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Home , Diplocarpon mespili, Maloideae, Mespilus germanica, Quince

1 Original Research Article 2 3 THE () STRUCTURAL AND FUNCTIONAL FEATURES 4 DETERMINING PASSIVE IMMUNITY TO MYCOSIS 5 6 7 With the help of microscopic methods the and surface tissues of of four 8 genera of the Maloideae subfamily were screened: Mill., Pyrus L., Cydonia Mill., 9 L., as model objects, and attempts were made to explain the dependence of mycosis 10 damage on microstructural features. The species composition of fungi that cause damage to the 11 Maloideae leaves and fruits in the Russia southern regions is analyzed. It is established that 12 among pathogens with different types of parasitism there are common excitants, as well as 13 highly specialized, more represented on . Higher resistance to the complex 14 of fungal diseases, in comparison with and , was found in and medlar. This 15 stability at the initial stage of the pathological process is associated with structural features such 16 as micromorphology of the fruits and stomata cuticle in the abaxial leaves epidermis. The leaves 17 stomatal cracks of the medlar are narrow with raised outgrowths, on the surface of the – the 18 layered structure of the cuticular layer. Quince has a powerful continuous cuticular cover. 19 Compared with Malus and Pyrus, Cydonia and Mespilus also have a large (30 % or more) 20 polyphenol content in the pericarp outer layer cells. In addition to the gender-specific differences 21 in the microstructure of the integumentary tissues and the content of polyphenols affecting the 22 resistance to pathogens at the stage of their penetration, general patterns of surface 23 formation, such as hypostomacy, anomocytic stomata, folded microrelief of the cuticular surface, 24 and the presence of single and multicellular trichomes are noted. Epidermal cells from the 4 25 genera examined contained most of the condensed polyphenols compared to the hypodermal 26 cells. 27 Key words: Maloideae; pathogenic fungi; resistance; ultrastructure; polyphenols. 28 29 Introduction 30 Maloideae, or Pomoideae is one of the subfamilies of Rosaceae, which includes the most ancient 31 cultivated representatives of angiosperms, which are widespread in many ecological and 32 geographical zones. It includes such famous fruit plants as pear (Pyrus L.), apple (Malus Mill.), 33 quince (Cydonia Mill.), medlar (Mespilus L.) and others. Their main feature is the fruit, 34 overgrown with a modified hypanthium – prefabricated leaflets or drupaceous modified carpels, 35 called in a broad sense (Tsitsin 1962; Kamelin 2006). The large (juicy) part of the fruit is 36 formed due to the succulentization of the tissues of the hippocampus. A typical fruit is typical for 37 apple, pear and quince, and the drupaceous apple is for the medlar germanica (Mespilus 38 germanica). 39 According to practical importance in world production, including in Russia, the apple 40 takes the leading place among fruit crops, the second is the pear tree. The enormous consumer 41 value of the fruits of these crops is widely known, which are extensively used in the food, 42 medical, perfume and other industries. Along with -bearing qualities, the special value of 43 apple and pear fruits is due to good antioxidant properties. Despite the huge number of varieties 44 currently available, there is a need to improve the assortment, since during the long selection 45 process many of the valuable traits that their wild relatives have got were lost. 46 Among the promising cultivated plants is quince common (Cydonia oblonga Mill.). It is 47 quite flexible to the conditions of growth, especially soil, can grow successfully on different 48 types of soils (alluvial, chernozemic, etc.). Also, quince has a high resistance to various stressors, 49 it is light-demanding, heat-resistant, salt-tolerant, fast-growing, durable and fruitful crops. 50 C. oblonga fruits are distinguished by high technological qualities, rich in , organic 51 acids, microelements, and other substances. Some researchers note that quince to a lesser

