- Email: [email protected]
An experimental system for characterizing isolates of Uncinula necator
675
Mycol. Res. 100 (6):675-680 (1996) Printed in Great Britain
An experimental system for characterizing isolates of Uncinula necafor
K. J. EVANS1,', D. L. WHISSONlr3A N D E. S. Cooperative Research Centre for Viticulture, PO BOX 154, Glen Osmond, South Australia 5064, Australia Department of Crop Protection, The University of Adelaide South Australian Research and Development Institute
Clonal lines of Uncinula necator were established using conidia from diseased Vitis vinifera leaves or berries collected from various Australian viticultural regions. Techniques for the establishment of single-conidial-chain isolates of U. necator on detached leaves and the subsequent maintenance of clonal lines on micropropagated grapevines in vitro are described. Conidia were successfully mass produced by the detached leaf technique and harvested efficiently using a cyclone separator device. Conidial yields were quantified for 14 clonal lines and ranged from 42 to 112 mg per 20 leaves at the first harvest. Nucleic acid extraction from conidia resulted in high-quality DNA suitable for restriction enzyme digestion and amplification by PCR, with yields ranging from 6 to 9 ng per mg conidia. This is the first report of a DNA extraction procedure for conidia of U. necator. Using the 18 base plant intron splice junction (ISJ) primer, RI, genetic variation among five South Australian clonal lines of U. necator was identified. Three of these clonal lines originated from vines grown within a 0.5 krn radius. This preliminary identification of genetic variation in U. necator and the system for handling different clonal lines provide the essential tools for further development of DNA markers and the molecular characterization of this economically important pathogen.
Powdery mildew of grapevine, caused by Uncinula necator (Schwein.) Burrill, is a chronic yield and quality loss problem in all viticultural regions of the world. Increased world-wide emphasis on the production of disease-free berries with minimal pesticide input provides a sound reason for exploring more efficient disease management strategies through a better understanding of U. necafor epidemiojogy and population biology. Little is known about the population structure of U. necator mainly because this haploid fungus has been difficult to study genetically. The obligately biotrophic nature and lack of morphological, physiological, biochemical and genetic markers contribute to the limited knowledge of U. necator population genetics. The genetic interaction of U. necafor with economically important Vitis spp. has not yet been characterized. However, the Blumeria (Eysiphe) graminis-Hordeum vulgare pathosystem has well-defined virulence and resistance genes plus a range of molecular markers which have been used to track the movement of genotypes in Europe for many years (Wolfe & McDermott, 1994). U. necator, in common with B. graminis, is a member of the Erysiphales (powdery mildews) and reproduces both asexually and sexually. Despite the difference in host biology, the similarities in these fungi make B. graminis-H. vulgare a useful model for studying genetic interactions and population biology of U. necator. The reproductive strategy of U. necator ensures the appearance of new genotypes and their potential rapid proliferation. Bipolar heterothallism (two mating types) was characterized relatively recently among isolates from New
York State (Gadoury & Pearson, 1991).In addition, ascospores appear to be the primary inoculum for powdery mildew epidemics in New York State vineyards (Pearson & Gadoury, 1987). The sexual stage was first recorded in Australia in 1984 (Wicks, Magarey & Emmett, 1985), whereas it has been known in North America since 1834 (Kapoor, 1967) and since 1893 in Europe (Bulit & Lafon, 1978). While a functional sexual stage creates significant opportunity for variation, nothing is known about the impact of sexual reproduction on the genetic diversity of U. necator. Population biology studies of this fungus will (a) determine the relative importance of the sexual stage in Australia and other regions, and (b) reveal the impact of disease management strategies, for example fungicide applications, on population dynamics. In order to characterize a U. necator population and to understand the genetic bases for variation, molecular-genetic and phenotypic markers must be developed for use as research tools. DNA techniques can provide abundant and selectively neutral genetic markers which can complement existing phenotypic markers (Michelmore & Hulbert, 1987). Here we describe an experimental system for manipulating different clonal lines of U. necator and a rapid, high-yielding DNA extraction procedure which results in high-quality DNA that can be restriction enzyme digested or amplified by the polymerase chain reaction (PCR). In addition, we identify genetic variation among clonal lines of U. necafor as a first step in the development of DNA markers for studying the population genetics of t h s economically important pathogen.
Characterization of Uncinula necator isolates
676
MATERIALS A N D M E T H O D S Isolation of clonal lines of Uncinula necator
Diseased leaves or berries were collected from various Australian vineyards in non-phylloxera infested regions and transported directly to the laboratory within 5 d of field sampling for the isolation of clonal lines of U . nerator (Table I). Each disease sample came from a single block or row of vines, usually from several adjacent panels of vines. A detached leaf technique was developed for the isolation of clonal lines of U. necator. Leaves were collected from glasshouse grown Vitis vinifera cv. Cabemet Sauvignon, clone G9V3. Grapevines were propagated from hardwood cuttings obtained from Registered Source Areas (Riverland Vine Improvement Committee, Barmera, South Australia) and maintained free from powdery mildew using vapours of penconazole (Szkolnik, 1983).Cuttings with roots and dormant buds were planted into 20 cm diam. pots containing U.C. potting mixture (Baker, 1957). After 6 wk, cuttings with shoots were fertilized fortnightly with a complete watersoluble fertilizer (1.6 g 1-' Aquasol@,Hortico, Laverton North, Victoria (NPK 23:4:18)). During the autumn and winter, active shoot growth was maintained by providing a photoperiod from 00.00 to 01.00 h, using 60 W incandescent bulbs 40 cm apart above the plants (K. Skene, pers. comrn.). For detached leaf culture, leaves were selected to fit into a Petri plate (10 cm diam., 2 cm deep) containing 20 ml of I 0 g I-I distilled water agar (Bitek, Difco Laboratories, Michigan). Four sterile toothpicks were placed parallel to each other at 1.5 cm intervals on the agar surface. Leaves were washed in distilled water and a fresh cut was made to the basal end of the petiole before surface sterilization in 0-5 g 1-I sodium hypochlorite for 3 min. The leaves were then rinsed three times in sterile distilled water and placed on the bed of toothpicks with the basal 2-4 mm of the petioie immersed in the agar. The leaves were placed within a laminar-flow hood until the upper surface was completely dry. The bed of
toothpicks raised the leaf slightly above the agar surface, providing a dry leaf surface for colonization by U. necator. A sterile artist's paint brush was used to brush conidia, from diseased leaves or berries, onto the detached leaf cultures described above. At least three detached leaf cultures were prepared to sample conidia randomly from the bulked disease material from each location. The lid of the Petri plate was replaced and the plate sealed with [email protected] plates were incubated at a slight angle (approx. 10') in an illuminated growth chamber (330 VE s-' m-2 from cool white fluorescent bulbs) with a 12 h photoperiod at 25 OC. Individual chains of conidia, which were free from visible contamination by other microbes, were transferred from 10-12-d-old colonies to healthy detached leaf cultures using the method described by Gadoury & Pearson (1991). A single artist's paintbrush hair attached to a dissecting needle with petroleum jelly was used to transfer individual chains of conidia. Six individual chains of conidia were selected at random from colonies growing on the detached leaves and transferred to a healthy detached leaf. This procedure was repeated at least twice. The resulting IC-12-d-old colonies, which were free from visible microbial contamination, were transferred individually, using a sterile artist's paint brush, either to a healthy detached leaf culture or directly to a micropropagated grapevine for culture maintenance in vitro. Conidia of isolates maintained on detached leaf cultures were transferred to healthy detached leaf cultures every 14 d. Clonal lines isolated during the 199213 growing season were maintained as detached leaf cultures for 2-5 months prior to being transferred to micropropagated grapevines because plantlets were not established in vifro at that time. There is no evidence of genetic change in clonal lines during maintenance (unpublished data). Culture maintenance and mass production and collection of conidia
Clonal U. necafor lines were maintained in vitro on V . vinifera cv. Cabemet Sauvignon, clone CW44. Micropropagated
Table 1. Origin of clonal lines of U. necator
Clonal llneb
Host cv. and organ
Locat~on
Date of collect~on of diseased matenal
9403 9404 9405 9407
Chenin Blanc leaves Chardonnay berries White Grenache berries Crouchon leaves V. amurensis leaves Sauvignon Blanc berries Chardonnay berries chardonnay berries Chardonnay berries Sauvignon Blanc berries Perlette berries CG 4320r berries Traminer berries Chardonnay berries
Waite Campus, Adelaide Plains, SA Nuriootpa, Barossa Valley, SA Nuriootpa, Barossa Valley, SA Nuriootpa, Barossa Valley, SA Home garden, Adelaide Plains, SA Morialta, Adelaide Hills, SA Block I, Summertown, Adelaide Hills, SA Block 2, Summertown, Adelaide Hills, SA Block 3, Summertown, Adelaide Hills, SA Yallingup. Margaret River, WA Swan Research Station, Swan Valley, WA Swan Research Station, Swan Valley, WA Lindemans, Coonawarra, SA Home garden, Launceston, Tas.
