Shoots of several table and wine grape cultivars were cultured in vitro on a medium supplemented with polysaccharides obtained from a culture filtrate of Botryotinia fuckeliana through differential ethanolic precipitations. The general effects of polysaccharides resulted in leaf yellowness and in a reduction of fresh and dry weight. Differential response of assayed cultivars to polysaccharides seemed to be not related to their bunch susceptibility to grey mould under field conditions

Bisignano, V.

Fanizza, G.

Faretra, F.

Miazzi, M.

Pollastro, S.

English

JKI Open Journal Systems (Julius Kühn-Institut)

Vitis 34 (1), 41-44 (1995) Effects of polysaccharides from Botryotiniafuckeliana (Botrytis cinerea) on in vitro culture of table and wine grapes (Vitis vinifera) by G. FANIZZA1), V. BISIGNANo2), S. PoLLASTRo3), M. MIAZZI3) and F. FARETRA3) 1) Istituto di Miglioramento Genetico, Universita di Bari, Italia 2) Istituto del Germoplasma del Consiglio Nazionale delle Ricerche, Bari, Italia 3) Dipartimento di Protezione delle Piante dalle Malattie, Universita di Bari, Italia S u m m a r y : Shoots of several table and wine grape cultivars were cultured in vitro on a medium supplemented with polysaccharides obtained from a culture filtrate of Botryotinia fuckeliana through differential ethanolic precipitations. The general effects of polysaccharides resulted in leaf yellowness and in a reduction of fresh and dry weight. Differential response of assayed cultivars to polysaccharides seemed to be not related to their bunch susceptibility to grey mould under field conditions. K e y w o r d s : grapevine, screening test, tolerance, grey mould, polysaccharides. Introduction Grey mould, induced by Botryotinia fuckeliana (de Bary) Whetz. (=Botrytis cinerea Pers.), causes heavy losses of yield in table and wine grapes in all temperate areas. In Southern Italy, the disease is severe on late ripening cultivars, especially when high relative humidity and rain-ing occur before harvesting time. Chemical control of grey mould is costly and is often hampered by development of fungicide resistance in the pathogen; in addition, chemical treatments may leave fun-gicide residues in grapes. Although biological control of grey mould is possible, the use of resistant cultivars might be an attractive alternative. Screening procedures repre-sent a crucial step in any selection method for disease re-sistance. Tissue culture techniques might be useful for such purpose (HAMMERSCHLAG 1984; DAUB 1986) because large numbers of genotypes might be screened in vitro in a lim-ited amount of space and time. Several papers have been published on in vitro selec-tion of Vitis for resistance to pathogens such as Plasmopara viticola (Berk. et Curt.) Berl. et De Toni (BARLASS et al. 1986), Uncinula necator (Schw.) Burr. (KLEMPKA 1984), Eutypa lata (Pers.: Fr.) Tul. et C. Tul. (MAURO et al. 1988) as well as B. fuckeliana (Hoos and BLAICH 1988). It is known that B. fuckeliana produces extracellular polysaccharides which include glucanes and rhamno-galacto-mannans (DusouRDIEU 1978; MoNTANT and THOMAS 1978; PIELKEN et al. 1990). They may play a role as elicitors of plant defence responses and seem to be toxic at higher dosages (DIXON and FuLLER 1977 ; KAMOEN 1984; LISWIDOWAT! et a/. 