Nectary Structure of Pear Cultivars and Its Relation to Fire Blight Susceptibility

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1 Nectary Structure of Pear Cultivars and Its Relation to Fire Blight Susceptibility Á. Farkas and Zs. Orosz-Kovács University of Pécs Department of Botany 7624 Pécs, Ifjúság u. 6 Hungary T. Bubán Research and Extension Centre for Fruitgrowing Újfehértó, 4244 Újfehértó Vadas tag 2 Hungary Keywords: Erwinia amylovora, glandular tissue, nectar, nectary, Pyrus communis L. Abstract Morphology and histology of the floral nectary was studied between 1999 and 2003 in 10 pear cultivars, susceptible to Erwinia amylovora to various degree. Samples were collected in a Hungarian commercial pear orchard and a cultivar collection. The nectary was receptaculo-ovarial in each pear cultivar studied, the gland was located on the adaxial side of the plate-like hypanthium and on the apical part of the ovary. The nectary was automorphic, protruding out of the hypanthial tissue on the apical, and in most cultivars on the basal part, too. In most cultivars the nectariferous tissue continued along the style, too, where the accumulating nectar could stay in the flower for a longer time, thus increasing the possibility of infection by E. amylovora. The size of the nectary was bigger in non-/less susceptible cultivars than in highly susceptible ones. No relationship was found, however, between the thickness of the nectary, the thickness of the glandular tissue and the degree of susceptibility. In most cultivars studied the nectary stomata were sunken below the level of epidermal cells, on the basis of which they belong to the xeromorphic ecological type. Thus nectar can accumulate in the small cavity above the guard cells, ensuring a favourable environment for the growth of bacteria, provided that both sugar composition and concentration of nectar is optimal. The size of nectar chambers, located under nectary stomata, was usually larger in tolerant cultivars than in susceptible ones. INTRODUCTION Fire blight, caused by the bacterium Erwinia amylovora (Burr.) Winslow et al., is one of the most threatening diseases of pome fruit trees (van der Zwet et al., 1988; Johnson and Stockwell, 1998). During the epiphytic phase the pathogen first colonises the stigma, which is followed by the external washing of cells from the stigma to the hypanthium. Hasler and Mamming (2002) found that 18 h after spraying E. amylovora on pear and apple blossoms, the highest populations were found on stigma, stylus and hypanthium. Bacteria gain entry to the plant through nectary stomata located on the hypanthial surface (Hattingh et al., 1986; Thomson, 1986; Wilson et al., 1989, 1990; Johnson and Stockwell, 1998). The nectary is mostly receptacular in the Rosaceae family, consisting of three histologically distinct parts: the epidermis, the glandular tissue and the nectary parenchyma (Frei, 1955; Kartashova, 1965). The intrafloral nectary of pear is receptaculo-ovarial. A narrow zone of the nectariferous tissue stretches along the style, allowing nectar accumulation in the gap between the style and the nectary. The surface of the nectary is covered by a smooth cuticle, lacking ornamentation. Guard cells of nectary stomata can be found either at the level of epidermal cells (mesomorphic type) or sunken a few cell rows below the epidermis (xeromorphic type). Below the stomata, among the cells of the glandular tissue, nectar chambers, i.e. nectar-storing intercellular cavities of varying size can be found (Farkas, 2004). In apple cultivars a relationship was found between the shape and surface of the hypanthium and susceptibility to fire blight. In the flowers of susceptible apple cultivars Proc. 10 th Intl. Workshop on Fire Blight Eds. C. Bazzi and U. Mazzucchi Acta Hort. 704, ISHS