Nov. 1992 Jan. 1993 Jan. 1993 Jan. 1993 Jan. 1993 Jan. 1993 Feb. 1993 Feb. 1993 Feb. 1993 Dec. 1993 Jan. 1994 Jan. 1994 Jan. 1994 Mar. 1994
Sourcea
-
All clonal lines except 9308 were obtained from Vitis uinifera. SA, South Australia; WA, Western Australia; Tas., Tasmania. Clonal lines are coded in chronological order of collection. " Accession No. IC758274.
a
K. J. Evans, D. L. Whisson and E. S. Scott
Fig. 1. Spomlating colonies of U.necator on a detached grapevine leaf on water agar in a 10 cm diam. Petri plate, 12 d after inoculation.
shoots were kindly provided by Dr M. Barlass. Shoots were cut into nodal segments and five segments placed upright into 50 ml of maintenance medium in 250 ml polycarbonate culture tubes with polypropylene breather lids (Disposable Products, South Australia). The maintenance medium consisted of MS medium (Murashige & Skoog, 1962) with all nutrients at half strength, 15 g I-' sucrose, 0.01 mg I-' anaphthaleneacetic acid (NAA) and 7 g 1-' agar (Bitek, Difco Laboratories, Michigan). The pH of the medium was adjusted to 5.8 before the addition of the agar and autoclaving. Cultures were maintained at 25' with a 16 h photoperiod (250-450 PE s-' mP2). After 10 d, most nodal segments had developed small roots and one segment per tube was transferred to 70 ml of the same maintenance medium, minus NAA, in a 500 ml culture tube with a breather lid. After approximately 21 d, the plantlet, which had developed at least five expanded leaves, was inoculated with U. necafor conidia. The four plantlets remaining in the 250 ml culture tube were cut into nodal segments approximately 31 d after the previous transfer and placed on fresh maintenance medium containing NAA. This culture cycle was repeated each month to provide a continuous supply of plantlets for dual culture with U. necator. Every 8-10 wk conidia were transferred aseptically to healthy in vifro plantlets by removing an infected leaf and brushing the conidia against a leaf of the plantlet being inoculated (Gadoury & Pearson, 1988). Conidia were mass produced by inoculating detached leaf cultures with aseptic conidia from infected micropropagated leaves in the same manner (Fig. 1). Following inoculation, conidia were harvested twice, after 12 and 16 d respectively, using a cyclone separator (Fig. 2) connected to a vacuum pump (Tervet et al., 1951; H. Wallwork, pers. comm.). The conidia were harvested inside a perspex hood that had been wiped with 70% ethanol to minimize contamination of the cultures and prevent cross-contamination between isolates.
Fig. 2. The cyclone separator device adapted for the collection of conidia into a 1-5 ml microfuge tube. A, the air intake is designed to
prevent host tissue bloclang the opening; B, air is drawn by a vacuum pump connected with tubing (not illustrated);C, conidia are separated from the air by a central tube extending below the point at which air enters the cyclone separator; D, the microfuge tube (length, 4 cm) is attached to the device with a piece of tubing (not illustrated) that encases both the microfuge tube and the arm of the device. The cyclone separator was rinsed with 70% ethanol and allowed to dry before each harvest. Conidia were harvested from sporulating colonies that were free from microbial contamination when examined at 50 x magnification. Occasional contamination of the water agar by fungi was reduced by incorporation of 10 mg I-' pimaricin. Conidia were collected directly into a 1.5 ml microfuge tube, frozen in liquid nitrogen, and stored at -80'. Conidia harvested on different days were stored separately.
DNA extraction from conidia The DNA extraction method was modified from that of Whisson (1996) for Blumeria (Eysiphe) graminis conidia and from that of Matthew, Herdina & Whisson (1995) for Rhiwctonia solani mycelia. A 700-900 ~1 volume of extraction buffer (150 mM sodium acetate, 20 mM EDTA, 3 % sarkosyl, pH 5.2, containing 2 mg ml-' predigested pronase) was added to 30-40 mg frozen conidia. The conidia were suspended,
Characterization of Uncinula necafor isolates pummelled with glass beads and incubated according to the method of Whisson (1996). The nucleic acids were separated from most of the other cell components by phenol-chloroform extraction. The volume of the final aqueous phase was increased to 500 yl with a solution containing 50 mM Tris-HC1, 20 mM EDTA, pH 8, and the nucleic acids were precipitated by the addition of 0.4 volumes (200 d) of 4 M ammonium acetate, pH 5.2, and 0.6 volumes (300 PI) of cold isopropanol and placing the tube on ice for 2 h. The nucleic acid pellet, obtained after centrifugation at 15 800 g (Eppendorf centrifuge) for 20 min, was washed briefly with cold 70% ethanol containing 10 mM magnesium acetate, and dissolved in 50 yl of TE b d e r (10 mM Tris-HC1, 1mM EDTA, pH 8).RNaseA was added to a final concentration of 50 pg ml-' and incubated at 37' for 30 min. The volume of the solution was then increased to 150 y1 with TE buffer and extracted with chloroform/isoamyl alcohol (24: 1).DNA was precipitated from the aqueous phase by the addition of two volumes of cold ethanol (99-7-100% v/v), overnight at - 20'. The solution was centrifuged as before for 20 rnin and the DNA pellet washed with cold 70% ethanol, 10 mM magnesium acetate for 30 min, pelleted again, and dissolved in 20 y1 TE buffer. The amount of DNA in each sample was estimated by running aliquots on a 1% agarose gel and visualizing the bands under uv light following ethidium bromide staining. The intensity of the staining of U. necafor bands was compared with that of known quantities of Rhiwcfonia solani DNA and Hind I11 digested lambda DNA.
