Int.J.Curr.Microbiol.App.Sci (2018) Special Issue-6: 714-728 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Special Issue-6 pp. 714-728 Journal homepage: http://www.ijcmas.com Original Research Article Isolation and Identification of Plasmopara viticola Associated with Grapevine from Marathwada Region M. A. Mane1*, S. S. Bodke1 and R. N. Dhawale2 1Department of Botany and Horticulture, Yeshwant Mahavidyalaya, Nanded, Swami Ramanand Teerth Marathwada University, Nanded-431 606, Maharashtra, India 2Vilasrao Deshmukh College of Agricultural Biotechnology, Latur, Vasantrao Naik Marathwada Agricultural University, Parbhani-431 402 (MS), India *Corresponding author ABSTRACT Grape (Vitis vinifera) is species of vitis, belongs to the family Vitaceae. It is native to Mediterranean region, central Europe and south western Asia from Morocco and Portugal north to southern Germany and east to northern Iran. A grape is a fruit, botanically a berry, of the deciduous woody vines of the flowering plant genus Vitis. Grapes can be eaten fresh as table grapes or they can be used for production of wine, jam jellies, grape seed extract, K e yw or ds raisin, vinegar etc. Presently, in India it is most commonly known as „Draksha‟ in Marathi Plasmopara and „Angur‟ in Hindi. Powdery mildew gives an undesired, off-flavor to wine but it is not a concern for grape juice. Mainly fungal plant diseases are usually managed with applications viticola , of chemical fungicides or heavy metals. In some cases, conventional breeding has provided Marathwada, fungus resistant cultivars. Genetic engineering enables new ways of managing infections. In Grape, RAPD, this study 10bp oligonucleotide primers (Operon kit) were tested. DNA samples isolated Microscopy, OPF -18 from Uncinula necator of grape sample and amplification were repeated at least thrice on 1.5% agarose gel and only bands reproducible on several runs were considered for analysis. For confirmation the specific RAPD product originated from the Plasmopara viticola resistant region was identified to this region. Isolated DNA was used in PCR reaction for amplification with primer OPF-18 and OPF -19. The specific band of both primers was also found in Plasmopara viticola. Introduction Downey mildew/Plasmopara viticola is “Peronospora viticola”. Plasmopara viticola endemic on wild Vitis species of North (Order: Peronosporales, Family: America. It was first observed in Europe in Peronosporaceae) is a heterothallic (Wong, 1878. It was probably introduced into et al., 2000) diploid obligate Biotrophico Europe with American grape cuttings used omycete. The biology was studied in detail to replant the French vineyards destroyed by beginning in 1900 and it was immediately phylloxera. Its appearance in Europe was clear that the pathogen overwintered as not a real surprise, as several voices had oospore in the leaf or berries residues on the expressed concern (Cornu, 1872 cited in surface layer (Gregory, 1915). The Muller and Sleumer, 1934) about the danger incidence of downy mildew has increased in of the accidental introduction of recent years in Europe, requiring costly crop 714 Int.J.Curr.Microbiol.App.Sci (2018) Special Issue-6: 714-728 protection treatments that have negative With the development of tools allowing the effects on the environment and that may genetic characterization of single genotypes adversely affect the quality of any wine and identification of the genotype of a single produced pevines (Clark and Spencer- lesion, it became possible to determine Phillips, 2000). This has led to the formation whether an epidemic was indeed caused by of groups of viticulturalists, researchers and one or a few clones deriving from a few winery owners devoted to the establishment primary infections, as had been assumed. of a more sustainable and environmentally- RAPDs were the first type of markers to be friendly type of viticulture that reduces the used for this work. Although the power of use of such treatments to a minimum. Such a this type of marker is limited, RAPDs can be goal requires the use of strategies based on used to detect genetic differences between an in-depth knowledge of the biology of P. genotypes in a single comparative viticola and the environmental conditions experiment. However, since P. viticola is an that favor its growth. Additionally, obligate biotroph, doubts remain as to knowledge of the susceptibility of different whether other genetic material collected grapevine varieties to downy mildew is with the sporangia may have been the source essential to select those more tolerant or of the observed differences. In 1998, Kump resistant to the disease in a growing area. reported a high level of variability between batches of sporangia collected from The conditions under which P. viticola can individual leaves with single oil spots in a sporulate in susceptible tissue under vineyard in northern Switzerland. controlled conditions were analyzed in detail by Blaeser (Blaeser, 1978; Blaeser and Attributing a specific genotype and, Weltzien, 1978; 1979). Most