EP0482130A1 - Production of mycorrhizal inoculum by static culture hydroponics - Google Patents

Abstract

The invention relates to a system of static hydroponic cultures for the production of vesicular mycorrhizae in the form of arbuscules. Good yields of fungal spores can be obtained as well as good growth of the host plant using a medium at high phosphorus concentrations, the initial association of mycorrhizae being started at lower phosphorus concentrations. The host plant species allowing the use of phosphorus should be selected. Several edible plant species can serve as host plants. Harvest and then store propagules of symbiotic mycorrhizal fungi, which can then be transmitted to plants.

Description

PRODUCTION OF MYCORRHIZAL INOCULUM BY STATIC CULTURE

HYDROPONICS

BACKGROUND OF THE INVENTION

Vesicular-arbuscular mycorrhizas (also VA mycorrhizas or VAM) are symbiotic associations occurring between certain naturally occurring soil fungi and the roots of higher plants. The mycorrhizal fungus forms a variety of structures within the plant root as well as extensive extramatrical (outside of the root) hyphae which grow through large volumes of soil The extramatrical hyphae take up nutrients from the soil which are then translocated back to the plant. The fungus obtains carbohydrates from, the plant in return. Consequently, the plant's nutrient uptake is increased. This is especially so for elements such as phosphorus which are usually relatively immobile in the soil. The increased nutrient uptake can, under many circumstances, give increased crop yields and may also lower fertilizer requirements. Furthermore, the symbiosis can impart increased disease resistance, transplant survivability, drought resistance and improved plant establishment in disturbed or polluted sites.

Although VAM fungi are ubiquitous, a variety of agricultural and horticultural practices destroy or reduce natural populations. Additionally, indigenous VAM fungi may not be best able to stimulate plant growth, especially if soil conditions, such as pH, have been altered.

Plant growth may be increased by introducing previously prepared cultures of VAM fungi to those plants. This may be done when the VAM inoculant is superior to the indigenous VAM species, or where increased VAM populations are desired.

The fungus cannot be commercially grown in pure culture. It must be grown with its symbiotic plant partner, or a portion of that higher plant, such as root tissue culture.

The traditional method of VAM inoculum production is pot culture. In this method, desired species of VAM fungi are first isolated from soils. The fungi are used to inoculate plants potted in soil, sand or a peat-based growing medium. VAM fungi are cultured in a variety of ways for inoculation. The plant is then grown for three to six months, during which time it is fertilized with a low phosphorus nutrient solution. When fungal development is extensive, the plant is harvested. The shoot is discarded and the entire contents of the pot can be used for inoculum. Alternatively, fungal spores can be extracted from the growing medium and used alone as the inoculum. The resultant inoculum is very bulky, because soil represents most of the inoculum's mass. An additional step of spore extraction must be undertaken when spores are used as the sole inoculum.

There are also many other problems with pot culture. It is very difficult and costly to precisely control conditions such a pH and nutrient levels especially in some media. These factors can seriously decrease the quality of an inoculum by inhibiting VAM colonization or sporulation. Solid media often also harbor contaminating microorganisms. Commercially produced pot cultures frequently become contaminated by pathogens. Techniques to ensure the cleanness of inocula are much more effective when the roots and extramatrical VAM structures are free of solid substrate. Such conditions are found in hydroponic culture.

Hydroponic VAM culture can be accomplished in two ways, by static culture or by the Nutrient Film Technique, which continuously circulates nutrient solution through a trough in which plants grow.

Static culture is rarely used, because it gives very poor yields of fungi and host plant tissue. This method is best exemplified by Crush and Hay in their note in the New Zealand Journal of Agricultural Research, Volume 24, pages 371 and 372. A rectangular receptacle is filled with an aerated nutrient solution. Growing plants are floated on supports, and their roots are immersed in the nutrient solution. The plant roots are previously infected with a vesicular arbuscular fungus, before placement in the nutrient solution.

The technique is not commercially practical. Phosphorus levels had to be kept very low to foster mycorrhizal growth. Stipulated levels were so low that there would be insufficient plant material obtained. Floating plant densities would also have to be low. The small amount of fungus and plant root material obtained was sufficient for the physiological and biochemical research on mycorrhizal associations that the authors performed.

Static culture is thought to give rise to root aeration problems, pH shifts and accumulation of plant and microbial metabolites. All of these perceived problems were thought to result in poor fungal development (in particular poor sporulation) and thus preclude commercial VAM fungus production.

Static culture has been generally used for experimental purposes only, in order to produce small amounts of fungi and plant material solely for study. Scientists have devised other methods for commercial production of VAM fungi.

Nutrient film technology, a flowing hydroponic system, has been popular. The method is exemplified in U.S. patent number 4,294,037 to B. Mosse and J. P. Thompson. Their nutrient film technique (NFT) system consists of a reservoir and trough. A nutrient solution is continuously circulated through the trough and reservoir by a system of pumps and valves. Plants are inoculated with a VAM fungus and subsequently transplanted into the trough. The plants are supported and their roots dangle in the flowing nutrient solution. Plant and VAM fungi yields are moderate. There are problems with this method. The tubing, pumps and valves must be cleaned frequently, and they are subject to breakdown. The flowing nutrient solution may inhibit VAM development because of turbulence in the rhizosphere.

Uncontaminated VAM fungus inoculum can be produced by root organ culture as disclosed in European Patent Application Number 0,209, 627A3, published on January 28, 1987. Roots of host plants are isolated and cultured on a sterile artifical medium such as agar. These plant roots are inoculated with surface-sterilized spores of VAM fungi. Root tissue and attendant VAM fungi are eventually harvested to be used as inoculum. Tissue culture and maintaining constant sterility are expensive. Both require highly trained staff.

SUMMARY OF THE INVENTION

The present invention provides for a static hydroponic system for the production of host plant and vesicular arbuscular mycorrhizae. The static hydroponic system of the invention gives a good, commercially-feasible yield of both the host plant and the VAM fungus.

It has surprisingly been discovered that a much higher concentration of phosphorus ion may be used in the static hydroponic growth medium than was previously thought possible. This higher concentration will still foster a mycorrhizal association. The phosphorus concentration is now high enough to foster good host plant growth and attendant mycelial and spore production. The fungi are also surprisingly tolerant to submersion in static culture solution, and will grow and establish a mycorrhizal association with the host plant roots. Fungus propagules produced from static hydroponic culture are infective, as seen in Table 1.

Legumes do not necessarily need addition of nitrogen when inoculated with Rhizobium, as the bacteria in their root nodules fix atmospheric nitrogen to nourish the plant.

Host plants such as lettuce, onions, beans and leeks support VAM fungus spore and hyphal production and may also give a usable crop for harvesting, if desired.

No soil, peat, inoculum or other solid substrates are required in the tanks. This reduces the chances of contaminating the cultures with pathogens, facilitates harvesting of spores which are clean of debris, and restricts the growth of saprophytes.

