purpose of this senior project was to prove the existence of Eutypa lata in Serbia by isolating the fungus [Senior project - part one]. The second purpose was to examine cultural characteristics of Eutypa lata on different nutrient media in order to determine which medium is the best for growing, sporulation i.e. fructification and isolation of this fungus [Senior project - part two].
Materials and methods
Collecting samples of necrotic (cankered) grapevine wood and visual inspection
Samples of necrotic (cankered) grapevine wood have been collected for three years: 2002, 2003. and 2004. Vines with typical shoot and leaf symptoms of Eutypa dieback have been selected for taking samples. Shoot, leaf, and cluster symptoms are not results of the fungus Eutypa lata in them. Toxine eutypine produced by E. lata is responsible for that symptoms. E. lata is situated only in old wood, while its toxine eutypine spreads through the vascular tissue and reaches the green organs. Wood necrosis (canker) is caused directly by the presence and growth of the pathogen in it. Because of that, wood parts which were taken as samples had necrosis that often appeared V- shaped in a cross-section of the perennial wood (Fig. 5, 8, 10 and 12). In order to inspect the samples same were transfered in a phytopathological laboratory at the Institute for Plant and Foodstuff Protection (Agricultural faculty in Belgrade).
Surface of the dead wood was observed by binocular. Purpose of this observation was discovering stromata of E. lata which could be located on the dead wood surface without bark as well as on the dead wood surface after removing the bark. Microscopic observation of different particles and the pycnidia-similar objects, found on the dead wood and bark surface, has been done too. The goal was to prove the existence of pycnidia formed by anamorph Libertella blepharis.
Isolation of Eutypa dieback causal agent
Isolation of Eutypa dieback causal agent was performed on potato dextrose agar (PDA). Smaller dead wood chips cut from the internal canker margin were used for the isolation. Before surface sterilization, wood chips were rinsed with plain water for about 20 min. Surface sterilization was performed with two chemical agents, in next order:
1. Ethanole (75 %) for 20 seconds
2. Mercuric chloride (0,1 %) for 40 seconds
3. Rinsing wood chips in sterile water 3 times for 5 - 10 seconds (each time).
After rinsing, chips had been placed aseptically on potato dextrose agar in Petri dishes. Than Petri dishes were placed in thermo-chamber on constant temperature of 25 şC. During the following days eye observations were performed in order to determine are there any colonies growth of on PDA especially around the chips.
Eutypa dieback symptoms in some grapevine growing regions in Serbia
1. The grapevine growing region of Vršac (Figures only)
Fig. 1. Eutypa dieback symptoms in an early stage (very low intensity) - slightly smaller leaves and slightly shortened internodes of green shoots.
Fig. 2. Eutypa dieback symptoms of various expression - from medium to severe level
of internode shortening on shoots with lightgreen and stunted leaves. Clusters and
grape bunches are not formed at all. In vineyards diseased vines like these
can be found often in groups or so called spots.
Fig. 5. Eutypa dieback symptoms on grapevine wood - V shaped necrosis (cankers) in a cross-section of the perennial wood, in TWO stages: EARLY STAGE - necrosis covers approximately Ľ of a wood cross-section surface or less (above, left and right) and MIDDLE STAGE - necrosis covers approximately ˝ of a wood cross-section surface (below, left and right).
2. The grapevine growing region of Fruška gora (Figures only)
Locality: Slankamenački Vinogradi
Fig. 7. Eutypa dieback symptoms inside grapevine wood - cankered (necrosed) wood around old pruning wounds becomes distorted and twisted after a number of years (above and below).
Fig. 8. Eutypa dieback symptoms on grapevine wood - V shaped necrosis (canker) in a MIDDLE STAGE - which covers approximately ˝ of a wood cross-section surface; on the outer side of
wood sample - along necrosis, a larger crack is visible (left) and magnified (in the middle).
Locality: Sremski Karlovci
Fig. 9. (a) Eutypa dieback symptoms in a middle stage - slightly shortened internodes of shoots, smaller chlorotic leaves and uneven grape bunches; (b) Intensified Eutypa dieback symptoms - severely shortened (stunted) internodes of shoots with few smaller chlorotic leaves and totally without grape bunches.