1991; KAMOEN and DUBOURDIEU 1990). Recent results (V ANNEL et al. 1991 a, b; BEssis et al. 1992) have suggested the use of culture filtrates and phytotoxic polysaccharides for in vitro selection of resist-ant plants. In this work we have used po1ysaccharides se-creted by B. fuckeliana and a large number of grapevine cultivars to evaluate the reliability of such in vitro screen-ing test. Materials and methods P I a n t m a t e r i a I : Several table or wine grape cultivars (Tab. 1) with different level of susceptibility to grey mould were cultured in vitro on a modified MS me-dium (HARRIS and STEYENSON 1982). The uppermost 30-35 mm portion of elongated shoots were transferred in vessels containing the same medium, with 1 mg/1 BAP and supplemented with polysaccharides obtained from B. fuckeliana culture filtrate (see below). Three vessels (replicates), each containing 5 shoots, were used for each cultivar and treatment. They were maintained in a growth chamber at 25 °C with a 16 h photoperiod. Fresh and dry weight of each replicate were measured and leaf colour (SPAD) was evaluated by a leaf greenness meter (SPAD-501, Minolta Corp.) (MARQUARD and TIPTON 1987; FANIZZA et al. 1991) after 2 months of in vitro culture. All data wei:e submitted to statistical analysis. Shoots of several cultivars were sectioned at different distance from their base by using a cryotome. Sections were mounted in glycerine and observed with a Zeiss Photo-microscope Ill equipped with epifluorescence system in-cluding mercury lamp HBO 100 W/2 and blue-violet filter set (exciter filter BP 436/8, beam splitter FT 460 and bar-rier filter LP 470). Obtainment of polysaccharides: Preliminary experiments showed that B. fuckeliana monoascosporic strain SAR2116 was a good producer of Correspondence to: Prof. G. FANIZZA, Universita di Bari, Istituto di Miglioramento Genetico delle Piante Agrarie, Via Amendola, 165/A, I-70126 Bari, Italy 42 G. FANIZZA, V. BrsrGNANO, S. PoLLASTRO, M. MrAzzr and F. FARETRA polysaccharides. The strain was grown on potato dextrose agar for 10 d at 21±1 oc and exposed to lighting from a combination of fluorescent and NUV lamps (12 hid) to obtain conidia. Conidia were scraped from the surface of cultures and suspended in water containing 0.05 % tween 20Aliquots (10 ml) ofthe conidial suspension were added to 2 I Erlenmeyer flasks containing 800 ml of Czapek-Dox medium, to give a final concentration of 2.5ool04 conidia/ml medium. The strain was then grown at 21±1 oc in still cultures in the darkness for 4 weeks. Cul-ture filtrate was collected and measured after mycelium removal by filtration on Miracloth (Calbiochem). Polysaccharides were obtained following the method of DuBOURDIEU (1978), with minor modifications. The filtrate was adjusted at pH 3 with IN hydrochloridic acid. Addi-tion of half volume of ethanol caused precipitation of P0.5 fraction (glucanes), which was removed by centrifugation (10,000 rpm, -10 °C, 20 min). The sumatant was then added with further 3.5 volumes ethanol and kept 24 h at 4 °C; the P4 fraction, which consists of rhamno-galacto-mannans was collected by centrifugation as above. Collected fractions were washed with ethanol, dried under vacuum, weighed, dissolved in distilled water, and then dialysed (membrane cut off: 1.2ool04 Da) for 48 h against water. The P4 frac-tion was then autoclaved before to be added (4 g/1) to modi-fied MS medium, just before dispensing to vessels. V ANNEL et al. (1991 a) found that the toxicity of culture filtrates is not reduced by autoclaving. The concentration of P4 frac-tion into the medium was made double of that in the fun-gal culture filtrate (VANNEL et al. 1991 b). Results and discussion Still culture of B. fuckeliana strains in large volumes of Czapek-Dox medium allowed only limited fungal growth. In this conditions a small amount of P0.5 fraction (glucanes) and a large amount of P4 fraction (rhamno-galacto-mannans) were secreted by the fungus into the fil-trate, as compared to shaken cultures on the same medium. For example, in experimental conditions described above, the strain SAR2116 produced 600-900 mg/1 of dried mycelium, 60-100 mg/1 of P0.5 and 2-2.5 g/1 of P4. The incorporation of