PROPAGATION & TISSUE CULTURE HORTSCIENCE 38(6):1163–1166. 2003. Berta and Bonfante-Fasolo, 1983; Bradley et al., 1981; Leake and Read, 1989). Other studies have attempted to evaluate the host Host Range of a Select Isolate of range of ericoid fungi, but have inoculated with unidentified ericoid fungal isolates, described, the Eri coid Mycorrhizal Fungus for example as, “dark, slow-growing cultures” (Pearson and Read, 1973b; Reed, 1987). Hymenoscyphus ericae To date there have been no studies that investigate the host range of a select isolate Nicole R. Gorman1 and Mark C. Starrett2 of the ericoid endophyte H. ericae. Therefore, Department of Plant and Soil Science, University of Vermont, Burlington, the objective of this study was to evaluate the VT 05405-0082 host range using select species within the Eri- caceae by inoculating with a specific iso late Additional index words. Ericaceae, Calluna, Enkianthus, Gaultheria, Kalmia, Leucothoe, of H. ericae. Oxydendrum, Pieris, Rhodo den dron, Vaccinium Materials and Methods Abstract. Studies were conducted to ex am ine the host range of a select isolate of the ericoid mycorrhizal fungus Hymenoscyphus ericae (Read) Korf and Kernan [American Type Seed of 15 ericaceous species was ob tained Culture Collection (ATCC) #32985]. Host status was tested for 15 ericaceous species, in- from commercial seed suppliers [Sheffield·s cluding: Calluna vulgaris (L.) Hull, Enkianthus campanulatus (Miq.) Nichols, Gaultheria Seed Co. (Lock, N.Y.) and F.W. Schumacher procumbens L., Kalmia latifolia L., Leucothoe fontanesiana Sleum., Oxydendrum arbo- Co. (Sandwich, Mass.)]. Seed was cleaned and reum (L.) DC., Pieris flo ri bun da (Pursh) Benth. & Hook., Rhodo den dron calendulaceum counted under a dissecting microscope to se- (Michx.) Torr., Rhodo den dron carolinianum Rehd., Rhododendron catawbiense Michx., lect seeds of similar size, color, and fullness. Rhodo den dronmax i mumL., Rhododendron mucronulatumTurcz., Vaccinium corymbosum Preliminary trials were conducted to determine L., and Vaccinium macrocarpon Ait. Arbutus unedo L., an eri ca ceous species that forms the approximate num ber of days to germination arbutoid, not ericoid, mycorrhizae, was used as a negative control. All of the species were for each species. Species were grouped (A–C) colonized by the eri coid isolate with the exception of Enkianthus campanulatus and the according to num ber of days to germination negative control. In oc u la tion with this isolate of H. ericae resulted in a significant increase (Table 1). Grouping species enabled all species in shoot growth. How ev er, intensity of root colonization was not correlated to amount of to be inoculated and transplanted at the same shoot growth. In fact, an increase in growth was observed in the two species that lacked growth stage. Simultaneous transplant allowed fungal colonization. for a single harvest date for each replication. Seed treatment and aseptic germination. Mycorrhizal symbioses are the rule rather fungi (Perotto et al., 1995). Select ericaceous On 30 Oct. 1998, seed from group A (Table 1) than the exception in the plant kingdom. Eri- species have been inoculated with unidenti- was surface-disinfested with a dilute so di um coid mycorrhizae, the most specialized type of fied ericoid fungi, often using peat originating hypochlorite solution (0.5% to 1.0%) also endomycorrhizae (Harley and Smith, 1983), from native habitats of ericaceous species, as an containing the surfactant polyoxyethylene are formed between a restricted number of inoculum (Allaway and Ashford, 1996; Brad- sor bi tan monolaurate [Tween 20 (0.005%)]. plants in the order Ericales and a small group ley et al., 1981; Leake and Read, 1989, 1991; Bleach concentrations were determined dur ing of ascomycetous fungi (Smith and Read, 1997). Mitchell and Read, 1981; Pearson and Read, preliminary trials to reduce contamination yet The most well known and widely researched 1973b; Powell and Bates, 1981; Reich et al., retain viability of the seed. As a result, dilution isolate is the ascomycete Hymenoscyphus 1982). The first ericoid fungus to be taxonomi- of the solution used in surface-dis in fes ta tion ericae (Read) Korf and Kernan (Peterson and cally identified, H. ericae (Read, 1974), is the varied between species (Table 1). Seed and Farquhar, 1994).Ericoid hosts have been iden- most intensely researched species (Peterson bleach solution were stirred vig or ous ly for 15 tified in the families Ericaceae, Epacridaceae and Farquahar, 1994). However, other studies min, the solution decanted, and seed rinsed with and, more recently, the nonvascular Hepaticeae have used di verse sources of fungal isolates sterile, distilled water. Seeds were transferred to (Duckett and Read, 1995; Straker, 1996). collected from all over the world (Currah et 60×15-mm poly sty rene disposable petri