Chapter X

Mycorrhiza AgricultureTechnologies

John A. MengeAssociate Professor

Department of Plant PathologyUniversity of California

Riverside, CA 92521

Contents

Page

Introduction—What Are Mycorrhizal Fungi? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185Ectomycorrhizae . . . . . . . . . . . . . . . . . . . . ,,, .,, ,, . . . .,.,.,... . . . . . . . . . . . . . 185Vesicular-Arbuscular (VA) Mycorrhizae . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . 185

How Do Mycorrhizal Fungi Improve the Growth of Agricultural Plants? . . . . . . . . 187Mycorrhizae as Substitutes for Fertilizers . . . . . . . . . . . . . . . . . . . . . . . .,.,., ,..,,, 189Current Commercial Use of Mycorrhizal Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191

Disturbed Sites ..............,,. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191Fumigated or Chemically Treated Sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191Greenhouses .. .,..., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .....,.,., . . . . . . . . . . 192

Commercial Production and Inoculation With Mycorrhizal Fungi . . . . . . . . . . . . . . 192Potential Uses of Mycorrhizal Fungi. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195Constraints on the Commercial Use of Mycorrhizal Fungi . . . . . . . . . . . . . . . . . . . . . 197Effects of Mycorrhizal Fungi on Agriculture. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198Conclusions and Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199

Table

Table No. Page

1. Estimated Cost of production of Vesicular-Arbuscular Mycorrhizal Inoculum onSudangrass 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 andat Different Nutrient Levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,. . . . . . , 188

4. Dry Weights of Mycorrhizal and Non-Mycorrhizal Brazilian Sour Orange andTroyer Citrange Seedlings Fertilized With Different Amounts of Phosphorus., . 190

5. Proposed Scheme for the Commercial Production of Vesicular-ArbuscularMycorrhizal Inoculum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193

Chapter X

Mycorrhiza Agriculture Technologies

INTRODUCTION-WHAT ARE MYCORRHIZAL FUNGI?

Mycorrhizal fungi are beneficial fungi thatare associated with plant roots via a symbioticassociation whereby both the host plant andthe fungus benefit. Mycorrhizae are the struc-tures formed by the symbiotic association be-tween plant roots and mycorrhizal fungi. My-corrhizae contain both plant roots and fungaltissues. In nature, mycorrhizae are far morecommon than non-mycorrhizal roots (24,92,94).Nearly all plant species are associated with my-corrhizal symbionts. Because of their impor-tance to plants and their widespread distribu-tion, mycorrhizae must be considered in allaspects of plant ecology, crop science, and agri-culture.

Mycorrhizal fungi are divided into four verydifferent types (66): ectomycorrhiza, vesicular-arbuscular mycorrhiza (abbreviated as VA my-corrhiza), ericaceous mycorrhiza, and or-chidaceous mycorrhiza. As indicated by theirnames, ericaceous mycorrhiza and orchida-ceous mycorrhiza are associated with erica-ceous plants (blueberries, cranberries, azaleas,etc.) and orchidaceous plants (orchids), respec-tively. Because of the relatively low economicimpact of these plants and the small amountof available data on these types of mycorrhiza,they will not be discussed further.

Ectomycorrhizae

Ectomycorrhizae are associated primarilywith trees such as pine, hemlock, spruce, fir,oak, birch, beech, eucalyptus, willow, and pop-lar, Ectomycorrhizae are formed by hundredsof different fungal species belonging to theBasidiomycetes (mushrooms and puffballs) andAscomycetes (cup fungi and truffles). Thesefungal symbionts are stimulated by root ex-udates and grow over the surface of host feederroots to form a thick fungal layer known as afungal mantle (figure 1). Hyphae of ectomycor-

Figure 1 .—Diagram of a Typical EctomycorrhizaIncluding the Hartig Net, Fungal Mantle, and

External Hyphae (courtesy D. H. Marx)

rhizal fungi penetrate between the cells of thehost root, develop around the root corticalcells, replace the host middle lamella, and formwhat is called the “Hartig net’’—the distin-guishing feature of ectomycorrhizae. In re-sponse to the fungal invasion, the host rootsusually swell substantially and may branch di-chotomously or in a coralloid manner. The rootcells are not injured, however, and function ofthe roots is enhanced, as we shall discuss.

Vesicular-Arbuscular (VA)Mycorrhizae

VA mycorrhizal fungi have the widest hostrange and form by far the most common typeof mycorrhizae. VA mycorrhizae occur onliverworts, mosses, ferns, some conifers, andmost broad-leaved plants. Only 14 families thatare considered primarily non-mycorrhizal (28).The important crop families that are non-my-corrhizal are Cruciferae (cabbage, broccoli,mustard, etc.); Chenopodiaceae (spinach, beet,

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etc.); Cyperaceae (sedges); and Caryophyllaceae(carnation, pinks, etc.). wetland rice also is usu-ally non-my corrhizal. Nearly all other impor-tant agronomic crops including wheat,potatoes, beans, corn, alfalfa, grapes, datepalms, sugar cane, cassava, and dryland riceare associated with VA mycorrhizal fungi. Al-though many trees have ectomycorrhizae, mosthave VA mycorrhizae. Sixty-three of sixty-sixtropical trees in Nigeria (77) are associated withVA mycorrhizae. So are most important treecrops such as cocoa, coffee, rubber, and cit-rus. Some trees such as juniper, apple, and pop-lar can have either ectomycorrhizae or VAmycorrhizae.

The fungi that form VA mycorrhizae, about80 species, are in a few genera in the Zygomy-cetes class of fungi. They are so common insoils that literally any field soil sample fromarctic to tropical regions will contain thesefungi (66).