polysaccharides in the growth medium resulted in a decrease of fresh and dry weight of grapevine shoots (Tab. 1) and in a yellowish colour of their leaves (Tab. 2); these effects were statistically significant and a different response was detected among cultivars. Mass decrease due to rhamno-galacto-mannans was more evident in Moscato Reale (92 %) than in Regina Bianca (51 %, Fig. 1). Leaf yellowness was observed in almost all cultivars; it was more pronounced in Trebbiano (SPAD decrease: 63 % ), and almost negligible in Copeta (SPAD decrease: 8 %, Tab. 2). Leaf yellowness was an evident symptom that stands out on in vitro grapevine cultures supplemented with the P4 fraction of polysaccharides. This is in agreement with findings of BEssrs et al. (1992), who observed chlorophyll destruction in leaves exposed to culture filtrate of Table 1 Mean values of fresh and dry (in parenthesis) weight in table (T) and wine (W) grape cultivars and rootstocks (R) after 2 months of in vitro culture on a medium supplemented with poly-saccharides produced by Botryotinia fuckeliana (P4 fraction) Cultivar Control P4 fraction Decrease (%) Malvasia Nera (W) 15.7 (0.88) ab 12.6 (0.69) a 20 (22) Baresana (T) 19.7 (0.97) a 11.3 (0.66) ab 43 (32) Copeta (T) 18.3 (0.93) a 7.2 (0.47) abc 61 (49) Regina Bianca (T) 12.7 (0.89) be 6.2 (0.45) be 51(44) Alphonse Lavalle (T) 12.9 (0.71) be 5.4 (0.38) be 58 (46) Uva di Troia (W) 9.9 (0.60) be 4.6 (0.25) be 54 (58) Trebbiano Dorato (W) 8.9 (0.65) c 4.2 (0.24) be 53 (63) Verdeca(W) 14.9 (0.82) ab 4.0 (0.20) be 73 (76) lnzolia (T) 12.1 (0.81)bc 3.0 (0.17) c 75 (79) Primitivo (W) 12.0 (0.75) be 2.8 (0.14) c 77 (81) Gloire M. (R) 7.4 (0.62) c 2.4 (0.12) c 68 (81) 140 R(R) 7.5 (0.61) c 2.0 (0.11) c 73 (82) Sangiovese (W) 8.9 (0.68) c 1.8 (0.11) c 80 (84) Bianco di Alessano (W) 15.4 (0.81) ab l.8(0.10)c 88 (88) Trebbiano (W) 12.1 (0. 73) be 1.4 (0.09) c 88 (88) Regina Nera (T) 15.7 (0. 73) ab 1.4 (0.09) c 91 (88) Regina dei Vigneti (T) 10.8 (0.53) be 1.2 (0.08) c 89 (85) Bombino Bianco (W) 9.0 (0.60) c 0.8 (0.06) c 91 (90) Negroamaro (W) 6.6 (0.47) c 0.7 (0.05) c 89 (89) Moscato Reale (W) 6.6 (0.48) c 0.5 (0.05) c 92 (90) Total mean 11.9J0.7!l 3.7_(0.2~ ** 69 (69) Means (fresh wetght) followed by same letters are not Significantly different at P-0.01level (Newman-Keuls test) ** P"--{).01 between treatments(t-test). Table 2 Mean values of SPAD (leaf greeness) in table (T) and wine (W) grape cultivars and rootstocks (R) after 2 months of in vitro cul-ture on a medium supplemented with polysaccharides produced by Botryotinia fuckeliana (P4 fraction) "C;iliivar------,-- P4 fracti~- -----·--·--Control Decrease (%) Baresana (T) 18.0 a 16.0 a ll Malvasia Nera (W) 14.6 ab 12.6 ab 14 Negroamaro (W) 19.0. 12.0 abc 37 Copeta(T) 12.5 abc 11.5 abc 8 Primitivo (W) 18.6 a 10.0 abc 46 Moscato Reale (W) 17,2 ab 10.0 abc 42 Regina Nera (T) 17.0 ab 10.0 abc 41 Bianco di Alessano (W) 16.3 ab 8.6bc 47 Regina Bianca (T) 13.0 abc 8.5 be 35 lnzolia (T) 10.6 be 7.0bc 34 Alphonse Lavalle (T) 15.3 ab 7.0bc 54 Verdeca (W) 13.0 abc 7.0bc 46 Trebbiano (W) 19.0 a 7.0bc 63 Bombino Bianco (W) 10.6 be 6.0bc 43 Gloire M. (R) 9.2 c 6.0bc 35 Uva di Troia (W) 12.6 abc 6.0bc 52 Sangiovese (W) 10.5 be 6.0bc 43 Regina dei Vigneti (T) 9.0 c 5.5 be 39 140R(R) 11.6 be 5.0bc 57 Trebbiano Dorato (W) 8.0c 4.3 c 47 Total mean 13.8 8.3** 40 Means followed by same letters are not Significantly different at P-0.01 level (Newman-Keuls test) ** P"--{).0 I between treatments (!