1 52 extent than other fruit crops from the subfamily Maloideae is damaged by pests and diseases 53 (Baskakova 2017). Despite the fact that quince is a promising fruit crop in its characteristics, 54 especially for the southern regions of Russia, until now it has not received due attention, and 55 industrial plantations do not exist yet. 56 To the group of promising fruit plants of the fruit (Maloideae) it is possible to include a 57 medlar germanica (Mespilus germanica), which is represented by two ecotypes: xerophilous, 58 confined to open habitats, and mesophilic, forest (Tsitsin 1962; Strasburger 2007). In Russia, it is 59 widespread in the mountain ecosystems of the southern regions, especially in the North 60 – from the lower zone to the upper limits of arboreal vegetation (300–2000 m above sea level), 61 as well as in the and adjacent territories, however, there are no industrial plantations. The 62 value of the medlar is known since ancient times. As a vitamin and medicinal , it entered the 63 culture of many countries of the Near and Middle Asia, the Eastern Mediterranean and Western 64 Europe. Many biologically active substances have been found in the vegetative and reproductive 65 organs of M. germanica, in particular, triterpenoids, tannins, phenolic carboxylic acids, 66 flavonoids, catechins, organic acids and their derivatives, aliphatic aldehydes, higher fatty acids, 67 significant amounts of fatty oil in (Stanimirović et al. 1963; Glew et al. 2003). 68 The development of the Maloideae of various habitats and the increased adaptability of 69 representatives are based primarily on the development of structural and physiological- 70 biochemical features that also play an important role in resistance to phytopathogenic organisms. 71 For research in this respect, the representatives of the above four genera were taken as model 72 objects. 73 To the anatomical and morphological features of passive immunity, or horizontal 74 stability, include the specificity of the deposition of wax deposits and the thickness of the cuticle 75 of integumentary tissues, the structure of stomata, and a number of other features (Kumakhova 76 and Melikyan 1989). The material on the structure of superficial tissues (epidermis and 77 hypoderma) accumulated by some Maloideae representatives for the vegetative and reproductive 78 organs, as well as for their mycobiota, is fragmentary (Kumakhova 2003; Shkalikov et al. 2005; 79 Pautov et al. 2008). There is insufficient information on the polyfunctionality of the epidermal 80 tissue, the specificity of the microrelief of the leaf epidermis (the formation of cuticular folds, 81 microtens in the stomata and trichomes, peristomatic rings, cork tissue and lentils on the fruits). 82 Their functional significance is not entirely clear to this day. In the literature there are 83 disagreements and assumptions about the mechanism of opening and closing stomata, and also 84 the interaction of the stomata with the surface structures of the leaf (Kumakhova and Melikyan 85 1989, Pautov et al. 2008). The role of the ultrastructure of the cuticle of leaves and fruits in 86 passive immunity for many pathogens also remains controversial, and direct dependence on the 87 type of structure and overall thickness is not revealed. 88 The chemical factors of passive immunity include, first, the content or absence in the 89 plants of substances necessary for the life of the pathogen, and secondly, the presence of 90 substances that oppressively act on it (phytoncides, or phytoantipypsins). The leading role in the 91 formation of the latter belongs to the synthesis and dynamics of various compounds of phenolic 92 nature (from relatively simple phenols to complex polyphenolic composites), toxic to many 93 phytopathogenic fungi (Upadyshev 2008). According to modern concepts of the significance of 94 the features of the structure of the surface and the subcellular structures of plants in protection 95 from phytopathogens, a major role belongs to the synthesis and dynamics of changes in 96 phytohormones (Kumakhova and Skorobogatova 2017). At the same time, the character of 97 distribution of lipophilic components, the specificity of the formation and localization of 98 substances of polyphenolic and terpenoid nature, as well as playing important protective 99 functions against the attack of fungal pathogens in the surface tissues have not been adequately 100 studied. It is shown that the phytoncidal effect of plants on specialized parasites is generally 101 poorly expressed. In most cases, it extends to microorganisms that do not affect this plant.