The hyphae of VA mycorrhizal fungi pene-trate directly into the root cortical cells of hostplants. Inside of the host plant cells, VA mycor-rhizal fungi form minute coralloid structuresknown as arbuscules (figure 2). Arbuscules arethought to be the site of nutrient transfer be-tween the symbiotic partners. The host plantsobtain fertilizer nutrients from the mycorrhizalfungus while the fungus obtains sugars or otherfood materials from the plant. Although the ar-buscule of VA mycorrhizal fungi occurs insideroot cells, they remain covered by the host cellmembrane and so are not in direct contact withthe host cytoplasm. Vesicles are balloon-likemycorrhizal fungus structures that usuallyform inside the host root. These structures arethought to be storage organs that the fungusproduces to store nutrient materials inside ofthe plant host.

VA fungi also produce abundant spores ei-ther inside or outside of host roots. These

Figure 2.—Diagram of a Typical Vesicular-Arbuscular Mycorrhiza Including Vesicles, Arbuscules,Spores, and External Hyphae

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spores are the survival structures of VA mycor-rhizal fungi. They are long-lived and extremelyresistant to most unfavorable soil conditions.These spores are responsible for the wide-spread occurrence of VA mycorrhizal fungi innearly all soils throughout the world. Despitethe intracellular penetration by VA mycorrhi-zal fungi, they do not affect the roots’ outwardappearance except by inducing a yellow col-oration in some hosts (4). Detection of VA my-corrhizal roots is best done by staining rootsand examining them microscopically for thepresence of hyphae, arbuscules, or vesicles (73).

Arbuscules of VA mycorrhizal fungi areshort-lived and generally survive for less than

2 weeks before they are digested by the hostplant (61,90). Plant roots normally release largequantities of chemical “exudates” into the rootzone (8). Since the arbuscules are covered bythe host membrane it is thought that the sym-biotic association is regulated by the host plantvia the cell membrane. The more nutrient ma-terials released by the plant membrane to thearbuscule of the mycorrhizal fungus, the moreabundant the mycorrhizal colonization (76). Byrestricting nutrients passing through the plantmembrane the plant is capable of restrictingmycorrhizal infection in roots. A similar mech-anism can be postulated for the regulation ofectomycorrhizae by plant roots.

HOW DO MYCORRHIZAL FUNGI IMPROVE GROWTH OFAGRICULTURAL PLANTS?

The VA mychorrhizal symbiosis results i nmarked increase in crop growth and develop-ment. For example, inoculation of fumigatedsand or soil with VA mycorrhizal fungi will in-crease the growth of citrus by as much as 1600percent (figure 3); (42), grapes by 4,900 percent(74), soybeans by 122 percent (84), pine by 323percent (100), and peaches by 80 percent (44).Growth responses due to VA mycorrhizal fungihave been observed in cotton (82), tomatoes(16), corn (27), wheat (41), clover (75), barley(5), potatoes (7), ornamental plants (99), and inmany other crops.

VA mycorrhizal fungi stimulate plant absorp-tion of phosphorus (85,74,28,62), zinc (44,61),calcium (84), copper (84,85,60,42), iron (60),magnesium (36,61), and manganese (84,61). In-creased uptake of phosphorus is perhaps themost important benefit provided by mycorrhi-zal fungi,

Most researchers agree that the increase ineffective nutrient absorbing surface providedby mycorrhizal fungi is primarily responsiblefor the increase in uptake of soil nutrients bymycorrhizal plants. Hyphae from figure 3 my-corrhizal plant roots can extend up to 8 cm intothe surrounding soil and transport nutrientsthis distance back to the roots (83).

VA mycorrhizal fungi may increase the ef-fective absorbing surface of a host root by asmuch as 10 times (6). Nutrient ions such asphosphorus, zinc, and copper do not diffusereadily through soil. Because of this poor dif-fusion, roots deplete these immobile soil nu-trients from a zone immediately surroundingthe root. Mycorrhizal hyphae extend into thesoil past the zone of nutrient depletion and canincrease the effectiveness of absorption of im-mobile elements by as much as 60 times (6).Others have calculated that approximately 50cm of mycorrhizal hyphae per cm root is nec-essary to account for the uptake of phosphorusby mycorrhizal plants (89), Experimental obser-vations indicate that plant roots can have morethan 80 cm of mycorrhizal hyphae, more thanthe amount necessary to account for the ob-served phosphorus uptake,

Plant uptake of mobile soil nutrients such asnitrogen and potassium is rarely improved bymycorrhizal fungi, Normal soil diffusion is ade-quate to supply roots of plants with these nu-trients whether the roots have a large absorb-ing surface or not. Generally, plants that aremost dependent on mycorrhizal fungi for nu-trient uptake are those having roots with a lowsurface to volume ratio; that is, plants withcoarse, fleshy roots with few root hairs (2).

38-846 0 - 85 - 7

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Although some scientists speculate that my-corrhizal fungi can solubilize and absorb nu-trients that are unavailable to plant roots, thereis little evidence to support this claim. Sandersand Tinker (88) showed conclusively with 32p-labelled phosphate that mycorrhizal fungi usethe same phosphorus sources as do plant rootsbut they are able to absorb from a larger soilvolume and so are responsible for the vast ma-jority of phosphorus absorption by crop plants.

Mycorrhizal fungi can also enhance watertransport in plants (87) and prevent water stressunder some conditions (54). This probably isnot a direct effect of mycorrhizal fungi, but in-stead is because of the improved nutrient statusprovided by the mycorrhizal fungi. Mycorrhi-zal fungi can endure much dryer soil condi-tions than can most plants and it is thought thatplants may benefit from mycorrhizal infection

under drought or water-stressed conditions(66,86). Ectomycorrhizae, in particular, withtheir mantle surrounding the roots, may pro-vide a physical barrier against root dessication.

Considerable evidence exists to suggest thatmycorrhizal plants may be better equipped towithstand the toxic effects of salt. Calcium,magnesium, and sodium concentrations innon-mycorrhizal citrus were 41 percent, 36 per-cent, and 150 percent greater than in mycor-rhizal citrus (55). Hirrel and Gerdemann (35)found that mycorrhizal fungus increased bellpeppers tolerance to salinity. Trappe, et al. (98),indicated that VA mycorrhizal fungi providedresistance to the toxic effects of arsenic. My-corrhizae may also provide tolerance to ex-cessive soil manganese and aluminum (34).

