WO2009009805A1

WO2009009805A1 - Micro-organism inoculum, method and compositions

Info

Publication number: WO2009009805A1
Application number: PCT/ZA2008/000060
Authority: WO
Inventors: Marianne Wilma Venter
Original assignee: Biocult (Pty) Ltd
Priority date: 2007-07-09
Filing date: 2008-07-08
Publication date: 2009-01-15
Also published as: ZA200808624B; EP2176196A4; EP2176196A1; WO2009009805A9

Abstract

A method of producing endomycorrhiza inoculum involves cultivating endomycorrhiza spores on roots of a host plant in a growing medium and sequences of steps to separate the spores and producing a spore concentrate on a carrier of exceptionally high spore count, excellent dispersibility and optionally satisfying 'organic' labelling requirements. The spore concentrate, optionally combined with growth-enhancing bacterial or fungal concentrates or metabolites, can be used as such or when converted into a variety of commercial products such as light weight granular forms for addition to the soil, or additives to irrigation water or compositions for seed impregnation in order to enhance plant growth and being antagonistic against pathogens and nematodes in agriculture, horticulture and forestry.

Description

Field of invention

This invention relates to a method of producing an endomycorrhiza inoculum, endomycorrhiza inoculum products produced or producible by such method and methods of using the aforegoing products.

Background to the invention

It is known that the fungi vesicular-arbuscular mycorrhiza (VAM) and the arbuscular mycorrhiza (AM) (hereinafter referred to jointly as endomycorrhiza) are generally present in the soil and on the roots of plants. VAM predominates over AM in the soil and in and on plant roots. Whilst VAM is almost invariably present in and on roots of an endomycorrhiza-infected plant, pure AM infection is quite rare. Mixed AM and VAM infections are more common than pure AM infections. Many plants which will become VAM infected will resist infection by AM.

The presence of endomycorrhiza on plant roots has a number of beneficial effects on the plant. The hyphae of endomycorrhiza grow into areas the coarser plant roots cannot reach, for example in clay soils, forming what can be described as an extended root system. Phosphate and calcium as well as other minerals and nutrients are absorbed by the hyphae and translocated into the plant roots. The endomycorrhiza receives carbohydrates from the plant. Both are fed by the exchange which is mutually beneficial. The presence of endomycorrhiza on a root system also has the effect of being antagonistic against pathogens and nematodes. Furthermore, endomycorrhiza excrete various growth hormones that stimulate plant growth. Fumigation, cultivation and excessive fertilisation have been found to deplete and/or destroy the endomycorrhiza populations on the plant roots and in the soil. It has, in view of the benefits of endomycorrhiza infection, and the depletion of endomycorrhiza in soil, been proposed that plant root systems should be deliberately infected with endomycorrhiza. A problem in the art is that of ensuring not only that the root system is infected with endomycorrhiza but is infected at a rate such that infection occurs before pathogens and nematodes have a deleterious effect on the plant.

A method is known of producing an endomycorrhiza inoculum which comprises growing a host plant in a growing medium which contains endomycorrhiza so that the roots of the host plant become endomycorrhiza-infected and recovering therefrom an inoculum.

Thus it was known to recover the infected roots, wash the roots, comminute the roots and mix the comminuted roots with said growing medium to produce an inoculum. Whilst this product is usable, it does not promote endomycorrhiza growth at a rate which will prevent all of the deleterious effects of pathogens and nematodes in the soil.

This method was improved according to South African patent no. 95/10628 by mixing the comminuted roots with all or part of the water with which the roots of the host plant were washed before the roots were comminuted, with a carbon source (e.g. sugar or CO₂ gas), with at least one microbial metabolite, with at least one live organism, with at least one enzyme and with at least one bacterial and fungi culture medium in which live micro-organisms have been grown.

Although the inoculum so produced was used successfully, it had a number of shortcomings. It was a heavy, bulky product of which large volumes needed to be used to be effective. The application of the inoculum for inoculating plants was cumbersome. A need has been recognized for an endomycorrhiza inoculum which is more concentrated, can be made available in water-soluble/dispersible form or in a lightweight granular form, or for seed coating, and the manufacture of which lends itself for satisfactory quality control.

