IIIHIIIHIIIHIIIHII US005178642A United States Patent (19) (11) Patent Number: 5,178,642 Janerette 45) Date of Patent: Jan. 12, 1993 54 INOCULUM FROM ECTOMYCORRHIZAL Glomus epigaeus' New Phytol, vol. 87, pp. FUNG FORMING ENDOMYCORRHZAL 345-354. NFECTION WITH HERBACEOUS PLANTS Crush, J. R., et al. (1975) "Preliminary Results on the 76 Inventor: Carol A. Janerette, 2218 Dickens Production of Vesicular-Arbuscular-Mycorrhizal In Terr., Newark, Del. 19702 oculum by Freeze Drying' (Sanders et al. Ed.) Ro thamsted Exper. Sta., Harpenden, U.K. pp. 485-493. (21) Appl. No.: 725,392 Piché, Y. et al. (1982) "Development of Mycorrhizae 22 Filed: Jun. 28, 1991 Extramatrical Myceliumand Sclerotia on Pinus Strobus Seedlings' New Phytol, vol. 92, pp. 128-137. Related U.S. Application Data Daniels Hetrick, B. A. (1984) " Germination and 63 Continuation of Ser. No. 385,515, Jul. 27, 1989, aban In Vitro Culture of Vesicular-Arbuscular Mycorrhizal doned, which is a continuation-in-part of Ser. No. Fungi', Applications in Mycorrhizal Fungi in Crop Pro 83,474, Aug. 10, 1987, abandoned. duction, Univ. of Fla., pp. 17-22. 51 Int. Cl...... A01G 1/00; A01N 63/00; Hile, N., et al. (1969) “In Vitro Culture of Pisolithus C12N 1/14 tinctorius Myceluim' Mycologia 61, pp. 195-198. 52 U.S. Cl...... 47/58; 424/93 N; Marks, D. H., (1975) "Mycorrhizal and Establishment 424/93 Q; 435/254; 435/911 of Freeson Stripmined Land” The Ohio Journal of Sci (58) Field of Search ...... 47/1.1, 58; 800/200; ences vol. 75, No. 6, pp. 288-297. 435/254, 520,911, 176; 7 1/5, 6, 7, 59; 424/93 Marx, D. H. (1980) “Role of Mycorrhizae in Foresta Q, 93 M, 93 N, 93 R tion of Surface Mines' Proc. Trees for Redamation, pp. 109-116. (56) References Cited Marks, G. C., et al. (1972) "Structure, Morphogenesis U.S. PATENT DOCUMENTS and Ultrastructure of Ectomycorrhizal" In Ectomycorr 2,813,821 11/1957 Updegraffet al...... 195/16 hizal Their Ecology and Physiology (Marks, G. C., ed.) 3,580,811 5/1971 Hidy et al...... 435/911 Academic Press Inc., New York, pp. 2-35. 4,294,037 10/1981 Mosse et al...... 47/59 Ruehle, J. L., et al., (1979) “Fiber, Food, Fuel, and 4,327,181 4/1982 Litchfield et al. 435/76 Fungal Symbionts" Science, vol. 206, pp. 419-422. 4,551,165 l/1985 Warner ...... 71/24 4,594,809 6/1986 Ower et al. .. 47/1, Tinker, P. B. H., (1975) Proc. of the 29th Symposium of the Soc. of Experimental Biology, pp. 325-349. 4,599,312 7/1986 Mugnier et al...... 35/1723 Snell and Dick, "A Glossary of Mycology", Cover FOREIGN PATENT DOCUMENTS page & pp. 96 & 193, Harvard U. Press (1957). 209627 1/1987 European Pat. Off...... 47/1.1 Marx, "Growth and Ectomycorrhizal Development of 207058 9/1971 France ...... 47/1.1 Loblolly Pine Seedlings in Fumigated Soil Infested with the Fungal Symbiont Pisolithus Tinctorius', For OTHER PUBLICATIONS est Science, vol. 22, No. 3, pp. 245-254 (1975). " Formation in Eucalyptus: I. Pure Watrud, "Methods and Principles of Mycorrhizal Re Culture Malajczuk N. et al., Synthesis, Host Specificity search', p. 81, The American Phytopathological Soci and Mycorrhizal Compatibility with ", 92 ety (1982). The New Rhytologist, (1982), pp. 467-482. Hudson, "Fungal Biology', pp. 218-219 (1986). Harley, J. L., (1972) The Biology of , 2nd ed., Smith & Douglas, "The Biology of Symbiosis', pp. Leonard Hall, p. 79. 152-153 (1987). Moore-Landecker, E. (1982) "Aeration' Fundamentals of the Fungi, Prentice-Hall, Inc., Englewood Cliffs, N.J., Primary Examiner-James R. Feyrer pp. 304 and 305. Attorney, Agent, or Firm-Darby & Darby Malloch, D. (1981), "Special Techniques' Moulds: (57) ABSTRACT Their Isolation, Cultivation and Identification Univ. of Toronto Press, Toronto pp. 38 and 39. Fungal inoculants for plants producing ectomycorr Alexopoulos, C. et al. (1979) "Mycorrhizal' Introduc hizae with woody plants or endomycorrhizae with leafy tory Mycology 3rd ed., John Wiley and Sons, New York, plants are formed in perlite from microsclerotia-produc pp. 450-452. ing fungi such as Cenococcum geophilium. Daniels, B. A. et al., (1981) "Evaluation of the Commer cial Value of the Vesicular-Arbuscular Mycorrhizal 16 Claims, No Drawings 5,178,642 1. 