2 102 Phytoncidal activity is not the same, it varies depending on the grade and age of the plant, as 103 well as on the time of day, the phase of development, weather conditions and other factors. 104 Thus, despite the availability of separate information from biology and disease affection 105 of the most valuable fruit representatives of the Apple Subfamily, no attempts have yet been 106 made to fully study their mechanisms of resistance. 107 In connection with the above, the purpose of this work was to conduct a comprehensive 108 search of micromorphological, anatomical and histochemical, and also ultrastructural and 109 physiological-biochemical features of plants of 4 genera of the Apple Subfamily, and an 110 evaluation of these indicators as markers of passive immunity to phytopathogenic fungi. 111 112 Materials and methods 113 The objects of research (botanical and phytopathological) were the leaves and fruits of 114 plants of different apple varieties (Malus domestica), () collected from the 115 experimental sites of the North Caucasian Research Institute of Mountain and Foothill 116 Gardening, as well as common quince (Cydonia oblonga Mill.) from planting of private plots, 117 and a wild medlar germanica (Mespilus germanica), which grows in high altitude zones in the 118 North Caucasus. Leaves and fruits for research were selected from the middle part of the crown 119 of model . To diagnose plant diseases and identify agents, the wet chamber methods, 120 microbiological with isolation of pathogens into a universal artificial nutrient medium (potato 121 glucose agar) followed by microscopy of infectious structures were used. 122 Histochemical studies of the fruit were carried out on an AxioImager D1 microscope 123 (CarlZeiss, Germany) in transmitted light. Sections 50 μm thick were made using a microtome 124 with a vibrating blade (Thermo Scientific, Microm HM 650V). To detect condensed 125 polyphenols, the sections were treated with potassium dichromate (K2Cr2O7) (Marin et al. 2010; 126 Kumachova et al. 2018). Microphotographs were obtained with an AxioCamMRc camera 127 (CarlZeiss, Germany); images were processed using the ZEN lite 2012 program (CarlZeiss, 128 Germany). 129 The microstructure of the surface of leaves and fruits was studied with the help of a 130 scanning electron microscope (SEM) in high vacuum mode and with gold deposition. 131 Autofluorescence of the surface structures of leaves and fruits was examined using the confocal 132 laser scanning microscope «Olympus FV1000D» under excitation with light 405, 473, 560 nm 133 (Marin et al. 2010). 134 Electron microscopic (TEM) studies of the polyphenols material content of in the cover 135 tissues was carried out according to a modified procedure. The material was fixed with 136 glutaraldehyde (0.1 M phosphate buffer pH = 7.2) and 1 % osmium tetroxide solution. Then the 137 samples were dewatered in a series of alcohols and acetones of increasing concentration and 138 poured into Epon-812. Ultrathin sections were made on ultramicrotome LKB-III-8801A. The 139 sections were contrasted with a 2 % aqueous solution of uranyl acetate (37°C) and lead citrate by 140 Reynolds (Kumakhova and Melikyan 1989). 141 142 Results and discussion 143 Based on the results of phytopathological studies in various plantings in the southern 144 region of Russia, as well as analysis of literature data (Shkalikov et al. 2005; Sorokopudov et al. 145 2017), a summary table of the main fungal diseases of the leaves and fruits of the Maloideae 146 subfamily was compiled (Table 1). 147 According to the obtained materials, on all the fruit crops under investigation, the leaves 148 and shoots were damaged by ( sp.) and powdery mildew (Podosphaera 149 sp.) caused by phytopathogenic fungi-obligatory highly specialized parasites, biotrophs 150 (Table 1). These plant genera are systematically affected by a number of pathogens related to 151 typical facultative parasites, necrothrophies, usually with a broad phylogenetic specialization. 152 Monilial fruit rot (Monilia fructigena Pers.), gray rot of and fruits (Botrytis cinerea