Mycorrhizal fungi also act to increase modu-lation by symbiotic nitrogen-fixing bacteria

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such as Rhizobium (64,69). Mycorrhizal fungimay stimulate other beneficial rhizosphere or-ganisms as well (1)0

Ectomycorrhizal fungi have been reported toprovide resistance to plant disease in manyplants (48). Although mycorrhizae never con-fer complete immunity, they often appear toreduce the severity of disease or symptom ex-pression. Resistance of ectomycorrhizae to dis-ease may result from (48):

● mechanical protection by the mantle,● better plant nutrition,Q production of antibiotics by the mycorrhi-

zal fungus,● competition for infection sites,● formation of phytoalexins, and● alteration of root exudates.

Evidence is accumulating that VA mycorrhi-zal fungi exert similar effects on plant patho-gens. Schenck, et al, (91), has reported mycor-rhizal resistance to root-knot nematodes.Schonbeck (93) has examined a variety of foliarand root pathogens on mycorrhizal plants andconcluded that root pathogens (Thielaviopsis,

Fusarium, nematodes, etc.) are usually inhib-ited by mycorrhizal fungi while foliar patho-gens (viruses, rusts, etc.) are often more severeon mycorrhizal plants. Davis, et al. (21,22), andDavis and Menge (20) concluded that the VAmycorrhizal fungus Glomus fasciculatus pro-duced little resistance to Phytophthora root rotin citrus and indeed increased Phytophthoraroot rot in avocado and Verticillium wilt in cot-ton. VA mycorrhizal effects on disease may re-sult from improved phosphorus nutrition be-cause of the increased absorbing surface of themycorrhizal hyphae. This effect is magnifiedwhen the roots’ normal absorbing capacity isreduced because the roots are partiallydecayed.

There have been reports of mycorrhizal fungiactually reducing growth of some plants (11,39,13). These parasitic effects are rare and thereason for them is not understood, but they ap-parently occur in grasses, cereals, andtomatoes at or above optimum soil nutrientlevels when the plant is actively regulatingmycorrhizal invasion,

MYCORRHIZAE AS SUBSTITUTES FOR FERTILIZERS

In the past 40 years the use of agriculturalfertilizers has more than doubled. Crop yieldshave risen dramatically as a result. However,because of shortages in some fertilizer suppliesand the high cost of energy, the cost of fertil-izers has risen tremendously. Agriculturaleconomists indicate that as energy costs risethe most responsive agricultural input is fer-tilizer. That is, as energy costs rise, fertilizeruse will decrease. This response is a dangerousone since chemical fertilizers are said to ac-count for one-third to one-half of the currentU.S. agricultural output (47).

Estimates indicate that agriculture uses be-tween 2.6 and 4.4 percent of all U.S. energyuse. Fertilizers and their application comprise30 to 45 percent of the total agricultural energyuse. Nitrogen is the main energy user, with

phosphorus and potassium accounting for only16 percent of the fertilizer energy use (47).

Because mycorrhizal fungi increase the effi-ciency of fertilizer use, they can be thought ofas “biotic fertilizers” and can indeed be sub-stituted for substantial amounts of some fer-tilizers (53,55). Mosse (61) maintains that 75percent of all phosphorus applied to crops isnot used during the first year and reverts toforms unavailable to plants. In soils high in pH,aluminum, or calcium carbonate, nearly 100percent of the phosphorus fertilizer can be im-mobilized to nonusable forms via chemical re-actions in the soil. Tropical oxisols and ultisolsare notorious for their capacity to immobilizephosphorus. Because mycorrhizal plants arebetter suited to exploiting soil with low amountsof available phosphorus, zinc, and copper, the

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addition of large amounts of these fertilizerseach year may be unnecessary. Menge, et al.(55), compared mycorrhizal citrus seedlingswith non-my corrhizal seedlings that receivedvarious amounts of phosphorus fertilizer (fig-ure 4).

Mycorrhizal Troyer citrange that received nofertilizer phosphorus were equal in size to non-mycorrhizal Troyer citrange that received 112kg phosphorus per hectare, Similarly, mycor-rhizal Brazilian sour orange that received nofertilizer phosphorus were equal in size to non-mycorrhizal plants that received 560 kg phos-phorus per hectare. Concentrations of phos-phorus in non-mycorrhizal Brazilian sourorange leaf tissue were never above 0.05 per-cent (less than 0.9 percent phosphorus in-dicates phosphorus deficiency) even whenseedlings were fertilized with 1,120 kg

Figure 4.—Dry Weights of Mycorrhizal andNon-Mycorrhizal Brazilian Sour Orange andTroyer Citrange Seedlings Fertilized With

Different Amounts of Phosphorus

phosphorus per hectare. Concentrations ofphosphorus in leaves of mycorrhizal Braziliansour orange were above deficiency levels in allseedlings fertilized with more than 56 kgphosphorus per hectare. Concentration ofphosphorus in leaves of mycorrhizal Troyercitrange were never in the deficiency rangeeven when plants were not fertilized withphosphorus.

Non-mycorrhizal Troyer citrange, on theother hand, required over 56 kg phosphorus perhectare before adequate phosphorus concen-trations were restored to the leaves. At 1980retail costs for triple super-phosphate, it ap-pears that use of mycorrhizal fungi could re-sult in savings of $111 to $558/ha ($45 to$226/acre) in the cost of phosphorus fertiliza-tion of citrus in fumigated nursery soil. In oneCalifornia citrus nursery, it was found that in-oculation with mycorrhizal fungi could reducephosphorus fertilization by two-thirds and save$652/ha ($264/acre). Similar savings in phos-phorus fertilizers have been shown by Kor-manik, et al. (43), in fumigated forest nurseriesin the production of sweetgum.

Mycorrhizal fungi also can be substituted forcopper fertilizer in the culture of citrus seed-lings (97). Other data has shown that mycor-rhizal fungi can be substituted for zinc fertilizerin the greenhouse culture of citrus and evennitrogen fertilization can be reduced by asmuch as 300 percent in the presence of mycor-rhizae (Menge, et al., unpublished data). Thisnitrogen savings effect is probably due to anincreased efficiency of nitrogen use resultingfrom improved phosphorus nutrition of theplant.

