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(12) United States Patent (10) Patent No.: US 6,576,457 B1 Hua (45) Date of Patent: Jun USOO6576457B1 (12) United States Patent (10) Patent No.: US 6,576,457 B1 Hua (45) Date of Patent: Jun. 10, 2003 (54) FUNGAL MEDIA AND METHODS FOR Douds, Jr., D.D., “A procedure for the establishment of CONTINUOUS PROPAGATION OF Glomus moSSeae in dual culture with Ri T-DNA-trans VESICULAR-ARBUSCULAR MYCORRHIZAL formed carrot roots.” Mycorrhiza (1997) 7:57-61. (VAM) FUNGI IN ROOT ORGAN CULTURE Harrison, M.J., “Molecular and Cellular Aspects of the Arbuscular Mycorrhizal Symbiosis,” Annu. Rev. Plant (75) Inventor: Sui-Sheng T. Hua, Orinda, CA (US) Physiol. Plant Mol. Biol. (1999) 50:361-389. (73) Assignee: The United States of America, as Hernández-Sebastia, C. et al., “Water relations of whole represented by the Secretary of strawberry plantlets in vitro inoculated with Glomus intrara Agriculture, Washington, DC (US) dices in a tripartite culture system.” Plant Sciences (1999) 143:81-91. (*) Notice: Subject to any disclaimer, the term of this Mugnier, J. et al., “Vesicular-Arbuscular Mycorrhizal Infec patent is extended or adjusted under 35 tion in Transformed Root-Inducing T-DNA Roots Grown U.S.C. 154(b) by 0 days. Axenically.” Phytopathology (1987) 77(7):1045-1050. Nuutila, A.M. et al., “Infection of hairy roots of strawberry (21) Appl. No.: 09/737,975 (Fragaria x Ananassa Duch.) with arbuscular mycorrhizal (22) Filed: Dec. 15, 2000 fungus.” Plant Cell Reports (1995) 14:505–509. (51) Int. Cl. .................................................. C12N 1/18 Rousseau, A. et al., “Mycoparasitism of the Extramatrical (52) U.S. Cl. ................................ 435/256.8; 435/254.1; Phase of Glomus intraradices by Trichoderna harzianum,” 47/1.1; 71/5 Phytopathology(1996) 86(5):434–443. (58) Field of Search ........................... 435/256.8, 254.1; Ruehle, J.L. et al., “Fiber, Food, Fuel, and Fungal Sym 47/1.1; 71/5 bionts,” Science (1979) 206:419–422. St-Arnaud, M. et al., “Enhanced hyphal growth and Spore (56) References Cited production of the arbuscular mycorrhizal fungus Glomus U.S. PATENT DOCUMENTS intraradices in an in vitro System in the absence of host roots.” Mycol. Res. (1996) 100(3):328-332. 3,011,951 A * 12/1961 Murray ........................ 435/56 4,144,133 A 3/1979 Dorn et al. Schreiner, R.P. et al., "Stimulation of Vesicular-Arbuscular 4.294,037 A 10/1981 Mosse et al. Mycorrhizal Fungi by Mycotrophic and Nonmycotrophic 4.550,527 A 11/1985 Hall et al. Plant Root Systems.” Applied and Environmental Microbi 4,551,165 A 11/1985 Warner ology (1993) 59(8):2750–2752. 4,599,312 A 7/1986 Mugnier et al. Wang, H. et al., “Vesicular-arbuscular Mycorrhizal Peat 4.945,059 A 7/1990 Okii et al. 5,002,603 A 3/1991 Safir et al. based Substrates Enhance Symbiosis Establishment and 5,075,332 A 12/1991 Haglund Growth of Three Micropropagated Species,” J. Amer: Soc. 5,096,481 A 3/1992 Sylvia et al. Hort. Sci. (1993) 118(6):896–901. 5,125.955 A 6/1992 Safir et al. 5,178,642 A 1/1993 Janerette * cited by examiner 5,262,381. A 11/1993 Tuse +e,uml +ee et al. 5,344,471 A 9/1994 Tise et al. Primary Examiner Irene Marx 5,554,530 A 9/1996 Fortin et al. (74) Attorney, Agent, or Firm- Margaret A. Connor; Thanda Wai; John Fado FOREIGN PATENT DOCUMENTS (57) ABSTRACT EP O 209 627 A2 1/1987 An in Vitro System using Agrobacterium-transformed OTHER PUBLICATIONS dicotyledon roots (such as carrot roots) and vesicular The Merck Index, p. 1271, 1996.* arbuscular mycorrhizal fungi (Such as Glomus intradices) Bécard, G. et al., “Early events of vesicular-arbuscular was developed to produce viable and aseptic Spores. The mycorrhiza formation on Ri T-DNA transformed roots,” entire life cycle from the growth of initial germ tubes to the New Phytol.(1988) 108:211–218. formation of external mycelium network and Spore produc Chabot, S. et al., “Life Cycle of Glomus Intraradix in Root tion was observed nondestructively using light microScopy. Organ Culture,” Mycologia (1992) 84(3):315-321. The system may be subcultured continuously for the pro Dehne, H.W., “Interaction Between Vesicular-Arbuscular duction of viable and clean Spores. Mycorrhizal Fungi and Plant Pathogens.” Phytopathology (1982) 72(8): 1115–1119. 