2 As far as known, however, inoculants suitable for NOCULUM FROM ECTOMYCORRHIZAL FUNGI leafy plants such as wheat or the common vegetables FORMING ENDOMYCORRHIZAL INFECTION corn, onion, asparagus and the like have not heretofore WITH HERBACEOUS PLANTS been produced. Compare Watrud, "Spore Germination 5 and Axenic Culture of Endomycorrhizae,' writing at This application is a continuation of Ser. No. page 81 of Methods and Principles of Mycorrhizal Re 07/385,515, filed Jul. 27, 1989, now abandoned, which, search, the American Phytopathological Society in turn, was a continuation-in-part of Ser. No. (1982): ". . . To date, successful axenic subculture of 07/083,474, filed Aug. 10, 1987, now abandoned. hyphae of vesicular-arbuscular mycorrhizae has yet to 10 be reported. . . .' See also Hudson, Fungal Biology, pp. BACKGROUND OF THE INVENTION 218 and 219, Edward Arnold (1986); Smith and Doug 1. Field of the Invention . las, The Biology of Symbiosis, pp. 152 and 153, Edward This invention relates to mycology, more specifi Arnold (1987); and Mugnier et al., U.S. Pat. No. cally, to a process for the production of inoculants for 4,599,312, "Method of Producing Endomycorrhizian herbaceous plants and to the inoculants so produced. 5 Fungi with Arbuscules and Vesicles in Vitro" (1986). 2. Prior Art As noted above, this invention relates to mycology, DETAILS OF THE INVENTION and the momenclature used is intended to be consistent In accordance with the present invention, it has been with Snell and Dick, A Glossary of Mycology, Harvard found that fungal inoculants for herbaceous plants can U. Press, Cambridge, Mass. (1957). 20 be made by a versatile process in which (1) mycelia of Mycorrhizae are symbiotic associations between the selected ectomycorrhizal fungi are grown from cultures hyphae of certain fungi and the absorbing organs-typi on a solid medium and (2) mycelia still in the solid ne cally the roots of plants. They are classified according dium (fungal plugs) are axenically added to perlite wet to the manner in which the fungus infects the root. The ted with a nutrient solution and incubated in vitro. After two main types are ectomycorrhizae in which fungal 25 about three months' incubation, the product is an inocu hyphae penetrate the intercellular spaces between root lant which can be used (3) either broadcast, e.g., with cells without entering the interior of the cells, and en forest trees (ectomycorrhizal), or axenically or other domycorrhizae where projections of the fungus enter wise with leafy plants (endomycorrhizal). the interior of the cell. The solid medium used in the growth of mycelia (1) Ectomycorrhizae are generally associated with trees 30 is generally hardened agar. Modess Modification of and other woody species and are formed by "higher Hagem Agar in the agar can be the nutrient. Other fungi" that are found in a number of families of basidio nutrients, such as a modified Melin and Rama Das Agar, mycetes and ascomycetes. In ectomycorrhizae, the nor can also be used. Three or four weeks in the dark at mal branching pattern in the roots is changed. Roots around 25 C. provide sufficient time for growth. infected with ectomycorrhizal fungi are short, swollen, 35 Fungal plugs taken from the agar growth medium are branched and lack root hairs. (2) axenically added to jars containing perlite and nutri Endomycorrhizae are generally associated with her ent solution and incubated