3 153 Pers.), black cancer (Sphaeropsis malorum Peck.) and anthracnose, or bitter rot of fruits 154 (Colletotrichum fructigenum (Berk.) Vassil.) occur on fruits all studied cultures (apple, pear, 155 quince and medlar). Alternaria (Alternaria spp.) during the vegetation period affects not only 156 their fruits, but also leaves. All four cultures are affected by fungi of the Phyllosticta, 157 causing phyllosticosis, or a brown leaf spot, with each of them parasitizing its pathogen species, 158 respectively, P. mali Pr.et Del., P. pirina Sacc., P. cydoniae var. cydoniicola (Allesch.) Cif. and 159 P. mespili Sacc. Brown spot caused by Entomosporium maculatum Lév. f. maculate Kleb. occurs 160 on the pear and quince, and the medlar is struck by a species specialized only in this culture E. 161 () mespili Sacc. Ascochyta piricola Sacc. parasitizes only on the leaves of 162 the pear and apple, a close species of A. mespili West. defeats medlar, on the quince pathogens 163 of this genus are not registered. 164 The analysis of the species composition of mycosis agents of representatives of the Apple 165 subfamily indicates the presence in the medlar M. germanica the most of the highly specialized 166 pathogens belonging to the group of facultative parasites and facultative saprotrophs. This may 167 be due to the physiological differences of these fungi that determine their different responses 168 when colonizing the surface tissues of plants and further development of mycelium and 169 sporulation. 170 Undoubtedly, the degree of defeat of representatives of this subfamily strongly depends 171 on the varietal characteristics and conditions of their growth. But in general, according to our 172 long-term observations, quince is more resistant to the complex of fungal diseases, and also a 173 medlar, whose wild forms and most varieties are practically not affected by diseases of fungal 174 etiology. 175 According to our observations, the features of the structure of integumentary tissues are 176 of great importance at the first stages of the pathological process. Study of Maloideae leaves 177 with SEM showed that a number of common morphogenetic characters are characteristic of their 178 surface. First of all, hypostomacy (the stomata is located only on the abaxial surface of the leaf) 179 and the anomocyte type of stomata (the cells adjacent to the stomata do not differ in form from 180 the epidermal ones themselves), the folded surface microrelief (Fig. 1, 2), the presence of single 181 and multicellular trichomes. In this case, the abaxial and adaxial surfaces of the epidermis of 182 leaves have different specific features of the microrelief, due to the functional load. Due to its 183 stability, including in different conditions of plant growth, the features of the microrelief of the 184 leaf surface can be considered as diagnostic features and used not only in plant systematics and 185 , but also as possible markers of resistance to pathogens at the stage of their 186 penetration. 187 The adaxial surface of the investigated representatives of the subfamily Maloideae is 188 characterized by a different morphology and degree of development of the cuticular folds 189 (Fig. 1). 190 However, for all four genera there is typically a rather weak development of epicuticular 191 wax, so they are strongly affected by agents that easily penetrate directly through the 192 integumentary tissues. Pathogens include agents of powdery mildew (Podosphaera sp.) and rust 193 (Gymnosporangium sp.), widespread and quite harmful to these plants (Table 1). Infection with 194 many other fungi occurs mainly through mechanical damage to the adaxial surface. 195 For the abaxial epidermis of the representatives of Maloideae, the different 196 micromorphology of the cuticular folds responsible for the regulation of stomatal movements is 197 characteristic (Fig. 2). For example, on the leaves of quince and medlar, the folds in the stomata 198 are arranged in the form of clearly pronounced high peristomatic rings encircling the closing 199 cells (Fig. 2 c, d). And on the leaves of the apple and pear they are less pronounced, and from 200 them numerous radial and diverging in all directions microstrands, often extending over the 201 borders of the actual epidermal cells (Fig. 2 a, b). 202 The high height of the cuticular folds changes the character of wettability of the leaf 203 surface (Kumakhova and Melikyan 1989). Water drops due to high surface tension only touch