2 the deep funnel-like hypanthium with wrinkled surface provided better environment for bacterial cells than the flat hypanthium with a smoother surface in the flowers of a tolerant apple cultivar. Sunken stomata were observed on the strongly divided nectary surface of the susceptible cultivars, whereas nectary stomata of the tolerant cultivar were located slightly above the level of the epidermis (Mihalik et al., 2004; Orosz-Kovács et al., 2004). The aim of the present investigation was to determine whether there is a relationship between the structural features of pear nectary and the possibility of infection by E. amylovora. MATERIAL AND METHODS Studied Cultivars Floral biological studies were carried out on six pear (Pyrus communis L.) cultivars in the cultivar collection of the Research and Extension Centre for Fruit Growing, Újfehértó, and four cultivars in a commercial orchard in Györgytarló (Sárospatak), between 1999 and The studied cultivars and their E. amylovora susceptibility, based on incidence of fire blight symptoms: Újfehértó: Non-susceptible Bőtermő Nyári Kálmán Nagyasszony Nyári, Dunaföldvár Susceptible Bajai 6 Fillér Zánkai magonc Györgytarló: Least susceptible Beurré Bosc Susceptible Olivier de Serres Conference Highly susceptible Hardenpont Histological Studies At least ten flowers of each cultivar were fixed in absolute ethanol: glycerine: distilled water 1:1:1. Flowers were embedded either in paraplast or synthetic resin. Before embedding in paraplast, samples were dehydrated in an ascending acetone series and xylol. Longitudinal sections (7-10 μm thick) were cut with a sledge or rotary microtome. Before embedding in the glycol methacrylate based resin, samples were dehydrated in 70% and 96% ethanol, then put into the infiltrating fluid (Technovit 7100). Longitudinal sections (5-8 μm thick) were cut with a rotary microtome. Staining was done with toluidine blue in both cases. In the medial longitudinal sections of the flowers the following parameters were measured with the software Image Tool 1.27: size of the nectary and nectar chambers, thickness of the whole nectary, the glandular tissue and the nectary cuticle. Mean values were calculated from data measured on 10 flowers from each cultivar, then standard deviation was determined. RESULTS AND DISCUSSION Topography and Morphology of the Nectary In accordance with previous studies (Farkas, 2004), the floral nectary of all studied pear cultivars was receptaculo-ovarial, lining the adaxial surface of the plate-like receptacle and the apical part of the ovary. The automorphic gland protruded out of the receptacular tissue at the apical part in each cultivar, and also at the basal part in some cultivars, e.g. Beurré Bosc (Fig. 1), Conference, Hardenpont and Olivier de Serres. 132

3 A part of the nectariferous tissue was located along the style. The nectar secreted here, together with the secretory product coming down from the exposed surface of the nectary, can accumulate in the gap between the style and the nectary, especially if nectar is abundant and dilute. Accumulated nectar can remain in the flower for a longer time, evaporating less readily than from the exposed gland surface. This nectar reservoir provides a suitable environment for growth of bacteria, like E. amylovora. Size and Thickness of the Nectary Based on data from 5 years, the size of the nectary was bigger in non-/less susceptible cultivars than in highly susceptible ones (Fig. 2 and 3). In two years of the study the highest values for nectary thickness were measured in one of the tolerant cultivars, Nagyasszony, whereas the lowest values were measured in the most susceptible Hardenpont. The latter cultivar could be characterised with the smallest values concerning thickness of the glandular tissue, as well. In 2002 the glandular tissue was twice as thick in the most tolerant cultivar Beurré Bosc as in the most susceptible one. However, data measured in other cultivars and/or years do not support the hypothesis that a relationship should exist between the thickness of the nectary, the thickness of the glandular tissue and the degree of susceptibility. Nectary Epidermis 1. Cuticle. The thickness of the nectary cuticle was measured in 1999 and Values showed great differences in the two years of study and no relationship was found between the thickness of the cuticle and the susceptibility of the cultivars. However, concerning apples, thin cuticle was characteristic to a susceptible cultivar, while a thick, waxy cuticle to a tolerant one (Mihalik et al., 2003, 2004). 2. Position of Nectary Stomata. According to the position of guard cells of nectary stomata in relation to epidermis cells, cultivars belonged either to the mesomorphic type (guard cells in the same level as epidermal cells) or the xeromorphic type (guard cells below the level of epidermis). A hygromorphic stoma position was not found in any of the studied cultivars, in contrast with a tolerant apple cultivar (Orosz-Kovács et al., 2004). Susceptible cultivars usually had a larger number of xeromorphic nectary stomata, where guard cells were sunken one, two or even more cell rows below the epidermis (Fig. 4). In less susceptible cultivars the mesomorphic or slightly xeromorphic stoma position (guard cells sunken to half of the epidermal cells) occurred more frequently (Fig. 5). Nectar can remain longer in the small cavity above the guard cells, if they are deeply sunken, thus providing favourable conditions for the growth of bacteria, supposing that the composition and concentration of nectar is also suitable for the pathogen. If conditions for growth are favourable, a greater number of bacteria can gain entry into flower tissues through nectary stomata. This process can be further enhanced if resorption takes place, which can be frequently observed in nectaries (Bielski and Redgwell, 1980; Davis, 1997; Torres and Galetto, 1998; Nepi et al., 2001). Nectar Chambers The size of nectar chambers, located below stomatal guard cells (Fig. 4), was usually larger in less susceptible cultivars than in highly susceptible ones (Fig. 6 and 7), except for year 2002, when the nectar-storing cavity was the largest in the most susceptible cultivar, Hardenpont. Glandular Tissue and Nectary Parenchyma In some of the pear cultivars studied (e.g. Fillér ) only a few sub-epidermal glandular cells were arranged into regular rows or columns, whereas in the deeper layers secretory cells could be observed together with parenchymatous cells, thus creating a mosaic-like structure. In other cultivars (e.g. Olivier de Serres, Fig. 8), however, the glandular tissue could be easily distinguished from the nectary parenchyma, because the smaller glandular cells formed several rows, below which the larger parenchymatous cells 133