in vitro for about three baceous plants such as grasses, corn, onions and many months in the dark under quiescent and substantially more, however there are some trees that also form en anerobic conditions. In this connection, "quiescent' domycorrhizae. Endomycorrhizae are formed from simply means without agitation and "substantially ane produced by "lower fungi," classified as zygo robic' means that air or oxygen need not be supplied to mycetes, and belong to one family, the Endogonales. the culture, not that they must be excluded. A useful The fungi that produce these spores are unknown. nutrient solution is Fowells and Krauss's pine nutrient From the outside, the infected roots look normal, and solution modified by the addition of glucose and thia the only way to detect the infection is by microscopic 45 mine (Fowells and Krauss, "The inorganic nutrition of examination. Then one can see that the hyphal projec Loblolly pine and Virginia pine with special reference tions of the fungus have invaded the cells forming small to nitrogen and phosphorus' Forest Science 5:95-112 branches (arbuscles) and/or swellings at tips of some (1959)). hyphae (vessicles). Many endomycorrhizae are called It should be noted that the inert carrier used in the "Vessicular-Arbuscular Mycorrhizae' or VAM, be 50 nutrient solution is somewhat selective. The most satis cause of the presence of vessicles and arbuscles inside factory results have been obtained with perlite as an root cells. Ectomycorrhizae do not have vessicles or effective carrier although some modified clays can be arbuscles. used. Vermiculite has been found ineffective under the Ectomycorrhizal fungal inoculants for woody plants conditions disclosed. The proportion of carrier to liquid such as pine have been produced. See, for example, 55 nutrient (carrier/nutrient weight ratio) is not critical but Litchfield et al., U.S. Pat. No. 4,327,181, "Aerobic Sub around at least 2/1 has been found satisfactory to pro merged Fermentation of Sporulating Ectomycorrhizal vide an effective saturated support. Fungi" (1982) disclosing liquid culture of selected fungi As noted, the inoculum prepared above can be used for broadcast over forest soil. Marx et al., "Growth and (3) by broadcast sowing with woody plants in the fash Ectomycorrhizal Development of Loblolly Pine Seed ion well known (ectomycorrhizae) or to facilitate the lings in Fumigated Soil Infested with the Fungal Symbi growth of leafy plants (endomycorrhizae). In the latter ont Pisolithus tinctorium,' Forest Science Vol. 22 No. 3, case, one axenically germinated seed can be placed on pp. 245-254 (1975), show the use of Pisolithus tinctorius top of one teaspoon full of the fungus inoculum about cultured in an agar/vermiculite/peat moss medium in three centimeters below the surface of a flower pot forest nurseries. See also Mosse et al., U.S. Pat. No. 65 filled with perlite. The plants are then grown in the 4,294,037, "Production of Mycorrhizal Fungi" (1981) greenhouse for four months. The pots are saturated and Warner, U.S. Pat. No. 4,551,165, "Mycorrhizal twice weekly with a low-phosphorus nutrient solution Seed Pellets” (1985). (Peter's Lo Phos Fertilizer). 5,178,642 3 4 Known ectomycorrhizal fungi that formed en C. in the dark in petri plates on Hagen's nutrient agar domycorrhizal infection with corn, wheat, onion, and modified by Modess (Modess, O. 1941. Zur Kenntnis /or asparagus plants include the following: der Mykorrhizabildner von Kiefer und Fichte. Syn TABLE I bolae Bot. Upsalienses 5(1):1-146) as shown in Table II. Name Source Rhizopogon roseolus John Melhuish TABLE II (Melhuish #20) U.S.D.A. Forest Service 2. Pisolithus tinctorius American Culture Formulation" of Modess Modification of Hagem Agar (ATCC 38054) Collection KH2PO 