The lack of soil or other solid substrates allows more precise control of all factors that influence the root environment. Non-flowing hydroponics has a number of advantages for inoculum production. The elimination of reservoirs, pumps and valves from the tanks reduces startup costs and makes more efficient use of growing space in the greenhouse. There is less turbulence in the hydroponicum that might disrupt the establishment of the extramatrical phase of the VAM infection.

The static culture nutrient solution has been designed to support very high plant densities. The use of an imbalanced nutrient solution (without lowering the phosphorus concentration to extreme deficiency) provides for good root growth and healthy shoots, while encouraging a mycorrhizal relationship.

Sporulation is sufficient to permit the collection of substantial quantities of spores, largely free from debris, simply by sieving the irrigant during tank drainage.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood from the following detailed description, given merely by way of example and therefore not intended in a limiting sense, taken in connection with the accompanying drawings wherein:

Fig. 1 is a plan schematic exploded view of a typical static hydroponic tank and prepared seedlings;

Fig. 2 is a cross-section of a tray and contents used to grow seedlings prior to infection with VAM fungi;

Fig. 3 is a cross-section of a receptacle for seedling germination, growth and support of plants in a hydroponic solution;

Fig. 4 is a schematic depiction of a plant and the disk used to support plants in a hydroponic tank lid.

DESCRIPTION OF THE INVENTION Hydroponics is the science of growing plants in a nutrient or culture solution composed of necessary nutrients dissolved in water. Usually this is accomplished by growing plants in a trough through which a nutrient solution is recirculated from a central reservoir: this is known as the Nutrient Film Technique. The static culture system, by contrast, uses non-flowing hydroponics. Plants are grown in tanks containing a certain volume of nutrient solution which remains in the tank for a set period of time before being drained and replaced with a fresh solution.

Specifically, this invention uses non-flowing hydroponics to grow plants colonized by vesiculararbuscular mycorrhizal fungi for the purpose of mass producing inocula of VAM fungi.

The process begins by establishing a plant, obtained from any source but preferably seed, colonized by one or more VAM fungi, in a static culture tank. Seedling production may be accomplished in one of three ways.

In the first method, depicted in Fig. 2, a shallow tray (1) is used. The tray (1) should have drainage holes (6) in its bottom. The tray may be made of biocompatible plastic. A paper towel lining (8) may be laid on the tray bottom, followed by a filling of a mixture (3) of, for example sand, soil-less potting mix, and vermiculite. Inoculum of one or more species of VAM fungus is layered in the mixture (3) below the seed (7) which is also placed in the mixture (3) . An optional covering of sand (4) may be layered on top of mixture (3).

After emergence, the seedlings (29) may then be suspended in lid (25) of static hydroponic container (19), of Fig. 1. This can be done in a number of ways. Fig. 4 shows one method. Seedling (29) may be attached along its stem to a disk (39) of, for example, styrofoam. The seedling (29) is placed in a notch (41) in the disk (39). The seedling (29) may be secured in the notch (41) by a suitable material such as a block of plasticine (43).

In the second method, seeds are sown in small receptacles (9), like those in Fig. 3, which are supported above the culture solution in the tank of Fig. 1. Inoculum (11) is again layered below each seed (13) in small receptacle (9). The bottom (15) of the receptacle (9) must be permeable to the growing roots, and allow them to grow through, but not permit any of the inoculum (11) or other contents (17) to fall into the nutrient solution of the static hydroponic system of Fig. 1. By this arrangement, no transplantation is required and colonized roots can immediately grow directly into the solution of tank (21). The contents of the receptacle (9) are variable, but should preferably contain some arrangement of sand and semisolid media such as agar.

One such arrangement of the receptacle layers could be, from the bottom to the top, 1 to 2 cm of foam sponge (17), then inoculum and a 1 cm of foam sponge (11), then 1 to 2 cm of sand (20). The seed (13) is planted in the sand (20). It is to be understood that other arrangements of the receptacle's (9) contents may be used in order to germinate seeds.

The receptacles (9) for seed germination may be designed as in Fig. 3. The size of the receptacle, composition and order of layers are variable.

These receptacles (9) may be placed directly in the lid (25) of hydroponicum (19) of Fig. 1. Receptacle (9) fits in hole (27) of lid (25). The roots dangle in the static hydroponic solution. The rest of the plant (29) rests above the lid (25).

In the third method, Petri plates, or similar glassware, not shown, containing moist paper towel or agar are autoclaved to eliminate contamination. Seeds are surface sterilized, inoculated with appropriate microbes such as Rhizobium species or VA mycorrhizas, and incubated in the petri plates until an adequate germination rate has been achieved.

Seedlings produced by this method may be suspended in lid (25) of static hydroponic container (19) of Fig. 1 using the means described for seedlings produced by the first method, (ie. secured in styrofoam disks), or receptacles described in the second method. The hydroponicum (19) is comprised of a tank (2) of variable area and depth. The tank (21) is made of any suitable non-permeable, inert, non-toxic material that can conveniently be fashioned into an appropriate shape to contain an aqueous solution. In particular, plastics such as PVC or acrylic are preferred.

The tank should be provided with means such as drain cock (23) to permit draining of the culture solution (not shown). A lid (25) with holes (27) for the plants (29) to fit through is preferred. This lid (25) supports the plants and prevents light from reaching the culture solution. Plants (29) are supported in the tanks by suitable means. Circular pieces of inch-thick styrofoam (31) serve the purpose, as do the plant germinating receptacles of Fig. 3.

The nutrient solution may also be aerated, preferably by bubbled air such as in Fig. 1. Air would course through line (33) to manifold (35) and out through nozzles such as air stones (37). Other aeration means may be used, where desired, to accomplish the same purpose.

Plant density in the tanks depends on the host species but is preferably greater than 80 plants per square metre and is usually about 120 plants per square metre.

Any host plant species that supports the growth of a VAM fungus may be used. Preferably, host plants should be chosen for their ability to be well colonized by the fungus and to ultimately provide highly infective inoculum. Plant species that have been used in the static culture system are varieties of Zea mays, Phaseolus vulgaris, Sorghum bicolor, Sorghum vulgare, Allium porrum, Allium cepa, Lactuca sativa, Panicum maximum, Panicum antidotale, Trifolium repens, Trifolium pratense, and Paspalum notatum as well as Sudan-Sorghum hybrids.

VAM fungi are used to inoculate the germinated seedlings, in all three seed germination methods. VAM inoculum is comprised of one or more species of fungi. Suitable inocula are mycorrhizal root fragments, VAM hyphae, VAM spores or any combination of these, all of which are capable of giving rise to a new growth of that organism under appropriate conditions. Inoculum is, therefore, any portion of a VAM fungus with or without attendant plant material that is capable of initiating new mycorrhizal infection with suitable host plants.

