Pacific Science (1995), vol. 49, no. 2: 181-191 © 1995 by University of Hawai'i Press. All rights reserved Vesicular-arbuscular Mycorrkizal Inoculation of Hawaiian Plants: A Conservation Technique for Endangered Tropical Species1 R. E. KOSKE AND J. N. GEMMA 2 ABSTRACT: Forty species of plants (including 28 species endemic to the Ha­ waiian Islands) were evaluated in the greenhouse for their response to inocula­ tion with the vesicular-arbuscular mycorrhizal fungus Glomus intraradices Schenck & Smith. Seedlings, cuttings, and established plants were inoculated. Several kinds of growth media were used. Increased growth and survival most frequently occurred when plants were grown in a gravel or fine sand medium that included calcined clay (up to 50% by volume) or sphagnum peat (up to 20%). Significant increases in height, weight, leaf number and size, and survival were noted in 10 of 14 species of seedlings grown in media in which peat con­ tent was 20% or less. Mycorrhizae were only rarely present in the non­ inoculated plants except for plants grown from cuttings. The latter routinely formed mycorrhizae in the absence of added inoculum. Addition of mycor­ rhizal fungi to potting mixes appears to have value as a conservation technique for some plants that are difficult to propagate. NUMEROUS GREENHOUSE STUDIES have shown 1985, Gagne 1988, Smith 1989, Stone 1989). the benefits of inoculation with vesicular­ Conservation of many Hawaiian species has arbuscular mycorrhizal (VAM) fungi to the been handicapped by our inability to cultivate growth of a wide variety of plant species species in the greenhouse. When plant species (Harley and Smith 1983). Agronomic crops can be established and temporarily main­ have received the most attention, but rela­ tained in a botanical garden, additional time tively few horticultural and wild species have is gained for the protection or acquisition of been tested for mycorrhizal responses (e.g., sites in which the plants can later be out­ Linderman 1978, 1981, Biermann and Lin­ planted (Theobald 1989). derman 1983a,b). Hawaiian species are notoriously difficult In the Hawaiian Islands, it is estimated to propagate in the greenhouse (Wooliams that 33% of the flora of ca. 1000 species is 1976). The frequent observations of the plant endangered or threatened (Vitousek et al. propagator, that seedlings "refused to grow 1987, Wagner et al. 1990). The major causes larger than 2", and after 3 months suddenly of this situation are the destruction of hab­ died" (Wooliams 1976: 74), could well be a itats (especially by humans, pigs, and goats), description of the behavior of an obligately damage from introduced pests, and com­ mycotrophic species grown in the absence of petition from alien plant species (Howarth mycorrhizal fungi (e.g., Janos 1980). Previous studies have shown the horti­ cultural benefits to be derived from the ap­ 1 This research was funded in part by a grant from propriate use of VAM in the greenhouse the Hawai'i State Division of Forestry and Wildlife ad­ (Barrows and Roncadori 1977, Linderman ~ste~ed tbrough the Hawaiian Co.nsef\lation Biology 1978, 1981,Johnsonetal.1980,Johnson 1981, Initiative program of The Nature Conservancy of Ha­ Biermann and Linderman 1983a,b), but their wai'i and a Faculty Fellowship from the University of Rhode Island. Manuscript accepted I3 April 1994. use in the propagation of rare and endan­ 2Department of Botany, University of Rhode Island, gered species has been overlooked. Indeed, Kingston, RI 02881. several standard greenhouse practices (e.g., 181 182 PACIFIC SCIENCE, Volume 49, April 1995 starting seeds and cuttings in "sterile" soil or TABLE I soilless mixes, extensive use of fungicides, PLANT SPECIES USED IN INOCULATION EXPERIMENTS and frequent fertilization) have the effect of suppressing or excluding VAM formation SPECIES STATUS' (Biermann and Linderman 1983a, Harley and Smith 1983). In a comment that could apply Agavaceae equally to effects of greenhouse practices on Pleomele aurea (H. Mann) N. E. Brown E Apiaceae VAM fungi, Wooliams (1976: 82) noted that Peucedanum sandwicensis Hillebr. E "Hawaiian plants are very susceptible to Araliaceae damage by ... fungicides and will often die Munroidendron racemosum (c. Forbes) more rapidly through their use than without! Sherif E The same is true to a lesser extent with Tetraplasandra hawaiensis A. Gray E Asteraceae fertilizers. " Dubautia scabra (DC) D. Keck E The consistent occurrence of mycorrhizae Remya kauaiensis Hillebr. E in Hawaiian plants collected from a variety Verbesina encelioides (Cav.) Benth. & Hook. I of habitats suggested that ca. 90% of native Wilkesia gymnoxiphium A. Gray E Hawaiian angiosperms are obligate myco­ Convolvu1aceae Ipomoea pes-caprae (L.) R. Br. subsp. trophs (i.e., dependent upon the association) brasiliensis (L.) Ooststr. (Koske et al. 1992). However, VAM were Euphorbiaceae essentially absent from > 100 potted plants Antidesma pulvinatum Hillebr. E grown in the greenhouses of the National Chamaescyce remyi var. remyi (A. Gray ex