Description of the invention

In accordance with the present invention there is provided a method of producing an endomycorrhiza inoculum which comprises growing a host plant in a growing medium which contains endomycorrhiza so that the roots of the host plant become endomycorrhiza-infected and recovering therefrom an inoculum, which further includes removing the host plant, including the roots, from the growth medium and processing the growth medium containing spores and hyphae by washing it thoroughly, e.g. repeatedly with water or aqueous liquid, separating any root matter and coarse debris from the resultant spore-containing water or aqueous liquid, separating the spores from the spore-containing water or liquid and mixing the spores with a fine absorbent carrier powder to obtain a nearly dry powder mixture being a concentrated mycorrhiza spore powder. This is preferably combined with the further features of including in said concentrated mycorrhiza-spore powder a carrier dispersing agent, being a substance having dispersing agent properties and being soluble/dispersible in water to hold the mycorrhiza spores in stable suspension. The absorbent carrier powder may itself be or may include this carrier dispersing agent.

The growth medium may be a mixture of sand on the one hand, and soil and clay or clay-like matter on the other hand in a suitable ratio, e.g. between 6:1 and 1 :1 by volume, preferably about 3:2. The soil and clay or clay-like matter may contain one or more of zeolite powder, vermiculite, coco peat, peat moss, montmorilonite, bentonite, apatite. Also present may be one or more of perlite, bark, horticultural peat and coir (the fibrous material from coconut husks). If zeolites are used, the preferred pore sizes are in the range of from about 3 to about 10 A in diameter. A suitable example is zeolite clino natural.

The composition of the growth medium depends also on the pH requirements of the mycorrhiza.

Typical examples are 3 parts sand and 2 parts vermiculite and/or peat (by volume).

The host plant may, for example, be selected from various grass species, legumes, such as peas, maize, sorghum, medico spp (e.g. medico sativa) or any other plant species sufficiently easy to grow rapidly and suitable as a host plant for the mycorrhiza.

Suitable grass host plants include Sudan grass, which grows best in summer, or rye (Korog), which grows best in winter.

The growth period of the host plant may be e.g. 1 to 6 months, preferably 2 to 5 months, e.g. about 3 to 4 months and this growth may be performed under shelter, e.g. in nursery tunnels or greenhouses. For quality control frequent control checks are performed to determine the number of spores in any production batch and thereby determine when the spores are ready for harvesting.

Washing of the mycorrhiza-infected growth medium may be performed with water, preferably with the minimum volume of water capable of retrieving substantially the entire mycorrhiza spore content, e.g., in practice, in a total volume of between about 1 and 100 times, preferably from 1 to 30 times, e.g. about twice the volume of growth medium, e.g. divided into a plurality of successive washings. Washing the growth medium is preferably performed by mixing it thoroughly with a portion of the water, allowing heavy sand particles to settle for a few seconds and decanting through a coarse screen, e.g. of from 150 to 300 micron, preferably 180 to 250 micron, e.g. 200 micron, to collect roots and coarse debris, and through one or more finer screens, preferably a plurality of finer screens to capture the spores.

This is repeated several times (e.g. 3 to 4 times) until the water is substantially clear.

For capturing the spores, preferably at least two of the screens are used in the range of between 20 and 90 microns, e.g. selected from the range of 38/45/53/60 microns, for example, one screen 38 to 45 microns and a second screen 53 to 60 microns.

The spores may then be washed from these finer screens with a small volume of water into a container, e.g. in a total volume of water equal to from 1 to 20% v/v, preferably from about 3 to 8% v/v, e.g. about 4 to 6% v/v, based on the original volume of growth medium, i.e. about 2% per screen. This volume of water, containing the spores is now taken up in from 5 to 100 times, preferably 6 to 80 times, e.g. about 10 times its weight of fine absorbent carrier powder. The mixture thus obtained is nearly dry (preferably about 10% moisture).