Generally, VAM fungus inoculum is placed near the developing roots of host plant seedlings . This is done, for example, in the seed germination tray (1) of Fig. 2 , the seed germination receptacle (9) of Fig. 3 or a suitable petri dish.

Suitable vesicular-arbuscular mycorrhiza fungi are, for example, those of the genus Glomus, species intraradices, fasiculatus, vesiculiferum. The invention is not limited to these species and genus. Other VAM genera and species may be successfully grown in the system.

Host plants are germinated, as discussed above, and infected with a VAM fungus. The infected seedlings are then transferred to the static culture hydroponicum (19), of Fig. 1. Further fungal inoculation may be made in the hydroponicum. These plants are allowed to grow until the inoculum is most infective, or spore production is optimised. This period varies, but is usually between 45 and 130 days.

The amount of nutrient solution in the tanks may be reduced significantly when the root mats are nearing maximum size. This enhances root aeration and may stimulate the production of extramatrical hyphae by the fungus.

The culture solution itself contains all of the elements required for plant growth and may also contain specific agents which improve inoculum quality. The individual concentrations and sources (chemical compounds) for each element are variable, some more so than others. The fundamental role of the nutrient solution is to provide for healthy plant growth as well as vigorous mycorrhizal colonization. Host plant species may have different nutritional needs so modifications may be made when changing host plant species. In particular, different hosts may have different optimal concentrations of nitrogen and phosphorus, depending on host needs. The most critical factors are nitrogen and phosphorus concentrations, for optimization of the system for each host.

The entire nutrient solution is generally replaced regularly. This is usually done by draining the tank contents and refilling with freshly prepared culture solution. Levels of specific nutrients, especially phosphorus and nitrogen, may be increased in the tanks at various intervals without replacing the entire nutrient solution. This is usually done by adding a small volume of a concentrated stock solution of the nutrient.

The culture solution is imbalanced. An imbalanced nutrient solution is a solution containing all of the elemental nutrients required for plant growth, but not in the proportions generally accepted as being balanced for optimal plant growth, as described in E.J. Hewitt, The composition of the Nutrient Solution Sand and Water Culture Methods Used in the Study of Plant Nutrition. Commonwealth Agricultural Bureau (1966), which is hereby incorporated by reference. Instead, elements that are generally thought not to affect VAM colonization are normal levels while those thought to inhibit colonization are at low to very low concentrations.

Generally it has been found that only phosphorus concentration need be limited for the purposed of this invention. The optimal concentrations on nutrients in the culture solution may be dependent upon the solution volume, the plant density and the rate at which nutrients are replace, (ie returned to their original levels.) More frequent replacement and lower plant densities per unit solution volume may reduce the nutrient concentrations needed for optimal production. This is particularly true of phosphorus and nitrogen, which are critical. Optimal nutrient concentrations may also be dependent upon host species and possibly VAM species.

All major elemental nutrients fall within the following preferred ranges in freshly prepared culture solution: magnesium 24 to 36 milligrams (mg) per litre (L); potassium 100 to 250 mg per L; calcium 80 to 120 mg per L; iron 2 to 15 mg per L; sulfur 32 to 150 mg per L; phosphorus 0.1 to 20 mg per L; nitrogen 1 to 250 mg per L. Preferred ranges of the latter two elements are phosphorus 0.5 to 12 mg per L and nitrogen 25 to 250 mg per L. More preferred phosphorus ranges are 4 to 12 mg per L.

There is some indication that higher phosphorus levels may be used when nitrogen levels are raised. Example 9 shows superior host plant growth and VAM spore production at elevated phosphorus and nitrogen levels.

Preferred levels of the other major nutrients are: potassium 150 mg per L; calcium 80 mg per L; magnesium 24 mg per L; sulfur 32 mg per L and iron 5 mg per L.

Trace element concentrations of the nutrient solution were: boron 0.50 mg per L; manganese 0.10 mg per L; copper 0.01 mg per L; cobalt 0.01 mg per L; molybdenum 0.02 mg per

L; and zinc 0.03 mg per L.

Table 2 gives the molar concentrations and sources of the nutrients.

The compound used to provide the required iron is preferably a chelate such as the ferric sodium salt of ethylenediamine-tetraacetic acid (EDTA). The iron requirements of different hosts may vary significantly.

The leguminous hosts may be inoculated with Rhizobium by soaking the surface sterilized seeds in cultures of appropriate strains prepared as described in Techical Handbook on Symbiotic Nitrogen Fixation: Legume/Rhizobium, FAO, Rome, 1983. Inoculation with nitrogen fixing bacteria permits a reduction in solution nitrogen levels without reducing host plant nitogen nutrition.

The pH of the culture solution is maintained, preferably nearly constant, at specific levels, usually between pH 4 and 8, through the use of a pH stat or chemical buffers. A pH stat is any device which is set up to automatically monitor solution pH and adjust the pH to within a preset range. Chemical buffers are included in the original nutrient solution at concentrations that will prevent a significant pH shift from its desired value. Examples of the buffers that may be used are bicarbonate, ( and other sources of the carbonate vuffer system such as CO₂ and CO₃) and any suitable Good buffer, as described in N. E. Good et al., Hydrogen Ion Buffering for Biological Research. Biochemistry 5(12) :467-477 (1966) and N. E. Good and S. Izawa, Hydrogen Ion Buffers. Methods in Enzymology Vol 24 (part B) : 53 (1968). Good buffers are a group of zwitterions which are commonly used as pH buffers in biological systems. These include MES [2-(N-Morpholino)ethanesulfonic acid], PIPES [piperazine-N,N'-Bis(2-ethanesulfonic acid)], HEPES [N- 2 - hydroxyethyl-piperazine-N'-2-ethanesulfonic acid], BES [N,N-bis(2-hydroxyethyl)-2-aminoethane-sulfonic acid], TES [N-tris(hydroxymethyl)methyl-2-aminoethane-sulfonic acid], choline [(2-aminoethyl) trimethylammonium chloride hydrochloride], acetamidoglycine [N-(2-acetamido)glycine], and MOPS [3-z(N-morpholino)propanesulfonic acid].

Spores can be collected without disturbing the cultures when the nutrients are replaced. As the tanks are drained the irrigant can be filtered through a 15-45 micrometer (um) sieve. This will collect all of the spores and they can be stored under appropriate conditions for later use as inoculum.

In harvesting mixed inoculum, the tanks are drained and the roots may be allowed to drip-dry for a few hours. The roots are cut from the shoots, finely chopped up, and mixed with an appropriate carrier material such as peat, clay or soil.

Example 1: Infectivity and spore production of Glomus intraradices inoculum produced by static culture hydroponics . Lettuce seeds were surface sterilized and pregerminated in petri plates containing nutrient agar. The germinated seeds were transferred into receptacles and inoculated with G. intraradices.

