Mycorrhiza Agriculture Technologies

Chapter X Mycorrhiza Agriculture Technologies

John A. Menge Associate Professor Department of Plant Pathology University of California Riverside, CA 92521 Contents

Page Introduction—What Are Mycorrhizal Fungi? ...... 185 Ectomycorrhizae ...... ,,, .,, ,, . . . .,.,.,...... 185 Vesicular-Arbuscular (VA) Mycorrhizae ...... , ...... 185 How Do Mycorrhizal Fungi Improve the Growth of Agricultural Plants? ...... 187 Mycorrhizae as Substitutes for Fertilizers ...... ,.,., ,..,,, 189 Current Commercial Use of Mycorrhizal Fungi ...... 191 Disturbed Sites ...... ,,...... 191 Fumigated or Chemically Treated Sites ...... 191 Greenhouses .. .,..., ...... ,.,., ...... 192 Commercial Production and Inoculation With Mycorrhizal Fungi ...... 192 Potential Uses of Mycorrhizal Fungi...... 195 Constraints on the Commercial Use of Mycorrhizal Fungi ...... 197 Effects of Mycorrhizal Fungi on Agriculture...... 198 Conclusions and Recommendations ...... 198 References ...... 199

Table Table No. Page 1. Estimated Cost of production of Vesicular-Arbuscular Mycorrhizal Inoculum on Sudangrass in 4 Inch Pots ...... 194

Figures

Figure No. Page l. Diagram of a Typical Ectomycorrhiza Including the Hartig Net, Fungal Mantle, and External Hyphae ...... 185 2. Diagram of a Typical Vesicular-Arbuscular Mycorrhiza Including Vesicles, Arbuscules, Spores, and External Hyphae ...... 186 3. The Growth of Citrus Seedlings With and Without VA Mycorrhizal Fungi and at Different Nutrient Levels ...... ,...... , 188 4. Dry Weights of Mycorrhizal and Non-Mycorrhizal Brazilian Sour Orange and Troyer Citrange Seedlings Fertilized With Different Amounts of Phosphorus., . 190 5. Proposed Scheme for the Commercial Production of Vesicular-Arbuscular Mycorrhizal Inoculum ...... 193 Chapter X Mycorrhiza Agriculture Technologies

INTRODUCTION-WHAT ARE MYCORRHIZAL FUNGI?

Mycorrhizal fungi are beneficial fungi that Figure 1 .—Diagram of a Typical Ectomycorrhiza are associated with plant roots via a symbiotic Including the Hartig Net, Fungal Mantle, and External Hyphae (courtesy D. H. Marx) association whereby both the host plant and the fungus benefit. Mycorrhizae are the structures formed by the symbiotic association between plant roots and mycorrhizal fungi. My- corrhizae contain both plant roots and fungal tissues. In nature, mycorrhizae are far more common than non-mycorrhizal roots (24,92,94). Nearly all plant species are associated with mycorrhizal symbionts. Because of their importance to plants and their widespread distribu- tion, mycorrhizae must be considered in all aspects of plant ecology, crop science, and agriculture. Mycorrhizal fungi are divided into four very different types (66): ectomycorrhiza, vesicular- arbuscular mycorrhiza (abbreviated as VA mycorrhiza), ericaceous mycorrhiza, and orchidaceous mycorrhiza. As indicated by their rhizal fungi penetrate between the cells of the names, ericaceous mycorrhiza and orchida- host root, develop around the root cortical ceous mycorrhiza are associated with erica- cells, replace the host middle lamella, and form ceous plants (blueberries, cranberries, azaleas, what is called the “Hartig net’’—the distin- etc.) and orchidaceous plants (orchids), respec- guishing feature of ectomycorrhizae. In re- tively. Because of the relatively low economic sponse to the fungal invasion, the host roots impact of these plants and the small amount usually swell substantially and may branch di- of available data on these types of mycorrhiza, chotomously or in a coralloid manner. The root they will not be discussed further. cells are not injured, however, and function of the roots is enhanced, as we shall discuss. Ectomycorrhizae Vesicular-Arbuscular (VA) Ectomycorrhizae are associated primarily Mycorrhizae with trees such as pine, hemlock, spruce, fir, oak, birch, beech, eucalyptus, willow, and pop- VA mycorrhizal fungi have the widest host lar, Ectomycorrhizae are formed by hundreds range and form by far the most common type of different fungal species belonging to the of mycorrhizae. VA mycorrhizae occur on Basidiomycetes (mushrooms and puffballs) and liverworts, mosses, ferns, some conifers, and Ascomycetes (cup fungi and truffles). These most broad-leaved plants. Only 14 families that fungal symbionts are stimulated by root ex- are considered primarily non-mycorrhizal (28). udates and grow over the surface of host feeder The important crop families that are non-my- roots to form a thick fungal layer known as a corrhizal are Cruciferae (cabbage, broccoli, fungal mantle (figure 1). Hyphae of ectomycor- mustard, etc.); Chenopodiaceae (spinach, beet,

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186 etc.); Cyperaceae (sedges); and Caryophyllaceae The hyphae of VA mycorrhizal fungi pene- (carnation, pinks, etc.). wetland rice also is usu- trate directly into the root cortical cells of host ally non-my corrhizal. Nearly all other impor- plants. Inside of the host plant cells, VA mycor- tant agronomic crops including wheat, rhizal fungi form minute coralloid structures potatoes, beans, corn, alfalfa, grapes, date known as arbuscules (figure 2). Arbuscules are palms, sugar cane, cassava, and dryland rice thought to be the site of nutrient transfer be- are associated with VA mycorrhizal fungi. Al- tween the symbiotic partners. The host plants though many trees have ectomycorrhizae, most obtain fertilizer nutrients from the mycorrhizal have VA mycorrhizae. Sixty-three of sixty-six fungus while the fungus obtains sugars or other tropical trees in Nigeria (77) are associated with food materials from the plant. Although the ar- VA mycorrhizae. So are most important tree buscule of VA mycorrhizal fungi occurs inside crops such as cocoa, coffee, rubber, and cit- root cells, they remain covered by the host cell rus. Some trees such as juniper, apple, and pop- membrane and so are not in direct contact with lar can have either ectomycorrhizae or VA the host cytoplasm. Vesicles are balloon-like mycorrhizae. mycorrhizal fungus structures that usually form inside the host root. These structures are The fungi that form VA mycorrhizae, about thought to be storage organs that the fungus 80 species, are in a few genera in the Zygomy- produces to store nutrient materials inside of cetes class of fungi. They are so common in the plant host. soils that literally any field soil sample from arctic to tropical regions will contain these VA fungi also produce abundant spores ei- fungi (66). ther inside or outside of host roots. These