Boiss.) Croizat & Degener E Tropical Botanical Garden (NTBG), Kaua'i, Fabaceae Hawai'i (unpubl. obs.), where standard Acacia koa A. Gray E greenhouse methods were in practice (e.g., Caesalpinia kavaiensis H. Mann E soilless mixes, frequent fertilization). The Erythrina berteroana Urban G discrepancy between the mycorrhizal status Erythrina sandwicensis Degener E Sesbania tomentosa Hook. & Amott E of Hawaiian plants thriving in the field and Sophora chrysophylla (Salisb.) Seem. E those barely surviving in the greenhouse led Scaevola gaudichaudiana Cham. E us to investigate the effect of VAM on the Scaevola sericea Yah! I growth of Hawaiian plants. If plants were Larniaceae Plectranthus parviflorus Willd. found to respond to VAM, an important step Lythraceae toward conservation of Hawaiian and other Lythrum maritimum Kunth I tropical species would be made. Malvaceae Abutilon eremitopetalum Caurn E Abutilon menziesii Seem. E MATERIALS AND METHODS Gossypium tomentosum Nutt. ex Seem. E Hedyotis sp. E A variety of experiments was conducted Hibiscadelphus hualalaiensis Rock E using seedlings, cuttings, and established Hibiscus clayi Degener & I. Degener E plants that had been growing in the green­ Hibiscus waimeae subsp. waimeae A. Heller E Kokia kauaiensis (Rock) Degener & Duvel E house (Table 1). Of the 40 species of plants Sidafallax Walp. I tested, 28 were endemic to the Hawaiian Myrtaceae Islands. A few species of non-Hawaiian, Metrosideros polymorpha Gaud. E tropical plants that were in the greenhouse Pittosporaceae also were included in the study. Where possi­ Pittosporum kauaiense Hillebr. E Primulaceae ble, sampling at the end of the experiment Lysimachia glutinosa Rock E was nondestructive, allowing the plants to be Rharnnaceae outplante_d at _a later date. _ Colubrina oppositifolia Brongn. ex H. Mann E Experiments were performed at NTBG RubIaceae Bobea elatior Gaud. E in a screened greenhouse between May 1989 Gardenia gordonii Baker G and June 1990 and at the University of Gardenia remyi H. Mann E 'W¥ijiM .. Mycorrhizal Inoculation of Hawaiian Plants-KosKE AND GEMMA 183 TABLE I (continued) plants (10 inoculated, 10 control). However, because of the scarcity of some species or of SPECIES STATUS' viable seed, fewer plants had to be used for some experiments. Data were analyzed using Sapindaceae Dodonaea viscosa Jacq. independent t tests (two-tailed), and signifi­ Solanaceae cance was assigned at P < 0.05. Differences Lycium sandwicense A. Gray in survival of plants were analyzed using the Verbenaceae adjusted chi-square test. Vitex rotundifolia L. fil. An index ("MjNM") to standardize 'E, endemic in Hawaiian Islands; I, indigenous to Hawaiian growth response was calculated by dividing Islands; G, greenhouse specimens, not native or naturalized in the average size of the inoculated (M) plants Hawai'i. by the average size of the uninoculated (NM) plants. Angiosperm systematics and nomenclature Rhode Island (URI) in glass-enclos~d, heated are those of Wagner et al. (1990). Endemic greenhouses between July 1990 and January species are those that occur naturally only in 1992. Daytime temperatures at URI were the Hawaiian Islands, and indigenous species 25-35°C (average 27°C) and nighttime tem­ are those that occur naturally in Hawai'i and peratures were 16-25°C (average 24°C). Ex­ elsewhere. periments performed before December 1990 at URI were carried out in a greenhouse Inoculation ofSeedlings without supplemental light. After that date, all plants (continuing experiments and new All experiments were performed at URI. experiments) received 16 hr of light per day Most experiments were performed in a gravel­ of sunlight supplemented with high-pressure calcined clay mix or a peat-calcined clay mix. sodium vapor lamps giving an intensity at the Seeds were germinated in the greenhouse in leaf surface of 350-1375 J.LEinjm 2 jsec. trays of a sphagnum peat: perlite mix (4: 1). Several types of growth media were used. Seedlings (two- to four-leaf stage) were se­ In all mixes listed below, ratios of ingredients lected for uniform size and vigor, sorted into are given on a volume basis. Based on results two groups (inoculated and control), and from the first part of our studies, the compo­ transplanted to containers filled with the ex­ sition of the media and growth conditions perimental mix. Experiments in 1989 were were varied during the investigation. carried out in plastic pots 10 cm square filled At the completion of each experiment, all with a 4: 1 mix of Pro-Mix BX: Oil Dri. plants were measured (typically, the dry Pro-Mix BX (premier Brands, Stamford, CT weight or height of shoots, although other 06902) is a peat-based mix (sphagnum peat, parameters sometimes were used), and roots vermiculite, and perlite), pH 5.5-6.2, con­ of 50-100% of the control and inoculated taining sufficient added macro- and micro­ plants in each experiment usually were nutrients for initial establishment of seed­ collected, cleared, and stained (Koske and lings. Oil Dri (Oil Dri Corp., Chicago, IL Gemma 1989).