simulation therefore, a unique oosporic origin to each models still rely on Blaeser's parameters, specific RAPD banding pattern, they including a minimum of 98% relative concluded that the ratio between primary humidity and 4 h of darkness, a minimal and secondary lesions was higher than temperature of 13°C and an optimal expected and recommended that the temperature of 19°C. quantitative role of oospores in epidemics of grape downy mildew be reconsidered Sporulation proceeds in darkness, but not in (Kump et al., 1998). the light, and is completed within 7 h. It is inhibited by irradiation with white light In the view of above constraint, the present (Rumbolz et al., 2002), near-UV light of study being proposed to take initiatives for 310–400 nm or green light (500–560 nm) at to develop isolation method for Plasmopara intensities >3–3.5 Wm-2 (Brook, 1979). The viticola from grape leaves and to identify the lifespan of the zoosporangia decreases as the isolated Plasmopara viticola by morphology water saturation deficit increases. The and molecular marker. zoosporangia are thought to be dispersed by rain splash, as they are detached by water. Materials and Methods So there is a need of rapid growth in agriculture sector not only for self-reliance, On the basis of symptoms and signs the but also to bring about equitable distribution Plasmopara viticola resistance and of income and wealth in rural areas as well susceptible leaves were collected from as to reduce poverty and improve the quality “Aurangabad, Osmanabad, Latur districts of of life (Davies, 2009). Marathwada region (Plate 1). 715 Int.J.Curr.Microbiol.App.Sci (2018) Special Issue-6: 714-728 Material collection Aseptic technique Plasmopara viticola infected leaves were In most microbiological procedures, it is collected from different locations of necessary to protect instruments, containers, Marathwada region. The same day as the lab media, and stock cultures from presentations on downy mildew of contamination by microorganisms grapevine, we were set up moist chambers constantly present in the environment. from which we were isolating Plasmopara viticola. Aseptic technique involves the sterilization of tools, glassware, and media before use as Preparation and procedure of petri plate well as measures to prevent subsequent moist chamber contamination by contact with non-sterile objects. Leaves or stem were made into small pieces and tape them onto a sterile slide. It is good Media preparation to leave a few pieces untreated because some pathogens are very sensitive to The media most commonly used are nutrient sterilization. Heavily damaged tissue was agar (bacteria), potato dextrose agar (fungi), not selected to avoid saprophytic organisms. and Sabouraud dextrose agar (fungi). Filter or blotting paper disk was placed on To prepare potato infusion, boil 200g sliced, the bottom and in the lid of petri dish and unpeeled potatoes in 1 liter distilled water moisten it thoroughly with sterile water. The for 30 min. slide was placed on the paper disk. The plate was closed and sealed with parafilm to hold The medium was filtered through cheese in moisture. cloth, saving effluent, which is potato infusion. Incubated at 18-220C under alternating cycles of light and darkness (10h light/14h Filtrate was mixed with dextrose, agar and darkness) (many fungus will not develop to water and boil to dissolve. the reproductive stage without this alternating light/darkness regimen). Final pH, 5.6 ± 0.2. Direct plating Autoclaved for 15 min at 121°C. Often it is most convenient to place fungal 20-25 ml of media dispensed into sterile 15 materials that are of interest directly on a × 100 mm petri dishes. nutrient agar medium, because it is widely used. It is a simple technique, requiring the Isolation and pure culture development placing of small bits of the leaf samples on the surface of the agar or the pouring of To obtain the pure culture of infecting melted but cooled agar over the fragments. microorganisms they should be cultured on suitable medium containing appropriate After a few days' incubation fungal growth amount of nutrients. For the development appear on the surface, and can be transferred fungal culture strains PDA i.e. Potato into pure culture. Dextrose Agar Medium is used. 716 Int.J.Curr.Microbiol.App.Sci (2018) Special Issue-6: 714-728 The infected leaf samples were cut into Lactophenol cotton blue technique for 3mm pieces with sterile razor blade, surface- fungus isolation sterilized in 1% hypochlorite solution for 2 minutes, then placed on Potato Dextrose Fungus is eukaryotic organisms and they are Agar (PDA) and incubated at room mainly classified into two main groups yeast temperature for 5 days. After incubation, and molds. Fungal structure includes colonies of different shape and colors were sporangiospores, mycelium, spores etc. The observed on the plates. A pure culture of lactophenol cotton blue wet mount is simply each colony type on each plate was obtained and widely used method for staining of and maintained (As per contamination fungus.