0.5 g 3. Ananita muscaria John Melhuish O MgSO4.7H2O 0.5 g (Melhuish #21) U.S.D.A. Forest Service NH4Cl 0.5 g 4. Astraeus hygrometricus American Type Culture FeCl3 (1% solution) .0 ml (ATCC #46449) Collection Glucose 5.0 g 5. Cenococcum geophilum American Type Culture Malt extract 5.0 g (ATCC #38052) Collection Agar 100 g 6. aurantium American Type Culture H2O (distilled) 1000.0 ml (ATCC #58507) Collection 15 7. Athelia neuhoffi John Melhuish Final pH = 4.7 (Melhuish #47) U.S.D.A. Forest Service 8. Boletinelius nerulioides John Melhuish Subsequently, 0.5 mm plugs were cut from the pe (Melhuish iE64) U.S.D.A. Forest Service 9. Hebelona anthracophilum John Melhuish riphery of the fungal colonies and axenically added to (Melhuish #54) U.S.D.A. Forest Service 20 separate test tubes containing 40 ml (8 g) of strained 10. Hebelona crustuliniforme John Melhuish perlite and 20 ml of Fowells and Krauss's modified (Melhuish #53) U.S.D.A. Forest Service 11. Paxillus involutus Americah Type Culture nutrient solution had previously been autoclaved at (ATCC #46218) Collection 120° C. and 15 lbs. pressure for 20 minutes. Inoculated 12. Piloderma bicolor John Melhuish test tubes were incubated for three months in the dark at (Melhuish #50) U.S.D.A. Forest Service 25 24 C. 13. Rhizopogon nigrescens John Melhuish (Melhuish #38) U.S.D.A. Forest Service TABLE III 14. Scleroderma albidum American Type Culture Modified Fowells and Krauss's Nutrient Solution Formulation (ATCC #58021) Collection 15. Scleroderma polyrhizum John Melhuish NH4NO3 0.68 ml of 1 molar stock solution (Melhuish #68) U.S.D.A. Forest Service 30 KH2PO4 0.15 ml of molar stock solution 6. Suilluscothurnatus John Melhuish MgSO4 0.95 ml of 1 molar stock solution (Melhuish #31) U.S.D.A. Forest Service CaCl2 0.95 ml of l molar stock solution 17. Alipova pachyploeus Dr. Donald Marx NaCl 0.95 ml of molar stock solution (Marx #258) U.S.D.A. Forest Service KC 2.92 ml of 1 molar stock solution 18. Boletus punctipes John Melhuish Feric Citrate 1.0 ml of stock solution (Melhuish #15) U.S.).A. Forest Service Combined minor elements 0.13 m of stock solution 19. Lactarius deliciosus American Type Culture 35 Thiamine 50 g (microgram) (ATCC #36647) Collection Glucose 2.5g HC 1.5 ml of 1N solution H2O (distilled) 1000 in While the reasons for the efficacy of the present inoc *Final pH = 5.7 ulum and its preparation are not completely under Stock Solution of Feric stood, it can theoretically be explained as follows. Citrate: Fungi are able to propagate from a number of sources Ferric Citrate 2.5 g or propagules including spores, infected root fragments, Citric Acid i.6 g. H2O 250 ml mycelia, hyphae, etc. Another propagule is sclerotia (cf. Stock Solution of Minor Elements: Willets, "Sclerotium Formation,” Filamentous Fungi 45 CuSO4.5H2O 8.0 mg 3:197-213 (1978)), and this is believed involved here. ZnSO4. 4H2O 21.8 mg The sclerotia are too small to be seen by the naked eye MnCl2.4H2O 180.0 mg but they can be seen with a microscope. Another propa H3BO3 285.5 mg gule is denoted "microsclerotia' (cf. Baard et al. H2M04. H2O 2.1 mg "Structure and Lysis of Microsclerotia ...,' Trans. Br. H2O 00 in Mycol. Soc. 77(2):251-260 (1981)). The development of 50 the mycelium in contact with the inert support and Five ml of Fowells and Krauss's nutrient solution was nutrient thus proceeds until sclerotia, microsclerotia, or added to each test tube and thoroughly mixed with the their initials are produced. Operable fungi for the pres incubated inoculum before use. ent inventions are, in any event, those fungi which pro 55 B. Growth of Endomycorrhizal Structures in Leafy duce sclerotia or microsclerotia. Spores are produced at Plants the same time, and the product, containing fungal hy Seeds of onion, wheat, corn and