Figure 2.—Diagram of a Typical Vesicular-Arbuscular Mycorrhiza Including Vesicles, Arbuscules, Spores, and External Hyphae 187 spores are the survival structures of VA mycor- 2 weeks before they are digested by the host rhizal fungi. They are long-lived and extremely plant (61,90). Plant roots normally release large resistant to most unfavorable soil conditions. quantities of chemical “exudates” into the root These spores are responsible for the wide- zone (8). Since the arbuscules are covered by spread occurrence of VA mycorrhizal fungi in the host membrane it is thought that the sym- nearly all soils throughout the world. Despite biotic association is regulated by the host plant the intracellular penetration by VA mycorrhi- via the cell membrane. The more nutrient ma- zal fungi, they do not affect the roots’ outward terials released by the plant membrane to the appearance except by inducing a yellow col- arbuscule of the mycorrhizal fungus, the more oration in some hosts (4). Detection of VA my- abundant the mycorrhizal colonization (76). By corrhizal roots is best done by staining roots restricting nutrients passing through the plant and examining them microscopically for the membrane the plant is capable of restricting presence of hyphae, arbuscules, or vesicles (73). mycorrhizal infection in roots. A similar mechanism can be postulated for the regulation of Arbuscules of VA mycorrhizal fungi are ectomycorrhizae by plant roots. short-lived and generally survive for less than

HOW DO MYCORRHIZAL FUNGI IMPROVE GROWTH OF AGRICULTURAL PLANTS?

The VA mychorrhizal symbiosis results in VA mycorrhizal fungi may increase the ef- marked increase in crop growth and develop- fective absorbing surface of a host root by as ment. For example, inoculation of fumigated much as 10 times (6). Nutrient ions such as sand or soil with VA mycorrhizal fungi will in- phosphorus, zinc, and copper do not diffuse crease the growth of citrus by as much as 1600 readily through soil. Because of this poor dif- percent (figure 3); (42), grapes by 4,900 percent fusion, roots deplete these immobile soil nu- (74), soybeans by 122 percent (84), pine by 323 trients from a zone immediately surrounding percent (100), and peaches by 80 percent (44). the root. Mycorrhizal hyphae extend into the Growth responses due to VA mycorrhizal fungi soil past the zone of nutrient depletion and can have been observed in cotton (82), tomatoes increase the effectiveness of absorption of im- (16), corn (27), wheat (41), clover (75), barley mobile elements by as much as 60 times (6). (5), potatoes (7), ornamental plants (99), and in Others have calculated that approximately 50 many other crops. cm of mycorrhizal hyphae per cm root is necessary to account for the uptake of phosphorus VA mycorrhizal fungi stimulate plant absorp- by mycorrhizal plants (89), Experimental obser- tion of phosphorus (85,74,28,62), zinc (44,61), vations indicate that plant roots can have more calcium (84), copper (84,85,60,42), iron (60), than 80 cm of mycorrhizal hyphae, more than magnesium (36,61), and manganese (84,61). In- the amount necessary to account for the ob- creased uptake of phosphorus is perhaps the served phosphorus uptake, most important benefit provided by mycorrhizal fungi, Plant uptake of mobile soil nutrients such as nitrogen and potassium is rarely improved by Most researchers agree that the increase in mycorrhizal fungi, Normal soil diffusion is ade- effective nutrient absorbing surface provided quate to supply roots of plants with these nu- by mycorrhizal fungi is primarily responsible trients whether the roots have a large absorb- for the increase in uptake of soil nutrients by ing surface or not. Generally, plants that are mycorrhizal plants. Hyphae from figure 3 my- most dependent on mycorrhizal fungi for nu- corrhizal plant roots can extend up to 8 cm into trient uptake are those having roots with a low the surrounding soil and transport nutrients surface to volume ratio; that is, plants with this distance back to the roots (83). coarse, fleshy roots with few root hairs (2).

38-846 0 - 85 - 7 188

Although some scientists speculate that my- under drought or water-stressed conditions corrhizal fungi can solubilize and absorb nu- (66,86). Ectomycorrhizae, in particular, with trients that are unavailable to plant roots, there their mantle surrounding the roots, may pro- is little evidence to support this claim. Sanders vide a physical barrier against root dessication. 32 and Tinker (88) showed conclusively with p- Considerable evidence exists to suggest that labelled phosphate that mycorrhizal fungi use mycorrhizal plants may be better equipped to the same phosphorus sources as do plant roots withstand the toxic effects of salt. Calcium, but they are able to absorb from a larger soil magnesium, and sodium concentrations in volume and so are responsible for the vast ma- non-mycorrhizal citrus were 41 percent, 36 per- jority of phosphorus absorption by crop plants. cent, and 150 percent greater than in mycorrhizal citrus (55). Hirrel and Gerdemann (35) Mycorrhizal fungi can also enhance water found that mycorrhizal fungus increased bell transport in plants (87) and prevent water stress peppers tolerance to salinity. Trappe, et al. (98), under some conditions (54). This probably is indicated that VA mycorrhizal fungi provided not a direct effect of mycorrhizal fungi, but in- resistance to the toxic effects of arsenic. My- stead is because of the improved nutrient status corrhizae may also provide tolerance to ex- provided by the mycorrhizal fungi. Mycorrhi- cessive soil manganese and aluminum (34). zal fungi can endure much dryer soil conditions than can most plants and it is thought that Mycorrhizal fungi also act to increase modu- plants may benefit from mycorrhizal infection lation by symbiotic nitrogen-fixing bacteria 189