The fine absorbent carrier powder may be a fine (10 to 150 micron) zeolite powder and/or one or more clay minerals, e.g. bentonite and/or montmorilonite and/or apatite and/or comminuted perlite. Perlite, when used is preferably comminuted by heating, e.g. to about 900⁰C.

In order to ensure the powderous consistency and dispersibility of this mixture, it is preferably sieved through a sieve, e.g. of from 80 to 200 micron, preferably 100 to 150 micron, most preferably 120 micron, depending on the spore sizes to be allowed to pass. 120 micron was found, in practice, to be the best mesh size to allow all mycorrhiza spores to pass through the sieve.

This mixture is now preferably mixed with the aforesaid carrier/dispersing agent, being a substance having dispersing agent properties and being soluble/dispersible in water and capable of holding the mycorrhiza spores in stable suspension. Further preferred properties thereof are that it is wholly organic (and suitable as an ingredient of a composition which is certified "organic"), readily biodegradable, on the one hand inert, on the other hand suitable as a nutrient for micro-organisms and generally advantageous for such micro-organisms as are relevant in the present context.

An example of such organic carrier is of plant origin, preferably a soya product, e.g. based on a by-product of soya meal manufacture.

A preferred absorbent carrier powder and carrier dispersing agent may be of a type described in South African patent 96/6318, the relevant contents of which are to be considered part of the present disclosure, or any organic soya powder product or mixtures of these. ZA Patent 96/6318 describes a microbial carrier comprising a comminuted inorganic silicious mineral, preferably heat- comminuted perlite. The suitability of this carrier for mycorrhiza spores and for keeping these in aqueous suspension was unforeseeable and surprising if it is borne in mind that mycorrhiza spores are about eight decimal orders of magnitude larger in size than the microbes for which the carrier is described in ZA Patent 96/6318 and for which that carrier had been used in practice in the past.

This preferred carrier substance is employed in a ratio of 10% to 80%, preferably 20% to 70%, e.g. 65% by weight of the final concentrate, which latter is considered to be a novel product in its own right. Thus, according to one aspect of the invention, there is provided a mycorrhiza concentrate, comprising endomycorrhiza spores and a powderous extender and carrier medium which includes a substance serving as a carrier/dispersing agent, and which concentrate contains said spores in a concentration of not less than 700 spores per gram, preferably not less than 1000 spores per gram. More particularly, the spore content is from 700 to 1300 spores per gram, more preferably from 800 to 1200 spores per gram and most preferably from 1000 to 1100 spores per gram. Preferably, this concentrate is wholly dispersible in water to form a stable suspension of the mycorrhiza spores.

The aforesaid spore concentrations are considerably higher than those which are standard prior art commercial concentrates of which applicant is aware, for which 20 spores/g are normal.

The powderous carrier is selected from any one of the aforegoing powders or a combination of two or more of these and has particle sizes to pass sieve sizes ranging from 20 to 200 micron, preferably from 50 to 150 micron, more preferably from 30 to 120 micron.

The carrier/dispersing agent may be as set out above.

This final concentrate may now be further converted into a variety of commercial products and be used in a variety of manners.

Thus, for example, the concentrate may be converted into a water-soluble or dispersible composition comprising 10 to 80% by weight, preferably 30 to 70% of the concentrate, the balance being one or more concentrates of a variety of growth promoting micro-organisms and/or their growth promoting metabolites, e.g. enzyme(s), more particularly a) Trichoderma fungi spp, e.g. Trichoderma harzianum, e.g. the T22 strain; b) bacteria, preferably nitrogen binding types, e.g. Azomonas spp, Pseudomonas spp, Azospirillum spp, Azotobacter spp, Rhizobium spp, Agrobacteria spp, Bazillus spp, Bavarea spp. For this purpose the aforesaid ingredients, optionally with carriers or extenders, may be mixed together, bagged or otherwise packaged, and preferably vacuum-sealed or alternatively stored under an inert atmosphere. Examples of the aforesaid bacteria are e.g. Actimomycetes thermophilus, Streptomyces spp, Azotobacter chococcum, Mixobacteria celvibrio, M. cytophaga, Bacillus ciculaus, B. subtilis, Fulvoviridis. A suitable enzyme which can be added is the bacterial enzyme known as Cyto-Turf cytozome as available from Mailco Turf, Umhlali, South Africa.