Amplification of D N A using the polymerase chain reaction
kbp
M
I
2
3
Fig. 3. U. necator DNA (approximately 100 ng lane-') digested with EcoR V, Lane M, 0.5 ~g lambda DNA digested with Hind 111; lane 1, line 9201 (Adelaide Plains); lane 2, line 9302 (Barossa Valley); lane 3, line 9313 (Adelaide Hills).
Amplification of U. necafor DNA was performed in a FTS-IS Capillary Fast Thermal Sequencer (Corbett Research, Sydney). The reaction was carried out in a 20 yl volume containing colony. When these colonies were free from visible microbial 1-3 ng DNA template, SO ng plant intron splice junction (ISJ) contamination, conidia were transferred to micropropagated primer, Rl (5'GTCCATTCAGTCGGTGCT3', Weining & grapevines in vifro, with greater than 99% of inoculations Langridge, 1991), 0.2 mM each of dATP, dCTP, dGTP and producing an infection without microbial contamination. dTTP, 2.5 mM MgCl,, 50 mM KCI, 10 mM Tris-HC1 (pH 8.8), The yield of conidia from 20 detached leaf cultures was 0.1% Triton X-100 and 1.5 units Taq DNA polymerase quantified for each of the 14 clonal lines of U. necafor. The (Promega).All reactions consisted of: (i) one cycle of 3 rnin at mean yield (n = 14) for the first harvest, at 12 d, was 70 mg 94', 30 s at 40' and 90 s at 72'. (ii) four cycles of 15 s at 95', conidia per 20 leaves (s.D. = 23.2), with a range of 41-112. 15 s at 40' and 90 s at 7Z0, (iii) forty cycles of 5 s at 95', 10 s The mean yield for the second harvest, at 16 d, was 19 mg per at 58' and 90 s at 72', and (iv) a final extension step of 5 rnin 20 leaves (s.D.= 11.7), with a range of 8-39 mg. Variation in at 72'. A ramp rate of 0.25' s-' was used between the conidial yields appeared to be due to clonal line fitness and/or annealing and primer extension temperatures. PCR products the health of the detached leaf. Conidia could be harvested a were analysed on 2% agarose gels and visualized under uv third time, up to 28 d after inoculation, but leaf senescence light following ethidium bromide staining. The size marker reduced the quantity of conidia collected to less than 10 mg used was pGEM@(Promega). per 20 leaves. The DNA extraction procedure resulted in high molecular weight DNA suitable for restriction enzyme digestion, and RESULTS amplification by PCR. DNA yields ranged from 170 to 280 ng Fourteen clonal lines were selected for this study (Table 1). per 30 mg of conidia. Figure 3 illustrates the restriction Diseased material was collected from non-phylloxera infected enzyme digestion of total U. necator DNA with EcoR V. At areas of New South Wales but the conidia were not viable least 16 distinct fragments are evident as brightly stained after transportation by air to South Australia. During clonal bands against the background smear of DNA. Clonal line isolation, at least one out of every six single-conidial chains 9313 (lane 3) has a 9.4 kbp fragment which appears to be placed individually on a detached leaf produced a sporulating absent from lines 9201 and 9302 (lanes 1 and 2, respectively).
K. J. Evans, D. L. Whisson and E. S. Scott Petri plates (Quim & Powell, 1981; Pearson & Gadoury, 1987; Gadoury & Pearson, 1991). The detached leaf technique is equaIly useful for this purpose but has the advantage of providing a sterilized leaf surface for the production of aseptic conidia. Aseptic conidia are required for (a) inoculation of micropropagated plantlets for maintenance of isolates, and (b) the mass production of conidia for DNA extraction and amplification. Using whole, surface sterilized leaves, we found it unnecessary to add benzimidazole to delay leaf senescence, in contrast to dual culture of B. graminis on barley leaf pieces (McDermott et al., 1994) and U. necator on grapevine leaf discs (Steva, 1994). The production of conidia using detached leaf cultures and the cyclone separator collection device allow conidia to be harvested from many different clonal lines. Conidia can be harvested from dual cultures on micropropagated plantlets, but manipulations are more time-consuming compared with dual culture on detached leaves. The detached leaves provide a large surface area for sporulation whereas conidia need to be collected from many small leaves of the in vifro plantlets to achieve the same yield (data not shown). However, microFig. 4. Amplification of U.necafor DNA with the ISJ primer, RI. The propagated plantlets with sporulating U. necator colonies are PCR products were fractionated on a 2 % agarose gel. Lane M, DNA the best source of aseptic inoculum for both culture size marker, [email protected] sizes are shown on the left. Lane I, a maintenance and for bulking conidia on detached leaves. In control where no genomic DNA was added to the reaction mixture; addition, dual cultures on plantlets can be maintained for up lane 2, line 9201 (Adelaide Plains); lane 3, line 9313 (Adelaide Hills); to 6 months before the plantlets senesce. We transferred lane 4, line 9310 (Adelaide Hills); lane 5, line 9312 (Adelaide Hills) conidia to healthy plantlets every 8-10 wk in order to lane 6, line 9301 (Barossa Valley). maintain vigorous dual cultures. Molecular characterization of U. necator required the development of a rapid DNA extraction method. This is the U. necafor DNA could be digested by a range of restriction first report of a DNA extraction procedure for conidia of U. endonucleases including EcoR I, BamH I, Psf I, SUM3A and necator. The typically small yields of aseptic conidia per clonal Taq I (data not shown). U. necator and the potential for population studies line of Figure 4 illustrates the amplification of U. necator DNA with involving many samples necessitated the development of an the ISJ primer, R1. There are two types of banding pattems efficient technique. Our DNA yield, which is in the range which identify genetic variation among these clonal lines of U. mg-' of conidia, is similar to that reported by O'Dell 6-9 ng necator from South Australian vineyards. The banding pattems ef al. (1989) for B. grarninis conidia. Using a large quantity, of lines 9201 and 9310 (lanes 2 and 4) differ from those of 20 g, of conidia they extracted approximately 5 ng mg-' of lines 9313, 9312 and 9301 (lanes 3,5 and 6, respectively). conidia. Furthermore, our acidic extraction buffer is simple and Lines 9310, 9313 and 9312 originate from diseased berries sampled from different blocks of Chardonnay vines within a preliminary experiments have revealed that the pH of 5.2 produced DNA which was less degraded than that obtained 0.5 km radius in an Adelaide Hills vineyard. using an extraction buffer of pH 8. Nucleic acid extraction from 30 mg of conidia yielded enough DNA for at least DISCUSSION 150 PCR reactions. In addition, the high quality of the U. We describe an experimental system for the clonal isolation of necafor DNA makes it useful for applications requiring U. necafor, the mass production and collection of conidia, and restriction enzyme digested DNA, for example ligation subsequent DNA extraction and amplification. Detached leaf reactions. U. necafor DNA was digested by a wide range of culture is a standard technique