such as Rhizobium (64,69). Mycorrhizal fungi Fusarium, nematodes, etc.) are usually inhib- may stimulate other beneficial rhizosphere or- ited by mycorrhizal fungi while foliar patho-

ganisms as well (1)0 gens (viruses, rusts, etc.) are often more severe on mycorrhizal plants. Davis, et al. (21,22), and Ectomycorrhizal fungi have been reported to Davis and Menge (20) concluded that the VA provide resistance to plant disease in many mycorrhizal fungus Glomus fasciculatus pro- plants (48). Although mycorrhizae never con- duced little resistance to Phytophthora root rot fer complete immunity, they often appear to in citrus and indeed increased Phytophthora reduce the severity of disease or symptom ex- root rot in avocado and Verticillium wilt in cot- pression. Resistance of ectomycorrhizae to dis- ton. VA mycorrhizal effects on disease may re- ease may result from (48): sult from improved phosphorus nutrition be- ● mechanical protection by the mantle, cause of the increased absorbing surface of the ● better plant nutrition, mycorrhizal hyphae. This effect is magnified Q production of antibiotics by the mycorrhi- when the roots’ normal absorbing capacity is zal fungus, reduced because the roots are partially ● competition for infection sites, decayed. ● formation of phytoalexins, and There have been reports of mycorrhizal fungi ● alteration of root exudates. actually reducing growth of some plants (11, Evidence is accumulating that VA mycorrhi- 39,13). These parasitic effects are rare and the zal fungi exert similar effects on plant patho- reason for them is not understood, but they ap- gens. Schenck, et al, (91), has reported mycor- parently occur in grasses, cereals, and rhizal resistance to root-knot nematodes. tomatoes at or above optimum soil nutrient Schonbeck (93) has examined a variety of foliar levels when the plant is actively regulating and root pathogens on mycorrhizal plants and mycorrhizal invasion, concluded that root pathogens (Thielaviopsis,

MYCORRHIZAE AS SUBSTITUTES FOR FERTILIZERS

In the past 40 years the use of agricultural phosphorus and potassium accounting for only fertilizers has more than doubled. Crop yields 16 percent of the fertilizer energy use (47). have risen dramatically as a result. However, Because mycorrhizal fungi increase the effi- because of shortages in some fertilizer supplies ciency of fertilizer use, they can be thought of and the high cost of energy, the cost of fertilizers has risen tremendously. Agricultural as “biotic fertilizers” and can indeed be sub- economists indicate that as energy costs rise stituted for substantial amounts of some fer- the most responsive agricultural input is fertilizers (53,55). Mosse (61) maintains that 75 percent of all phosphorus applied to crops is tilizer. That is, as energy costs rise, fertilizer not used during the first year and reverts to use will decrease. This response is a dangerous forms unavailable to plants. In soils high in pH, one since chemical fertilizers are said to ac- aluminum, or calcium carbonate, nearly 100 count for one-third to one-half of the current percent of the phosphorus fertilizer can be im- U.S. agricultural output (47). mobilized to nonusable forms via chemical re- Estimates indicate that agriculture uses be- actions in the soil. Tropical oxisols and ultisols tween 2.6 and 4.4 percent of all U.S. energy are notorious for their capacity to immobilize use. Fertilizers and their application comprise phosphorus. Because mycorrhizal plants are 30 to 45 percent of the total agricultural energy better suited to exploiting soil with low amounts use. Nitrogen is the main energy user, with of available phosphorus, zinc, and copper, the 190 addition of large amounts of these fertilizers phosphorus per hectare. Concentrations of each year may be unnecessary. Menge, et al. phosphorus in leaves of mycorrhizal Brazilian (55), compared mycorrhizal citrus seedlings sour orange were above deficiency levels in all with non-my corrhizal seedlings that received seedlings fertilized with more than 56 kg various amounts of phosphorus fertilizer (fig- phosphorus per hectare. Concentration of ure 4). phosphorus in leaves of mycorrhizal Troyer citrange were never in the deficiency range Mycorrhizal Troyer citrange that received no even when plants were not fertilized with fertilizer phosphorus were equal in size to non- phosphorus. mycorrhizal Troyer citrange that received 112 kg phosphorus per hectare, Similarly, mycor- Non-mycorrhizal Troyer citrange, on the rhizal Brazilian sour orange that received no other hand, required over 56 kg phosphorus per fertilizer phosphorus were equal in size to non- hectare before adequate phosphorus concen- mycorrhizal plants that received 560 kg phos- trations were restored to the leaves. At 1980 phorus per hectare. Concentrations of phos- retail costs for triple super-phosphate, it ap- phorus in non-mycorrhizal Brazilian sour pears that use of mycorrhizal fungi could re- orange leaf tissue were never above 0.05 per- sult in savings of $111 to $558/ha ($45 to cent (less than 0.9 percent phosphorus in- $226/acre) in the cost of phosphorus fertiliza- dicates phosphorus deficiency) even when tion of citrus in fumigated nursery soil. In one seedlings were fertilized with 1,120 kg California citrus nursery, it was found that inoculation with mycorrhizal fungi could reduce phosphorus fertilization by two-thirds and save Figure 4.—Dry Weights of Mycorrhizal and $652/ha ($264/acre). Similar savings in phos- Non-Mycorrhizal Brazilian Sour Orange and phorus fertilizers have been shown by Kor- Troyer Citrange Seedlings Fertilized With manik, et al. (43), in fumigated forest nurseries Different Amounts of Phosphorus in the production of sweetgum. Mycorrhizal fungi also can be substituted for copper fertilizer in the culture of citrus seedlings (97). Other data has shown that mycorrhizal fungi can be substituted for zinc fertilizer in the greenhouse culture of citrus and even nitrogen fertilization can be reduced by as much as 300 percent in the presence of mycorrhizae (Menge, et al., unpublished data). This nitrogen savings effect is probably due to an increased efficiency of nitrogen use resulting from improved phosphorus nutrition of the plant. Since mycorrhizal fungi are present in most soils, their unique fertilizer-absorbing abilities are normally already being used by most crops. If mycorrhizal fungi are removed or damaged in any way, then the amount of fertilizer required by a crop increases enormously. This is demonstrated by reports that citrus grown in fumigated soil or in hydroponic solutions often require massive phosphorus applications for adequate growth compared to field grown citrus (55). Citrus in the field can absorb phos- 191 phorus from phosphorus-deficient soils more When and if the cost of fertilizer becomes ex- efficiently than either corn or tomatoes, and orbitant, we must devise the most efficient fer- citrus orchards do not normally require phos- tilizer supply systems possible—to minimize phorus fertilization (9), Differences in phospho- costs while conserving energy and nonrenew- rus absorption by citrus grown in fumigated able resources. I submit that mycorrhizal fungi soil and citrus grown in nonfumigated soils can could be one alternative that might increase be reconciled if mycorrhizal fungi, which are crop yields and yet reduce fertilizer costs and present in nearly all citrus orchards (52), are energy demands. the equivalent of 100 to 500 pounds phosphorus per acre.