A preferred ratio of mixing is, for example, from 50 to 100, e.g. 75 parts by weight Mycorrhiza concentrate, from 70 to 150, e.g. 116 parts by weight Trichoderma concentrate (spore count 1 x 10⁹/g) and from 5 to 15, e.g. 9 parts by weight Azobacter concentrate (spore count 6 x 10⁸/g). The final mix may, for example, contain not less than 375 mycorrhiza spores per gram. This is achieved by careful quality control.

Methods of using the water-soluble/dispersible formulation include dissolving/dispersing it in water and applying the dispersion through an irrigation system or for dipping into it the roots of plants or applying it to plants with a watering can or the like. It has been found that the solutions/dispersions of the formulation are stable for prolonged periods without agitation and do not clog irrigation systems.

Typically, this formulation is dispersed in water in a weight ratio of powder to water of from 1 : 20 to 1 : 100, e.g. about 1 : 50 and this may then be diluted further with water depending on the crop to be irrigated.

According to a further preferred embodiment the mycorrhiza concentrate is converted into granules. It is a particular advantage that the granules can be made of very low bulk density such as from 500 to 800, preferably 550 to 750, more particularly about 600 to 700 g/l of granules.

The mycorrhiza spore count of the granules may, for example, be from 1 to 100 spores per gram, e.g. 30 to 40 spores/g.

The granules comprise the spores applied onto a solid carrier substance, preferably together with growth-enhancing micro-organisms or substances as described above in the context of the water-soluble/dispersible formulation.

The solid carrier substance is preferably biodegradable. Suitable solid carrier substances include farinaceous substances, e.g. starch or rice, although sago is particularly preferred. However, it is also possible to use as solid carrier substance particles sterilised round seeds, e.g. sterilised by boiling, e.g. sorghum seed. The granule size (e.g. the sago or seed size) is selected so that the product can be readily scattered on lands.

Preferred embodiments comprise from 100 to 800, preferably 200 to 450, e.g. about 300 parts by weight mycorrhiza concentrate, and from 5000 to 15 000, preferably 7000 to 12 000, e.g. about 9000 parts by weight sago. The preferred embodiments may contain additions of 50 to 150, preferably 70 to 120, e.g. 90 parts by weight Azotobacter and/or from 50 to 250, preferably 80 to 200, e.g. 135 parts by weight Trichoderma spp.

The granules may be manufactured by mixing the solid carrier, e.g. sago (or other farinaceous substance), e.g. in a mixer, with the remaining dry ingredients and with moistening, e.g. while turning the mixer. Any suitable dye, preferably a vegetable dye, may be added as well. After thorough mixing, the granulate is packaged, e.g. in bags, and sealed. The granulate may be applied in planting holes with plants placed on top or it may be mixed with seed and sown.

According to yet a further preferred embodiment, the endomycorrhiza concentrate is incorporated in a seed-coating powder, including a suitable carrier or mixture of carriers and extenders. Preferably, the seed-coating powder also contains growth-enhancing micro-organisms and/or their metabolites as aforesaid, Trichoderma spp being preferred.

The preferred carrier comprises rock phosphate and the carrier/dispersing agent composition referred to above.

Preferred proportions are from 3 to 20 parts by weight, preferably 5 to 12 parts by weight, e.g. about 7 parts by weight of mycorrhiza concentrate; Trichoderma spp (preferably Trichoderma harzianum, e.g. T22) 15 to 120, preferably 20 to 80, e.g. about 40 parts by weight; rock phosphate 2 to 20, preferably 3 to 10, e.g. about 5 parts by weight; carrier/dispersing agent composition 60 to 300, preferably 100 to 200, e.g. about 150 parts by weight. The mixture is packaged, e.g. in bags, and sealed.