5 Claims, 7 Drawing Sheets U.S. Patent Jun. 10, 2003 Sheet 1 of 7 US 6,576,457 B1 U.S. Patent Jun. 10, 2003 Sheet 2 of 7 US 6,576,457 B1 FIG. 2 U.S. Patent Jun. 10, 2003 Sheet 3 of 7 US 6,576,457 B1 U.S. Patent Jun. 10, 2003 Sheet 4 of 7 US 6,576,457 B1 U.S. Patent Jun. 10, 2003 Sheet 5 of 7 US 6,576,457 B1 U.S. Patent Jun. 10, 2003 Sheet 6 of 7 US 6,576,457 B1 U.S. Patent Jun. 10, 2003 Sheet 7 of 7 US 6,576,457 B1 US 6,576,457 B1 1 2 FUNGAL MEDIA AND METHODS FOR Hormone accumulation in host tissue is affected by mycor CONTINUOUS PROPAGATION OF rhizal infection, with changes in the levels of cytokinin, VESICULAR-ARBUSCULAR MYCORRHIZAL abscisic acid, and gibberellin-like Substances and alteration (VAM) FUNGI IN ROOT ORGAN CULTURE in biomass partitioning (Barker et al., 1998; Danneberg et al., 1992; Dixon, 1992; Goicoechea, 1996). The effect of VAM fungal infection on the drought FIELD OF THE INVENTION resistance of plants is probably also due to the improvement of nutrient uptake. A mycorrhizal pathway of nutrient acqui The invention relates to novel fungal growth media and Sition would become much more important in dry Soil, methods for the in Vitro aseptic mass production of endo because the nutrients become much less mobile. There mycorrhizal fungal propagules, mainly Spores, using trans Seems little doubt that, like plant growth changes, mycor formed root organ culture. rhizal fungi can cause changes in plant water relations and BACKGROUND OF THE INVENTION may, at least in Some cases, improve drought resistance of the plant (Al-Karaki et al., 1998; Goicoevhea et al., 1996; Vesicular-arbuscular mycorrhizal (VAM) fungi form ben 15 Nelson, 1987). eficial Symbiotic associations with roots of a wide range of Morton and Benny (1990) described 149 species of VAM plants (Harley and Smith, 1983). The term “mycorrhiza,” fungi. The VA mycorrhizal fungi are all classified as belong which literally means “fungus root,” was first used in 1885 ing to the family Endogonaceae possessing coenocytic by A. B. Frank to describe the intimate associations between hyphae (with only rare septa or none at all), which contain fungal hyphae and the roots of forest trees. The hyphae of many nuclei. They have not propagated independently from VA mycorrhizal fungi penetrate and form arbuscules within host plant roots, and no Sexual reproduction has observed, root cortical cells and intercellular vesicles, but the external although both have been demonstrated in Some non hyphae extend further into the soil for mineral nutrient mycorrhizal, Saprophytic members of the Endogonaceae. uptake. A simple interpretation of mycorrhizal Symbiosis is Genera known to form VA mycorrhizae are Glomus, that the plant Supplies carbon compounds to increase fungal 25 Gigaspora, Acaulospora, Sclerocystis, and Entrophospora. growth and the plant benefits by having its root System Knowledge of the phylogenetic relationships of VAM fungi effectively extended. The Surface area of fungal hyphae can is fragmentary. There is Scant literature available on cyto be much greater than that of the plant roots (Smith and logical or Sexual processes involved in Spore formation for Gianinazzi-Pearson; 1992, Smith and Read, 1997). these fungi. The taxonomic position of the VAM fungi is, The importance of VA mycorrhizae in the growth and therefore, inferred from developmental processes and Spore nutrition of plants has recently been reviewed (Gianinazzi morphology (Schenck and Yvonne, 1992; Morton, 1995). and Schhepp, 1994; Smith and Read, 1997; Harrison, 1999). The VA mycorrhizal fungi produce large vegetative Spores On Soils low in available phosphate, the improved growth (often greater than 100 um in diameter) usually on hyphae observed in VA mycorrhizal plants compared with uninocu external to the roots. The genera have been classified accord lated controls is thought to be due largely to an improved 35 ing to the appearance of the Spores. Spores may be recovered Supply of phosphate. The external hyphae in Soil absorbs from the Soil around infected plants by wet-sieving through non-mobile nutrients such as phosphate (P), potassium (K), a Series Sieves of different sizes, ranging from 1,000 um to and ammonium (NH) beyond the root Zone and transfer 75 um. Either spores or fragments of infected roots may be phosphate from the fungus to the root cells. Thus, any used to inoculate further plants. Spores produced by process that was previously limited by the availability of 40 Gigaspora, Acaulospora, and Entrophospora are called azy phosphate will increase in rate. Improved phosphate gospores. Spores produced by Glomus and Sclerocystis, nutrition, as well as direct fungal effects, may be implicated termed chlamydospores, are presumably asexual, thick in the enhanced uptake of other macronutrients and micro walled resting cells. Additional morphological characters nutrients by plants. that are useful in Separating genera and Species of VAM Many VA mycorrhizal plants have increased resistance to 45 fungi are (i) the method of Spore germination; (ii) presence disease compared to non-mycorrhizal controls, although the or absence of Sporocarps; (iii) presence or absence of converse has also been reported. It has been Suggested that auxiliary cells; (iv) spore dimensions, (v) spore color; (vi) physical protection of potential entry points on the root spore ornamentation; (vii) number and type of Spore walls; Surface by the mycorrhizal fungi, and the reduced availabil and (viii) histochemical reactions (Hall, 1984; Morton, ity of carbon compounds to pests and pathogens (due to their 50 1988, 1995; Schenck and Yvonne, 1992).
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