CURRENT COMMERCIAL USE OF MYCORRHIZAL FUNGI

Although nearly all plants require mycorrhi- harmful to mycorrhizal development, Flood- zal fungi for maximum growth, the widespread ing, planting non-my corrhizal crops, or remov- occurrence of these fungi in nearly all soils ing topsoil, may also reduce the population of limits the immediate needs for inoculation with mycorrhizal fungi to a level requiring reinocu- mycorrhizal fungi. Mycorrhizal fungi are cur- lation (7,78). rently commercially usable in only three ma- Fumigation with the biocide methyl bromide jor agricultural areas: 1) disturbed sites, 2) to remove soil-borne pests is required by reg- fumigated soils, 3) greenhouses. ulation for the production of many nursery crops. It is also regularly used in many field agricultural situations. This chemical is ex- Disturbed Sites tremely toxic to mycorrhizal fungi and most Mycorrhizal fungi have been conclusively field fumigations are sufficient to destroy the shown to improve revegetation of coal spoils, native mycorrhizal inoculum (56). Stunting of strip mines, waste areas, road sites, and other crops following fumigation with methyl bro- disturbed areas (18,19,15,49,81). In these stressed mide is common and is due to the destruction sites, mycorrhizal fungi are usually lacking and of mycorrhizal fungi. Although a relatively adding mycorrhizal fungi provides a nutrition- small amount of land is treated with this chem- al advantage to associated plants in addition ical, less than 100,000 acres annually in the to providing possible resistance to low pH, United States, stunting following fumigation heavy metal toxicants, and high temperature, with methyl bromide has been reported in the United States, Africa, Spain, Peru, Venezuela, and many other countries (52). Crops that are Fumigated or Chemically Treated Sites routinely grown in methyl bromide fumigated Fumigation with biocides or pesticides such soils include strawberries, tomatoes, tobacco, as methyl bromide (56), chloropicrin (72), nursery crops, tree crop replants, and some dazomet (50), 1,3-D (72), vapam (71), and vorlex vegetable crops. For many of these crops the (71) may destroy or inhibit root infection by addition of mycorrhizal fungi following fumi- mycorrhizal fungi, Application of many soil gation with methyl bromide is not only recom- fungicides such as arasan (71), banzot (95), mended but is imperative. benomyl (96), botran (71), carbofuran (3), It appears that inoculating methyl bromide chloramformethane (37), dichlofluanid (37), fumigated crops is economically possible. The ethirimol (37), lanstan (71), mylone (71), PCNB cost for inoculating nursery-grown citrus with (96), sodium azide (3), thiabendazole (37), mycorrhizal fungi is about $288/acre, while the thiram (96), triademifon (37), tridemorph (37), cost for phosphorus fertilizer alone is $338/ and vitavax (96) have also been reported to be acre. Fumigated tomatoes receive $51 worth 192

of phosphorus per acre while the cost for mycorrhizal fungi rather than leaving mycor- corrhizal inoculation is less than $28/acre. My- rhizal infection to chance; and corrhizal fungi can provide additional benefits ● mycorrhizal plants may be more resistant to the crop other than just improved phos- or tolerant to some plant diseases. phorus nutrition. For nursery plants grown in methyl bromide Greenhouses fumigated soil, inoculation with mycorrhizal Greenhouse culture uses growth media such fungi should be imperative for the following as pine bark, vermiculite, perlite, builders sand, reasons: and peat moss and these are devoid of mycor- ● the plants grow better (prevents stunting rhizal fungi. In addition, most greenhouse following fumigation); operators steam, pasteurize, or chemically treat ● there is a decreased need for fertilization, their mixes to eradicate harmful pathogens. specifically phosphorus, zinc, and copper, Nurserymen have compensated for the absence resulting in decreased fertilizer cost and of beneficial mycorrhizal fungi by applying lux- energy conservation; ury amounts of fertilizer and water to achieve ● there is decreased chance for water stress desired growth. Inoculation of container and therefore reduced transplant injury; grown plants to reduce irrigation, fertilizer, ● mycorrhizal plants survive better especial- and pesticide applications and cost can be done ly if transplanted to fumigated, poorly fer- as demonstrated by Chatfield, et al. (10), Lin- tilized, or disturbed soil; derman (46), and Crews, et al. (12). ● plants will be inoculated with effective my-