The mycorrhiza spore count of the product may be e.g. from 300 to 1500 spores per g, preferably about 700 spores/g.

The seed-coating powder is applied to seed by mixing together with an adhesive substance, e.g. sugar or molasses solution, or even beer, or compatible fungicides and pesticides in proportions depending on the crop and seed-planting density. For example, wheat may be treated in a proportion of 200 g seed- coating powder to 100 kg wheat seed/hectare.

Examples of seeds to which the powder may be applied are generally seeds not less than 2 mm in size and may include wheat, maize, barley, sorghum, mustard, cotton, luzerne, sun flowers, peas, beans, onions, to mention but a few examples, e.g. in a ratio of 200 g per 10 to 50 kg, e.g. per 25 kg of seed.

Seed coated as aforesaid falls within the scope of the present invention.

Examples

Example 1 - Preparation of inoculum

A growth medium is prepared consisting of 3 parts sand and 2 parts soil mixture containing zeolite powder, vermiculite, coconut fibre, peat, bentonite and apatite. This is placed in crates and Sudan grass or Korog is sown therein and grown for 3 to 4 months in nursery tunnels, the time being determined by frequent control checks on spore counts in the growth medium.

The grass, including the roots, is removed and the growth medium which contains spores and hyphae is then processed as follows: mixed in water and decanted through a series of sieves after allowing heavy sand particles to settle for a few seconds. The washing and decanting process is repeated until the water is clear (3 to 4 times). Roots and coarse debris are collected on a coarse 200 micron screen while spores are captured on 2 finer screens i.e. one screen from 38 to 45 microns and the other screen from 53 to 60 microns. Decant the spores on these 2 screens in water into a container. Add this water containing the spores to fine zeolite powder (10 to 150 micron size) / powder from any clay e.g. apatite and bentonite and work it through with the hands to obtain a nearly dry mixture (10 % moisture). Sieve the mixture through a 120 micron sieve and mix with carrier/dispersing agent powder, i.e. any inorganic mineral (calcitic clay, silicious mineral, in particular perlite as also described in ZA patent 96/6318) or an organic soya powder. The resulting product is the water-dispersible mycorrhiza concentrate to be used in the following examples. It has a spore count of not less than 1000 spores/g. Example 2 - "Water-soluble" formulation

75 g of mycorrhiza concentrate (75 000 spores) are mixed with 116 g Trichoderma harzianum T22 concentrate (spore count 1 x 10⁹/g and 9 g Azotobacter concentrate (spore count 6 x 10⁸/g), bagged and sealed. Mycorrhiza spore count not less than 375 spores/g.

The formulation is suspended in water in a concentration depending on the crop and manner of application. For potatoes 200 g of the composition are suspended in 10 L of water and then further diluted in 200 to 800 litres of water to be spread over 0,5 hectares.

The Trichoderma concentrate used in this and the following examples contains 1 x 10⁹ active spores/g on a carrier mixture. The Azotobacter concentrate contains 6 x 10⁸ minimum colony-forming units/g on a carrier mixture. In both cases, the carrier mixture may be any suitable inorganic mineral (calcitic clay, silicious mineral, perlite as described in ZA patent 96/6318) or any suitable organic soya powder product.

Example 3 - Granulate

414 g mycorrhiza concentrate (414,000 spores) are mixed with 9 kg sago (size 1 to 5 mm), 90 g Azotobacter concentrate and 135 g Trichoderma harzianum T22 concentrate in a mixer. During mixing an aqueous solution of vegetable dye is added to moisten the mixture just sufficiently for the active ingredients to adhere to the sago. The mixture is bagged and sealed. It has a bulk density of 650 g/l. The individual granules have a mass density of 1 g/ml. The mycorrhiza spore count is 40 spores/g. Example 4 - Seed-coating powder

Mix 7 g mycorrhiza concentrate (7 000 spores), 40 g Trichoderma harzianum T22 concentrate (spore count 1 x 10⁹/g), 5 g rock phosphate powder and 148 g soya bean-based carrier/dispersing agent powder. Bag the mixture and seal. Mycorrhiza spore count not less than 35 spores/g. 200 g of the powder are mixed with 100 kg wheat seed and moistened with a molasses solution to cause adherence to the seed. Enough for 1 ha.

