Arbuscular Mycorrhizas: Producing and Applying Arbuscular Mycorrhizal Inoculum M. Habte and N. W. Osorio Department of Tropical Plant and Soil Sciences Acknowledgments the United States Department of Agriculture, nor the We are grateful to Dr. Mark Brundrett, who generously author shall be liable for any damage or injury resulting gave us permission to use his drawings, including the from the use of or reliance on the information contained one on the cover, in this publication. in this publication or from any omissions to this publi­ This work was funded by the Hawaii Renewable cation. Mention of a company, trade, or product name Resources Extension Program, supported by RREA or display of a proprietary product does not imply ap­ Smith-Lever 3D funds from CSREES/USDA. proval or recommendation of the company or product to the exclusion of others that may also be suitable. About this publication This information may be updated in more recent The information contained herein is subject to change publications posted on the CTAHR Web site, <www2. or correction. Procedures described should be consid­ ctahr.hawaii.edu>. For information on obtaining addi­ ered as suggestions only. To the knowledge of the au­ tional copies of this book, contact the Publications and thor, the information given is accurate as of July 2001. Information Office, CTAHR–UHM, 3050 Maile Way Neither the University of Hawaii at Manoa, the UH (Gilmore Hall 119), Honolulu, HI 96822; 808-956-7036; College of Tropical Agriculture and Human Resources, 808-956-5966 (fax); e-mail <ctahrpub@ hawaii.edu>. Table of contents Arbuscular mycorrhizal associations .......................................................................... 3 AMF functions............................................................................................................ 4 Sources of AMF inoculum.......................................................................................... 5 Producing crude inoculum .......................................................................................... 9 Producing root inoculum .......................................................................................... 12 Producing hydroponic and aeroponic inoculum ....................................................... 13 Inoculum storage ...................................................................................................... 13 Amount of inoculum to apply................................................................................... 15 Evaluating effectiveness of AMF inoculum ............................................................. 16 Appendixes 1. Extracting AMF spores from soil or crude inoculum ........................................ 22 2. Extracting spores from a crude inoculum and determining their viability ........ 25 3. A modified Hoagland’s solution II for use in AMF inoculum production ......... 27 4. Hydroponic production of AMF inoculum ........................................................ 28 5. Aeroponic production of AMF inoculum .......................................................... 30 6. Detecting and quatifying AMF colonization of roots ........................................ 31 7. Determining the abundance of infective propagules in crude inoculum and in soil ........................................................................................................... 33 8. Determining AMF symbiotic effectiveness by the pinnule technique and similar nondestructive approaches .............................................................. 35 9. Determining the P-sorption capacity of soils ..................................................... 39 10. Monitoring the symbiotic effectiveness of indigenous AMF............................. 41 11. Table of most probable numbers for use with 10-fold dilutions, five tubes per dilution ........................................................................................ 42 Copyright 2001 © College of Tropical Agriculture and Human Resources, University of Hawaii at Manoa; ISBN 1-929325-10-X Published by the College of Tropical Agriculture and Human Resources (CTAHR) and issued in furtherance of Cooperative Extension work, Acts of May 8 and June 30, 1914, in cooperation with the U.S. Department of Agriculture. Andrew G. Hashimoto, Director/Dean, Cooperative Extension Service/CTAHR, University of Hawaii at Manoa, Honolulu, Hawaii 96822. An Equal Opportunity / Affirmative Action Institution providing programs and services to the people of Hawaii without regard to race, sex, age, religion, color, national origin, ancestry, disability, marital status, arrest and court record, sexual orientation, or veteran status. CTAHR publications can be found on the Web site <http://www2.ctahr.hawaii.edu> or ordered by calling 808-956-7046 or sending e-mail to [email protected]. 