Fig. 10. Eutypa dieback symptoms on grapevine wood - V shaped necrosis (canker) in a
MIDDLE STAGE - which covers approximately ˝ of a wood cross-section surface.
3. The grapevine growing region of Kruševac (Figures only)
Fig. 11. Eutypa dieback symptoms on leaves and shoots of grapevine: shortened (stunted)
internodes, small, curled, chlorotic leaves - with smaller necrotic edges (A and B) and larger
necrotic edges (C and D). D picture was borrowed for comparision (Source: Ferreira, 2000).
Fig. 12. Eutypa dieback symptoms on grapevine wood - V shaped cankers in a cross-section of the perennial wood: (A) and (B) in EARLY, (C) and (D) in MIDDLE and (E) in LATE STAGE of development.
Visual inspection of necrotic (cankered) grapevine wood samples
By performing visual inspection of wood samples V shaped cankers were found on cross-sections (Figures above). V shaped necrosis (canker) overtook floem, cambium and xylem tissue parts. Between cankered (dead) wood and live wood tissue there is a definite canker margin. However there are some samples from Kruševac with blured (indefinite) canker margins and some resemblances with cankers caused by Botryosphaeria spp. (Fig. 12 A and D). It stays undetermined which fungus exactly causes these cankers. Isolation was not performed from these samples. It is not excluded even a possible existence of mixed infection like in ESCA disease. Shoots and leaves symptoms from these vines are also indicating that other causal agents might be present (Fig. 11 B and C; also Fig. 9. from the locality of Sremski Karlovci in the grapevine growing region of Fruška gora).
On dead wood surface without bark, as well as on the dead wood surface after removing bark, no stromata areas which resemble to those of Eutypa lata were found by binocular. Microscopic observation of different particles and the pycnidia-similar objects found on the dead wood and bark surface, did not confirm the existence of Libertella blepharis pycnidia.
Fungi isolated from necrotic grapevine wood samples
Already after 3 - 4 days from wood chips placeing, smaller colonies developed on PDA medium around chips. When the colonies spreaded on medium so covering approximately ˝ of the Petri box size, visual inspection was performed. It has been established the existence of four fungal species whose colonies differed in growth and appearance (coloure and shape). Each fungus particulary was re-seeded on a new PDA medium for pure culture obtaining. After 7 days on 25 şC colonies developed and covered approximately ˝ of the Petri box size (Fig. 13). Fungi were determined by microscopic observation but no Eutypa lata existence was confirmed.
Fig. 13. Fungi isolated from necrotic grapevine wood tissue: (a) and (b) are two isolates
which belong to genus Fusarium; (c) and (d) are the rest two isolates which belong
to division BASIDIOMYCOTA.
(at this moment available only in Serbian language)
Control of Eutypa lata - recommendations
(at this moment available only in Serbian language)
The author would like to thank everyone who unreservely helped and supported the working on this project and especially to his mentor - Assistant Professor Dr Goran Delibašić and Teaching Assistant - Instructor MPhil. Dragutin Antonijević. Help afforded Dragomir Jokić BSc, Dr Novica Miletić, Prof. Dr Pascal Lecomte, Prof. Dr Mirko Ivanović, Prof. Dr Petar Vukša, Prof. Dr Branka Krstić, Bojan Duduk BSc, Prof. Dr Ibrahim Elezović, friend Savo Strika and students Nemanja Bojčić and Dana Dobriković.
The most gratitude of all, for endless support during studying and senior project working, I owe to my parents Ivana and Goran, brother Žarko, aunt Antica, grandfather Spasoje and grandmother Jelena.
1. Avramov, L. (1997): Vinogradarstvo za IV razred poljoprivredne škole, Zavod za udžbenike i nastavna sredstva, Beograd.