Since mycorrhizal fungi are present in mostsoils, their unique fertilizer-absorbing abilitiesare normally already being used by most crops.If mycorrhizal fungi are removed or damagedin any way, then the amount of fertilizer re-quired by a crop increases enormously. Thisis demonstrated by reports that citrus grownin fumigated soil or in hydroponic solutionsoften require massive phosphorus applicationsfor adequate growth compared to field growncitrus (55). Citrus in the field can absorb phos-

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phorus from phosphorus-deficient soils moreefficiently than either corn or tomatoes, andcitrus orchards do not normally require phos-phorus fertilization (9), Differences in phospho-rus absorption by citrus grown in fumigatedsoil and citrus grown in nonfumigated soils canbe reconciled if mycorrhizal fungi, which arepresent in nearly all citrus orchards (52), arethe equivalent of 100 to 500 pounds phosphorusper acre.

When and if the cost of fertilizer becomes ex-orbitant, we must devise the most efficient fer-tilizer supply systems possible—to minimizecosts while conserving energy and nonrenew-able resources. I submit that mycorrhizal fungicould be one alternative that might increasecrop yields and yet reduce fertilizer costs andenergy demands.

CURRENT COMMERCIAL USE OF MYCORRHIZAL FUNGI

Although nearly all plants require mycorrhi-zal fungi for maximum growth, the widespreadoccurrence of these fungi in nearly all soilslimits the immediate needs for inoculation withmycorrhizal fungi. Mycorrhizal fungi are cur-rently commercially usable in only three ma-jor agricultural areas: 1) disturbed sites, 2)fumigated soils, 3) greenhouses.

Disturbed Sites

Mycorrhizal fungi have been conclusivelyshown to improve revegetation of coal spoils,strip mines, waste areas, road sites, and otherdisturbed areas (18,19,15,49,81). In these stressedsites, mycorrhizal fungi are usually lacking andadding mycorrhizal fungi provides a nutrition-al advantage to associated plants in additionto providing possible resistance to low pH,heavy metal toxicants, and high temperature,

Fumigated or Chemically Treated Sites

Fumigation with biocides or pesticides suchas methyl bromide (56), chloropicrin (72),dazomet (50), 1,3-D (72), vapam (71), and vorlex(71) may destroy or inhibit root infection bymycorrhizal fungi, Application of many soilfungicides such as arasan (71), banzot (95),benomyl (96), botran (71), carbofuran (3),chloramformethane (37), dichlofluanid (37),ethirimol (37), lanstan (71), mylone (71), PCNB(96), sodium azide (3), thiabendazole (37),thiram (96), triademifon (37), tridemorph (37),and vitavax (96) have also been reported to be

harmful to mycorrhizal development, Flood-ing, planting non-my corrhizal crops, or remov-ing topsoil, may also reduce the population ofmycorrhizal fungi to a level requiring reinocu-lation (7,78).

Fumigation with the biocide methyl bromideto remove soil-borne pests is required by reg-ulation for the production of many nurserycrops. It is also regularly used in many fieldagricultural situations. This chemical is ex-tremely toxic to mycorrhizal fungi and mostfield fumigations are sufficient to destroy thenative mycorrhizal inoculum (56). Stunting ofcrops following fumigation with methyl bro-mide is common and is due to the destructionof mycorrhizal fungi. Although a relativelysmall amount of land is treated with this chem-ical, less than 100,000 acres annually in theUnited States, stunting following fumigationwith methyl bromide has been reported in theUnited States, Africa, Spain, Peru, Venezuela,and many other countries (52). Crops that areroutinely grown in methyl bromide fumigatedsoils include strawberries, tomatoes, tobacco,nursery crops, tree crop replants, and somevegetable crops. For many of these crops theaddition of mycorrhizal fungi following fumi-gation with methyl bromide is not only recom-mended but is imperative.

It appears that inoculating methyl bromidefumigated crops is economically possible. Thecost for inoculating nursery-grown citrus withmycorrhizal fungi is about $288/acre, while thecost for phosphorus fertilizer alone is $338/acre. Fumigated tomatoes receive $51 worth

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of phosphorus per acre while the cost for my-corrhizal inoculation is less than $28/acre. My-corrhizal fungi can provide additional benefitsto the crop other than just improved phos-phorus nutrition.

For nursery plants grown in methyl bromidefumigated soil, inoculation with mycorrhizalfungi should be imperative for the followingreasons:

●

●

●

●

●

the plants grow better (prevents stuntingfollowing fumigation);there is a decreased need for fertilization,specifically phosphorus, zinc, and copper,resulting in decreased fertilizer cost andenergy conservation;there is decreased chance for water stressand therefore reduced transplant injury;mycorrhizal plants survive better especial-ly if transplanted to fumigated, poorly fer-tilized, or disturbed soil;plants will be inoculated with effective my-

●

corrhizal fungi rather than leaving mycor-rhizal infection to chance; andmycorrhizal plants may be more resistantor tolerant to some plant diseases.

Greenhouses

Greenhouse culture uses growth media suchas pine bark, vermiculite, perlite, builders sand,and peat moss and these are devoid of mycor-rhizal fungi. In addition, most greenhouseoperators steam, pasteurize, or chemically treattheir mixes to eradicate harmful pathogens.Nurserymen have compensated for the absenceof beneficial mycorrhizal fungi by applying lux-ury amounts of fertilizer and water to achievedesired growth. Inoculation of containergrown plants to reduce irrigation, fertilizer,and pesticide applications and cost can be doneas demonstrated by Chatfield, et al. (10), Lin-derman (46), and Crews, et al. (12).