COMMERCIAL PRODUCTION AND INOCULATION WITH MYCORRHIZAL FUNGI

Many ectomycorrhizal fungi can be readily duced inoculum. The inoculum will be tested cultured on artificial media and inoculum can in nearly 100 tree nursery test sites through- be grown under standard laboratory conditions out the United States. Results will be available (49). Experimentally, sterilized vermiculite and within 4 years and will indicate the commer- peat moss is frequently saturated with a liquid cial feasibility of producing and using mycor- nutrient medium (49) and is infested with a de- rhizal inoculum in fumigated tree nurseries. sirable ectomycorrhizal fungus. Ectomycorrhi- zal fungi generally grow quite slowly and may Ectomycorrhizal inoculum can best be ap- take several months to colonize the vermiculite- plied in the nursery. Once the trees become peat moss mixture. This material can be used infected, the benefits can be transferred to on a small scale to inoculate nurseries and wherever the trees are grown. In the nursery, greenhouses with mycorrhizal fungi. Abbott mycorrhizal inoculum can be distributed by Laboratories, North Chicago, Illinois, has pro- hand and rototilled into the soil before plant- duced massive amounts of inoculum of the ec- ing seed. Special machinery has already been tomycorrhizal fungus Pisolithus tinctorius (86). built and is being used to incorporate ectomy- Abbott Laboratories produced the peat moss- corrhizal inoculum. vermiculite-nutrient solution inoculum under Commercial production of mycorrhizal in- large-scale commercial conditions using com- oculum for use in sterilized or fumigated soil mercial fermenters. is being attempted at several locations in the Under the direction of D. H. Marx, the U.S. United States. Currently, the only way to pro- Forestry Service has undertaken a massive duce suitable quantities of a mycorrhizal in- testing program using the commercially pro- oculum is on roots of susceptible host plants. 193

The possibility of pathogenic organisms will probably be produced on the roots of suit- contaminating mycorrhizal inoculum is an ex- able host plants. tremely serious problem when growing VA With proper safeguards, mycorrhizal inocu- mycorrhizal inoculum in semi-sterile cultures lum, free of plant pathogens, can be produced in the greenhouse. For this reason, many scien- on plants in the greenhouse. Figure 5 illustrates tists will consider mass production of VA a proposed scheme for producing mycorrhizal mycorrhizal fungi only if it is done axenically inoculum [53). VA mycorrhizal fungi can be (one organism only). isolated by using bits of roots or soil from the Realistically, however, not only must these field to inoculate roots of “trap plants” grow- obligate parasites be grown in vitro, but they ing in sterilized soil in the greenhouse. Sudan- must produce large quantities of spores in grass (Sorghum vulgare Pers.) is frequently culture which will survive under soil condi- used, but other plants such as tomato, soybean, tions and infect plants in nature. Information corn, and safflower may be equally suitable. gained from the culture of other formerly The soil used throughout is a low nutrient sand obligate parasites suggests that the possibility fertilized once per week with one-half the of realizing this goal in the near future is standard Hoagland’s solution minus phospho- unlikely. Even if mycorrhizal fungi are cultured rus. After production of VA mycorrhizal spores axenically, mycorrhizal inoculum for field use in the “pot cultures,” the spores can be 194 removed by wet sieving (29), elutriation (25), water may be effective in increasing spore pro- or centrifugation (85). These spores must be duction. When spores are mature, plant tops surface disinfested with substances such as are removed and roots, soil, and spores can be chloroamine T or sodium hypochlorite and ground up and partially dried (7 to 20 percent streptomycin to assure that pathogens do not moisture content) and stored at 40 C until used. accompany the spores (68). If concentrated spore suspensions are desired, spores can be concentrated by wet sieving (38), are to These surface disinfested spores used elutriation (25), or centrifugation (85) before roots were ger- inoculate the of plants that storage. VA mycorrhizal inoculum can be minated and grown under aseptic conditions freeze-dried if desired (38). Inoculum produced in growth chambers. The containers illustrated in this manner should be consistently infective are made from plastic petri plates and filled and yet pathogen free. with the low nutrient sand. After 1 to 4 weeks when the mycorrhizal fungi have infected roots Using the method described above, the esti- grown under aseptic conditions root pieces can mated costs for producing mycorrhizal in- be removed and stained (73) to observe infec- oculum are shown in table 1. These figures are tion, root pieces are carefully removed and derived from production costs of a foliage plant used to infect suitable host plants grown in greenhouse and could be reduced considerably sterilized soil in the greenhouse. Similar root pieces can be removed, examined, and plated Table 1 .—Estimated Cost of Production of on agar to observe pathogenic organisms. Vesicular-Arbuscular Mycorrhizal Inoculum on Sudangrass in 4 Inch Pots If no pathogens are observed, the greenhouse “pot culture” may be used as a “mother cul- Item Cost/pot ture” to produce inoculum that will be used in 1. Labor: a. to prepare the soil mix ...... $0.03 the field. Inoculum should be produced on se- b. potting, inoculating, and seeding...... 0.05 lected hosts that have no root diseases in com- c. moving pots to growing area ...... 0.03 mon with the host plant for which the in- d. pruning ...... 0.02 e. spraying (insecticides and fungicides) . . . . . 0.03 oculum is intended. For instance, inoculum for f. watering ...... 0.02 citrus could be produced on sudangrass but g. harvesting...... 0.01 never on citrus. In this way the wide host range h. grinding and packaging ...... 0.03 i. quality control ...... 0.02 of most VA mycorrhizal fungi can be used. As j. maintenance of mother cultures ...... 0.05 another precaution against propagating path- Labor cost ...... $0.29 ogens along with mycorrhizal inoculum, the 2. Materials: field inoculum should be drenched several a. pots 4 ' ...... $0.07 b. seed...... 0.002 times with pesticides chosen to eliminate path- c. fertilizer ...... 0.02 ogens known to infect the host for which the d. shipping containers ...... 0.025 inoculum is intended. Mycorrhizal inoculum e. insecticides and fungicides ...... 0.03 intended for citrus should be drenched with Materials ...... $0.147 3. Overhead expenses: a nematicide to control the citrus nematode a. heat ...... $0.08 and fungicides to control Phytophthora and b. depreciation on greenhouse ...... 0.008 Rhizoctonia. Suggested pesticides are Ethazole c. depreciation on boilers ...... 0.003 d. maintenance allowance ...... 0.006 and PCNB. PCNB reduces the population of e. office supplies ...... 0.002 mycorrhizal spores but the other pesticide can f. management and office work ...... 0.03 actually increase spore production (57). Several g. return on investment ...... 0.01 other pesticides can be used without harming h. loss due to undeveloped plants ...... 0.001 i. taxes ...... 0.06 mycorrhizal fungi (96). j. laboratory, incubator, etc...... 0.04 Total for overhead ...... $0.24 Horticultural practices also could be used at Total cost ...... $0.677 this point to maximize spore production. Elim- Selling price ... ..$0.90 inating fertilization and slowly reducing the 0.18¢/500 spores 195 since mycorrhizal inoculum quality is of im- sponses of citrus in a fumigated nursery after portance and not plant quality, A reasonably inoculating citrus seed with mycorrhizal in- generous estimate of the cost of mycorrhizal oculum. Gaunt (26) inoculated onion and production, including technical labor and tomato seeds with a VA mycorrhizal fungus quality control, together with a small margin and reported that seed inoculated plants grew of profit, indicates that consumers may pay as well as plants that were inoculated by mix- about 0.180/500 spores of VA mycorrhizal ing VA mycorrhizal inoculum into the soil. fungi. Such a cost could reasonably be borne Commercial applications of mycorrhizal in- by consumers such as greenhouse operators or oculum using fertilizer banding machinery nurserymen. were successfully carried out in citrus nurseries in California (23). A similar method to that outlined above has been patented in England and is being per- Commercial VA mycorrhizal inoculum is fected for large-scale commercial use (34). In produced using the method described above in this method plants are grown in peat blocks two citrus nurseries—Brokaw Nursery, Saticoy that are standing in a shallow nutrient-flow California and the Thermal Ranch, Thermal, culture. After VA mycorrhizal spores are pro- California. Experimental VA mycorrhizal induced in the peat blocks they are ground up, oculum is being produced and distributed on roots and all, for inoculation. The finished a large scale by Abbott Laboratories, North Chi- product is not only excellent mycorrhizal in- cago, Illinois. Other major corporations that oculum but is light and easy to ship. are supporting or carrying out research on VA mycorrhizal fungi include Dow Chemical Co., Although many methods have been used to Rohm & Haas Co., Dupont, Monsanto Co., and inoculate plants with VA mycorrhizal fungi in Ceiba-Geigy Chemical Co. greenhouse trials, few inoculation methods are acceptable for large-scale commercial inocu- Plants growing in all soils do not respond fa- lation. Several different methods to inoculate vorably to VA mycorrhizal inoculum. If soil nu- corn have been studied and layering inoculum trition is optimum, mycorrhizal fungi will not under the seed was superior to seed inocula- enhance growth of plants. A method for detect- tion or banding the inoculum (38), Hall (30) de- ing which soils require mycorrhizae for max- veloped a method for pelleting seed with a my- imum production of citrus was devised by corrhizal infection and determined that Menge, et al. (58). In soils with less than 34 ppm mycorrhizal fungi could survive up to 28 days available P (Olson analysis), 12 ppm available under these conditions. Menge, et al. (53), Zn, 27 ppm available Mn, or 3 percent organic found that layering inoculum below the seed matter, citrus trees will probably require my- and banding inoculum were superior to seed corrhizal fungi for maximum growth. Mycor- inoculations. Crush and Pattison (14) experi- rhizal inoculations are recommended only in mented with several means of inoculating soils with these characteristics, It is estimated seeds with VA mycorrhizal fungi, but again that this includes approximately 85 percent of found that sowing seed above pelleted mychor- the southern California citrus soils. Similar rhizal inoculum was the most effective method studies could be done with other crops to de- for obtaining mycorrhizal infection. Hattingh termine which soils require mycorrhizal in- and Gerdemann (31) reported growth re- festation.