4 204 the upper edges of the cuticular ridges and therefore easily slide down from the epidermis. It can 205 be assumed that, blowing out or washing away by rain sporadic spores (conidia) of 206 phytopathogenic fungi, having a rounded shape, occurs similarly at which point they cannot 207 firmly hold onto the folds of the cuticle before the germination of the hypha germ begins. The 208 nature of the surface of Cydonia leaves and, to a lesser extent, Mespilus, contribute to increased 209 resistance to fungal pathogens at the stage of their entry onto the surface of the affected organ, 210 preventing further penetration. 211 The structure of stomata and the number of them on the leaf are also of great importance 212 if the pathogen penetrates through them the tissues. Narrow gaps of the stomata (lenticles) with 213 their rarer location delay the infection of plants with fungal and bacterial infection. The 214 peculiarity of the structure of stomata in the representatives of the genus Mespilus – narrow 215 stomatal gaps with raised outgrowths above the surface (Fig. 2 d), with a high degree of 216 probability, complicates penetration of pathogens. 217 In representatives of the genus Pyrus, the availability of fungal penetration through the 218 stomata is due to the absence of these features and the depth of the stomata in the epidermal 219 tissue (Fig. 2 b). In this case, a funnel is formed, which protects spores from rapid removal by air 220 or water streams under natural plant growth conditions. 221 It is also important to note that the penetration of pathogenic fungi in plants of the genus 222 Pyrus in many cases occurs through the spaces between the closing and proper epidermal cells 223 (Fig. 3 a, b) rather than the traditional methods through the stomatal slit, as in Malus (Fig. 3 e, f). 224 Penetration of pathogenic fungi into leaf tissue can also be carried out through the bases of fallen 225 trichomes, as, for example, we noted for the genus Mespilus (Fig. 3 c, d). 226 When studying the surface of the tissues of fruits of representatives of this subfamily, we 227 found that in apple and pear they are covered with a continuous layer of cuticle with a thickness 228 of 13.7 ± 2.7 and 11.5 ± 1.6 μm, respectively. The fruits of quince also have a continuous 229 cuticular cover on their surface, while it is 80 % larger than that of apple and pear (22.6 ± 230 4.0 μm). Unlike the listed representatives of Maloideae, the fruits of the medlar germanica 231 (M. germanica) do not have such a continuous cuticular layer in their mature state. 232 Morphologically it is represented by separate areas remaining between the crests of the 233 microrelief of the fetal surface. If you take this feature into account, then the medlar barrier that 234 prevents the penetration of pathogens should be much worse than that of apple, pear, especially 235 quince. But the structural and functional difference of the cuticular layer in the genus Mespilus is 236 its peeling surface, the properties of which contribute to the removal (spillage) of spores of 237 pathogens at the stage of their germination and penetration into tissues. Also in this genus 238 Mespilus, below the thick-walled cells with suberinized walls, limit the penetration and 239 localization of microorganisms in the surface tissues of the host plant. This, in many respects, 240 explains the high resistance of medlar germanica fruit to the defeat of various fungal diseases 241 during the growing season. Stomata of quince on fruits are larger and more numerous than in 242 other cultures (from Fig. 4 d), however, the fruits of this culture are less often affected by 243 mycosis. It can be assumed that some gaseous compounds located directly in the under stomatal 244 cavity are toxic to pathogens. 245 The study of regularities in the distribution of phytopathogenic fungi on the surface of 246 leaves and fruits, as well as the ways of their penetration into internal tissues, in our opinion 247 allows us to relate the features of the microstructure of the integumentary tissues with the disease 248 affection and partially explain the mechanism of the formation of resistance. 249 When examining the surface of apple fruit in the cuticle in the SEM, microcracks with 250 emerging from them conidia of fungi – scab pathogens were noted, as well as numerous lenticles 251 (especially in Pyrus), often filled with conidia of various fungi (Fig. 4 b, c). 252 The hyphae of the pathogenic fungus, presumably of the genus Fusicladium, were also 253 found in the lenticule cavity by the method of confocal microscopy (Fig. 5 a, b). Moreover, the

5 254 hyphalic cell walls possessed an intense autofluorescence in the blue part of the spectrum, due to 255 this they were clearly distinguished among the cells of the performing tissue of these structures. 256 It has been established that flavonoids and flavonoid glycosides of quercetin and 257 anthocyanins are widely represented in the fruits of the plants of the Apple subfamily, as well as 258 polyphenolic compounds – catechin and epicatechin (Budantsev 2009). Phenolic compounds 259 with antioxidant activity are present in large numbers in plant tissues as precursors of lignin and 260 other complex flavonoids (polyphenols) performing various functions (Solovchenko and 261 Schmitz-Eiberger 2003; Merzlyak et al. 2005; Assumpçãoa et al. 2018). They are part of the 262 growth regulators, pigments, structural elements of the . Phenols are stress metabolites, 263 the synthesis of which increases dramatically when wounding or infected with pathogens 264 (Menden et al. 1996; Khokhryakov et al. 2003). These compounds have an overwhelming 265 majority of fungitoxicity. Many flavonoids, even with minimal effect on the growth of 266 phytopathogenic micromycetes, are able to inhibit the production from certain fungi, for 267 example, aspergillus species Aspergillus flavus and A. parasiticus, mycotoxins, which are 268 dangerous to human health and animals (Mabrouk and El-Shayeb 1992; Duarte et al. 1998; 269 Goncalez et al. 2001; Dzhavakhiya et al. 2016). Due to their high inhibitory activity and 270 potential value as food products, biflavonoids may be promising candidates for the role of 271 inhibitors of aflatoxin genesis (De Luca et al. 1995). 272 As a result of histochemical study of the surface tissues of apple, pear, quince and medlar 273 fruits, it was found out that the common feature of all studied representatives of 4 genera of the 274 Maloideae subfamily is the presence of condensed polyphenols in epidermal cells and 275 hypodermis (Fig. 6 a2–d2). These compounds are well detected by microscopy when staining 276 transverse sections of the pericarp with potassium dichromate. If flobaphenes are present in the 277 cells – products of oxidation of tanning substances related to polyphenols, they become saturated 278 dark brownish- reddish brown in color. Control cells that did not contain condensed polyphenols 279 in sufficient quantities had a pale brown hue after staining (Fig. 6). 280 According to our data, epidermal cells contained most of the condensed polyphenols, 281 which is one of the chemical factors of field (horizontal) resistance to phytopathogenic fungi. 282 Moreover, in the deeper layers of the cells of the pericarp, especially the hypoderm, the number 283 of cells with condensed polyphenols is significantly less than in the epidermis (Fig. 7). The 284 exception was the pear fruit of some varieties, which, on average, had 10 % more cells with 285 polyphenols in the hypodermis. 286 We found that in a medlar, more than 80 % of the area of a mature fetus is covered with 287 plug-like cells containing condensed polyphenols. Probably, this can be a compensatory 288 mechanism due to the absence of a continuous cuticular cover. Thus, in a medlar the passive 289 immunity can be caused by at least two factors: first of all, a high concentration of polyphenols 290 in the surface tissues, and also by the peculiarities of the exfoliated cuticle layer and the 291 suberinized cells beneath it. 292 The presence of condensed polyphenols in the cells of the epidermis and the hypoderma 293 of the Apple fruit was also revealed by us using the transmission electron microscope (TEM), 294 since in this method the fixation of the material with osmium oxide (OsO4) allows us to visualize 295 complexes of with polyphenols in the form of black electronically dense material 296 (EDM). The presence of electronically dense material in the fetal cells observed by us in the 297 transmission electron microscope is consistent with the results obtained in the histochemical 298 study of the pericarp tissue (when staining with potassium dichromate) and indicates the identity 299 of the observed structures and their relationships in representatives of the Apple subfamily 300 (Fig. 8). 301 As can be seen on the TEM micrographs, most of the cells in the outer layer of the 302 pericarp, the medlar (Mespilus) are completely clogged with polyphenols, unlike the apple tree 303 (Malus), in which the polyphenols are deposited only in the form of separate rounded formations

