Keywords. Podosphaera leucotricha, scanning electron microscopy. Summary

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1 Scanning electron microscopy of apple powdery mildew (Podosphaera leucotricha, Ell and Ev.) fungi infecting susceptible Jonathan apple cultivar leaf mesophyll ZS. JAKAB-ILYEFALVI 1 1 Fruit Research and Development Station Bistrita,Romania 3, Drumul Dumitrei Nou Street, Bistrita,420127, Romania Phone : +40 (0 ) , zsolt.jakab@yahoo.com Keywords. Podosphaera leucotricha, scanning electron microscopy. Summary The aim of the study was to investigate the three dimensional (3D) structure through scanning electron microscopy of the hypha haustoria of apple powdery mildew (Podosphaera leucotricha, Ell and Ev.) fungi, the mode of infection and entering the apple leaf mesophyll in late autumn at the susceptible cultivar Jonathan. Results showed that the haustoria enters the leaf perpendicularly like a drill causing a whole in the leaf mesophyll, the conection part of the haustoria is thickened having a diameter at the base of μm. The haustoria are convoluted and folded in multiple ways having a vavy surface, they entered directly through the epidermis, the stomata were not affected. In late autumn the infected leaves were attacked massively. A group of sexual spores with ascospores were identified as a larger group of tight sacks with an average diameter of 2-3 μm being connected at the bottom of the haustoria. Several new conidiophores were identified also in the SEM images at secondary infections. Introduction Podosphaera leucotricha (Ell and Ev.) is an ascomycete fungi from the Erisiphaceae family and it is one of the most destructive pathogen agent of the apple orchards round over the world causing economic damages in susceptible cultivars by reducing tree vigor in nurseries and orchards infecting blossoms, young shoots and massively attacking the leaves thus reducing the photosynthetic activity and the fruit yield. Several researchers studied this fungus, Biggs et al. (2009) ; Hickey et al. (1990) ; Turechek (2004), Savulescu et al.(1965), Lazar et al., (1980) general observation was that apple powdery mildew represents a problem which appears yearly depending on climatic conditions and biological mycelium reserve. The conidia resulting from overwintered mycelium germinates in high air humidity (Ur=70 %) at C temperature. These conidia are producing hyphal outgrows called haustoria which penetrate the epidermis of leaves and absorb mineral and organic nutrients. These haustoria forms a colony which is infectious and absorbs all available nutrients from the vegetal tissue until plant leaf dries out and no longer lives. The aim of the study was to investigate the three dimensional structure through scanning electron microscopy (SEM) of the haustoria and the secondary infections mode in late autumn at the susceptible cultivar Jonathan. Materials and methods The experiment was established in September 2015 at an intensive apple orchard, planted with 1250 trees/ha with two apple cultivars Golden delicious and Jonathan. The study was focused on the apple powdery mildew susceptible cultivar Jonathan, there were sampled several shoots with infected leafs which were put in a plastic bag. The leaves were cut into appropriate samples and were 15

2 subjected to fixation. Prefixation was made with glutar-aldehyde 2,7 % in phosphate buffer 0,1M, ph 7,4 for 90 minutes, dehydration ( room temperature). The samples were put on brass supports with two carbon adhesive discs, metallization effectuated with gold (10nm layer) using an agar sputter coater. The examination of samples was made in high vacuum condition using a JEOL JSM 5510LV Scanning Electron Microscope. Results and discussions First observation on the scanning electron microscopy (SEM) images of the infection method of apple powdery mildew (Podosphaera leucotricha Ell.and Ev) is that the fungus penetrates deeply the mesophyll of the leaves through the haustoria. In late autumn a massive recurrent attack occurred in a plot planted with Jonathan cultivar. (Fig.1). Fig. 2. Leaf surface with infecting apple mildew haustoria. When haustoria and mycelium is well developed and the disease is in its massive virulence form, hyphal haustoria are folded in bunches (Fig.3). Fig. 3. Bunches of apple powdery mildew hyphal outgrows. Fig.1. Apple powdery mildew hyphal haustoria massively attacking Jonathan apple cultivar leaf mesophyll. In the disease epidemics the late mycelium and haustoria attack will provide elements for the life cycle of the fungi in the next year. On the outer leaf surface on the top it is visible a great amount of haustoria (Fig 2). Outer surface of the haustoria is rough and wavy with several protuberancies (Fig.4). The outside membrane of the haustoria is not even, it has a wavy surface. The haustoria are folded in many patches forming a complex web which almost completely covers the leaf. 16