COMMERCIAL PRODUCTION AND INOCULATION WITHMYCORRHIZAL FUNGI

Many ectomycorrhizal fungi can be readilycultured on artificial media and inoculum canbe grown under standard laboratory conditions(49). Experimentally, sterilized vermiculite andpeat moss is frequently saturated with a liquidnutrient medium (49) and is infested with a de-sirable ectomycorrhizal fungus. Ectomycorrhi-zal fungi generally grow quite slowly and maytake several months to colonize the vermiculite-peat moss mixture. This material can be usedon a small scale to inoculate nurseries andgreenhouses with mycorrhizal fungi. AbbottLaboratories, North Chicago, Illinois, has pro-duced massive amounts of inoculum of the ec-tomycorrhizal fungus Pisolithus tinctorius (86).Abbott Laboratories produced the peat moss-vermiculite-nutrient solution inoculum underlarge-scale commercial conditions using com-mercial fermenters.

Under the direction of D. H. Marx, the U.S.Forestry Service has undertaken a massivetesting program using the commercially pro-

duced inoculum. The inoculum will be testedin nearly 100 tree nursery test sites through-out the United States. Results will be availablewithin 4 years and will indicate the commer-cial feasibility of producing and using mycor-rhizal inoculum in fumigated tree nurseries.

Ectomycorrhizal inoculum can best be ap-plied in the nursery. Once the trees becomeinfected, the benefits can be transferred towherever the trees are grown. In the nursery,mycorrhizal inoculum can be distributed byhand and rototilled into the soil before plant-ing seed. Special machinery has already beenbuilt and is being used to incorporate ectomy-corrhizal inoculum.

Commercial production of mycorrhizal in-oculum for use in sterilized or fumigated soilis being attempted at several locations in theUnited States. Currently, the only way to pro-duce suitable quantities of a mycorrhizal in-oculum is on roots of susceptible host plants.

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The possibility of pathogenic organismscontaminating mycorrhizal inoculum is an ex-tremely serious problem when growing VAmycorrhizal inoculum in semi-sterile culturesin the greenhouse. For this reason, many scien-tists will consider mass production of VAmycorrhizal fungi only if it is done axenically(one organism only).

Realistically, however, not only must theseobligate parasites be grown in vitro, but theymust produce large quantities of spores inculture which will survive under soil condi-tions and infect plants in nature. Informationgained from the culture of other formerlyobligate parasites suggests that the possibilityof realizing this goal in the near future isunlikely. Even if mycorrhizal fungi are culturedaxenically, mycorrhizal inoculum for field use

will probably be produced on the roots of suit-able host plants.

With proper safeguards, mycorrhizal inocu-lum, free of plant pathogens, can be producedon plants in the greenhouse. Figure 5 illustratesa proposed scheme for producing mycorrhizalinoculum [53). VA mycorrhizal fungi can beisolated by using bits of roots or soil from thefield to inoculate roots of “trap plants” grow-ing in sterilized soil in the greenhouse. Sudan-grass (Sorghum vulgare Pers.) is frequentlyused, but other plants such as tomato, soybean,corn, and safflower may be equally suitable.The soil used throughout is a low nutrient sandfertilized once per week with one-half thestandard Hoagland’s solution minus phospho-rus. After production of VA mycorrhizal sporesin the “pot cultures,” the spores can be

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removed by wet sieving (29), elutriation (25),or centrifugation (85). These spores must besurface disinfested with substances such aschloroamine T or sodium hypochlorite andstreptomycin to assure that pathogens do notaccompany the spores (68).

These surface disinfested spores are used toinoculate the roots of plants that were ger-minated and grown under aseptic conditionsin growth chambers. The containers illustratedare made from plastic petri plates and filledwith the low nutrient sand. After 1 to 4 weeks

when the mycorrhizal fungi have infected rootsgrown under aseptic conditions root pieces canbe removed and stained (73) to observe infec-tion, root pieces are carefully removed andused to infect suitable host plants grown insterilized soil in the greenhouse. Similar rootpieces can be removed, examined, and platedon agar to observe pathogenic organisms.

If no pathogens are observed, the greenhouse“pot culture” may be used as a “mother cul-ture” to produce inoculum that will be used inthe field. Inoculum should be produced on se-lected hosts that have no root diseases in com-mon with the host plant for which the in-oculum is intended. For instance, inoculum forcitrus could be produced on sudangrass butnever on citrus. In this way the wide host rangeof most VA mycorrhizal fungi can be used. Asanother precaution against propagating path-ogens along with mycorrhizal inoculum, thefield inoculum should be drenched severaltimes with pesticides chosen to eliminate path-ogens known to infect the host for which theinoculum is intended. Mycorrhizal inoculumintended for citrus should be drenched witha nematicide to control the citrus nematodeand fungicides to control Phytophthora andRhizoctonia. Suggested pesticides are Ethazoleand PCNB. PCNB reduces the population ofmycorrhizal spores but the other pesticide canactually increase spore production (57). Severalother pesticides can be used without harmingmycorrhizal fungi (96).

Horticultural practices also could be used atthis point to maximize spore production. Elim-inating fertilization and slowly reducing the

water may be effective in increasing spore pro-duction. When spores are mature, plant topsare removed and roots, soil, and spores can beground up and partially dried (7 to 20 percentmoisture content) and stored at 40 C until used.If concentrated spore suspensions are desired,spores can be concentrated by wet sieving (38),elutriation (25), or centrifugation (85) beforestorage. VA mycorrhizal inoculum can befreeze-dried if desired (38). Inoculum producedin this manner should be consistently infectiveand yet pathogen free.