for the cereal powdery mildew restriction endonucleases and the distinct fragments evident pathogen Blumeria (Eysiphe)grarninis (Yarwood, 1946; Wolfe, after digestion with EcoR V (Fig. 3) may indicate the existence 1965; Jorgensen, 1988; McDermott ef al., 1994), but has not of repeated DNA and/or mitochondria1 DNA. Using the 18 base ISJ primer, Rl, we identified genetic been described previously for the mass production of conidia of U. necator. Grapevines, being perennial, are propagated by variation among South Australian clonal lines of U. necator. RI cuttings. Leaves must be collected from young shoots which is based on the consensus sequences for the intron-exon splice are pruned to maintain active growth. Compared with barley junctions of plants and generates products from the exon plants, more grapevines are required to obtain the same leaf region (5' splice site, exon targeting, Weining & Langridge, surface area. Grapevines with active shoot gowth can be 1991). This primer has also been used to amplify DNA from maintained in the glasshouse for up to 1yr provided they are Gaeumannomyces graminis (Harvey, 1993) and Rhiwcfonia pruned carefully. The establishment of single conidial chain solani (Duncan, Barton & O'Brien, 1993; Whisson, Herdina & colonies can be achieved by using detached leaves in double Francis, 1995). The amplification of U. necafor DNA using RI kbp
M
1
2
3
4
5
6
Characterization of Uncinula necator isolates as a primer indicates that R1 has some homology with U. necator DNA and implies the existence of conserved sequences at the junctions to plant exons. The different banding patterns among the five clonal lines examined demonstrates genetic variation among these isolates from different grape growing regions in South Australia. Most interestingly, variation was identified between clonal lines originating from the same vineyard, giving a preliminary indication of fine-scale variation in this fungus. More isolates need to be characterized within and between vineyards and regions, and with a range of molecular markers, in order to determine population structures and population dynamics (e.g. gene flow, reproductive mode and selective pressures). Thus, it will be worthwhile to develop other DNA markers, including other PCR primers and DNA probes. We are currently screening cloned U . necator sequences to identify U. necator specific DNA probes that show polymorphisms between clonal lines. Markers could then be developed to characterize populations of U . necator using total DNA from infected grapevine tissue. We thank Dr M. Barlass for supplying V. vinifera cultures and advice on micropropagation, Dr R. Cirami for supplying the first batch of V. vinifera hardwood cuttings, Mr P. Wood, Mr B. Wagner, Mr T. Somers, Ms B. Hall and Dr T. Wicks for supplying diseased grapevine material, Dr H. Wallwork for supplying the basic design for the cyclone separator device, Mr R. Balali for supplying Rkizoctonia solani DNA and Dr P. Langridge for supplying the ISJ primer, RI. We also thank Professor R. Symons (Programme Leader) for helpful discussions and comments on the manuscript.
REFERENCES Baker, K. F. (1957). The U.C. System for Producing Healthy Container Grown Plants. Manual 23, California Agricultural Experimental Station Extension Service: California. Bulit, J. & Lafon, R. (1978). Powdery mildew of the vine. In 7'he Powdery Mildews (ed. D. M . Spencer), pp. 525-548. Academic Press: London. Duncan. S., Barton, 1. E. & O'Brien, P. A. (1993). Analysis of variation in isolates of Rhizoctonia solani by random amplified polymorphic DNA assay. Mycological Research 97, 1075-1082. Gadoury, D. M. & Pearson, R. C. (1988). Initiation, development, dispersal, and survival of cleistothecia of Uncinula necator in New York vineyards. Phytopathology 78, 1413-1421. (Accepted 13 November 1995)
680 Gadoury, D. M. & Pearson, R. C. (1991). Heterothallism and pathogenic specialization in Uncinula necator. Phytopathology 81, 1287-1293. Harvey, P. (1993). Genetic diversity among populations of the take-all fungus Gaeumannomyces graminis. PhD Thesis, University of Adelaide. mildew of cereals and Jorgensen, J. H. (1988). Eysiphe graminis, grasses. Advances in Plant Pathology 6, 137-157. Kapoor, 1. N. (1967). Uncinula necator. In C M I Descriptions of Pathogenic Fungi and Bacteria. No. 160. Commonwealth Mycological Institute, The Eastern Press Ltd: London. Matthew, J., Herdina & Whisson D. (1995). DNA probe specific to Rhiwctonia solani anastomosis group 8. Mycological Research 99, 745-750. McDermott, J. M., Briindle, U., Dutly, F., Haemmerli, U. A,, Keller, S., Miiller, K. E. & Wolfe, M. S. (1994). Genetic variation in powdery mildew of barley: development of RAPD, SCAR, and VNTR markers. Phytopathology 84, 1316-1321. Michelmore, R. W. & Hulbert, S. H. (1987). Molecular markers for genetic analysis of phytopathogenic fungi. Annual Review of Phytopathology 25, 383-404. Murashige, T. & Skoog, K. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15, 473-497. O'Dell, M., Wolfe, M. S., Flavell, R. B., Simpson, C. G. & Summers, R. W. (1989). Molecular variation in populations of Eysiphe graminis on barley, oats and rye. Plant Pathology 38, 340-351. Pearson, R. C. & Gadoury, D. M. (1987). Cleistothecia, the source of primary inoculum for grape powdery mildew in New York. Phytopathology 77, 1509-15 14. Quinn, J. A. & Powell, C. C., Jr. (1981). Identification and host range of powdery mildew of begonia. Plant Disease 65, 68-70. Steva, H. (1994). Evaluating anti-resistance strategies for control of Uncinula necator. In Fungicide Resistance (ed. S. Heany, D. Slawson, D. W. Hollomon, M. Smith, P. E. Russell & D. W. Parry), pp. 59-66. British Crop Protection Council Monograph No. 60: Surrey, U.K. Szkolnik, M. (1983). Unique vapor activity by CGA-64251 (Vangard) in the control of powdery mildews roomwide in the greenhouse. Plant Disease 67, 36G366. Tervet, I. W., Rawson, A. J., Cheny, E. & Saxon, R. B. (1951). A method for the collection of microscopic particles. Phytopathology 41, 282-285. Weining, S. & Langridge, P. (1991). Identification and mapping of polymorphisms in cereals based on the polymerase chain reaction. Theoretical and Applied Genetics 82, 209-216. Whisson. D. L. (1996). Molecular characterization and pathogenicity of Erysiphe graminis f. s p hordei in Australia. Australasian Plant Pathology (in press). Whisson D. L., Herdina, & Francis, L. (1995). Detection of Rhizoctonia sohni AG-8 in soil using a specific DNA probe. Mycological Research 99, 1299-1302. Wicks, T. J., Magarey, P. & Emmett, R. W. (1985). First report of Uncinula necator cleistothecia on grapevines in Australia. Plant Disease 69, 727. Wolfe, M. S. (1965). Physiologic specialization of £.graminis fritici in the U.K. Transactions of the British Mycological Society 48, 315-326. Wolfe, M. S. & McDermott, J. M. (1994). Population genetics of plant pathogen interactions: the example of the Erysiphe gramini~Hordeum vulgare pathosystem. Annual Review of Phytopathology 32, 89-113. Yarwood, C. E. (1946). Detached leaf culture. Botanical Review 12, 1-56.