Receptacles were transferred into a static culture tank at the rate of 9 plants per tank. The tank had dimensions of 29cm wide x 25 cm long x 11.5 cm deep and contained 5L of nutrient solution. The nutrient regime was as described in Table 2. The tank was set up in a controlled environment chamber with a 14 hour photoperiod. Daytime temperature was 23°C and nighttime was 18°C.

The pH of the culture solution was maintained at pH 6.3 ± 0.3 by including 2.0 mM MES. The culture solution was changed weekly by draining the tank completely and refilling with freshly prepared nutrient solution. At 49 days the volume of the culture solution was reduced to 3L. At 61 days, all roots were harvested and mixed with peat for storage.

Thick mycelia of extramatrical hyphae were macroscopically observed to be covering many of the roots in the tank. After lowering, the culture solution was filtered through a 45 um sieve during tank drainage. 60 mature spores typical of G. fasiculatus were isolated.

Plants were harvested by draining the tank and allowing the roots to drip dry for a few hours. The roots were then cut from the shoots and finely chopped up. The chopped roots were mixed with 3 volumes of peat and stored at 4 C. A standard dilution assay initiated a few days later according to procedures based on those described in C. LI. Powell, Mycorrhizal Infectivity of Eroded Soils, Soil Biology and Biochemistry, 12:247-250 (1980) found the root and peat mixture to have an infectivity of 9200 propagules per gram.

Example 2 : A sowing medium used to initiate Phaseolus vulgaris seedlings prior to transplantation to static culture tanks.

A sowing medium composed of a 1-part soilless potting mix, 1-part vermiculite, 5-part sand mixture was set up as depicted in figure 2. The medium was autoclaved at 250 F for 1 hour before being placed in a plastic tray which had been rinsed with 70% ethanol. 50g of Glomus fasiculatum inoculum were layered 2 cm below the seeds. Phaseolus vulgaris seeds were prepared by washing off the Captan*¹ fungicide coating and sterilizing the seed surface in 3% hydrogen peroxide for 5 minutes. The seeds were rinsed 6 times in distilled water and soaked for 1 hour in a mixture of 1 part shaker-cultured Rhizobium inoculum, prepared as described in Technical Handbook on Symbiotic Nitrogen Fixation: Legume/Rhizobium. FAO, Rome, 1983, combined with 2 parts 8.5g per L sodium chloride solution. The seeds were then sown in the tray at a rate of 1 seed per 24 square cm.

¹ Denotes trademark. All trays were fertilized by adding 250 mL of 3.8M KC1, 1.0M MgSO4.7H2O and a small quantity of trace elements. The total volume of the sowing medium in the tray was 5L. All seeds germinated within 5 days and the plants were harvested after 26 days. Average shoot and root masses were 3.21 g and 1.35 g; the average percent infection of roots was 13% as determined by staining according to a variation of the method described by Kormanik and McGraw in Quantification of vesiculararbuscular mycorrhizae in plant roots, in Methods and Principles of Mycorrhial Research, N.C. Schenck, Ed., American Phytopathological Soc, 1982. and assay by the grid intersect method described by Giovanetti and Mosse in An evaluation of techniques for measuring vesicular arbuscular mycorrhizal infection in roots, New Phytol. 84: 489-500, 1980. The plants showed no deficiency symptoms when harvested.

At this point the plants would normally be transplanted to the tanks.

As a variation of this procedure the seeds can be sterilized in 1 part Javex*² bleach to 3 parts water for 1 hour. The seeds are rinsed and soaked in Rhizobium inoculum and germinated in sterile petri plates lined with wet paper towel. When germination is complete, the seedlings can be transplanted into a tray containing sowing medium.

² Denotes trademark. Example 3 : Lettuce colonized by Glomus vesiculiferum

A sowing medium was prepared as previously defined, except that the ratio of components was 4 parts sand to 1 part soil, and Glomus vesiculiferum inoculum was used.

Lactuca sativa seeds were layered into the trays.

Germination was complete within 4 days and after 44 days, root samples were stained and assayed for VAM colonization. All roots were found to be heavily colonized. On day 46,

20 plants were transplanted into a 10 L static culture tank. The dimensions of the tank were 28.5 cm x 51 cm x

11.5 cm

deep. The tank was situated in a controlled environment growth chamber with a photoperiod of 14 hours, daytime temperature of 22 °C and nighttime temperature of 17°C.

The nutrient solution was substantially as in Table 2 with the following changes (all concentrations given in mg per L): N=10, Fe=5, P=1, Ca=80, K=150, Mg=24. No buffers were included in the medium; rather, a pH stat was used to adjust the pH to 6.2 daily. The pH generally fell to near

4 overnight.

The plants were allowed to grow for 98 days in the tank. Upon harvest, fresh masses of shoots and roots, and percent infection of roots were determined. Percent infection was determined by the grid intersect method, by observing 100 intersections on root segments collected randomly from all over the root mat of each plant. Root segments were stained as in example 2. The average percent infection was 20%.

The roots were chopped into approximately 1 cm lengths at harvest and mixed with 3 volumes of peat. The peat had been sieved through a 4 mm sieve and steam sterilized for 1 hour at 230 F. The peat had a pH of 3.9.

After 6 months of storage at 10°C, a dilution assay found the inoculum to have an infectivity of 72 propagules per gram.

Example 4: Comparison of static culture inoculum to pot culture inoculum for the inoculation of Grape seedlings with Glomus fasiculatus

Seedlings of the hybrid grape variety Marechal Foch were obtained by rooting 2 bud cuttings from established plants. When the cuttings had well developed root mats they were transplanted into 1L pots containing Nova-Mix 300-S*³. Nova-Mix*³ (made by Annapolis Valley Peat Moss Co. Ltd.) is a commercial growing medium which is high in nitrogen and moderate in phosphorus. The plants were divided into high and low fertility. The high fertility

³ Denotes trademark.

³ Denotes trademark plants received weekly additions of soluble nutrients at the rate of 20 mg each of elemental nitrogen and phosphorus and 24 mg of elemental potassium plus trace elements per pot. The low fertility treatments received no further nutrients.

Within each fertility level there were 3 treatments: uninoculated, inoculated with pot culture inoculum, and inoculated with static culture inoculum. The pots were inoculated at transplant by placing inoculum in the pots in close contact with the plant roots. 25 g of pot culture inoculum or 6 g of static culture inoculum were used per pot. Both inocula had been recently prepared and originated from the same stock culture of G. fasiculatus. There were nine replicates in each treatment, 3 of which were harvested at 3, 6 and 8 weeks after inoculation.

The experiment was conducted in a greenhouse in July and August. The pots were organized according to a randomized design and were watered manually.

At harvest the potting mix was shaken and gently washed from the roots. The free water was blotted off the roots using paper towel and the fresh masses of all plants were determined. The amount of growth of each plant .was expressed as the harvest mass minus the mass at transplant. Percent infection of each plant was determined by randomly selecting roots from over the root mass and staining. The grid intersect method was used to estimate the percentage infection by observing 100 intersects per plant. The results are presented in Table 1.