Using the method described above, the esti-mated costs for producing mycorrhizal in-oculum are shown in table 1. These figures arederived from production costs of a foliage plantgreenhouse and could be reduced considerably

Table 1 .—Estimated Cost of Production ofVesicular-Arbuscular Mycorrhizal Inoculum

on Sudangrass in 4 Inch Pots

Item Cost/pot1. Labor:

a. to prepare the soil mix . . . . . . . . . . . . . . . . . . $0.03b. potting, inoculating, and seeding. . . . . . . . . . 0.05c. moving pots to growing area . . . . . . . . . . . . . 0.03d. pruning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.02e. spraying (insecticides and fungicides) . . . . . 0.03f. watering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.02g. harvesting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.01h. grinding and packaging . . . . . . . . . . . . . . . . . . 0.03i. quality control . . . . . . . . . . . . . . . . . . . . . . . . . . 0.02j. maintenance of mother cultures . . . . . . . . . . 0.05

Labor cost . . . . . . . . . . . . . . . . . . . . . . . . . . . .2. Materials:

a. pots 4 ' . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .b. seed. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .c. fertilizer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .d. shipping containers . . . . . . . . . . . . . . . . . . . . .e. insecticides and fungicides . . . . . . . . . . . . . .

Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3. Overhead expenses:

a. heat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .b. depreciation on greenhouse . . . . . . . . . . . . . .c. depreciation on boilers . . . . . . . . . . . . . . . . . .d. maintenance allowance . . . . . . . . . . . . . . . . . .e. office supplies . . . . . . . . . . . . . . . . . . . . . . . . .f. management and office work . . . . . . . . . . . . .g. return on investment . . . . . . . . . . . . . . . . . . . .h. loss due to undeveloped plants . . . . . . . . . . .i. taxes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .j. laboratory, incubator, etc. . . . . . . . . . . . . . . . .

$0.29

$0.070.0020.020.0250.03

$0.147

$0.080.0080.0030.0060.0020.030.010.0010.060.04

Total for overhead . . . . . . . . . . . . . . . . . . . . . . $0.24Total cost ... ... .$0.677Selling price ... ..$0.900.18¢/500 spores

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since mycorrhizal inoculum quality is of im-portance and not plant quality, A reasonablygenerous estimate of the cost of mycorrhizalproduction, including technical labor andquality control, together with a small marginof profit, indicates that consumers may payabout 0.180/500 spores of VA mycorrhizalfungi. Such a cost could reasonably be borneby consumers such as greenhouse operators ornurserymen.

A similar method to that outlined above hasbeen patented in England and is being per-fected for large-scale commercial use (34). Inthis method plants are grown in peat blocksthat are standing in a shallow nutrient-flowculture. After VA mycorrhizal spores are pro-duced in the peat blocks they are ground up,roots and all, for inoculation. The finishedproduct is not only excellent mycorrhizal in-oculum but is light and easy to ship.

Although many methods have been used toinoculate plants with VA mycorrhizal fungi ingreenhouse trials, few inoculation methods areacceptable for large-scale commercial inocu-lation. Several different methods to inoculatecorn have been studied and layering inoculumunder the seed was superior to seed inocula-tion or banding the inoculum (38), Hall (30) de-veloped a method for pelleting seed with a my-corrhizal infection and determined thatmycorrhizal fungi could survive up to 28 daysunder these conditions. Menge, et al. (53),found that layering inoculum below the seedand banding inoculum were superior to seedinoculations. Crush and Pattison (14) experi-mented with several means of inoculatingseeds with VA mycorrhizal fungi, but againfound that sowing seed above pelleted mychor-rhizal inoculum was the most effective methodfor obtaining mycorrhizal infection. Hattinghand Gerdemann (31) reported growth re-

sponses of citrus in a fumigated nursery afterinoculating citrus seed with mycorrhizal in-oculum. Gaunt (26) inoculated onion andtomato seeds with a VA mycorrhizal fungusand reported that seed inoculated plants grewas well as plants that were inoculated by mix-ing VA mycorrhizal inoculum into the soil.Commercial applications of mycorrhizal in-oculum using fertilizer banding machinerywere success fu l ly ca r r i ed ou t in c i t rusnurseries in California (23).

Commercial VA mycorrhizal inoculum isproduced using the method described above intwo citrus nurseries—Brokaw Nursery, SaticoyCalifornia and the Thermal Ranch, Thermal,California. Experimental VA mycorrhizal in-oculum is being produced and distributed ona large scale by Abbott Laboratories, North Chi-cago, Illinois. Other major corporations thatare supporting or carrying out research on VAmycorrhizal fungi include Dow Chemical Co.,Rohm & Haas Co., Dupont, Monsanto Co., andCeiba-Geigy Chemical Co.

Plants growing in all soils do not respond fa-vorably to VA mycorrhizal inoculum. If soil nu-trition is optimum, mycorrhizal fungi will notenhance growth of plants. A method for detect-ing which soils require mycorrhizae for max-imum production of citrus was devised byMenge, et al. (58). In soils with less than 34 ppmavailable P (Olson analysis), 12 ppm availableZn, 27 ppm available Mn, or 3 percent organicmatter, citrus trees will probably require my-corrhizal fungi for maximum growth. Mycor-rhizal inoculations are recommended only insoils with these characteristics, It is estimatedthat this includes approximately 85 percent ofthe southern California citrus soils. Similarstudies could be done with other crops to de-termine which soils require mycorrhizal in-festation.

POTENTIAL USES FOR MYCORRHIZAL FUNGI

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

196

Under these conditions, any growth benefitdue to mycorrhizal inoculation would dependprimarily on the superiority and/or placementof the mycorrhizal inoculum. Beneficial re-sponses under these conditions would be pre-dicted to be far less than the responses obtainedin fumigated or partially sterilized soil. How-ever, greenhouse and field experiments inwhich plants were inoculated with mycorrhizalfungi in nonfumigated soils have demonstratedthat growth responses due to mycorrhizal fungican occur under these circumstances.