Mycol. Res. 100 (6):675-680 (1996) Printed in Great Britain
An experimental system for characterizing isolates of Uncinula necafor
K. J. EVANS1,', D. L. WHISSONlr3A N D E. S. Cooperative Research Centre for Viticulture, PO BOX 154, Glen Osmond, South Australia 5064, Australia Department of Crop Protection, The University of Adelaide South Australian Research and Development Institute
Clonal lines of Uncinula necator were established using conidia from diseased Vitis vinifera leaves or berries collected from various Australian viticultural regions. Techniques for the establishment of single-conidial-chain isolates of U. necator on detached leaves and the subsequent maintenance of clonal lines on micropropagated grapevines in vitro are described. Conidia were successfully mass produced by the detached leaf technique and harvested efficiently using a cyclone separator device. Conidial yields were quantified for 14 clonal lines and ranged from 42 to 112 mg per 20 leaves at the first harvest. Nucleic acid extraction from conidia resulted in high-quality DNA suitable for restriction enzyme digestion and amplification by PCR, with yields ranging from 6 to 9 ng per mg conidia. This is the first report of a DNA extraction procedure for conidia of U. necator. Using the 18 base plant intron splice junction (ISJ) primer, RI, genetic variation among five South Australian clonal lines of U. necator was identified. Three of these clonal lines originated from vines grown within a 0.5 krn radius. This preliminary identification of genetic variation in U. necator and the system for handling different clonal lines provide the essential tools for further development of DNA markers and the molecular characterization of this economically important pathogen.
Powdery mildew of grapevine, caused by Uncinula necator (Schwein.) Burrill, is a chronic yield and quality loss problem in all viticultural regions of the world. Increased world-wide emphasis on the production of disease-free berries with minimal pesticide input provides a sound reason for exploring more efficient disease management strategies through a better understanding of U. necafor epidemiojogy and population biology. Little is known about the population structure of U. necator mainly because this haploid fungus has been difficult to study genetically. The obligately biotrophic nature and lack of morphological, physiological, biochemical and genetic markers contribute to the limited knowledge of U. necator population genetics. The genetic interaction of U. necafor with economically important Vitis spp. has not yet been characterized. However, the Blumeria (Eysiphe) graminis-Hordeum vulgare pathosystem has well-defined virulence and resistance genes plus a range of molecular markers which have been used to track the movement of genotypes in Europe for many years (Wolfe & McDermott, 1994). U. necator, in common with B. graminis, is a member of the Erysiphales (powdery mildews) and reproduces both asexually and sexually. Despite the difference in host biology, the similarities in these fungi make B. graminis-H. vulgare a useful model for studying genetic interactions and population biology of U. necator. The reproductive strategy of U. necator ensures the appearance of new genotypes and their potential rapid proliferation. Bipolar heterothallism (two mating types) was characterized relatively recently among isolates from New
York State (Gadoury & Pearson, 1991).In addition, ascospores appear to be the primary inoculum for powdery mildew epidemics in New York State vineyards (Pearson & Gadoury, 1987). The sexual stage was first recorded in Australia in 1984 (Wicks, Magarey & Emmett, 1985), whereas it has been known in North America since 1834 (Kapoor, 1967) and since 1893 in Europe (Bulit & Lafon, 1978). While a functional sexual stage creates significant opportunity for variation, nothing is known about the impact of sexual reproduction on the genetic diversity of U. necator. Population biology studies of this fungus will (a) determine the relative importance of the sexual stage in Australia and other regions, and (b) reveal the impact of disease management strategies, for example fungicide applications, on population dynamics. In order to characterize a U. necator population and to understand the genetic bases for variation, molecular-genetic and phenotypic markers must be developed for use as research tools. DNA techniques can provide abundant and selectively neutral genetic markers which can complement existing phenotypic markers (Michelmore & Hulbert, 1987). Here we describe an experimental system for manipulating different clonal lines of U. necator and a rapid, high-yielding DNA extraction procedure which results in high-quality DNA that can be restriction enzyme digested or amplified by the polymerase chain reaction (PCR). In addition, we identify genetic variation among clonal lines of U. necafor as a first step in the development of DNA markers for studying the population genetics of t h s economically important pathogen.
Characterization of Uncinula necator isolates
676
MATERIALS A N D M E T H O D S Isolation of clonal lines of Uncinula necator
Diseased leaves or berries were collected from various Australian vineyards in non-phylloxera infested regions and transported directly to the laboratory within 5 d of field sampling for the isolation of clonal lines of U . nerator (Table I). Each disease sample came from a single block or row of vines, usually from several adjacent panels of vines. A detached leaf technique was developed for the isolation of clonal lines of U. necator. Leaves were collected from glasshouse grown Vitis vinifera cv. Cabemet Sauvignon, clone G9V3. Grapevines were propagated from hardwood cuttings obtained from Registered Source Areas (Riverland Vine Improvement Committee, Barmera, South Australia) and maintained free from powdery mildew using vapours of penconazole (Szkolnik, 1983).Cuttings with roots and dormant buds were planted into 20 cm diam. pots containing U.C. potting mixture (Baker, 1957). After 6 wk, cuttings with shoots were fertilized fortnightly with a complete watersoluble fertilizer (1.6 g 1-' Aquasol@,Hortico, Laverton North, Victoria (NPK 23:4:18)). During the autumn and winter, active shoot growth was maintained by providing a photoperiod from 00.00 to 01.00 h, using 60 W incandescent bulbs 40 cm apart above the plants (K. Skene, pers. comrn.). For detached leaf culture, leaves were selected to fit into a Petri plate (10 cm diam., 2 cm deep) containing 20 ml of I 0 g I-I distilled water agar (Bitek, Difco Laboratories, Michigan). Four sterile toothpicks were placed parallel to each other at 1.5 cm intervals on the agar surface. Leaves were washed in distilled water and a fresh cut was made to the basal end of the petiole before surface sterilization in 0-5 g 1-I sodium hypochlorite for 3 min. The leaves were then rinsed three times in sterile distilled water and placed on the bed of toothpicks with the basal 2-4 mm of the petioie immersed in the agar. The leaves were placed within a laminar-flow hood until the upper surface was completely dry. The bed of