Example 5: Effect of phosphorus concentration on VAM

colonization of highly nitrogen deficient P. vulgaris

Phaseolus vulgaris seeds were surface sterilized and pregerminated. The germinated seeds were inoculated with Rhizobium and transplanted into receptacles. A layer of Glomus fasiculatus inoculum was included in each receptacle.

Six receptacles containing bean seedlings were transplanted into each of 6 trays. The plastic trays had a volume of 5L and a depth of 12cm. The trays were painted black and covered by a styrofoam lid which supported the receptacles. The lid was covered with aluminum foil to reduce light penetration. Compressed air was bubbled into the culture solution. 3.5 L of nutrient solution were added to each tank. The nutrient solution contained (in mg per L) : K=100, Mg=24, S=32, Ca=80, Fe=3 , N=4 (with 3 parts nitrate to 1 part ammonium) and trace elements at the same concentration as in Table 2.

The trays were situated in a growth chamber with a daytime temperature of 23°C and a nighttime temperature of 18°C. A 14 hour photoperiod was used and the light intensity was approximately 18,000 lux. For the first two weeks, all plants were at the same phosphorus level of 0.5 mg/L. Then each tank was given a different phosphorus level, of 1, 4, 8, 12, 16 or 20 mg/L. Phosphorus was replaced 3 times every two weeks and nitrogen was replaced weekly. The entire nutrient solution was drained and replaced every two weeks. The nutrient solution also contained 2mM Pipes buffer for pH control.

Although all plants became nodulated, the plants appeared quite nitrogen deficient throughout the experiment. Symptoms included stunting and leaf chlorosis followed by abscission. There were no differences in plant size among tanks of different phosphorus levels; therefore, nitrogen was the limiting factor to plant growth.

On day 40, the nutrient solution volume in each tank was lowered to 2L. On day 47, the nitrogen concentration in the culture solution was increased to 20 mg/L in order to improve plant health. On day 69, the plants were harvested. Four root samples were collected from around the root mat of each tray, stained and assayed by the plate estimation method. The average root length colonized by mycorrhizae in each tank was: 1 mg/L P tank = 30-40% infection; 4 mg/L P tank = 10-20% infection; 8 mg/L P tank = 10-20% infection; 12 mg/L P tank = 20-30% infection; 16 mg/L P tank = 40-50% infection; 20 mg/L P tank = 20-30% infection.

Thus all plants had a significant percentage of their root length colonized, and macroscopic mycelia of VAM, independent of phosphorus concentration. This lack of dependence on phosphorus concentration is explained by the extreme nitrogen deficiency of the hosts, which disposes the host to becoming mycorrhizal.

Example 6: Effect of phosphorus concentration on VAM

colonization of Lactuca sativa

Lactuca sativa (lettuce) seeds were surface sterilized and pregerminated. The germinated seeds were placed in receptacles. Tanks (identical to those used in Example 5) containing six plants each were set up in a growth room. The growth room had a constant temperature of 80°+ 2°F and a 14 hour photoperiod was used. Compressed air was bubbled into the tank to aerate the culture solution.

Initially, 3.5 L of the following nutrient solution were placed in each tank (all in mg/L): potassium = 120, magnesium = 24, sulfur = 32, calcium = 80, iron = 5, trace elements were as in Table 2, nitrogen = 25 (with 3 parts nitrate to 1 part ammonium), and phosphorus = 0.5. On day 20, the phosphorus levels were adjusted in the tanks to 1, 5, 10, 15, 20, or 25 mg/L. As in Example 5, phosphorus was replaced 3 times every 2 weeks, nitrogen was replaced weekly and the entire solution was drained and replaced every 2 weeks. 2 mM Pipes buffer was included for pH control. On day 41, the nutrient solution volume in each tank was reduced to 2L. On day 70, the plants from all tanks were harvested. Only the plants from the 1 mg/L tank showed visible extramatrical hyphae. The VAM fungus was Glomus fasiculatus. The root samples were collected from each tank. Each sample was stained for VAM and assayed by the plate estimation method for percent root length colonized. The 1 mg/L phosphorus tank had 20-30% of its root length colonized by VAM; all other tanks had less than 1% root length colonized.

Because the infectivity of the inoculum is largely dependent upon the quantity of extramatrical hyphae, only the 1 mg/L phsphorus inoculum would have reasonable infectivity.

Example 7. Improved spore production by Glomus fasiculatus on Zea mays in response to increased phosphorus and nitrogen concentration.

Zea mays (corn) seeds were surface sterilized, pregerminated and sown in receptacles. The receptacles were inoculated with G. fasiculatus and were transferred to tanks at a plant density of 9 per tank. The tanks had the same design as described in Example 1 , including the use of bubbled air to aerate the nutrient solution. Each tank was filled with 5L of nutrient solution containing (all in mg/L): potassium = 150, magnesium = 24, calcium = 80, iron

= 15, trace elements as in Table 2, and sulfur = 32. Five tanks were used, containing 0.3 mg/L phosphorus/ 30 mg/L nitrogen, 0.6P/30N, 1.2P/30N, 1.2P/40N, 1.2P/50N, respectively. Each tank also contained 2.0 mM Mes buffer for pH control.

The nutrient solution was drained and replaced every two weeks with phosphorus being replaced three times over two weeks and nitrogen being added weekly. The tanks were situated in a growth chamber with the same conditions as described in Example 7.

On day 58, the volume of the culture solution in each tank was reduced to 3L. On day 98, the corn was harvested. At harvest, the culture solution was sieved through a 45um filter and the residue examined. The spores in the residue were counted using a dissecting microscope. Root samples were taken from each plant and assayed for percent infection using the grid intersect method. Both statistics are presented for each tank below:

P and N

Conc. 0.3P/30N 0.6P/30N 1.2P/30N 1.2P/40N 1.2P/50N (mg/L)

Number of

Spores 80 450 1500 2000 5600 Percent

Infection 33% 38% 37% 54% 41.5%

Example 8. Spore production at various nitrogen and phosphorus concentrations in static culture using corn, colonized by Glomus intraradices.

Corn seeds were sown in sand and allowed to germinate and grow for two weeks. The seedlings were then tranferred to receptacles such as described in other examples where they were inoculated with Glomus intraradices. Sixteen, 5 litre tanks, containing 8 receptacles each were set up. Each tank received the same nutrient regime of 1 mg/L phosphorus and 30 mg/L nitrogen for the first two weeks while the corn got established in the receptacles and began to grow. A 4X4 factorial set of treatments was then imposed upon the tanks in which phosphorus concentrations of 1.2, 2, 4 and 8 mg/L were tested with nitrogen concentrations of 50, 75, 100 and 150 mg/L. Other nutrient concentrations were similar to those used in other examples. Phosphorus was added to the tank, at the proper concentration, three times every two weeks, and nitrogen was added weekly. The entire solution was drained and replaced every two weeks.