In greenhouse experiments, using untreatedsoil, Mosse and her colleagues (62,63,65,67,70)demonstrated that preinoculation with mycor-rhizal fungi could provide the following growthincreases:

Crop Growth increaseCentrosema spp. . . . . 34 percentcorn . . . . . . . . . . . . . . . 306 percentMelinis spp. . . . . . . . . 41-60 percentonions . . . . . . . . . . . . . 48-3155 percentstrawberries . . . . . . . . 250 percentStylosanthes spp. . . . 85-88 percentsweetgum . . . . . . . . . . 45 percentViola spp. . . . . . . . . . . 527 percentOther studies have noted similar growth increases in untreatedsoil:corn . . . . . . . . . . . . . . . 14 percent (Gerdemann, 1964)corn . . . . . . . . . . . . . . . . 0-53 percent (Jackson, et al., 1972)mahogany . . . . . . . . . . 151 percent (Redhead, 1975)sudangrass . . . . . . . . . . 0-18 percent (Jackson, et a]., 1972)white clover . . . . . . . . 80-100 percent (Powell, 1977)

In a large-scale field experiment conductedin nonsterile, virgin, infertile fields, wheat pre-inoculated with a mycorrhizal fungus pro-duced 220 percent more grain than non-mycor-rhizal wheat (41). In a similar experiment (40),corn inoculated with a mycorrhizal fungus was122 percent larger than non-mycorrhizal corn.Hayman (33) reported white clover growth in-creases in the field due to inoculation with amycorrhizal fungus. Black and Tinker, in anextremely well-documented field experiment,found that fallow field inoculation with a my-corrhizal fungus increased potato yield 20percent.

Not all mycorrhizal inoculations in nonster-ile soil result in increased growth. Hayman (33)indicated that mycorrhizal fungi did not stim-ulate growth of white clover at several loca-tions. Powell (75) obtained significant growth

increases of white clover after inoculation withmycorrhizal fungi in only three of nine sites.Jackson, et al. (38), indicated that with certainmycorrhizal inoculation methods, growth ofcorn, sudangrass, and soybeans was not stim-ulated in nonsterile soil. Mosse (65) obtainedsignificant growth responses of Stylosanthesspp. due to mycorrhizae in 6 of 11 nonsterilesoils. Ross and Harper (85) reported no growthstimulation of soybeans in nonsterile soil.

Mosse (65) indicated that the inoculum po-tential of indigenous mycorrhizal fungi is themajor determinant governing growth responsesof plants to mycorrhizal fungi in nontreatedsoil. Powell (75) indicated that many indige-nous mycorrhizal fungi are “inefficient” sym-bionts, and that inoculation by more efficientmycorrhizal fungi will result in growth in-creases even in nonsterile soil that contain highpopulations of “inefficient” mycorrhizal fungi.placement of mycorrhizal inoculum is equallyimportant in affecting a plant growth response(38). Certainly, plants infected early in thegrowing season by mycorrhizal fungi are bet-ter than plants that do not become infected un-til later (82).

Huge expanses of tropical soils (e.g., the Bra-zilian Cerrado) are either deficient in phos-phorus or immobilize phosphorus fertilizers.These marginal agricultural lands could be pro-ductive if mycorrhizal fungi, with the abilityto efficiently use extremely small quantities offertilizer, were developed and added to the soil.Cheap but readily available rock phosphatecould be added as the phosphorus source. Thisphosphorus source is a poor fertilizer but re-leases small quantities of phosphorus for longperiods of time. Some mycorrhizal fungi userock phosphate much better than others andcan tremendously improve growth of plantsgrowing in poor soils fertilized with this ma-terial (59,66).

Mycorrhizal fungi have been proposed as un-stable soil or sand dune stabilizers (96). Finallyectomycorrhizal fungi have been shown to im-prove rooting of a wide variety of non-hostplants and the possibility of using them as acommercial root stimulant has been proposed(45).

197

CONSTRAINTS ON THE COMMERCIAL USE OFMYCORRHIZAL FUNGI

The current major obstacles to the commer-cial use of mycorrhizal fungi are:

c the lack of large-scale field experimentsunder normal agricultural conditions,

Q the lack of cost-benefit analysis to deter-mine the economics of mycorrhizal appli-cations, and

● the trend toward excessive fertilization tosubstitute for the lack of mycorrhizalfungi.

Perhaps the most important deterrent of com-mercial use of mycorrhizal fungi is the lack oflarge-scale field tests in a variety of agriculturalsoils and locations. The program initiated byD. H. Marx and the U.S. Forest Service willcorrect this deficiency for ectomycorrhizal fun-gi and within 4 years it will be known if thesemycorrhizal fungi will indeed be economicallyfeasible to use on a wide scale in the produc-tion of forest trees.

This type of program remains to be establishedfor VA mycorrhizal fungi. Without such datait is difficult to establish a potential market formycorrhizal inoculum. Without a market thereis little incentive for industry to initiate the pro-duction of commercial inoculum. Without com-mercial inoculum it is difficult to carry outlarge-scale field tests. With the recent establish-ment of several commercial sources of mycor-rhizal inoculum perhaps this cycle will be bro-ken and more field tests will result.

Once large-scale field tests are seen to be suc-cessful, light-weight commercial mycorrhizalformulations will develop and new applicationmethods will be devised, Most importantly,from large-scale field tests, cost benefit analy-sis can be accurately done to determine theeconomic benefit derived from the use of my-corrhizal fungi. In the end, this will be the de-termining factor in the commercial applicationof mycorrhizal fungi. Biological scientists arerarely able to critically assess the economic fac-tors involved in the application of a new tech-nique and I recommend that agricultural econ-omists should be asked to participate in the

cost-benefit assessment of VA mycorrhizal in-oculation.

Heavy phosphorus fertilization severely in-hibits mycorrhizal infections (17,68). Morerecently, it is becoming evident that heavy ni-trogen and zinc applications are also inhibitoryto mycorrhizal fungi (32,51). Daily applicationsof 100 ppm nitrogen under greenhouse condi-tions have been shown to completely eliminatemycorrhizal infections (J.A. Menge, unpub-lished data). Many commercial greenhousesadd over 200 ppm nitrogen daily to their plants.In greenhouse and fumigated nursery condi-tions, growers are using excessive fertilizationto substitute for the lack of mycorrhizal fungi.Under these conditions, not only do mycorrhi-zal fungi not benefit their host plants, but it isdifficult to successfully establish mycorrhizalinfections so that the plants will be mycorrhizalonce they leave the supraoptimal fertilityregime. As long as fertilizer is relatively avail-able and not excessively expensive, it will takea major educational program to convince manygrowers to change their standard operatingprocedures and use mycorrhizal fungi that willnot only be cheaper but will conserve fertilizerand energy.