toothpicks raised the leaf slightly above the agar surface, providing a dry leaf surface for colonization by U. necator. A sterile artist's paint brush was used to brush conidia, from diseased leaves or berries, onto the detached leaf cultures described above. At least three detached leaf cultures were prepared to sample conidia randomly from the bulked disease material from each location. The lid of the Petri plate was replaced and the plate sealed with [email protected] plates were incubated at a slight angle (approx. 10') in an illuminated growth chamber (330 VE s-' m-2 from cool white fluorescent bulbs) with a 12 h photoperiod at 25 OC. Individual chains of conidia, which were free from visible contamination by other microbes, were transferred from 10-12-d-old colonies to healthy detached leaf cultures using the method described by Gadoury & Pearson (1991). A single artist's paintbrush hair attached to a dissecting needle with petroleum jelly was used to transfer individual chains of conidia. Six individual chains of conidia were selected at random from colonies growing on the detached leaves and transferred to a healthy detached leaf. This procedure was repeated at least twice. The resulting IC-12-d-old colonies, which were free from visible microbial contamination, were transferred individually, using a sterile artist's paint brush, either to a healthy detached leaf culture or directly to a micropropagated grapevine for culture maintenance in vitro. Conidia of isolates maintained on detached leaf cultures were transferred to healthy detached leaf cultures every 14 d. Clonal lines isolated during the 199213 growing season were maintained as detached leaf cultures for 2-5 months prior to being transferred to micropropagated grapevines because plantlets were not established in vifro at that time. There is no evidence of genetic change in clonal lines during maintenance (unpublished data). Culture maintenance and mass production and collection of conidia
Clonal U. necafor lines were maintained in vitro on V . vinifera cv. Cabemet Sauvignon, clone CW44. Micropropagated
Table 1. Origin of clonal lines of U. necator
Clonal llneb
Host cv. and organ
Locat~on
Date of collect~on of diseased matenal
9403 9404 9405 9407
Chenin Blanc leaves Chardonnay berries White Grenache berries Crouchon leaves V. amurensis leaves Sauvignon Blanc berries Chardonnay berries chardonnay berries Chardonnay berries Sauvignon Blanc berries Perlette berries CG 4320r berries Traminer berries Chardonnay berries
Waite Campus, Adelaide Plains, SA Nuriootpa, Barossa Valley, SA Nuriootpa, Barossa Valley, SA Nuriootpa, Barossa Valley, SA Home garden, Adelaide Plains, SA Morialta, Adelaide Hills, SA Block I, Summertown, Adelaide Hills, SA Block 2, Summertown, Adelaide Hills, SA Block 3, Summertown, Adelaide Hills, SA Yallingup. Margaret River, WA Swan Research Station, Swan Valley, WA Swan Research Station, Swan Valley, WA Lindemans, Coonawarra, SA Home garden, Launceston, Tas.
Nov. 1992 Jan. 1993 Jan. 1993 Jan. 1993 Jan. 1993 Jan. 1993 Feb. 1993 Feb. 1993 Feb. 1993 Dec. 1993 Jan. 1994 Jan. 1994 Jan. 1994 Mar. 1994
Sourcea
-
All clonal lines except 9308 were obtained from Vitis uinifera. SA, South Australia; WA, Western Australia; Tas., Tasmania. Clonal lines are coded in chronological order of collection. " Accession No. IC758274.
a
K. J. Evans, D. L. Whisson and E. S. Scott
Fig. 1. Spomlating colonies of U.necator on a detached grapevine leaf on water agar in a 10 cm diam. Petri plate, 12 d after inoculation.
shoots were kindly provided by Dr M. Barlass. Shoots were cut into nodal segments and five segments placed upright into 50 ml of maintenance medium in 250 ml polycarbonate culture tubes with polypropylene breather lids (Disposable Products, South Australia). The maintenance medium consisted of MS medium (Murashige & Skoog, 1962) with all nutrients at half strength, 15 g I-' sucrose, 0.01 mg I-' anaphthaleneacetic acid (NAA) and 7 g 1-' agar (Bitek, Difco Laboratories, Michigan). The pH of the medium was adjusted to 5.8 before the addition of the agar and autoclaving. Cultures were maintained at 25' with a 16 h photoperiod (250-450 PE s-' mP2). After 10 d, most nodal segments had developed small roots and one segment per tube was transferred to 70 ml of the same maintenance medium, minus NAA, in a 500 ml culture tube with a breather lid. After approximately 21 d, the plantlet, which had developed at least five expanded leaves, was inoculated with U. necafor conidia. The four plantlets remaining in the 250 ml culture tube were cut into nodal segments approximately 31 d after the previous transfer and placed on fresh maintenance medium containing NAA. This culture cycle was repeated each month to provide a continuous supply of plantlets for dual culture with U. necator. Every 8-10 wk conidia were transferred aseptically to healthy in vifro plantlets by removing an infected leaf and brushing the conidia against a leaf of the plantlet being inoculated (Gadoury & Pearson, 1988). Conidia were mass produced by inoculating detached leaf cultures with aseptic conidia from infected micropropagated leaves in the same manner (Fig. 1). Following inoculation, conidia were harvested twice, after 12 and 16 d respectively, using a cyclone separator (Fig. 2) connected to a vacuum pump (Tervet et al., 1951; H. Wallwork, pers. comm.). The conidia were harvested inside a perspex hood that had been wiped with 70% ethanol to minimize contamination of the cultures and prevent cross-contamination between isolates.
Fig. 2. The cyclone separator device adapted for the collection of conidia into a 1-5 ml microfuge tube. A, the air intake is designed to
prevent host tissue bloclang the opening; B, air is drawn by a vacuum pump connected with tubing (not illustrated);C, conidia are separated from the air by a central tube extending below the point at which air enters the cyclone separator; D, the microfuge tube (length, 4 cm) is attached to the device with a piece of tubing (not illustrated) that encases both the microfuge tube and the arm of the device. The cyclone separator was rinsed with 70% ethanol and allowed to dry before each harvest. Conidia were harvested from sporulating colonies that were free from microbial contamination when examined at 50 x magnification. Occasional contamination of the water agar by fungi was reduced by incorporation of 10 mg I-' pimaricin. Conidia were collected directly into a 1.5 ml microfuge tube, frozen in liquid nitrogen, and stored at -80'. Conidia harvested on different days were stored separately.
DNA extraction from conidia The DNA extraction method was modified from that of Whisson (1996) for Blumeria (Eysiphe) graminis conidia and from that of Matthew, Herdina & Whisson (1995) for Rhiwctonia solani mycelia. A 700-900 ~1 volume of extraction buffer (150 mM sodium acetate, 20 mM EDTA, 3 % sarkosyl, pH 5.2, containing 2 mg ml-' predigested pronase) was added to 30-40 mg frozen conidia. The conidia were suspended,
Characterization of Uncinula necafor isolates pummelled with glass beads and incubated according to the method of Whisson (1996). The nucleic acids were separated from most of the other cell components by phenol-chloroform extraction. The volume of the final aqueous phase was increased to 500 yl with a solution containing 50 mM Tris-HC1, 20 mM EDTA, pH 8, and the nucleic acids were precipitated by the addition of 0.4 volumes (200 d) of 4 M ammonium acetate, pH 5.2, and 0.6 volumes (300 PI) of cold isopropanol and placing the tube on ice for 2 h. The nucleic acid pellet, obtained after centrifugation at 15 800 g (Eppendorf centrifuge) for 20 min, was washed briefly with cold 70% ethanol containing 10 mM magnesium acetate, and dissolved in 50 yl of TE b d e r (10 mM Tris-HC1, 1mM EDTA, pH 8).RNaseA was added to a final concentration of 50 pg ml-' and incubated at 37' for 30 min. The volume of the solution was then increased to 150 y1 with TE buffer and extracted with chloroform/isoamyl alcohol (24: 1).DNA was precipitated from the aqueous phase by the addition of two volumes of cold ethanol (99-7-100% v/v), overnight at - 20'. The solution was centrifuged as before for 20 rnin and the DNA pellet washed with cold 70% ethanol, 10 mM magnesium acetate for 30 min, pelleted again, and dissolved in 20 y1 TE buffer. The amount of DNA in each sample was estimated by running aliquots on a 1% agarose gel and visualizing the bands under uv light following ethidium bromide staining. The intensity of the staining of U. necafor bands was compared with that of known quantities of Rhiwcfonia solani DNA and Hind I11 digested lambda DNA.