Spores were sieved from the culture solution upon draining at 6 and 8 weeks after the receptacles had been transplanted into the tanks. Spores were counted at each seiving and the cumulative number of spores found from each tank are presented below.

[P]/[N] # of Spores

1.2/50 13,390 1.2/75 5,490 1.2/100 2,450 1.2/150 260

2/50 6,270

2/75 5,220

2/100 7,160

2/150 6,160

4/50 8,130

4/75 8,050

4/100 1,410

4/150 5,700

8/50 9,240

8/75 15,480

8/100 18,350

8/150 4,541 This is a Continuation-in-Part of U.S. Patent Application 07/380,098, filed July 14, 1989.

The present invention relates to the production of Vesicular Arbuscular Mycorrhizae (V.A.M.). V.A.M. fungi are symbiotic with plants and can enhance the host plant's nutrient uptake from soil.

The present invention provides for V.A.M. production in static hydroponic culture. Such culture supports growth of the host plant and the mycorrhizal fungus. Good yields of V.A.M. fungus spores may be obtained by the process and apparatus of the invention.

It is generally believed in the art that high phosphorus levels in static culture nutrient solutions results in little or no yield of V.A.M. fungus propagules, such as spores.

Surprisingly, it has been found that good yields of spores may be obtained with medium and high phosphorus concentrations in the growth medium.

Elevated phosphorus levels inhibit fungus growth in the host root tissue, but exterior hyphal growth is possible at medium phosphorus concentration. Eight to twenty parts per million seems to be the concentration where internal V.A.M. fungus growth tends to be suppressed depending on host species, nutrient replacement schedule and plant density. Surprisingly, V.A.M. spore production does not seem to be inhibited by medium or high phosphorus levels and good spore yields have been obtained with essentially high phosphorus concentrations. V.A.M. sporulation may occur in phosphorus ion concentration levels that are very high. These levels may extend up to the level of toxicity in a particular host plant. It should be noted that high phosphorus ion concentrations - in the medium - will inhibit the initial V.A.M. colonization of host plants.

Initial V.A.M. infection may be done in conditions of low or moderate phosphorus concentration. Phosphorus levels may be raised after the infection process has occurred. The raised phosphorus level does not seem to eliminate V.A.M. sporulation and those spores may be harvested as described previously.

Alternatively, newly sprouted host plants may grow in pots in a low phosphorus environment inoculated with V.A.M.. The pots may be suspended above a static solution high in phosphorus. The roots can grow down to the solution and be well infected before they reach the solution.

The collected V.A.M. spores may be used to infect higher plant roots, such as those of field crops.

It has been found that the roots of many seedlings become infected by V.A.M. as they grow through the matrix. The fungus spreads through the host plant root before those roots grow down into the liquid culture medium of the static hydroponic system. That liquid culture medium may have a relatively high phosphorus concentration. An initial low phosphorus concentration of the liquid culture medium may be skipped while still yielding large numbers of spores.

Generally, V.A.M. production facilities will want to propagate specific selected strains of V.A.M. fungi with their host plants. Consequently, host plants would be surface sterilized and then be infected with selected V.A.M. fungi.

Raised phosphorus levels do not work equally well with all host plants. Some plants use relatively little nutrient, and it can then accumulated in the culture medium and adversely affect plant growth. Lettuce is one such host plant species, and lower phosphorus concentrations should be used in that specific case. Example 6 shows this phenomenon.

A selected host plant species should readily utilize the nutrients in the static hydroponic system. Such plants include tomato, cucumber and others.

Example nine shows the two level phosphorus treatment of corn plants with Glomus intraradices V.A.M. inoculation. Plant roots grew down through the matrix. Two weeks later, phosphorus levels and nitrogen levels were raised when those roots grew into the solution. Spore production does not drastically drop with a rise in phosphorus and nitrogen levels. Phosphorus levels could be raised higher and still give practical spore yields.

Example ten shows spore production of Glomus intraradices and leeks. Four levels each of phosphorus and nitrogen were used. The rise in phosphorus and nitrogen was done after three weeks. It should be noted that although spore yields dropped where phosphorus and, nitrogen levels were high, they were still respectable. High phosphorus concentrations gave considerable spore yield with 50 and 100 mg/L of nitrogen. The phosphorus level would be expected to continue to give good spore yields at higher concentrations. Example 11 shows that other V.A.M. species behave in a similar manner to Glomus intraradices of examples nine and ten.

Five species showed greater spore production at higher phosphorus and nitrogen levels whereas three had lower spore productions. Example twelve features cucumbers as the host plants. The greater phosphorus and nitrogen levels resulted in a significant gain in host plant mass.

Example 9 : Spore production by Glomus intraradices on corn, grown under high concentrations of phosphorus and nitrogen.

Corn seeds were germinated in trays containing moist sand and allowed to grow for approximately two weeks. The seedling were then transplanted into receptacles where they were inoculated with Glomus intraradices inoculum. The receptacles were then transferred into tanks with a density of nine plants per tank, and 5L of nutrient solution were added to each tank (the tank dimensions were the same as in Example 1). For the first two weeks, while the corn roots grew out of the receptacles and into the tanks, a common nutrient solution with 1 mg/L phosphorus and 50 mg/L nitrogen was used (as well as other nutrients as described in Table 2). When the plants had become established in the tanks, the composition of the nutrient solution was as follows (all in mg per L) : magnesium=36, calcium=80, iron=15, trace elements as in Table 2, and potassium concentration was equal to the nitrogen concentration in each tank. Nitrogen and phosphorus treatments were imposed after the initial two weeks and were varied among tanks according to a 4 x 4 factorial design. Nitrogen concentrations of 150, 200, 250 and 300 mg/L, and phosphorus concentrations of 8, 12, 16 and 20 mg/L were used; therefore, there were sixteen tanks in total. MES at a concentration of 2.0 millimolar was added to the tanks to maintain a pH around 6.2.

Phosphorus was added at the concentrations given, three times every two weeks, while nitrogen was replaced at the given concentration weekly. The entire nutrient solution was drained every two weeks and replaced. Beginning after six weeks, the nutrient solution was sieved upon draining and the spores were collected and counted. When the sieving of the tanks began, the tanks were allowed to dry overnight between draining and re[placement of the nutrient solution. After 12 weeks in the tanks, the corn was harvested. Spores were collected by sieving the nutrient solution, as well as by gently washing the roots and sieving the washings. The yields of typical, tan-coloured Glomus intraradices spores were as follows:

Phosphorus Nitrogen concentration (in mg/L)

conc. (mg/L) 150 200 250 300

8 6,430 14,220 56,160 3,660

12 5,540 71,130 32,270 3,900

16 4,730 35,230 12,020 4,610

20 980 320 9,980 14,300

The percent root length colonized by mycorrhizae in each tank was, on average, well under 1% in all treatments and there were no differences detected between the treatments.