asparagus that had phae, microsclerotia, spores, sclerotia, or their initials is been soaked in distilled water overnight were washed an inoculum with a good shelf life. for 1 minute in 1% HgCl2 with 1 ml of Tween 20 per There follow examples illustrating the invention in liter and rinsed three times with sterile distilled water. detail: Seeds were then axenically placed in sterile petri plates EXAMPLES 1-6 containing moistened filter paper and allowed to germi nate in the dark at 24' C. When emerged radicles were A. Preparation of Inoculum between 1 cm and 3 cm long (within 1 week for all Selected fungi (Cenococcum geophilum, Pisolithus 65 seeds) they were planted in the greenhouse in 4-inch tinctorius, Astraeus hygrometricus, Amanita muscaria, pots filled with strained horticultural grade perlite. A Rhizopogon roseolus, and Scleroderma aurantium) were hole was dug 3 cm from the surface, and one teaspoon axenically grown from mycelia for three weeks at 24 full of the fungus inoculum was put in the hole with one 5,178,642 5 6 germinated seed placed on top of the inoculum and Results of these studies indicate that inocula pro covered with perlite. Twenty pots were prepared for duced from ectomycorrhizal fungi by procedures out each fungus being tested along with twenty control lined above can be used to induce endomycorrhizal plants of each seed type. Each pot was placed on top of infection in herbaceous plants. Furthermore, all the an inverted, empty 5-inch pot to eliminate cross-con 5 inocula produced were shown to form mycorrhizae tamination on the green-house bench. All pots were with loblolly pine seedlings. saturated with the Fowells and Krauss's solution. The pots were watered daily and, in addition, Saturated EXAMPLES 7-19 twice a week with Peter's Lo Phos Fertilizer. A. Preparation of Inoculum Each of the six fungi being tested was used as inocu 10 lum for the onion seeds. Only Cenococcum geophilum The preparation of inocula as in Examples 1-16 was and Pisolithus tinctorius were used to inoculate the corn, substantially repeated except that different fungal spe wheat, and asparagus seeds. cies were employed and in a different agar medium. Inoculated plants were grown under normal green Selected fungi (Athelia neuhofii, Boletinellus meruli house conditions with the day length extended to 12 15 oides, Hebeloma anthracophilum, Hebeloma crus hours with incandescent light bulbs. tuliniforme, Paxillus involutus, Piloderma bicolor, After 8 weeks, plants were periodically removed Rhizopogon nigrescens, Scleroderma albidum, Sclero from the greenhouse and the roots and perlite in which derma polyrhizum, Suillus cothurnatus, Alpoya pachyplo they were grown was examined as follows. Roots were eus, Boletus punctipes, and Lactarius deliciosus) were individually washed in distilled water until free of per 20 axenically grown from mycelia for four weeks at 24' C. lite. The perlite that the plant grew in was agitated in 1 in the dark in petri plates on a modification of Melin and liter of distilled water and filtered along with the root Rama Das nutrient agar (Melin and Rama Das "Influ washings through one layer of cheesecloth. This filtrate ence of root metabolites on the growth of tree mycor was then refiltered through nested 230 mesh and 325 rhizal fungi" Physiol. Plant. 7:851-858 (1954)). mesh sieves. The precipitate on the 325 mesh sieve was 25 TABLE IV saved and examined microscopically. The cleaned roots Formulation of Modified Melin and Rama Das Agar were cleared and stained using the procedure of Kor KH2PO4 1.0 gm manik et al. (Kormanik, Bryan and Schultz. "Procedure MgSO4. 