Amplification of D N A using the polymerase chain reaction
kbp
M
I
2
3
Fig. 3. U. necator DNA (approximately 100 ng lane-') digested with EcoR V, Lane M, 0.5 ~g lambda DNA digested with Hind 111; lane 1, line 9201 (Adelaide Plains); lane 2, line 9302 (Barossa Valley); lane 3, line 9313 (Adelaide Hills).
Amplification of U. necafor DNA was performed in a FTS-IS Capillary Fast Thermal Sequencer (Corbett Research, Sydney). The reaction was carried out in a 20 yl volume containing colony. When these colonies were free from visible microbial 1-3 ng DNA template, SO ng plant intron splice junction (ISJ) contamination, conidia were transferred to micropropagated primer, Rl (5'GTCCATTCAGTCGGTGCT3', Weining & grapevines in vifro, with greater than 99% of inoculations Langridge, 1991), 0.2 mM each of dATP, dCTP, dGTP and producing an infection without microbial contamination. dTTP, 2.5 mM MgCl,, 50 mM KCI, 10 mM Tris-HC1 (pH 8.8), The yield of conidia from 20 detached leaf cultures was 0.1% Triton X-100 and 1.5 units Taq DNA polymerase quantified for each of the 14 clonal lines of U. necafor. The (Promega).All reactions consisted of: (i) one cycle of 3 rnin at mean yield (n = 14) for the first harvest, at 12 d, was 70 mg 94', 30 s at 40' and 90 s at 72'. (ii) four cycles of 15 s at 95', conidia per 20 leaves (s.D. = 23.2), with a range of 41-112. 15 s at 40' and 90 s at 7Z0, (iii) forty cycles of 5 s at 95', 10 s The mean yield for the second harvest, at 16 d, was 19 mg per at 58' and 90 s at 72', and (iv) a final extension step of 5 rnin 20 leaves (s.D.= 11.7), with a range of 8-39 mg. Variation in at 72'. A ramp rate of 0.25' s-' was used between the conidial yields appeared to be due to clonal line fitness and/or annealing and primer extension temperatures. PCR products the health of the detached leaf. Conidia could be harvested a were analysed on 2% agarose gels and visualized under uv third time, up to 28 d after inoculation, but leaf senescence light following ethidium bromide staining. The size marker reduced the quantity of conidia collected to less than 10 mg used was pGEM@(Promega). per 20 leaves. The DNA extraction procedure resulted in high molecular weight DNA suitable for restriction enzyme digestion, and RESULTS amplification by PCR. DNA yields ranged from 170 to 280 ng Fourteen clonal lines were selected for this study (Table 1). per 30 mg of conidia. Figure 3 illustrates the restriction Diseased material was collected from non-phylloxera infected enzyme digestion of total U. necator DNA with EcoR V. At areas of New South Wales but the conidia were not viable least 16 distinct fragments are evident as brightly stained after transportation by air to South Australia. During clonal bands against the background smear of DNA. Clonal line isolation, at least one out of every six single-conidial chains 9313 (lane 3) has a 9.4 kbp fragment which appears to be placed individually on a detached leaf produced a sporulating absent from lines 9201 and 9302 (lanes 1 and 2, respectively).
K. J. Evans, D. L. Whisson and E. S. Scott Petri plates (Quim & Powell, 1981; Pearson & Gadoury, 1987; Gadoury & Pearson, 1991). The detached leaf technique is equaIly useful for this purpose but has the advantage of providing a sterilized leaf surface for the production of aseptic conidia. Aseptic conidia are required for (a) inoculation of micropropagated plantlets for maintenance of isolates, and (b) the mass production of conidia for DNA extraction and amplification. Using whole, surface sterilized leaves, we found it unnecessary to add benzimidazole to delay leaf senescence, in contrast to dual culture of B. graminis on barley leaf pieces (McDermott et al., 1994) and U. necator on grapevine leaf discs (Steva, 1994). The production of conidia using detached leaf cultures and the cyclone separator collection device allow conidia to be harvested from many different clonal lines. Conidia can be harvested from dual cultures on micropropagated plantlets, but manipulations are more time-consuming compared with dual culture on detached leaves. The detached leaves provide a large surface area for sporulation whereas conidia need to be collected from many small leaves of the in vifro plantlets to achieve the same yield (data not shown). However, microFig. 4. Amplification of U.necafor DNA with the ISJ primer, RI. The propagated plantlets with sporulating U. necator colonies are PCR products were fractionated on a 2 % agarose gel. Lane M, DNA the best source of aseptic inoculum for both culture size marker, [email protected] sizes are shown on the left. Lane I, a maintenance and for bulking conidia on detached leaves. In control where no genomic DNA was added to the reaction mixture; addition, dual cultures on plantlets can be maintained for up lane 2, line 9201 (Adelaide Plains); lane 3, line 9313 (Adelaide Hills); to 6 months before the plantlets senesce. We transferred lane 4, line 9310 (Adelaide Hills); lane 5, line 9312 (Adelaide Hills) conidia to healthy plantlets every 8-10 wk in order to lane 6, line 9301 (Barossa Valley). maintain vigorous dual cultures. Molecular characterization of U. necator required the development of a rapid DNA extraction method. This is the U. necafor DNA could be digested by a range of restriction first report of a DNA extraction procedure for conidia of U. endonucleases including EcoR I, BamH I, Psf I, SUM3A and necator. The typically small yields of aseptic conidia per clonal Taq I (data not shown). U. necator and the potential for population studies line of Figure 4 illustrates the amplification of U. necator DNA with involving many samples necessitated the development of an the ISJ primer, R1. There are two types of banding pattems efficient technique. Our DNA yield, which is in the range which identify genetic variation among these clonal lines of U. mg-' of conidia, is similar to that reported by O'Dell 6-9 ng necator from South Australian vineyards. The banding pattems ef al. (1989) for B. grarninis conidia. Using a large quantity, of lines 9201 and 9310 (lanes 2 and 4) differ from those of 20 g, of conidia they extracted approximately 5 ng mg-' of lines 9313, 9312 and 9301 (lanes 3,5 and 6, respectively). conidia. Furthermore, our acidic extraction buffer is simple and Lines 9310, 9313 and 9312 originate from diseased berries sampled from different blocks of Chardonnay vines within a preliminary experiments have revealed that the pH of 5.2 produced DNA which was less degraded than that obtained 0.5 km radius in an Adelaide Hills vineyard. using an extraction buffer of pH 8. Nucleic acid extraction from 30 mg of conidia yielded enough DNA for at least DISCUSSION 150 PCR reactions. In addition, the high quality of the U. We describe an experimental system for the clonal