2 Arbuscular Mycorrhizas: Producing and Applying Arbuscular Mycorrhizal Inoculum o one degree or another, most plants in their vide information that will enable interested individu­ natural habitats function under the influence of als to produce and then evaluate AMF inocula with Ta special group of soil fungi known as arbuscular minimal external assistance. mycorrhizal fungi (“AM fungi” or AMF). The exist­ ence of these fungi has been recognized for more than Arbuscular mycorrhizal associations a century, although they did not receive the attention The term “mycorrhiza” was coined by A. B. Frank, a they deserve until approximately 40 years ago. World­ researcher in Germany, more than 100 years ago. It wide, interest in AM fungi has now reached a point means “fungus-root,” and stands for the mutualistic wherein any discussion of agricultural biotechnology association existing between a group of soil fungi and that does not include their role in plant productivity higher plants. There are many types of mycorrhizal can hardly be considered complete. associations,(47) of which the endomycorrhizal associa­ Interest in AM fungi has been gradually growing in tion of the vesicular arbuscular (VA) type are the most Hawaii over the past 18 years. Many individuals and widespread geographically as well as within the plant organizations concerned with managing native plant kingdom. VA mycorrhizal fungi invade cortical cells species, restoring natural ecosystems, and producing inter- and intra-cellularly and form clusters of finely agronomic, horticultural, and forest plants with mini­ divided hyphae known as arbuscules in the cortex. They mal chemical inputs are interested in applying AMF also form membrane-bound organelles of varying technology. But a major, recurring challenge to large­ shapes known as vesicles inside and outside the corti­ scale utilization of AMF is the lack of availability of cal cells. Arbuscules are believed to be sites of exchange large quantities of high-quality AMF inoculum. The of materials between the host and the plant. Vesicles problem is largely due to the fact that AM fungi are generally serve as storage structures, and when they obligate symbionts—they require the presence of ac­ are old, they could serve as reproductive structures. tively growing plants during their reproduction. They Vesicles and arbuscules together with large spores con­ therefore cannot be cultured on laboratory media in the stitute the diagnostic feature of the VA mycorrhizal as­ same manner as other beneficial soil microorganisms sociations (Figure 1). Because vesicles are absent in such as Rhizobium bacteria. Fortunately, specialized two of the seven genera containing these fungi, the term techniques for AMF inoculum production have been in that is currently preferred by many researchers to rep­ development at the University of Hawaii and elsewhere. resent the association is arbuscular mycorrhizal (AM) During the past few years, we have received nu­ fungi rather than vesicular-arbuscular (VA) mycorrhizal merous inquiries from people in Hawaii and beyond fungi. Arbuscular mycorrhizal fungi occur on a wide about AMF and their inocula. This publication will try spectrum of temperate and tropical plant species and to answer common questions about AM fungi and pro­ are absent in less than 30 plant families(68, 99). 3 Figure 1. Diagram of a longitudinal section of a root showing the characteristic structures of arbuscular mycorrhizal fungi. (Adapated from M. Brundrett.(57)) Fungal appresorium Root epidermis at the point of entry into the root Hypodermis Intracellular hyphae Cortex Intracellular hyphae Arbuscules Vesicle AMF functions Roles in plant nutrition AM fungi absorb N, P , K, Ca, S, Fe, Mn, Cu, and Zn cause of its antagonistic interactions with other nutri­ from the soil and then translocate these nutrients to the ents, or because it inhibits mycorrhizal formation(71). plants with whose roots they are associated.(33, 49, 80, 101) Studies with the forage tree Leucaena leucocephala, Their most consistent and important nutritional effect which is highly dependent on mycorrhizal association, is to improve uptake of immobile nutrients such as P, have shown that the AMF symbiosis can decrease the Cu, and Zn.(73, 84) AM fungi have their greatest effect plant’s external P requirement, reducing it to as much when a host plant not associated with them is deficient as 40 times less than the plant would require for good in P. They are also very useful to plant species that in­ growth in the absence of AMF (MH, unpublished). herently lack either morphological or physiological The ability of AMF to reduce plants’ external P mechanisms for efficient P uptake.(68,