6 304 – tannoglobules (Kumakhova et al. 2018). In cells of the epidermis and hypoderma of fruits of 305 medlar also contained a significant amount of condensed polyphenols. 306 Based on the results of the conducted studies, it can be assumed that in the formation of 307 functional passive immunity in Maloideae fruits the most important role belongs to the surface 308 layer of the cells – the epidermis. Its cells form a sufficient powerful cuticle on the surface, 309 which may well be a mechanical barrier to penetration of pathogens, and also contain most of all 310 condensed polyphenols, which are one of the chemical factors of field (horizontal) resistance to 311 phytopathogenic fungi. 312 It was established that representatives of the genera Malus, Pyrus, Cydonia and Mespilus 313 among the phytopathogenic fungi with different types of parasitism have common pathogens, as 314 well as highly specialized ones, more represented on Mespelus germanica. A higher, in 315 comparison with apple and pear, resistance to a complex of fungal diseases is established in 316 quince and medlar. This stability in the penetration of phytopathogens, as the initial stage of the 317 pathological process, is associated with the morphology of stomata in the abaxial epidermis of 318 leaves, features of the layered structure of the cuticle, the presence of suberinized cells of the 319 medlar fruit, a powerful continuous cuticular cover in quince, and a large polyphenol content in 320 the cells of the outer layer of their pericarp. 321 Using the example of representatives of the Maloideae subfamily as model objects, the 322 structural features of the epidermis, such as hypostomacy, anomocytic stomata, folded 323 microrelief, the presence of single and multicellular trichomes, were revealed by different 324 electron microscopic methods, but also the specific differences in the abaxial and adaxial surface 325 of the leaves, as well as ultrasculpture of the fruit surface. In the genera studied, the content of 326 condensed polyphenols in the epidermis and hypoderm of the fetuses was different. In our 327 opinion, the established genera-specific microstructural and functional features of integumentary 328 tissues determine the different degree of determinism of passive immunity to mycosis within the 329 subfamily Maloideae. 330 The prior information on the correlation between microstructure characteristics of the 331 surface tissues, their functional state and damage by fungal pathogens obtained with the use of 332 electron microscopy methods can be useful and important in carrying out selection work on the 333 breeding of resistant varieties. These data are also of practical interest for assessing the 334 physiological and phytosanitary status of vegetating plants, as well as their production during 335 storage, for determining the rates and stages of development of pathogens. Timely monitoring of 336 the surface of plants with modern microscopic methods will help to identify the primary signs of 337 infection and determine the timing of preventive protective measures, as well as reduce the toxic 338 load and adverse effects on the environment by reducing the use of pesticides. 339 340 References 341 1. Assumpçãoa CF, Hermesa VS, Pagnoa C, Castagna A, Mannucci A, Sgherri C, Pinzino 342 C, Ranieri A, Flôres SH, Rios AO (2018) Phenolic enrichment in apple skin following 343 post-harvest fruit UV-B treatment. Postharvest Biology and Technology 138:37–45 344 https://doi.org/10.1016/j.postharvbio.2017.12.010 345 2. Baskakova VL (2017) Creation of varieties of quince for industrial gardening. Collection 346 of scientific works of GRBS 1:98–102 347 3. Budantsev AL. (2009) Vegetable resources of Russia: Wild flowering plants, their 348 component composition and biological activity. Families Actinidiaceae – Malvaceae, 349 Euforbiaceae – Haloragaceae SPb. and Moscow: Assoc. of Scien. Pub. KMK p 513 350 4. De Luca C, Passi S, Fabbri AA, Fanelli C (1995) Ergosterol oxidation may be considered 351 a signal for fungal growth and aflatoxin production in Aspergillus parasiticus. J Food 352 Addit. Contam. 12:445–50