isolation of necafor DNA makes it useful for applications requiring U. necafor, the mass production and collection of conidia, and restriction enzyme digested DNA, for example ligation subsequent DNA extraction and amplification. Detached leaf reactions. U. necafor DNA was digested by a wide range of culture is a standard technique for the cereal powdery mildew restriction endonucleases and the distinct fragments evident pathogen Blumeria (Eysiphe)grarninis (Yarwood, 1946; Wolfe, after digestion with EcoR V (Fig. 3) may indicate the existence 1965; Jorgensen, 1988; McDermott ef al., 1994), but has not of repeated DNA and/or mitochondria1 DNA. Using the 18 base ISJ primer, Rl, we identified genetic been described previously for the mass production of conidia of U. necator. Grapevines, being perennial, are propagated by variation among South Australian clonal lines of U. necator. RI cuttings. Leaves must be collected from young shoots which is based on the consensus sequences for the intron-exon splice are pruned to maintain active growth. Compared with barley junctions of plants and generates products from the exon plants, more grapevines are required to obtain the same leaf region (5' splice site, exon targeting, Weining & Langridge, surface area. Grapevines with active shoot gowth can be 1991). This primer has also been used to amplify DNA from maintained in the glasshouse for up to 1yr provided they are Gaeumannomyces graminis (Harvey, 1993) and Rhiwcfonia pruned carefully. The establishment of single conidial chain solani (Duncan, Barton & O'Brien, 1993; Whisson, Herdina & colonies can be achieved by using detached leaves in double Francis, 1995). The amplification of U. necafor DNA using RI kbp
M
1
2
3
4
5
6
Characterization of Uncinula necator isolates as a primer indicates that R1 has some homology with U. necator DNA and implies the existence of conserved sequences at the junctions to plant exons. The different banding patterns among the five clonal lines examined demonstrates genetic variation among these isolates from different grape growing regions in South Australia. Most interestingly, variation was identified between clonal lines originating from the same vineyard, giving a preliminary indication of fine-scale variation in this fungus. More isolates need to be characterized within and between vineyards and regions, and with a range of molecular markers, in order to determine population structures and population dynamics (e.g. gene flow, reproductive mode and selective pressures). Thus, it will be worthwhile to develop other DNA markers, including other PCR primers and DNA probes. We are currently screening cloned U . necator sequences to identify U. necator specific DNA probes that show polymorphisms between clonal lines. Markers could then be developed to characterize populations of U . necator using total DNA from infected grapevine tissue. We thank Dr M. Barlass for supplying V. vinifera cultures and advice on micropropagation, Dr R. Cirami for supplying the first batch of V. vinifera hardwood cuttings, Mr P. Wood, Mr B. Wagner, Mr T. Somers, Ms B. Hall and Dr T. Wicks for supplying diseased grapevine material, Dr H. Wallwork for supplying the basic design for the cyclone separator device, Mr R. Balali for supplying Rkizoctonia solani DNA and Dr P. Langridge for supplying the ISJ primer, RI. We also thank Professor R. Symons (Programme Leader) for helpful discussions and comments on the manuscript.
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680 Gadoury, D. M. & Pearson, R. C. (1991). Heterothallism and pathogenic specialization in Uncinula necator. Phytopathology 81, 1287-1293. Harvey, P. (1993). Genetic diversity among populations of the take-all fungus Gaeumannomyces graminis. PhD Thesis, University of Adelaide. mildew of cereals and Jorgensen, J. H. (1988). Eysiphe graminis, grasses. Advances in Plant Pathology 6, 137-157. Kapoor, 1. N. (1967). Uncinula necator. In C M I Descriptions of Pathogenic Fungi and Bacteria. No. 160. Commonwealth Mycological Institute, The Eastern Press Ltd: London. Matthew, J., Herdina & Whisson D. (1995). DNA probe specific to Rhiwctonia solani anastomosis group 8. Mycological Research 99, 745-750. McDermott, J. M., Briindle, U., Dutly, F., Haemmerli, U. A,, Keller, S., Miiller, K. E. & Wolfe, M. S. (1994). Genetic variation in powdery mildew of barley: development of RAPD, SCAR, and VNTR markers. Phytopathology 84, 1316-1321. Michelmore, R. W. & Hulbert, S. H. (1987). Molecular markers for genetic analysis of phytopathogenic fungi. Annual Review of Phytopathology 25, 383-404. Murashige, T. & Skoog, K. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15, 473-497. O'Dell, M., Wolfe, M. S., Flavell, R. B., Simpson, C. G. & Summers, R. W. (1989). Molecular variation in populations of Eysiphe graminis on barley, oats and rye. Plant Pathology 38, 340-351. Pearson, R. C. & Gadoury, D. M. (1987). Cleistothecia, the source of primary inoculum for grape powdery mildew in New York. Phytopathology 77, 1509-15 14. Quinn, J. A. & Powell, C. C., Jr. (1981). Identification and host range of powdery mildew of begonia. Plant Disease 65, 68-70. Steva, H. (1994). Evaluating anti-resistance strategies for control of Uncinula necator. In Fungicide Resistance (ed. S. Heany, D. Slawson, D. W. Hollomon, M. Smith, P. E. Russell & D. W. Parry), pp. 59-66. British Crop Protection Council Monograph No. 60: Surrey, U.K. Szkolnik, M. (1983). Unique vapor activity by CGA-64251 (Vangard) in the control of powdery mildews roomwide in the greenhouse. Plant Disease 67, 36G366. Tervet, I. W., Rawson, A. J., Cheny, E. & Saxon, R. B. (1951). A method for the collection of microscopic particles. Phytopathology 41, 282-285. Weining, S. & Langridge, P. (1991). Identification and mapping of polymorphisms in cereals based on the polymerase chain reaction. Theoretical and Applied Genetics 82, 209-216. Whisson. D. L. (1996). Molecular characterization and pathogenicity of Erysiphe graminis f. s p hordei in Australia. Australasian Plant Pathology (in press). Whisson D. L., Herdina, & Francis, L. (1995). Detection of Rhizoctonia sohni AG-8 in soil using a specific DNA probe. Mycological Research 99, 1299-1302. Wicks, T. J., Magarey, P. & Emmett, R. W. (1985). First report of Uncinula necator cleistothecia on grapevines in Australia. Plant Disease 69, 727. Wolfe, M. S. (1965). Physiologic specialization of £.graminis fritici in the U.K. Transactions of the British Mycological Society 48, 315-326. Wolfe, M. S. & McDermott, J. M. (1994). Population genetics of plant pathogen interactions: the example of the Erysiphe gramini~Hordeum vulgare pathosystem. Annual Review of Phytopathology 32, 89-113. Yarwood, C. E. (1946). Detached leaf culture. Botanical Review 12, 1-56.