Claims

1. A method of producing an endomycorrhiza inoculum which comprises growing a host plant in a growing medium which contains endomycorrhiza so that the roots of the host plant become endomycorrhiza-infected and recovering therefrom an inoculum, characterised by the combination of the following steps:-

(1) removing the host plant, including the roots, from the growth medium and processing the growth medium containing spores and hyphae by washing it thoroughly with water or aqueous liquid;

(2) separating any root matter and coarse debris from the resultant spore-containing water or aqueous liquid;

(3) separating the spores from the spore-containing water or liquid and

(4) mixing the spores with a fine absorbent carrier powder to obtain a nearly dry powder mixture being a concentrated mycorrhiza spore powder.

2. The method as claimed in claim 1 , characterised by including in said concentrated mycorrhiza-spore powder, as or as part of the absorbent carrier powder, a carrier dispersing agent, being a substance having dispersing agent properties and being soluble/dispersible in water to hold the mycorrhiza spores in stable suspension.

3. The method as claimed in claim 1 or 2, characterised in that the growth medium is a mixture of sand on the one hand, and soil and clay or clay- like matter on the other hand in a suitable ratio.

4. The method as claimed in claim 3, characterised in that said ratio is between 6:1 and 1 :1 by volume, preferably about 3:2.

5. The method as claimed in claim 3 or 4, characterised in that the soil and clay or clay-like matter contains one or more of zeolite powder, vermiculite, coco peat, peat moss, montmorilonite, bentonite, apatite and optionally one or more of perlite, bark, horticultural peat and coir (the fibrous material from coconut husks).

6. The method as claimed in claim 5, wherein a zeolite or zeolites is/are used, characterised in that a zeolite has pore sizes in the range of from about 3 to about 10 A in diameter.

7. The method as claimed in any one of claims 1 to 6, characterised in that the host plant is selected from various grass species, legumes, such as peas, maize, sorghum, medico spp (e.g. medico sativa).

8. The method as claimed in any one of claims 1 to 7, characterised in that the growth period of the host plant is 1 to 6 months, preferably 2 to 5 months, e.g. about 3 to 4 months and this growth is performed under shelter, e.g. in nursery tunnels or greenhouses.

9. The method as claimed in any one of claims 1 to 8, characterised in that for quality control, control checks are performed to determine the number of spores in any production batch and thereby determine when the spores are ready for harvesting.

10. The method as claimed in any one of claims 1 to 9, characterised in that washing of the mycorrhiza-infected growth medium is performed with the minimum volume of water capable of retrieving substantially the entire mycorrhiza spore content, e.g., in practice, in a total volume of between about 1 and 100 times, preferably from 1 to 30 times, e.g. about twice the volume of growth medium, e.g. divided into a plurality of successive washings.

11. The method as claimed in any one of claims 1 to 10, characterised in that washing of the mycorrhiza-infected growth medium is performed by mixing it thoroughly with a portion of the water, allowing heavy sand particles to settle for a few seconds and decanting through a coarse screen, e.g. of from 150 to 300 micron, preferably 180 to 250 micron, e.g. 200 micron, to collect roots and coarse debris, and through one or more finer screens, preferably a plurality of finer screens to capture the spores, repeated several times (e.g. 3 to 4 times) until the water is substantially clear.

12. The method as claimed in any one of claims 1 to 11 , characterised in that for capturing the spores at least two of the finer screens are used in the range of between 20 and 90 microns, e.g. selected from the range of 38/45/53/60 microns, for example, one screen 38 to 45 microns and a second screen 53 to 60 microns.