Example 10 : Spore production by Glomus intraradices on leeks, grown under high concentration of phospohorus and nitrogen.

Leek receptacles were prepared by both germinating seeds in the receptacles, and by transplanting seedlings grown in a sowing medium into receptacles. The grown in a sowing medium into receptacles. The receptacles were inoculated with Glomus intraradices inoculum and then transferred into tanks at a density of nine per tank. five litres of nutrient solution were added to each tank ( with the dimensions as in Example 1) containing 2 mg/L phosphorus and 30 mg/L nitrogen (as well as other nutrients as described in Table 2). After three weeks, when leek roots had begun to grown down into the solution, the nutrient level treatments were imposed. The composition of the nutrient solution was as follows (all in mg/L): magnesium=24, calcium=80, iron=6, trace elements as. in Table 2, and potassium concentration was equal to the nitrogen concentration in each tank. Nitrogen and phosphorus treatments were varied among tanks according to a 4 x 4 factorial design. Nitrogen concentrations of 50, 100, 200, and 300 mg/L, and phosphorus concentrations of 2, 4, 8 and 16 mg/L were used; therefore, there were sixteen tanks in total. Each tank's culture solution contained MES at 2.0 millimolar concentration to control pH variation

Phosphorus was added at the concentrations given, three times every two weeks, while nitrogen was replaced at the given concentration weekly. The entire nutrient solution was drained every two weeks and replaced. Beginning after six weeks, the nutrient solution was sieved upon draining and the spores were collected and counted. When the sieving of the tanks began, the tanks replacement of the nutrient solution. After 12 weeks in the tanks, the leeks were harvested. Spores were collected by sieving the nutrient solution, as well as by gently washing the roots and sieving the washings. The yields of typical, tan-coloured Glomus intraradices spores were as follows:

Phosphorus Nitrogen concentration (in mg/L)

conc. (mg/L) 50 100 200 300

2 33,400 8,800 16,300 34,200

4 21,600 11,300 5,000 23,300

8 27,500 22,800 12,200 4,600

16 25,200 15,900 6,000 7,000

Example 11 : Inoculum production of eight different species of V.A.M. on leeks in static culture.

Leek seeds were sown in large trays containing established leek seedlings infected with one of eight species of V.A.M., growing in a vermiculite medium. The seeds were allowed to germinate and grow until a strong shoot had developed, and the roots were well colonized by V.A.M.. During the growth period, the trays were regularly watered with a complete nutrient solution to regularly watered with a complete nutrient solution to support the growth of the seedlings. Seedling infected with Gigaspora margarita, Glomus aggregatum, Glomus fasiculatum, Glomus vesiculiferum, Glomus monosporum, Glomus clarum, Glomus caledonium, or Glomus versiforme were transplanted into receptacles.

For each species of V.A.M., there were two tanks, one with a nutrient solution containing 2 mg/L phosphorus and 100 mg/L nitrogen, and the other with 4 mg/L phosphorus and 150 mg/L nitrogen; therefore, there were 16 tanks in total. All other nutrients were as described in Table 2, except for potassium at 100 mg/L, and iron at 6 mg/L. The nutrient solution contained 2 millimolar MES for pH control, and the plant density was 9 per tank (tanks had the same dimensions as in Example 1 and contained 5L of nutrient solution). Nitrogen was replaced at the concentrations given every week, and phosphorus was replaced three times every two weeks. The entire nutrient solution was drained and replaced every two weeks. After 12 weeks in the tanks, the leeks were harvested. The nutrient solution and root washings were sieved, and spores were counted. The results are summarized below: Total spores at harvest

V.A.M. species 2 mg/L P 4 mg/L P

100 mg/L N 150 mg/L N

Gigaspora margarita 22,700 55,500

Glomus aggregatum 149,600 43,300

Glomus fasiculatum 261,800 195,900

Glomus vesiculiferum 45,500 232,700

Glomus monosporum 560 9,670

Glomus clarum 45,100 9,000

Glomus caledonium 5,800 9,400

Glomus versiforme 4,600 11,100 Representative root samples were collected from each plant of each tank as well, and were assayed for percent: infection, root samples from almost every plant in all tanks showed V.A.M. infection, although levels were low. Also, sporocarps containing from 50 to 100 spores each were observed in the sievings from the G. clarum tanks: 700 sporocarps were produced in the 2 mg/L phosphorus; 100 mg/L nitrogen tank and 1600 sporocarps were produced in the 4 mg/L phosphorus; 150 mg/L nitrogen tank. Example 12 : Cucumber plants permit the production of

V.A.M. inoculum and a vegetable for harvesting. Cucumber seedlings were produced by germinating seeds in a sowing medium as in Figure 2. About a week after germination, the cucumber seedlings were transplanted into receptacles and inoculated with Glomus intraradices. The receptacles were placed into two tanks at a density of 9 per tank. Each tank received 5L of culture solution as in Table 2, except for the nitrogen and phosphorus values. In one tank, phosphorus was at 2 mg/L and nitrogen was at 50 mg/L, while in the other, phosphorus was at 4 mg/L and nitrogen was at 100 mg/L. Phosphorus was replaced three times every two weeks and nitrogen was replaced weekly. The entire culture solution was drained and replaced every two weeks. 2 millimolar MES was included in the culture solution as well for pH control.

Fifteen weeks after transplanting, the cucumber tanks were harvested. Data on percent infection, spore production, and vegetable production are given in the table below. Note that all spores were obtained from sieving the culture solution at final harvest.

2 mg/L P 4 mg/L P

Statistic 50 mg/L N 100 mg/L N Mean % V.A.M. infection 50-60% 20-30%

Mass of cucumbers produced 494 grams 621 grams

Number of spores produced 2800 200 Table 2

Composition of Nutrient Solution in Example 1

Element Conc., mg/L Conc., mM Source

P 1.00 0.0312 KH2PO4 + K2HPO4.H2O

K 150.0 3.836 KCI + both P sources

Ca 80.0 2.00 CaCI2

Mg 24.3 1.00 MgSO4.7H2O

S 32.2 1.00 MgSO4.7H2O + trace elem.

Fe 5.0 0.090 FeNaEDTA

B 0.50 0.046 H3BO3

Mn 0.10 1.8E-03 MnCI2.4H2O

Cu 0.010 1.6E-04 CuSO4

Mo 0.020 2.1 E-04 Na2MoO4.2H2O

Co 0.010 1.7E-04 CoSO4.7H2O

N 25.0 1.78 NH4NO3

Zn 0.03 4.6E-04 ZnSO4.7H2O

Claims

We Claim:

1. A process for the production of Vesicular arbuscular mycorrhizal fungi on host plant roots wherein at least a portion of said process is carried out in static culture containing a nutrient solution wherein the phosphorus concentration is chosen from the range comprising from 0.1 to 100 mg/L.