POTENTIAL USES FOR MYCORRHIZAL FUNGI

Because mycorrhizal fungi occur on most Large-scale field inoculations with mycorrhizal agronomic crop plants and improve the growth fungi are rare because of limited inoculum, and of these plants, the potential use of these fungi natural field soils usually contain adequate as commercial “biotic fertilizers” is enormous. populations of indigenous mycorrhizal fungi. 196

Under these conditions, any growth benefit increases of white clover after inoculation with due to mycorrhizal inoculation would depend mycorrhizal fungi in only three of nine sites. primarily on the superiority and/or placement Jackson, et al. (38), indicated that with certain of the mycorrhizal inoculum. Beneficial re- mycorrhizal inoculation methods, growth of sponses under these conditions would be pre- corn, sudangrass, and soybeans was not stim- dicted to be far less than the responses obtained ulated in nonsterile soil. Mosse (65) obtained in fumigated or partially sterilized soil. How- significant growth responses of Stylosanthes ever, greenhouse and field experiments in spp. due to mycorrhizae in 6 of 11 nonsterile which plants were inoculated with mycorrhizal soils. Ross and Harper (85) reported no growth fungi in nonfumigated soils have demonstrated stimulation of soybeans in nonsterile soil. that growth responses due to mycorrhizal fungi Mosse (65) indicated that the inoculum po- can occur under these circumstances. tential of indigenous mycorrhizal fungi is the In greenhouse experiments, using untreated major determinant governing growth responses soil, Mosse and her colleagues (62,63,65,67,70) of plants to mycorrhizal fungi in nontreated demonstrated that preinoculation with mycor- soil. Powell (75) indicated that many indige- rhizal fungi could provide the following growth nous mycorrhizal fungi are “inefficient” sym- increases: bionts, and that inoculation by more efficient mycorrhizal fungi will result in growth in- Crop Growth increase Centrosema spp. . . . . 34 percent creases even in nonsterile soil that contain high corn ...... 306 percent populations of “inefficient” mycorrhizal fungi. Melinis spp...... 41-60 percent placement of mycorrhizal inoculum is equally onions ...... 48-3155 percent strawberries ...... 250 percent important in affecting a plant growth response Stylosanthes spp. . . . 85-88 percent (38). Certainly, plants infected early in the sweetgum ...... 45 percent growing season by mycorrhizal fungi are bet- 527 percent Viola spp...... ter than plants that do not become infected un- Other studies have noted similar growth increases in untreated soil: til later (82). corn ...... 14 percent (Gerdemann, 1964) corn ...... 0-53 percent (Jackson, et al., 1972) Huge expanses of tropical soils (e.g., the Bra- mahogany ...... 151 percent (Redhead, 1975) zilian Cerrado) are either deficient in phos- sudangrass ...... 0-18 percent (Jackson, et a]., 1972) white clover ...... 80-100 percent (Powell, 1977) phorus or immobilize phosphorus fertilizers. These marginal agricultural lands could be pro- In a large-scale field experiment conducted ductive if mycorrhizal fungi, with the ability in nonsterile, virgin, infertile fields, wheat pre- to efficiently use extremely small quantities of inoculated with a mycorrhizal fungus pro- fertilizer, were developed and added to the soil. duced 220 percent more grain than non-mycor- Cheap but readily available rock phosphate rhizal wheat (41). In a similar experiment (40), could be added as the phosphorus source. This corn inoculated with a mycorrhizal fungus was phosphorus source is a poor fertilizer but re- 122 percent larger than non-mycorrhizal corn. leases small quantities of phosphorus for long Hayman (33) reported white clover growth in- periods of time. Some mycorrhizal fungi use creases in the field due to inoculation with a rock phosphate much better than others and mycorrhizal fungus. Black and Tinker, in an can tremendously improve growth of plants extremely well-documented field experiment, growing in poor soils fertilized with this ma- found that fallow field inoculation with a my- terial (59,66). corrhizal fungus increased potato yield 20 Mycorrhizal fungi have been proposed as un- percent. stable soil or sand dune stabilizers (96). Finally Not all mycorrhizal inoculations in nonster- ectomycorrhizal fungi have been shown to im- ile soil result in increased growth. Hayman (33) prove rooting of a wide variety of non-host indicated that mycorrhizal fungi did not stim- plants and the possibility of using them as a ulate growth of white clover at several loca- commercial root stimulant has been proposed tions. Powell (75) obtained significant growth (45). 197