2. Avramov, L., Nakalamić, A., Žunić, D. (1999): Vinogradarstvo, Poljoprivredni fakultet, Beograd.
3. Balasubramaniam, R., Harvey I. C., Braithwaite, M., Jordan, D. (1993): Grapevine Disease Identification Workshop. HortResearch Client Report 93/36., Marlborough Research Centre, New Zealand, online: HortFACT - Grapevine Diseases in New Zealand (http://www.hortnet.co.nz/publications/hortfacts/Eutypa Dieback).
4. Cindrić, P., Sidor, M., Korać, N. (2002): Bolesti drveta vinove loze - Eutypioza i ESCA, Zbornik rezimea radova sa XII simpozijuma o zaštiti bilja i Savetovanja o primeni pesticida, Društvo za zaštitu bilja Srbije, Beograd.
5. Delibašić, G., Babović, M., Jakovljević, D. (2001): Eutypa lata - prouzrokovač propadanja čokota, nova opasnost u gajenju vinove loze, Zbornik rezimea radova sa V jugoslovenskog savetovanja o zaštiti bilja, Društvo za zaštitu bilja Srbije, Beograd.
6. Ferreira, J. H. S. (2000): Dieback Disease in Vines caused by Eutypa lata, Wynboer - Technical Guide for Wine Producers online: http://www.wynboer.co.za, WineLand, South Africa.
7. Gubler, W. D. (2004): Organic Grape Production in California, Department of Plant Pathology, University of California, Davis, online.
8. Halleen, F., Fourie, P., Calitz, F. (2000): In vitro sensitivity of Eutypa lata to various fungicides, Wynboer - Technical Guide for Wine Producers online: http://www.wynboer.co.za, WineLand, South Africa.
9. Halleen, F., Volkmann, A., Fourie, P. (2000): Incidence of Eutypa-like symptoms in Cabernet Sauvignon vineyards in the greater Stellenbosch area, Wynboer - Technical Guide for Wine Producers online: http://www.wynboer.co.za, WineLand, South Africa.
10. Ivanović, M., Ivanović, D. (2001): Mikoze i pseudomikoze biljaka, Poljoprivredni fakultet, Beograd.
11. Lecomte, P., Péros, J-P., Blancard D., Bastien. N., Délye, C. (2000): PCR Assays That Identify the Grapevine Dieback Fungus Eutypa lata, Applied and Environmental Microbiology (AEM), p. 4475-4480, Vol. 66, No. 100099-2240/00/$04.00+0, American Society for Microbiology online: http://www.asmusa.org, USA.
12. Marković, S. (1979): Osnove fitopatoloških laboratorijskih metoda, Savezni komitet za poljoprivredu (granična služba za zaštitu bilja i biljni karantin), Beograd.
13. Munkvold, G. P. (2001): Eutypa Dieback of Grapevine and Apricot, Plant Health Progress online: http://www.plantmanagementnetwork.org/pub/diagnosticguide/dieback.
14. Naseva, Ž. (2002): Prouzrokovači sušenja i propadanja vinove loze (diplomski rad), Poljoprivredni fakultet, Beograd.
15. Pscheidt, J. W. (2004): Eutypa Dieback, An Online Guide to Plant Disease Control, Oregon State University online: http://www.plant-disease.ippc.orst.edu.
16. Rolshausen, P. E., Trouillas, F., Gubler, W. D. (2004): Identification of Eutypa lata by PCR-RFLP, Plant Disease 88: 925 - 929, Department of Plant Pathology, University of California online, Davis (Corresponding author: P. E. Rolshausen, E-mail: firstname.lastname@example.org).
17. Stojanović, S., Kuzmanović, S., Starović, M., Rajković, S., Tomić, T. (2002): Sušenje vinove loze, Zbornik rezimea radova sa XII simpozijuma o zaštiti bilja i Savetovanja o primeni pesticida, Društvo za zaštitu bilja Srbije, Beograd.
18. Tilcher, R., Schmidt C., Lorenz D., Wolf G. A. (2001): About the use of antagonistic bacteria and fungi (Untersuchungen zur Anwendung von antagonistischen Bakterienund Pilzen), online publication, Germany.