-test). B. fuckeliana The mechanism through which poly-saccharides causes leaf colour degradation is not known. The leaf color change is a symptom of the beginning of leaf senescence, a phenomenon which may be caused by different factors which induce stress (abiotic or biotic). The influence of nutrients and cytokinins on leaf senescence has been reported by different authors (RICHMOND and LANG 1957; THIMANN 1987; SIGN et al. 1992). Thus, leaf yellow-ness in grape shoots might be due either to direct toxicity Effects of Botrytis cinerea on in vitro culture of grapes 43 of P4 fraction or to interactions of polysaccharides with cytokinins contained in the culture medium. Fig. 1: Shoot growth of Regina Bianca (a), and Moscato Reale (b) after two months of in vitro culture on control medium (left) and medium supplement with 4 gll P4 fraction (right). Observations at the microscope of cross sections from stunted shoots grown on medium containing the P4 frac-tion revealed that large parts of tissues were brownish in colour. Tissue browning was limited to the downmost part of shoots at direct contact with the medium. Brown tis-sues, but not healthy ones, emitted a strong yellow autofluorescence when excited with blue-violet light (Fig. 2). This indicates that the P4 fraction probably in-duced accumulation of phenolic substances, which are well known for their role in defence mechanisms (LANGCAKE and PRYCE 1976). Our results indicate that the P4 fraction of culture fil-trate from B. fuckeliana causes leaf yellowness and growth reduction in vitro cultures of grapevines, with a different response among cultivars. Similar results were previously obtained by VANNEL et al. (1991 a, b) and BESSIS et al. (1992), who suggested that a bioassay based on differen-tial response of grapevines to P4 fraction might be exploited to screen in vitro cultivars tolerant to grey mould. In our research, screening a larger number of genotypes, we have detected a low relationship between the cultivar response in vitro and their susceptibility to grey mould under field conditions; for instance some cvs such as Baresana and Moscato Reale are both highly susceptible to B. fuckeliana under field conditions, as it is well known, but in vitro they showed a different response. In fact the cv. Baresana presented little or no sensitivity while the cv. Moscato Reale high sensitivity to P4 fraction (Tab. 1). Further and more extensive investigations are sug-gested to carry out in vitro and field experimental condi-tions in order to evaluate the possibility to apply the in vitro culture as a screening test for the tolerance to B. fuckeliana in table and wine grapes. Fig. 2: Cross sections from basal parts of shoots (Regina Bianca) grown on control medium (a) and on medium added with 4 gll P4 fraction (b). Browned tissues in (b) emitted a yellow auto-fluorescence when exited by blue-violet light (c). Acknowledgements Researches supported by the Italian Ministero delle Risorse Agricole, Alimentari e Forestali in the framework of the project "Resistenze genetiche delle piante agrarie agli stress biotici ed abiotici". Literature cited BARLASS, M.; MILLER, R. M.; ANTCLIFF,A. J.; 1986: Development of meth-ods for screening grapevines for resistance to infection by downy mildew. I. Dual cultures in vitro. Amer. J. Enol. Viticult. 37, 61-66. 8ESSIS, R.; VANNEL, D.; BARBIER, M.; 1992: In vitro bioassays to evaluate the relationship between grapevines and Botrytis cinerea. In: 44 G. FANIZZA, V. BisiGNANO, S. PoLLASTRo, M. MIAZZI and F. FARETRA VERHOEFF, K.; MALATHRAKIS, N. E.; WILLIAMSON, B. (Eds.): Recent Advances in Botrytis Research, I 04- I 08. Pudoc Scientific Publisher, Wageningen. DAUB, M. E.; 1986: Tissue cultures and the selection of resistance to patho-gens. Ann. Rev. Phytopathol. 24, 159-186. DrxoN, R. A.; FuLLER, K. W.; 1977: Characterisation of components from culture filtrates of Botrytis cinerea which stimulate phaseolin biosynthesis in Phaseolus vulgaris cell suspension cultures. Physiol. Plant Pathol. 