7H2O 0.5 gm and equipment for staining large numbers of plant root NH4 Tartrate 5.0 gm 30 ZnSO4 (1:500) 0.5 ml samples for endomycorrhizal assay." Can. J. Microbiol. Fe Citrate (1%) 0.5 ml 26:536-538 (1980)). This procedure allows the micro Thiamin 50.0 ug (microgram) scopic visualization of fungi and fungal propagules Glucose 20.0 gm within the intact root. Agar 10.0 gm When roots were examined after 8 weeks in the Distilled H2O to 1000 In greenhouse, there was no apparent internal fungal infec 35 tion in those observed; however, mycelia were present Subsequently, 0.5 mm plugs were cut from the periph on the exterior of the roots. The perlite washings did ery of the fungal colonies and two plugs from each reveal the presence of microsclerotia in every instance. fungus were axenically added to a jar containing 100 ml When plants were similarly treated for examination of strained perlite and 55 ml of Fowells and Krauss's after 16 weeks, endomycorrhizal infection was well modified nutrient solution (Fowells and Krauss. "The established in plants treated with all six fungi, and nu inorganic nutrition of loblolly pine and Virginia pine merous microsclerotia were present on and around the with special reference to nitrogen and phosphorus', roots. Root samples from plants grown for 16 weeks or For. Sci. 5:95-112)(1959)) as shown above in Table III). more were excised and fixed, dehydrated, and embed 45 The jars containing the perlite and nutrient solution had ded in epoxy resin according to methods in Pizzolato, previously been autoclaved at 120° C. and 15 lbs. pres T. D., 1978, "A tannic acidferric chloride-toluidine blue sure for 20 minutes. The inoculated jars were incubated stain for wood amyloplasts embedded in epoxy resin," for 3 months in the dark at 24 C. Forest Science 24:49-51, and subsequently stained in Alsops reagent for 1 minute at 130° C. by the methods 50 B. Ectomycorrhizal Infection in Asparagus Plants of Alsop (1974) and Pizzolato (1984) (Alsop, D. 1974, Asparagus seeds that had been soaked in distilled "Rapid single solution polychrome and staining of se water overnight were washed for one minute in 1% mithin epoxy sections by polyethylene glycol 200 (PEG HgCl2 with 1 ml of Tween 20 per liter and rinsed three 200) as a stain solvent,' Stain Technology 49:265-272. times with sterile distilled water. Seeds were then axeni Pizzolato, T. D. 1984, "Vascular system of the fertile 55 cally placed in sterile petri plates containing moistened floret of Anthoxanthium odoratum L.,' Botanical Ga filter paper and incubated in the dark at 24' C. Six days zette 145(3):358-371.) The light microscope sections of later, the germinated seeds were planted in 4-inch pots internal root cells obtained with these procedures re filled with strained horticultural grade perlite. A hole vealed the presence of intercellular and intracellular was dug 3 cm from the surface, and one teaspoon full of hyphae, vessicles, and spores with all fungi and plants the fungus inoculum was put in the hole with one germi tested. Control plants yielded no evidence of infection nated seed placed on top of the inoculum and covered or microsclerotia. with perlite. Ten pots were prepared for each fungus An examination of the fungus inocula used to infect being tested along with twenty control plants. Each pot the plants showed the presence of numerous sporulating was placed on top of an inverted empty 5 inch pot to hyphae and spores but no discernable microsclerotia, a 65 eliminate contamination on the greenhouse bench. All situation indicating that microsclerotia initials were pots were then saturated with the Fowells and Krauss's present and required more time, plant exudates or both solution. The pots were watered daily and, in addition, for maturation. saturated twice a week with Peter's Lo Phos Fertilizer. 