CONSTRAINTS ON THE COMMERCIAL USE OF MYCORRHIZAL FUNGI

The current major obstacles to the commer- cost-benefit assessment of VA mycorrhizal in- cial use of mycorrhizal fungi are: oculation. c the lack of large-scale field experiments Heavy phosphorus fertilization severely in- under normal agricultural conditions, hibits mycorrhizal infections (17,68). More Q the lack of cost-benefit analysis to deter- recently, it is becoming evident that heavy ni- mine the economics of mycorrhizal appli- trogen and zinc applications are also inhibitory cations, and to mycorrhizal fungi (32,51). Daily applications ● the trend toward excessive fertilization to of 100 ppm nitrogen under greenhouse condi- substitute for the lack of mycorrhizal tions have been shown to completely eliminate fungi. mycorrhizal infections (J.A. Menge, unpublished data). Many commercial greenhouses Perhaps the most important deterrent of com- add over 200 ppm nitrogen daily to their plants. mercial use of mycorrhizal fungi is the lack of In greenhouse and fumigated nursery condi- large-scale field tests in a variety of agricultural tions, growers are using excessive fertilization soils and locations. The program initiated by to substitute for the lack of mycorrhizal fungi. D. H. Marx and the U.S. Forest Service will Under these conditions, not only do mycorrhi- correct this deficiency for ectomycorrhizal fun- zal fungi not benefit their host plants, but it is gi and within 4 years it will be known if these difficult to successfully establish mycorrhizal mycorrhizal fungi will indeed be economically infections so that the plants will be mycorrhizal feasible to use on a wide scale in the produc- once they leave the supraoptimal fertility tion of forest trees. regime. As long as fertilizer is relatively available and not excessively expensive, it will take This type of program remains to be established a major educational program to convince many for VA mycorrhizal fungi. Without such data growers to change their standard operating it is difficult to establish a potential market for procedures and use mycorrhizal fungi that will mycorrhizal inoculum. Without a market there not only be cheaper but will conserve fertilizer is little incentive for industry to initiate the pro- and energy. duction of commercial inoculum. Without commercial inoculum it is difficult to carry out In my opinion, granting agencies such as the large-scale field tests. With the recent establish- National Science Foundation, Rockefeller ment of several commercial sources of mycor- Foundation, USDA competitive grants, and the rhizal inoculum perhaps this cycle will be bro- Israeli-U.S. granting agency BARD have effec- ken and more field tests will result. tively provided adequate funding for basic mycorrhizal research. The number of scientific Once large-scale field tests are seen to be suc- papers on mycorrhizal fungi has quadrupled cessful, light-weight commercial mycorrhizal since 1960, which is evidence that there is great formulations will develop and new application interest and money available for basic mycor- methods will be devised, Most importantly, rhizal research. However, there are few agen- from large-scale field tests, cost benefit analy- cies that will fund the final applied steps in a sis can be accurately done to determine the biological commercialization project. Research economic benefit derived from the use of my- money for large-scale “applied” or “demonstra- corrhizal fungi. In the end, this will be the de- tion” experiments is unavailable. Funding for termining factor in the commercial application small-scale pilot projects is also not available. of mycorrhizal fungi. Biological scientists are It remains for private industry to pick up the rarely able to critically assess the economic fac- projects from this point, but they have been re- tors involved in the application of a new tech- luctant to do so. The transition is not going nique and I recommend that agricultural econ- smoothly and seems to be proceeding slowly omists should be asked to participate in the if at all, 198

FUNGI ON AGRICULTURE

It is very difficult for a scientist to speculate with a less well-developed agricultural system, on the effects of a new procedure on the social mycorrhizal fungi have not been altered and and economic structure of an agricultural so- are probably functioning effectively and need ciety. Frequently good ideas do not receive the not be applied under such conditions. Fertilizer acclaim they deserve because of prejudices, ig- in most underdeveloped countries is probably norance, religious preferences, social mores, applied sparingly as manure and therefore my- and other reasons not fully understood by sci- corrhizal fungi will not result in a great sav- entists. In my opinion, the effects of mycor- ings either of fertilizer or energy. rhizal technology would most alter the socio- If superior strains of mycorrhizal fungi are economic structure in areas of intensive agri- developed, marginal agricultural land could be culture. These situations would be more prev- made productive. Huge amounts of marginal alent in agriculture in developed nations. agricultural land exists in Africa and South Mycorrhizal fungi are most useful in reclaim- America and the proper use of this land may ing sites disturbed by heavily mechanized in- well decide the future of some countries. In- dustries or soil fumigation. Mycorrhizal fungi creased use of agricultural land will provide can reduce energy and fertilizer and increase for a greater economic base, larger agricultural the efficiency of crops grown intensively. productivity, and a better way of life for large Therefore, mycorrhizal fungi can be viewed as populations in underdeveloped countries. Edu- conservation measures or as substitutes for cating agriculturists to the importance of my- high energy uses in developed nations. corrhizal fungi may allow developing countries In less developed nations, growers would to avoid the excessive use of energy, fumigants, have to be educated to the methods of produc- and fertilizers associated with intensive agri- ing, handling, and inoculating living micro- culture. organisms. This may be difficult. In countries