11, 287-296. DuBOURDIEU, D.; 1978: Etude des polysaccharides secretees par Botrytis cinerea dans la baie de raisin. Ph.D. Thesis Univ. Bordeaux 11, France. FANIZZA, G.; DELLA GATTA, C.; BAGNULo, C.; 1991:Anon destructive deter-mination of leaf chlorophyll in Vttis vinifera. Ann. Appl. Bioi. 119, 203-205 . HAMMERSCHLAG, F. A.; 1984: In vitro approaches to disease resistance. In: CoLUNS, G. B.; PETDuNo J. G. (Eds.): Application of genetic engi-neering to crop improvement, 453-490. Martinus Nishoff Junk, Dordrecht, Netherlands. HARRIS, R. E.; STEVENSON, J. H.; 1982: In vitro propagation of Vttis. Vitis 21, 22-32. Hoos, G.; BLAICH, R.; 1988: Metabolism of stilbene pytoalexins in grape-vines: Oxidation of resveratrol in single-cell cultures. Vitis 27, I -2. KAMOEN, 0.; 1984: Secretions from Botrytis cinerea as elicitors of necrosis and defence. Rev. Cytol. Bioi. Veg. Bot. 7, 241-248. - -; DusouRDIEU, D.; 1990: Phytotoxic secretions from Botrytis cinerea in grapes. Meded. Fac. Landbouwwet. Rijksuniv. Gent 53, 799-804. KLEMPKA, K. C.; MEREDITH, C. P.; SALL, M. A.; 1984: Dual culture of grape powdery mildew (Uncinula necator Burr.) on its hosts (Vttis vinifera L.). Amer. J. Enol. Viticult. 35, 170-174. LANGCAKE, P.; PRICE, R. J.; 1976: The production of resveratrol by Vttis vinifera and other member of the Vitaceae as a response to infection or injury. Physiol. Plant Pathol. 9, 77-86. LrswrooWATI F. M.; HoHMANN, F.; ScHWER, B.; KINDL, H.; 1991: Induction of stilbene synthase by Botrytis cinerea in cultured grapevine cells. Planta, 183,307-314. MARQUARD, R. D. J.; TrPTDN, J . L.; 1987: Relationship between extractable chlorophyll and in situ method of estimate leaf greenness. HortScience 22, 1327. MAURO, M. C.; VAILLANT, V.; TEv-RuLH, P.; MATHIEU, Y.; FALLOT, J.; 1988: In vitro study of the relationship between Vitis vinifera and Eutypa lata (Pers.: Fr.) Tu!. I. Demonstration of toxic compounds secreted by the fungus. Amer. J. Enol. Viticult. 39, 200-204. MONTANT, C. ; THOMAS, L. ; 1978: Proprietes physico-chemiques du !3(1,3) B(l ,6) glucane exocellulaire produit par le Botrytis cinerea (Pers .) (2"dnote). Ann. Sci. Nat. Bot. Bioi. Veg. Paris 19, 39-43. PrELKEN, P.; STAHMANN, P.; SAHM, H.; 1990: Increase in glucan formation by Botrytis cinerea and analysis of the adherent glucan. A pp. Microbiol. Biotechnol. 33, 1-6. RICHMOND, A. E.; LANG, A.; 1957: Effect of kinetin on protein content and survival of detached xanthium leaves. Science 125, 650-651 . SIGN, S.; LETHAM, D. S.; ZHANG, Xue-ooNG; PALMI, L. M. S.; 1992: Cytokinin biochemistry in relation to leaf senescence. VI Effect of nitrogenous nutrients on cytokinin levels on senescence of tobacco leaves. Physiol. Plant 84, 262-268. THIMANN, K. V.; 1987: Plant senescence: a proposed integration of the con-stituent processes. In: THOMPSON W. W., NATHNAGEL, E. A.; HUFFAKER, R. C. (Eds.): Plant seQescence. Amer. Soc. Plant Physiol. 31, 83-111. VANNEL, D.; BARBIER, M.; Bessrs, R.; 1991 a: Etude de la toxicite du filtrat de culture de Botrytis cinerea sur des vitroplant de vignes: I. Effet du filtrat brut. Vitis 30, 167-175. - -; - -; - -; 1991 b: Etude de la toxicite du filtrat de culture de Botrytis cinerea sur des vitroplant de vignes: 11. Effet des differentes fractions isolees a partir du filtrat brut. Vitis, 30, 213-2 I 8. Received August 9, 1994 

Effects of polysaccharides from Botryotinia fuckeliana (Botrytis cinerea) on in vitro culture of table and wine grapes (Vitis vinifera)

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Effects of polysaccharides from Botryotinia fuckeliana (Botrytis cinerea) on in vitro culture of table and wine grapes (Vitis vinifera)

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