2. A process according to Claim 1 wherein said phosphorus concentration is chosen from the range comprising from 0.1 to 50 mg/L.

3. A process according to Claim 1 wherein said host plant seedling are first colonized by vesicular arbuscular mycorrhizal fungi and then transplanted to a static hydroponicum wherein said phosphorus concentration is chosen from the range comprising from 0.1 to 50 mg/L.

4. A process according to Claim 2 wherein said host plant seedling are first colonized by vesicular arbuscular mycorrhizal fungi and then transplanted to a static hydroponicum wherein said phosphorus concentration is chosen from the range comprising from 0.1 to 30 mg/L.

5. A process according to Claim 1 wherein said phosphorus concentration is selected from the range comprising from 2.0 to 20 mg/L.

6. A process according to Claim 1 wherein said phosphorus concentration is selected from the range comprising from 0.5 to 12 mg/L.

7. A process according to Claim 1 wherein nitrogen concentration is initially chosen from the range comprising from 1 to 300 mg/L.

8. A process according to Claim 2 wherein nitrogen concentration is chosen from the range comprising from 1 to 300 mg/L.

9. A process according to Claim 5 wherein nitrogen concentration is chosen from the range comprising from 50 to 200 mg/L.

10. A process according to Claim 1 wherein said vesicular arbuscular mycorrhizal fungus is selected from the group comprising Gigaspora margarita. Glomus aggregatum, Glomus fasiculatus, Glomus vesiculiferum, Glomus monosporum, Glomus clarum, Glomus caledonium, Glomus versiforme, Glomus intraradices and Gicraspora margarita.

11. A process according to Claim 2 wherein said vesicular arbuscular mycorrhizal fungus is selected from the group comprising Giσaspora marcrarita, Glomus aggregatum, Glomus fasiculatus. Glomus vesiculiferum, Glomus monosporum, Glomus clarum, Glomus caledonium. Glomus versiforme, Glomus intraradices and Gigaspora margarita.

12. A process according to Claim 7 wherein said vesicular arbuscular mycorrhizal fungus is selected from the group comprising Giqaspora margarita. Glomus agqreqatum, Glomus fasiculatus, Glomus vesiculiferum, Glomus monosporum. Glomus clarum, Glomus caledonium. Glomus versiforme, Glomus intraradices and Gigaspora margarita.

13. A process according to Claim 2 wherein said host plant is selected from the group comprising Zea mays, Phaseolus vulgaris, Allium porrum, Allium cepa. Panicum maximum, Curcumis sativus, and Lycopersicum esculentum.

14. A process according to Claim 8 wherein said host plant is selected from the group comprising Zea mays, Phaseolus vulgaris, Allium porrum. Allium cepa, Panicum maximum, Curcumis sativus, and Lycopersicum esculentum.

15. A process according to Claim 11 wherein said host plant is selected from the group comprising Zea mays, Phaseolus vulgaris, Allium porrum, Allium cepa, Panicum maximum, Curcumis sativus, and Lycopersicum esculentum.

16. A process according to Claim 12 wherein said host plant is selected from the group comprising Zea mays, Phaseolus vulgaris. Allium porrum, Allium cepa, Panicum maximum, Curcumis sativus, and Lycopersicum esculentum.

17. A process according to Claim 2 wherein said phosphorus concentration is initially chosen from the range comprising from 0.1 to 4 mg/L and is subsequently changed to a concentration chosen from the range comprising from 4.1 to 50 mg/L and said host is a plant adapted to utilize a substantial portion of said phosphorus.

18. A process according to Claim 2 wherein said host is a legume and is further inoculated with symbiotic nitrogen-fixing bacteria.

19. A process according to claim 18 wherein said symbiotic nitrogen-fixing bacteria is a species of Rhizobium.

20. A process according to Claim 1 wherein the pH is kept between 4 and 8 by the use of a buffer selected from the group comprising, bicarbonate buffers, HEPES (N- 2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid), BES (N,N-bis(2-hydroxyethyl)-2-aminoethane sulfonic acid), TES [N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid], choline [(2-aminoethyl)trimethyl- ammonium chloride hydrochloride], acetamidoglycine [N- (2-acetamido)glycine], and MOPS [3-z(N- morpholino)propanesulfonic acid].

21. A process according to Claim 2 wherein the pH is kept between 4 and 8 by the use of a buffer selected from the group comprising, bicarbonate buffers, HEPES (N- 2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid), BES (N,N-bis(2-hydroxyethyl)-2-aminoethane sulfonic acid), TES [N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid], choline [(2-aminoethyl) trimethyl- ammonium chloride hydrochloride], acetamidoglycine [N- (2-acetamido)glycine], and MOPS [3-z(N- morpholino)propanesulfonic acid].

22. A process according to Claim 1 wherein the pH is kept between 4 and 8 by the use of a buffer selected from the group comprising bicarbonate buffers, HEPES (N-2- hydroxyethyl-piperazine-N'-2-ethanesulfonic acid), and MOPS [3-z(N-morpholino)propanesulfonic acid],

23. A process according to Claim 2 wherein the pH is kept between 4 and 8 by the use of a buffer selected from the group comprising bicarbonate buffers, HEPES (N-2- hydroxyethyl-piperazine-N'-2-ethanesulfonic acid), and MOPS [3-z(N-morpholino)propanesulfonic acid].

24. A process according to Claim 1 wherein said nutrient solution has a mineral concentration range of phosphorus from 0.1 to 20 mg per L, nitrogen from 1 to 250 mg per L, calcium from 80 to 120 mg per L, magnesium from 24 to 36 mg per L, iron from 2 to 15 mg per L, potassium from 100 to 250 mg per L and sulfur from 32 to 150 mg per L.

25. A process according to Claim 1 wherein said nutrient solution has a mineral concentration range of phosphorus from 0.5 to 12 mg per L, nitrogen from 20 to 250 mg per L, calcium from 80 to 120 mg per L, magnesium from 24 to 36 mg per L, iron from 2 to 15 mg per L, potassium from 100 to 250 mg per L and sulfur from 32 to 150 mg per L.

26. A process according to Claim 1 wherein said nutrient solution has a mineral concentration range ,of phosphorus from 0.1 to 4 mg per L, nitrogen from 20 to 50 mg per L, calcium from 80 to 120 mg per L, magnesium from 24 to 36 mg per L, iron from 2 to 15 mg per L, potassium from 100 to 250 mg per L and sulfur from 32 to 150 mg per L.

27. A process for the production of vesicular arbuscular mycorrhizal fungi comprising the steps of;

a. infecting host plants with vesicular

arbuscular mycorrhizal fungus,

b. transplanting said infected host plants to a static culture hydroponicum,

c. culturing said host plants in an imbalanced nutrient solution wherein phosphorus concentration is 0.5 to 20 mg per L,

d. maintaining the pH between 4 and 8,

e. harvesting said vesicular arbuscular mycorrhizal fungus.