3 Fig. 4. Outer surface of the Podosphaera leucotricha haustoria. Haustoria are penetrating the leaf as a drilling machine resulting at the side parts pieces of mesophyll which are folded at the bottom of the haustoria (Fig.5). Fig. 6. Haustoria entering in the mesophyll. In a close up detail it can be observed that the haustoria is causing a whole (Fig.6.) in the leaf mesophyll through which enters in the lower deeper level under the upper epidermis, probable between the palisade mesophyll cells and spongy mesophyll area or near the xylem and phloem. Further investigations will be made in order to elucidate this fact. What is until now clear and observable that the leaf mesophyll haustoria deeply penetrates the upper epidermis (Fig.7). Fig. 5. Penetration of the Podosphaera leucotricha haustoria in the leaves. The convuluted haustoria deeply penetrate the leaf mesophyll in many points infecting the entire leaf surface (Fig.6). Fig. 7. Deep penetration of the upper epidermis. The haustoria are convoluted and folded in multiple way. They entered perpendicularly the leaf from the top (Fig.7). There are visible several conidiphores formed, preparing new primary infections for the next year (Fig.8- bottom center). 17

4 Fig. 8. Apple leaf with obvious stomata and at the bottom conidiophores with attached conidia. In the SEM images there are visible several stomata with a length of 20 μm, they are not penetrated with haustoria ( Fig. 8). The entering zone of the haustoria, the hyphal part is thickened, being like a connection tube to the mesophyll (Fig.9). Fig. 9 Conection of the apple powdery mildew haustoria to the leaf. A group of sexual spores were also observed (ascospores) being evident like groups of sacks being tight attached to the leaf surface. The average diameter of the sacks were 2-3 μm. Fig. 10. Groups of ascospores being attached to the leaf surface. Conclusions Hyphal outgrows and haustoria are the specialized organs through which Podosphaera leucotricha massively attacks plant leaf. In our study we documented the three dimensional form of these structures presenting the mode of entering and penetration of the leaf and the complex net system formed on the outer surface of the leaf. The plant protection products (organic or chemical) should prevent or limit these haustoria in order to control or eliminate the attachment and penetration in the leaf. Results are important in order to understand and see how these specialized organs enter and infect the leaf mesophyll and how it is formed the mycelium which colonizes the complete surface of leaves and provides elements of the future infections thus the multiplication of the fungus. References Biggs, A. R., Yoder, K. S., and Rosenberger, D. A.: Relative susceptibility of selected apple cultivars to powdery mildew caused by Podosphaera leucotricha. Online. Plant Health Progress doi: /php rs, 2009 Hickey, K. D. and K. S. Yoder. : Apple powdery mildew. Pages 9-10 in A. L. Jones and H. S. Aldwinckle, ed. Compendium of Apple and Pear Diseases. American Phytopathological Society, St. Paul, MN.,

5 Savulescu, Olga, Eugenia Eliade, Victoria Sevcenco, Elena Popa Margaritescu, Lucrari practice de fitopatologie,p , 1965 Lazar,Al, Hatman, M., Bobes,I.,Perju,T., Sapunaru, T., Goian,M., Protectia plantelor, Editura pedagogica, Buuresti, p. 298, 1981 Turechek, W. W., J. E. Carroll, and D. A. Rosenberger. : Powdery mildew of apple. Tree Fruit Factsheet, Cornell University,