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8 402 23. Stanimirović SG, Stanimirović D, Nastić DR (1963) Identivicaton des acides organiques 403 et saccharides dans le fruits et la feuille de Neflier (Mespilus germanica). Bull. Soc. 404 Chim. Beograd 28(5–6):327–335 405 24. Strasburger E. (2007) Botanica. Moscow: pub. «Academy» p 362. 406 25. Tsitsin NV (1962) Atlas of medicinal plants of the USSR. Medical literature p 702 407 26. Upadyshev MT (2008) The role of phenolic compounds in the processes of life of garden 408 plants. Moscow: Publishing House of SMEs p 318 409

9 410 Table 1. Fungal diseases of leaves and fruits and their agents in representatives of the subfamily 411 Apple (Maloideae)

Apple Pear Quince Medlar (Malus Mill.) (Pirus L.) (Cydonia Mill.) (Mespilus L.)

1a1, 2b, 4d, 5e, 1a2, 2b,4d, 5e, 6f1, 3c, 4d, 6f1, 8g4, 11i2, 4d, 7f2, 9g5, 10h3, 8g1,2,3, 10h1, 12j1, 8g3, 10h2, 11i1, 12j1, 14l3, 15m2, 16n, 17o1, 11i3, 12j2, 13k, 14l3, 14l1, 15m1, 16n, 14l2, 15m1, 16n, 22o6, 23p,24s 15m2, 16n, 18o2, 19o3, 21o5, 22o6, 23p, 24s 21o5, 22o6, 23p, 24s 20o4, 22o6, 23p, 24s 412 1 – Scab (a1 – Fusicladium dendriticum (Wallr.) Fuck., ascigerous stage of Venturia inaequalis 413 (Cooke) Wint, a2 – F. Pirinum Fuck., ascigerous stage V. pirina Aderh.); 2 – Olive mold (b – 414 Cladosporium herbarum Link., Alternaria tenius Nees.); 3 – Anthracnose (c – Cylindrosporium 415 cydoniae (Mont.) Schoschiaschwili (= GloeosporiumcydoniaeMont.)); 4 – Anthracnose, bitter rot 416 of fruit (d – Colletotrichum fructigenum (Berk.) Vassil. (= Gloeosporium fructigenum Berk.) 417 ascigerous stage – Glomerella cingulata (Ston.) Sp. Et Schr.); 5 – Sooty blotch (e – 418 Leptothyrium pomiSacc.); 6 – Brown spot (f1 – Entomosporium maculatumLév. f. maculate 419 Kleb. ascigerous stage Fabraea maculata (Lév.) Atk.); 7 – Dark brown spot (f2 – E. mespili 420 Sacc.); 8 – Phallosticosis, brown spot (g1 – Phyllosticta mali Pr.et Del.; g2 – P. Briardi Sacc; g3 – 421 P. Pirina Sacc; g4 – P. Cydoniae var.cydoniicola (Allesch.) Cif.); 9 – Phallostictosis, brown 422 spotting (g5 – P. Mespili Sacc.); 10 – Spotting (h1 – Hendersonia mali Trum, h2 – H. Piricola 423 Sacc, h3 – H. Mespili West.); 11 – Septoria, white spot (i1 – Septoria piricola Desm.; i2 – S. 424 Cydoniicola Trum; i3 – S. Mespili Sacc.); 12 – Ascochitis (j1 – Ascochyta piricola Sacc, j2 – A. 425 Mespili West.); 13 – Brown spot (k – Asteroma mespili Rob., Et Desm.); 14 – Rust (l1 – 426 Gymnosporangium juniperinum (L.) Mart. = G. Tremelloides Hartig, l2 – G. sabinae (Dicks) 427 Wint, l3 – G. confusumPlowr.); 15 – Powdery mildew (m1 – Podosphaera leucotricha Salm; m2 428 – P. oxyacanthae DB.); 16 – Gray rot of flowers and fruits (n – Botrytis cinerea Pers.); 17 – 429 Moniliosis, leaf spot (o1 – Monilia cydoniae Schell.); 18 – Light brown spot (o2 – M. Linhartiana 430 Sacc.); 19 – Yellow spot (o3 – M. foliicola Woronich.); 20 – Brown spot (o4 – M. Necans Ferr.); 431 21 – Monilial burn, moniliolysis (o5 – M. laxaHer. = M. cinereaBonord); 22 – Fruit rot, 432 moniliasis (o6 – Monilia fructigenaPers.); 23 – Black cancer (p – Sphaeropsis malorum Peck.), 433 24 – (s – Alternaria spp). 434