Mycorrhizal fungi may be one alternative fertilizer system to a new, but cheaper, energy that can immediately improve revegetation of conservative system using mycorrhizal fungi. disturbed sites, increase crop growth in fumi- Recommendations that could substantially gated soils and greenhouses, and yet reduce increase the commercial use of’ mycorrhizal fertilizer costs and energy demands. If superior fungi (in relative order of importance) are as strains of mycorrhizal fungi were developed, follows: they could potentially improve growth of nearly all agronomic crops in a wide variety of soils 1. Improved availability of grant funds for throughout the world. Both ectomycorrhizal large-scale field applications of mycorrhi- fungi and vesicular-arbuscular mycorrhizal zal fungi in a wide variety of soils through- fungi are in commercial production on a small out the world. It would be useful to estab- scale. The greatest obstacles to the commercial- lish several pilot projects in various less ization of mycorrhizal fungi appears to be: 1) developed countries. These pilot projects the lack of large-scale field tests under typical could produce and distribute mycorrhizal agricultural conditions in a variety of locations; inoculum on a variety of crops growing 2) adequate cost-benefit analysis to determine under different soil conditions. Cost-ben- the economics of the utilization of mycorrhizal efit analysis on such projects could ade- fungi; and 3) a reluctance on the part of grow- quately assess the economics of inocula- ers to switch from an energy dependent, heavy tion with mycorrhizal fungi. 199

2. Funds should be made available to create tists have been trying to artificially culture a worldwide bank of beneficial mycorrhi- VA mycorrhizal fungi since 1900, this ob- zal fungi. The establishment of such a jective may be difficult to achieve and in- facility is being investigated by the mycor- centives to work on such a problem are dif- rhizal community and the National Sci- ficult to justify. ence Foundation has agreed to entertain a proposal for such a facility. The Univer- sity of Florida has agreed to supply the fa- References cilities as well as substantial operating costs for such an establishment. A second 1. Bagyaraj, D. J., and Menge, J. A., “Interaction idea would be to add the responsibility for Betweeen VA Mycorrhiza and Azotabacter and Their Effects on Rhizosphere Microflora maintaining mycorrhizal cultures to the and Plant Growth, ” New I%ytol. 80: 567-573, already established government facility 1978. called the American Type Culture Collec- 2, Baylis, G. T. S., “Experiments on the Ecologi- tion which maintains many important cal Significance of Phycomycetous Mycorrhi- fungal cultures. zas,” New l%yto~. 66: 231-243, 1967. 3. It would be desirable to establish a USDA- 3, Backman, P. A., and Clark, E, M., “Effect of supported vesicular-arbuscular mycorrhi- Carbofuran and Other Pesticides on Vesicular- zal research center that would be respon- Arbuscular Mycorrhizae in Peanuts, ” Nema- sible for maintaining and coordinating tropica 7: 13-15, 1977. U.S. research on mycorrhizal fungi. This 4. Becker, W, N., “Quantification of Onion Vcsic- ular-Arbuscular Mycorrhizae and Their Resis- facility would complement the Mycorrhi- tance to Pyrenochaeta tarrestris, ” Ph. D. thesis zal Institute in Athens, Georgia, which was (University of Illinois at Champaign-Urbana, created by the Forest Service to coordinate 1976). mycorrhizal research on forest trees. 5. Benians, G. J., and Barber, D. A., “Influence 4. A world survey should be conducted to of Infection With Endogone Mycorrhiza on collect and test as many different vesicu- the Absorption of Phosphate by Barley Iar-arbuscular mycorrhizal species as pos- Plants, ” Letcombe Lab. Ann. Rpt., 1971: 7-9, sible. The discovery of a superior mycor- 1972. rhizal strain with a wide host range could 6. Bieleski, R, L., “Phosphate Pools, Phosphate tremendously increase agricultural pro- Transport and Phosphate Availability, ” Ann. ductivity throughout the world. Rev. P]ant Physiol 24: 225-252, 1973, 5. 7, Black, R. L. B., and Tinker, P. B., “Interaction Research is necessary to elucidate the ex- Between Effects of Vesicular-Arbuscular My- act role of mycorrhizal fungi play in im- corrhiza and Fertilizer Phosphorus on Yields proving plant growth under stress condi- of Potatoes in the Field, ” Nature 267: 510-511, tions such as drought, salt, toxic soil 1977. materials, or in marginal agricultural 8. Bowen, G. D., “Nutrient Status Effects on Loss lands. of Amides and Amino Acids From Pine 6. Research is necessary to elucidate the ge- Roots,” Plant and Soil 30: 139-143, 1969. netics of mycorrhizal fungi. Virtually 9. Chapman, H. D., and Rayner, D. S., “Effect of nothing is known on this subject. The Various Maintained Levels of Phosphate on ability to breed these organisms could re- the Growth, Yield Composition and Quality of sult in tremendously increased agricul- Washington Naval Oranges, ” Hilgardia 20:325-358, 1951. tural productivity. 7. 10. Chatfield, J. A., Rhodes, L. H., and Powell, C. Efforts should be intensified to grow ve- C., “Mycorrhiza in Floriculture, the Potential sicular-arbuscular mycorrhizal fungi in the Use of Beneficial Fungi in Flower Crops, ” laboratory using artificial media. A Ohio Florists’ Assn., Bull, No. 594: 5-6, 1979. breakthrough in this area could improve 11. Cooper, K. M., “Growth Responses to the the feasibility of attaining all of the above Formation of Endotrophic Mycorrhizas in recommendations, However, since scien- Solarium, Leptospermum and New Zealand 200

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