4 could be seen. No relationship was found between this structural feature of the nectary and the susceptibility of the cultivar. ACKNOWLEDGEMENTS We are grateful to E. Obzsut-Truskovszky for providing the study material in Györgytarló and to R. Zorn and M. Kocsis for their technical assistance. Literature Cited Bielski, R.L. and Redgwell, R.J Sorbitol metabolism on nectaries from flowers of Rosaceae. Austr. J. Plant Physiol. 7: Davis, A.R Influence of floral visitation on nectar-sugar composition and nectary surface changes in Eucalyptus. Apidologie 289: Farkas, Á Morphology and histology of the nectary in Hungarian local pear cultivars. Abstract 7, Proc. 9 th Intl. Pear Symposium, Stellenbosch, South Africa, 1-5 February. p.14. Frei, E Die Innervierung der floralen Nektarien dikotyler Pflanzenfamilien. Diss. E.T.H. Zürich. Ber. Schweiz. Bot. Ges. 65:60. Hasler, T. and Mamming, L Population dynamics of Erwinia amylovora on different blossom elements of pear and apple. Acta Hort. 590: Hattingh, M.J., Beer, S.V. and Lawson, E.W Scanning electron microscopy of apple blossoms colonized by Erwinia amylovora and E. herbicola. Phytopathology 76: Johnson, K.B. and Stockwell, V.O Management of fire blight: A case study in microbial ecology. Annu. Rev. Phytopathol. 36: Kartashova, N.N Sztroenije i funkcija nektarnikov cvetka dvudolnüh rasztenij. Izdatelsztvo Tomszkogo Universziteta, Tomszk. [in Russian]. Mihalik, E., Radvánszky, A., Dorgai, L. and Bubán, T Scanning electron microscopy and epifluorescence microscopy in studying Erwinia amylovora colonization and migration on blossoms of sensitive and tolerant apple cultivars. Proc. 6 th Multinational Congress on Microscopy, Pula, Croatia, June 1-5. p Mihalik, E., Radvánszky, A., Dorgai, L. and Bubán, T Study of Erwinia amylovora colonization and migration on blossoms of susceptible and tolerant apple cultivars. Int. J. Hort. Sci. 10(2): Nepi, M., Guarnieri, M. and Pacini, E Nectar secretion, reabsorption, and sugar composition in male and female flowers of Cucurbita pepo. Int. J. Plant Sci. 162(2): Orosz-Kovács, Zs., Farkas, Á., Bubán, T., Bukovics, P., Nagy Tóth, E. and Déri, H Floral biological investigations of apple cultivars in relation to fire blight. Int. J. Hort. Sci. 10(2):9-14. Thomson, S.V The role of the stigma in fire blight infections. Phytopathology 76: Torres, C. and Galetto, L Patterns and implications of floral nectar secretion, chemical composition, removal effects and standing crop in Mandevilla pentlandiana (Apocynaceae). Bot. J. Linnean Soc. 127: van der Zwet, T., Zoller, B.G. and Thomson, S.V Controlling fire blight of pear and apple by accurate prediction of the blossom blight phase. Plant Dis. 72: Wilson, M., Sigee, D.C. and Epton, H.A.S Erwinia amylovora infection of hawthorn blossom: II. The stigma. J. Phytopathol. 127: Wilson, M., Sigee, D.C. and Epton, H.A.S Erwinia amylovora infection of hawthorn blossom: III. The nectary. J. Phytopathol. 128:

5 Figures Fig. 1. Automorphic nectary of pear cultivar Beurré Bosc. Fig. 2. Nectary size of pear cultivars, Újfehértó,

Fig. 3. Nectary size of pear cultivars, Györgytarló, 2001-2003. Fig. 4.

6 Fig. 3. Nectary size of pear cultivars, Györgytarló, Fig. 4. Deeply sunken stomata of the susceptible cultivars Olivier de Serres. 136

7 Fig. 5. Slightly sunken stomata of the tolerant cultivars Bőtermő Nyári Kálmán. Fig. 6. Size of nectar chambers in pear cultivars, Újfehértó,

Fig. 7. Size of nectar chambers in pear cultivars, Györgytarló, 2001-2003. Fig. 8.

8 Fig. 7. Size of nectar chambers in pear cultivars, Györgytarló, Fig. 8. The glandular tissue and the nectary parenchyma can be well distinguished in cultivars Olivier de Serres. 138

Blossoms by Pseudomonas syringae pv. syringae

Blossoms by Pseudomonas syringae pv. syringae APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1989, p. 533-538 0099-2240/89/020533-06$02.00/0 Copyright ) 1989, American Society for Microbiology Vol. 55, No. 2 Scanning Electron Microscopy of Invasion