In my opinion, granting agencies such as theNational Science Foundation, RockefellerFoundation, USDA competitive grants, and theIsraeli-U.S. granting agency BARD have effec-tively provided adequate funding for basic my-corrhizal research. The number of scientificpapers on mycorrhizal fungi has quadrupledsince 1960, which is evidence that there is greatinterest and money available for basic mycor-rhizal research. However, there are few agen-cies that will fund the final applied steps in abiological commercialization project. Researchmoney for large-scale “applied” or “demonstra-tion” experiments is unavailable. Funding forsmall-scale pilot projects is also not available.It remains for private industry to pick up theprojects from this point, but they have been re-luctant to do so. The transition is not goingsmoothly and seems to be proceeding slowlyif at all,

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It is very difficult for a scientist to speculateon the effects of a new procedure on the socialand economic structure of an agricultural so-ciety. Frequently good ideas do not receive theacclaim they deserve because of prejudices, ig-norance, religious preferences, social mores,and other reasons not fully understood by sci-entists. In my opinion, the effects of mycor-rhizal technology would most alter the socio-economic structure in areas of intensive agri-culture. These situations would be more prev-alent in agriculture in developed nations.Mycorrhizal fungi are most useful in reclaim-ing sites disturbed by heavily mechanized in-dustries or soil fumigation. Mycorrhizal fungican reduce energy and fertilizer and increasethe efficiency of crops grown intensively.Therefore, mycorrhizal fungi can be viewed asconservation measures or as substitutes forhigh energy uses in developed nations.

In less developed nations, growers wouldhave to be educated to the methods of produc-ing, handling, and inoculating living micro-organisms. This may be difficult. In countries

FUNGI ON AGRICULTURE

with a less well-developed agricultural system,mycorrhizal fungi have not been altered andare probably functioning effectively and neednot be applied under such conditions. Fertilizerin most underdeveloped countries is probablyapplied sparingly as manure and therefore my-corrhizal fungi will not result in a great sav-ings either of fertilizer or energy.

If superior strains of mycorrhizal fungi aredeveloped, marginal agricultural land could bemade productive. Huge amounts of marginalagricultural land exists in Africa and SouthAmerica and the proper use of this land maywell decide the future of some countries. In-creased use of agricultural land will providefor a greater economic base, larger agriculturalproductivity, and a better way of life for largepopulations in underdeveloped countries. Edu-cating agriculturists to the importance of my-corrhizal fungi may allow developing countriesto avoid the excessive use of energy, fumigants,and fertilizers associated with intensive agri-culture.

Mycorrhizal fungi may be one alternativethat can immediately improve revegetation ofdisturbed sites, increase crop growth in fumi-gated soils and greenhouses, and yet reducefertilizer costs and energy demands. If superiorstrains of mycorrhizal fungi were developed,they could potentially improve growth of near-ly all agronomic crops in a wide variety of soilsthroughout the world. Both ectomycorrhizalfungi and vesicular-arbuscular mycorrhizalfungi are in commercial production on a smallscale. The greatest obstacles to the commercial-ization of mycorrhizal fungi appears to be: 1)the lack of large-scale field tests under typicalagricultural conditions in a variety of locations;2) adequate cost-benefit analysis to determinethe economics of the utilization of mycorrhizalfungi; and 3) a reluctance on the part of grow-ers to switch from an energy dependent, heavy

fertilizer system to a new, but cheaper, energyconservative system using mycorrhizal fungi.

Recommendations that could substantiallyincrease the commercial use of’ mycorrhizalfungi (in relative order of importance) are asfollows:

1. Improved availability of grant funds forlarge-scale field applications of mycorrhi-zal fungi in a wide variety of soils through-out the world. It would be useful to estab-lish several pilot projects in various lessdeveloped countries. These pilot projectscould produce and distribute mycorrhizalinoculum on a variety of crops growingunder different soil conditions. Cost-ben-efit analysis on such projects could ade-quately assess the economics of inocula-tion with mycorrhizal fungi.

199

2.

3.

4.

5.

6.

7.

Funds should be made available to createa worldwide bank of beneficial mycorrhi-zal fungi. The establishment of such afacility is being investigated by the mycor-rhizal community and the National Sci-ence Foundation has agreed to entertaina proposal for such a facility. The Univer-sity of Florida has agreed to supply the fa-cilities as well as substantial operatingcosts for such an establishment. A secondidea would be to add the responsibility formaintaining mycorrhizal cultures to thealready established government facilitycalled the American Type Culture Collec-tion which maintains many importantfungal cultures.It would be desirable to establish a USDA-supported vesicular-arbuscular mycorrhi-zal research center that would be respon-sible for maintaining and coordinatingU.S. research on mycorrhizal fungi. Thisfacility would complement the Mycorrhi-zal Institute in Athens, Georgia, which wascreated by the Forest Service to coordinatemycorrhizal research on forest trees.A world survey should be conducted tocollect and test as many different vesicu-Iar-arbuscular mycorrhizal species as pos-sible. The discovery of a superior mycor-rhizal strain with a wide host range couldtremendously increase agricultural pro-ductivity throughout the world.Research is necessary to elucidate the ex-act role of mycorrhizal fungi play in im-proving plant growth under stress condi-tions such as drought, salt, toxic soilmaterials, or in marginal agriculturallands.Research is necessary to elucidate the ge-netics of mycorrhizal fungi. Virtuallynothing is known on this subject. Theability to breed these organisms could re-sult in tremendously increased agricul-tural productivity.Efforts should be intensified to grow ve-sicular-arbuscular mycorrhizal fungi in thelaboratory using artificial media. Abreakthrough in this area could improvethe feasibility of attaining all of the aboverecommendations, However, since scien-

tists have been trying to artificially cultureVA mycorrhizal fungi since 1900, this ob-jective may be difficult to achieve and in-centives to work on such a problem are dif-ficult to justify.

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