13. The method as claimed in claim 12, characterised in that the spores captured are washed from these finer screens with a small volume of water into a container, e.g. in a total volume of water equal to from 1 to 20% v/v, preferably from about 3 to 8% v/v, e.g. about 4 to 6% v/v, based on the original volume of growth medium, i.e. about 2% per screen, whereafter this volume of water, containing the spores is now taken up in from 5 to 100 times, preferably 6 to 80 times, e.g. about 10 times its weight of fine absorbent carrier powder so that the mixture thus obtained is nearly dry (preferably about 10% moisture).

14. The method as claimed in claim 13, characterised in that the fine absorbent carrier powder is a fine (10 to 150 micron) zeolite powder and/or one or more clay minerals, e.g. bentonite and/or montmorilonite and/or apatite and/or comminuted perlite.

15. The method as claimed in claim 13 or 14, characterised in that the nearly dry mixture is sieved through a sieve, e.g. having a mesh size of from 80 to 200 micron, preferably 100 to 150 micron, most preferably 120 micron, depending on the spore sizes to be allowed to pass.

16. The method as claimed in any one of claims 13 to 15, characterised in that it includes the feature of claim 2.

17. The method as claimed in claim 16, characterised in that the carrier dispersing agent includes a comminuted inorganic silicious mineral, preferably heat-comminuted perlite and/or an organic powder, preferably a soya powder product.

18. The method as claimed in claim 16 or 17, characterised in that the carrier substance is employed in a ratio of 10% to 80%, preferably 20% to 70%, e.g. 65% by weight of the final concentrate.

19. A mycorrhiza concentrate, comprising endomycorrhiza spores and a powderous extender and carrier medium characterised in that the extender and carrier medium includes a substance serving as a carrier/dispersing agent, and that the concentrate contains said spores in a concentration of not less than 700 spores per gram, preferably not less than 1000 spores per gram.

20. The concentrate as claimed in claim 19, characterised in that the spore content is from 700 to 1300 spores per gram, more preferably from 800 to 1200 spores per gram and most preferably from 1000 to 1100 spores per gram.

21. The concentrate as claimed in claim 19 or 20, characterised in that it is wholly dispersible in water to form a stable suspension of the mycorrhiza spores.

22. The concentrate as claimed in any one of claims 19 to 21 , characterised in that the powderous carrier is selected from any one of zeolite powder and/or one or more clay minerals, e.g. bentonite and/or montmorilonite and/or apatite or a combination of two or more of these and has particle sizes to pass sieve sizes ranging from 20 to 200 micron, preferably from 50 to 150 micron, more preferably from 30 to 120 micron.

23. The concentrate as claimed in any one of claims 19 to 22, characterised in that the carrier/dispersing agent is as set out in claim 17.

24. The concentrate as claimed in any one of claims 19 to 23, characterised in that it is produced by a method as claimed in any one of claims 1 to 18.

25. Use of the concentrate as claimed in any one of claims 19 to 24, characterised in that the concentrate is converted into a water-soluble or dispersible composition comprising 10 to 80% by weight, preferably 30 to 70% of the concentrate, the balance being one or more concentrates of a variety of growth promoting micro-organisms and/or their growth promoting metabolites, e.g. enzyme(s), more particularly a) Trichoderma fungi spp, e.g. Trichoderma harzianum, e.g. the T22 strain; b) bacteria, preferably nitrogen binding types, e.g. Azomonas spp, Pseudomonas spp, Azospirillum spp, Azotobacter spp, Rhizobium spp, Agrobacteria spp, Bazillus spp, Bavarea spp.

26. The use as claimed in claim 25, characterised in that the bacterium/a is/are selected from the group consisting of Actimomycetes thermophilus, Streptomyces spp, Azotobacter chococcum, Mixobacteria celvibrio, M. cytophaga, Bacillus ciculaus, B. subtilis, Fulvoviridis or more than one of these.

27. The use as claimed in claim 25 or 26, characterised by mixing from 50 to 100, e.g. 75 parts by weight Mycorrhiza concentrate, from 70 to 150, e.g. 116 parts by weight Trichoderma concentrate (spore count 1 x 10⁹/g) and from 5 to 15, e.g. 9 parts by weight Azobacter concentrate (spore count 6 x 10⁸/g).