10 435 Figures legends

436 Fig. 1. The microrelief of the adaxial epidermis of the Apple subfamily (Maloideae) leaves: a – 437 Malus Mill.; b – Pyrus L.; c – Cydonia Miil.; d – Mespilus L. Arrows indicate the different 438 structure (height and location features) of the cuticular folds. 439 440 Fig. 2. Cuticular microstrands and peristomatic rings on the abaxial epidermis of the Apple 441 subfamily (Maloideae) leaves: a – Malus Mill.; b – Pyrus L.; c – Cydonia Miil.; d – Mespilus L. 442 Designation: s – stomata. Arrows indicate: cuticular microstrands (one arrow) and peristomatic 443 rings (two arrows). 444 445 Fig. 3. The surface of the abaxial epidermis of Apple subfamily (Maloideae) leaves with various 446 pathogenic fungi: a, b – Cydonia oblonga Mill.; c, d – Mespilus germanica L.; e, f – Malus Mill., 447 mycelium of the fungus in the stomatal gap. Arrows indicate hyphae of fungi. 448 449 Fig. 4. Fragments of the surface of the fruit of Apple subfamily (Maloideae) with the infectious 450 structures of fungi in SEM: a – conidia Fusicladium dendriticum (Wallr.) Fuck. In microcracks 451 of the cuticle Malus Mill.; b, c – conidia of F. pirinum Fuck in Pyrus L lentils; d – stomata with 452 mycelium on the surface of the fruit Cydonia Mill. Arrows indicate conidia of the fungus. 453 454 Fig. 5. Fragments of the surface of the fruit Malus Mill. with lentils in a confocal microscope: a – 455 general view; b – lenticular cavity with hyphae of the fungus in an enlarged form. Designation: 456 ln – lenticels. Arrows indicate the hyphae of the fungus. 457

458 Fig. 6. Sections of Apple subfamily fruits (Maloideae): a – Malus Mill.; b – Pyrus L.; c –

459 Cydonia Miil.; d – Mespilus L. (1) – control (not stained); (2) – painted with K2Cr2O7. Black 460 arrows indicate the cuticle on the surface of the epidermal cells, white – the cells with 461 polyphenols. Bar = 100 µm.

462 Fig. 7. The percentage of the area of cells containing condensed polyphenols from the area of the 463 entire outer layer of Apple subfamily fruits (Maloideae). The mean values of measurements of at 464 least 50 micrographs and their standard deviations are given. 465 466 Fig. 8. Fragments of cells of the outer layer of Apple subfamily fruits (Maloideae) with 467 polyphenols in TEM in the form of electron dense material (EDM): a – apple (Malus Mill.); b – 468 medlar (Mespilus L.). Arrows indicate EDM. 469

11 470 Fig.1

471 472

12 473 Fig. 2

474 475

13 476 Fig. 3

477 478

14 479 Fig. 4

480 481

15 482 Fig. 5

483 484

16 485 Fig. 6

486 487

17 488 Fig. 7

489 490

18 491 Fig. 8

492

19