5,178,642 7 8 Inoculated plants were grown under normal green trient solution including sources of carbon, nitro house conditions. After 4 months, plants were removed gen, potassium, magnesium, zinc, sulfur and iron, from the greenhouse and the roots and perlite in which and they were grown was examined as follows: Roots were maintaining said contact in the absence of living plant individually washed in distilled water until free of per 5 matter in darkness and without aeration for a per lite. The perlite that the plant grew in was agitated in iod of time effective to stress the mycelia, inducing one liter of distilled water and filtered along with the it to produce at least microsclerotia or its initials root washings through one layer of cheesecloth. This among said mycelia, and filtrate was then refiltered through nested 230 mesh and obtaining an ectomycorrhizal inoculum containing at 325 mesh sieves. The precipitate on the 325 mesh sieve 10 least microsclerotia or the initials of microsclerotia, was saved and examined microscopically. The cleaned which inoculum is capable of producing intracellu roots were cleared and stained by the procedure of lar hyphae when contacted with herbaceous plants Kormanik et al. (Kormanik, Bryan and Schultz, "Proce and; is effective to form mycorrhizal symbiotic dure and equipment for staining large numbers of plant associations with the roots of woody plants. root samples for endomycorrhizal assay.' Can. J. Mi 5 5. The process of claim 4 wherein said maintaining is crobiol. 26:536-538) (1980). This procedure allows the conducted for about three months at about room tem microscopic visualization of fungi and fungal propa perature. - gules within the intact root. When plants were exam 6. The process of claim 4 wherein said mycelia of ined, endomycorrhizal infection was well established ectomycorrhizal fungi are selected from the fungus with all of the fungi tested, and numerous microscle 20 species consisting of Rizopogon roseolus, Pisolithus tinc rotia were present on and inside the roots. torius, Amanita muscaria, Astraeus hygrometricus, Ceno An examination of the fungus inocula used to infect coccum geophilum, Scleroderma aurantium, Athelia neu the plants showed the presence of numerous sporulating hoffii, Boletinellus merulioides, Hebeloma anthraco hyphae and spores but no discernable microsclerotia philum, Hebeloma crustuliniforme, Paxillus involutus, were present indicating that microsclerotia initials were 25 Piloderma bicolor, Rhizopogon nigrescens, Scleroderma present and required more time, plant exudates or both albidum, Scleroderma polyrhizum, Suillus cothurnatus, for maturation. Alpova pachyploeus, Boletus punctipes, and Lactarius Each of these fungi forms mycorrhizae with Virginia deliciosus. Pine and/or Loblolly Pine in axenic culture. 7. The process of claim 4 wherein said carrier is se Results of these studies indicate that inocula pro 30 lected from the group consisting of perlite and modified duced from ectomycorrhizal fungi by procedures out clays. lined above can be used to induce endomycorrhizal 8. The process of claim 7 wherein in said contacting infection in herbaceous plants. and maintaining steps the weight ratio of said particu Having described my invention, I claim: late carrier to said growth medium is at least 2:1. 