28. The use as claimed in any one of claims 25 to 27, characterised in that the final mix contains not less than 375 mycorrhiza spores per gram.

29. A method of using a water-soluble/dispersible formulation prepared as claimed in any one of claims 25 to 28, characterised by dissolving/dispersing it in water and applying the dispersion through an irrigation system or for dipping into it the roots of plants or applying it to plants with a watering can or the like.

30. The method as claimed in claim 29, characterised in that the formulation is dispersed in water in a weight ratio of powder to water of from 1 : 20 to 1 : 100, e.g. about 1 : 50 and this may then be diluted further with water depending on the crop to be irrigated.

31. A use of the concentrate as claimed in any one of claims 19 to 24, characterised in that the mycorrhiza concentrate is converted into granules.

32. The use as claimed in claim 31 , characterised in that the granules are made of a bulk density in the range of from 500 to 800, preferably 550 to 750, more particularly about 600 to 700 g/l of granules.

33. The use as claimed in claim 31 or 32, characterised in that the mycorrhiza spore count of the granules is from 1 to 100, preferably 30 to 40 spores/g.

34. The use as claimed in claim 32 or 33, characterised in that the granules comprise the spores applied onto a solid carrier substance, together with growth-enhancing micro-organisms or substances as set out in claim 25 or 26.

35. The use as claimed in claim 34, characterised in that the solid carrier substance is biodegradable.

36. The use as claimed in claim 35, characterised in that the solid carrier substance is a farinaceous substance, e.g. starch, rice or sago.

37. The use as claimed in claim 35, characterised in that solid carrier substance particles sterilised round seeds, e.g. sterilised by boiling, e.g. sorghum seed are used.

38. The use as claimed in any one of claims 31 to 37, characterised in that the granules comprise from 100 to 800, preferably 200 to 450, e.g. about 300 parts by weight mycorrhiza concentrate, and from 5000 to 15 000, preferably 7000 to 12 000, e.g. about 9000 parts by weight sago or other farinaceous substance.

39. The use as claimed in claim 38, characterised by additions of 50 to 150, preferably 70 to 120, e.g. 90 parts by weight Azotobacter concentrate (assuming a spore count of 6 x 10⁸/g) and/or from 50 to 250, preferably 80 to 200, e.g. 135 parts by weight Trichoderma spp concentrate (assuming a spore count of 1 x 10⁹/g).

40. A use of the mycorrhiza concentrate as claimed in any one of claims 19 to 24, characterised in that the concentrate is incorporated in a seed-coating powder, including a suitable carrier or mixture of carriers and extenders, preferably further containing other growth-promoting micro-organisms and/or their growth-promoting metabolites.

41. The use as claimed in claim 40, characterised in that the carrier comprises rock phosphate and the carrier/dispersing agent composition, in proportions from 3 to 20 parts by weight, preferably 5 to 12 parts by weight, e.g. about 7 parts by weight of mycorrhiza concentrate; Trichoderma spp concentrate (preferably Trichoderma harzianum, e.g. T22) 15 to 120, preferably 20 to 80, e.g. about 40 parts by weight; rock phosphate 2 to 20, preferably 3 to 10, e.g. about 5 parts by weight; carrier/dispersing agent composition 60 to 300, preferably 100 to 200, e.g. about 150 parts by weight.

42. The use as claimed in claim 40 or 41 , characterised in that the mycorrhiza spore count of the product is from 300 to 1500 spores per g, preferably about 700 spores/g.

43. The use as claimed in any one of claims 40 to 42, characterised in that the seed-coating powder is applied to seed by mixing together with an adhesive substance, e.g. sugar or molasses solution, or beer, or compatible fungicides and pesticides.

44. The use as claimed in any one of claims 40 to 43, characterised in that the powder is applied to seeds, generally not less than 2 mm in size in a ratio of 200 g per 10 to 50 kg, e.g. per 25 kg of seed.