dishes Previous studies of ericoid mycorrhizae al., 1993; Leake and Read, 1991; Pearson and containing 10 mL sterile water agar (6 g·L–1). have produced varied growth responses in host Read, 1975; Straker and Mitchell, 1985). Many There were five seeds per dish and a total of plants (Bonfante-Fasolo et al., 1984; Bra d ley of these studies failed to identify a specific six dishes per species. The dishes were sealed et al., 1981; Currah et al., 1993; Leake and fungal strain (Bannister and Norton, 1974; with Parafilm “M” and enclosed in 0.95-L re- Read, 1991; Powell and Bates, 1981; Read, 1995; Reich et al., 1982; Singh, 1974; Star- Table 1. Seed treatment protocol for asep tic germination of select ericaceous species. rett, et al., 2001; Stribley et al., 1975). These inconsistent results may have resulted from the Ericaceous species Abbreviation of species Groupz Sodium hypochlorite treat ment diverse sources of ericoid mycorrhizae utilized Calluna vulgaris CALL A 0.5% fl in each study. Research indicates that consider- Pieris oribunda PIER A 1.0% Vaccinium corymbosum able genetic diversity may exist among ericoid VACC A 0.5% Arbutus unedoy ARBU B 0.5% Gaultheria procumbensx GAUL B 0.5% Received for pub li ca tion 17 July 2002. Ac cept ed for Rhododendron calendulaceum RCAL B 0.8% publication 2 Jan. 2003. This research was funded Rhododendron carolinianum RCAR B 0.5% by a Fed er al Hatch Grant. Use of trade names in Rhododendron catawbiense RCAT B 0.5% this pub li ca tion does not imply endorse ment by the Rhododendron maximum RMAX B 0.8% Vermont Ag ri cul tur al Experiment Station or the U.S. Rhododendron mucronulatum RMUC B 0.5% Dept. of Agriculture–Agricultural Re search Service Vaccinium macrocarpony VACM B 1.0% of products named nor crit i cism of similar ones not Enkianthus campanulatus ENKI C 1.0% mentioned. Sta tis ti cal assistance of Gary Badger and Kalmia latifolia KALM C 0.5% John Aleong is gratefully ac knowl edged. From a Leucothoe fontanesiana LEUC C 0.5% thesis sub mit ted by N.R.G. in partial fulfillment of Oxydendrum arboreum OXYD C 1.0% the requirements of an MS degree. zSpecies are grouped according to number of days to germination. Grouping allowed all species to be 1Former Graduate Assistant. transplanted and inoculated at the same growth stage. A at 28 d; B at 21 d; C at14 d. 2Associate Professor; to whom reprint requests should ySeeds were cold stratified for 30 d at 1 to 5 °C after surface disinfestation procedure. be addressed. E-mail: [email protected] xSeeds were cold stratified for 60 d at 1 to 5 °C after surface disinfestation procedure. HORTSCIENCE, VOL. 38(6), October 2003 1163 24-7399, p1163-1166 1163 10/16/03, 1:02:10 PM PROPAGATION & TISSUE CULTURE sealable plastic bags. The bags were placed in media were used as a guard-row and placed variance assumption as so ci at ed with the analy- a controlled-environment chamber maintained around the perimeter of the rack. The racks sis of variance, data cor re spond ing to percent at 22 °C day/20 °C night temperature. A 16- were placed in a controlled-environment colonization was arc sine transformed and leaf h pho to pe ri od was provided by cool-white chamber main tained at 25 °C day/22 °C night number was square root transformed prior to fluorescent lamps and incandescent bulbs. temperature. A 16-h photoperiod was provided analysis. Although statistical significance was Lamps and bulbs provided an average pho- by cool-white florescent tubes suspended 2 cm based on trans formed data, means presented tosynthetic pho ton flux [PPF (400–700 nm)] above the tops of the vials. Lamps provided an in tables and figures represent nontransformed of 251 µmol·m–2·s–1 as measured at the top of averagePPFof 58 µmol·m–2·s–1as measured at data. All F tests corresponding to treatment the dishes. These and all other light measure- the top of the vials. These light and temperature comparisons utilized the Replicate × Treat- ments were taken with a LI-COR Quantum conditions were used for the remainder of the ment mean square as the error term. Pairwise Sensor and recorded with a LI-COR 1000 study. This procedure was repeated for each comparisons are based on Fisher·s LSD proce- Data Logger (LI-COR, Lincoln, Nebr.). Petri subsequent replication. dure. Statistical significance was determined dishes were scanned weekly for contamination. Transplant. After incubation, seedlings using P < 0.05. Con tam i nat ed dishes were removed from the with ful ly ex tend ed cotyledons were chosen bags and discarded. for trans plant ing. Seedlings were aseptically Results Three species, Arbutus unedo, Gaulth e ria trans ferred to the vials. An additional 1.0 mL procumbens, and Vaccinium macrocarpon, of sterile, liquid WPM was added to each vial All of the ericaceous species tested, in- re quired cold stratification fol low ing sur- at the time of transplant.