1. A method of growing plants having mycorrhizae 35 9. The process of claim 8 wherein said carrier is per associated with their roots comprising the steps of: lite. obtaining mycelia of an ectomycorrhizal, microscle 10. The process of claim 4 wherein the propagule rotia-producing fungus, used in the contacting step are obtained by axenically contacting propagule of said mycelia with a growth growing said mycelia in the dark in petri dishes on a medium containing a particulate carrier and a nu nutrient-containing solid second medium. trient solution including sources of nitrogen, potas 11. The process of claim 10 wherein the contacting sium, magnesium, ammonia, zinc, sulfur, iron, step comprises cutting plugs from said petri dishes on Sugar and thiamine, which said mycelia have been grown and axenically maintaining said contact in the absence of living plant contacting the plugs with the growth medium and par matter in darkness and without aeration for a per 45 ticulate carrier. iod of time effective to stress the mycelia, inducing 12. The process of claim 11 wherein said carrier is it to produce at least microsclerotia or its initials, perlite and said solid medium is agar. obtaining an ectomycorrhizal inoculum from said 13. The method of claim 4 wherein the growth me mycelia which is capable of producing intracellular dium is Fowells and Krauss's pine nutrient solution hyphae when contacted with herbaceous plants, 50 modified by the addition of glucose and thiamine, and germinating a seed to produce a seedling, pH balanced with acid. infecting said seedling with said inoculum and con 14. A process of producing a mycorrhizal inoculant tacting said seedling with a second nutrient solu of a normally ectomycorrhizal fungi for use with either tion, and woody or herbaceous plants, comprising the steps of: obtaining plants having a symbiotic association with 55 obtaining mycelia of an ectomycorrhizal fungus, said fungi. contacting a propagule of said mycelia with a Fo 2. The method of claim 1 wherein said seed is of a wells and Krauss's nutrient solution modified by herbaceous plant and wherein said symbiotic associa the addition of glucose and thiamine and a particu tion is of endomycorrhizal morphology. late carrier, and 3. The method of claim 1 wherein the nutrient solu 60 maintaining said contact in the absence of living plant tion is Fowells and Krauss's pine nutrient solution and matter in darkness and without aeration for a per the growth medium is pH balanced with acid. iod of time effective to stress the mycelia, inducing 4. A process of producing a mycorrhizal inoculant of it to produce at least microsclerotia or its initials a normally ectomycorrhizal fungi for use with either among said mycelia, and woody or herbaceous plants, comprising the steps of: 65 obtaining an ectomycorrhizal inoculum containing at obtaining mycelia of an ectomycorrhizal fungus, least microsclerotia or the initials of microsclerotia, contacting a propagule of said mycelia with a growth which inoculum is capable of producing intracellu medium containing a particulate carrier and a nu lar hyphae when contacted with herbaceous plants. 5,178,642 10 15. A mycorrhizal inoculant for either woody or 16. The inoculant of claim 15 wherein said mycelia of herbaceous plants produced from mycelia of at least one isolated ectomycrorrhizal fungi are selected from the isolated and cultivated ectomycorrhizal fungi in the group consisting of Rizopogon roseolus, Pisolithus tinc absence of living plant matter, said inoculant consisting torius, Ananita muscaria, Astraeus hygrometricus, Ceno coccum geophilum, Scleroderma aurantium, Athelia neu essentially of said fungi and including at least microscle hofii, Boletinellus merulioides, Hebelona anthraco rotia or the initials of microsclerotia of said fungi, said philum, Hebeloma crustuliniforme, Paxillus involutus, inoculant being capable of producing intracellular hy Piloderma bicolor, Rhizopogon nigrescens, Scleroderma phae when contacted with herbaceous plants which are albidum, Scleroderma polyrhizum, Suillus cothurnatus, effective to form endomycorrhizal symbiotic associa 10 Alpova pachyploeus, Boletus punctipes, and Lactarius tions with herbaceous plants and producing mycorrhi deliciosus. zal symbiotic associations with woody plants. k k k

15

20

25

30

35

45

50

55

65