AU649898B2 - Method of preparing VA mycorrhizae inoculant - Google Patents

Description

AUSTRALIA

Patents Act COMPLETE SPECIFICATION (ORIGINAL) 49898 Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: i cc 4* *0

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0 0 0* 0 0* Name of Applicant: Agricultural Genetics Company Limited Actual Inventor(s): Ingrid Arias de Williams Jonathan Day Address for Service: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mar Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Invention Title: METHOD OF PREPARING VA MYCORRHIZAE INOCULANT Our Ref 311403 POF Code: 1286/189789 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): -1- 6006 METHOD OF PREPARING VA MYCORRHIZAE INOCULANT BACKGROUND OF THE INVENTION The present invention relates to a method of preparing a VA mycorrhizae inoculant which is useful in the field of agriculture and horticulture and, more precisely, to a method of preparing a VA mycorrhizae inoculant substance having a high VA mycorrhizal spore density and having a well stabilized activity.

VA mycorrhizae (Vesicular Arbuscular mycorrhizae) are known to live together with plants by symbiosis to promote the growth of plants as infected Stherewith and to improve the disease resistance of the infected plants (N.

Kobayashi, Utilization of Mycorrhizae to Soil Diseases, Prevention of Plant Diseases, Vol. 42, No. 5, pages 259 to 266, 1988); and cultivation of plants with utilizing such a natural power is desired.

Therefore, various methods of artificially propagating VA mycorrhizae S" for preparing a VA mycorrhizae inoculant substance have heretofore been proposed.

S For instance, there have been proposed a method of separating and recovering spores of VA mycorrhizae as propagated in a soil, blending the spores with a carrier such as vermiculite, attapulgite or diatomaceous earth along with an adhesive substance such as carboxviethyl cellulose, and granulating the resulting blend (JP-A 1-165369); and a method of using a mixture with charcoal (JP-A 3-103124). (The term "JP-A" as used herein means an "unexamined published Japanese patent application".) In accordance with the former method, however, spores of VA mycorrhizae are damaged during the step of separating them or during the step of granulating the spores-containing inoculant. In addition, in the step of granulation, the inoculant is forcedly dried so that many spores would die with considerable frequency. As a result, it was difficult to obtain a VA mycorrhizae inoculant of high quaiitv by the method. The same shall apply to the latter mathod of using a mixture with charcoal, in which many spores would also die during the drying step.

On the other hand, other methods have also been proposed. For instance, there are mentioned a method of using a soil (JP-A 3-58715, 3-76572); a method of using a porous structure such as a foamed clay or pumice (JP-A 60-237987, S118390); and a method of using a medium soil containing a porous ampho-ion exchanger (JP-A 63-87973). In these methods, roots of plants and VA mycorrhizae are made to live together by symbiosis so as to propagate the VA mycorrhizae, and the VA mycorrhizae as adhered to the carrier are directly used as they are as a VA mycorrhizae inoculant.

In these methods, however, where a natural soil is used, there would have a problem of contamination with pathogenic microbes.

With respect to the method of using a porous structure such as a foamed clay or pumice, a high-density VA mycorrhizae inoculant could not be obtained.

In the method of using a medium soil containing a porous ampho-ion exchanger, host plants are limited to only potato and the like plants and the carrier of itself is an expensive one such as DEAE-cellulose. Therefore, the method is not practical.

The object of the present invention is to overcome these drawbacks in the above related arts and to provide a method of preparing a VA mycorrhizae inoculant having a high spore density of VA mycorrhizae and having a stabilized activity of them.

SUMMARY OF THE INVENTION Specifically, the present invention provides a method of preparing a VA mycorrhizae inoculant substance characterized by cultivating plants as infected with VA mycorrhizae of the genus Glomus in a medium containing calcined attapulgite or calcined montomorillonite so as to propagate the VA mycorrhizae.

*o DETAILED DESCRIPTION OF THE INVENTION VA mycorrhizae are a kind of Zygomycetes living in soil, and it is known .I that the mycelium of them infect into roots of various plants to form mycorrhizal organs therein so that the both live together symbiotically.

VA mycorrhizae to be used in the present invention are those of the genus Glomus.

As VA mycorrhizae of the genus Glomus to be used in the present invention, more concretely, there are mentioned Glomus fasciculatum, Glomus mosseae, Glomus etunicatum, Glomus intraradicies, Glomus manifotis and Glomus caledonium.

These VA mycorrhizae of the genus Glomus are collected from the field(Suzuki, Problems on VA Mycorrhizae 5, Agriculture and Horticulture, Vol. 62, No. 3, pp. 28 to 33, 1987); or they may be artificially propagated by a nutrient thin film culture method (JP-A 55-118390) or by a method of using roots as cultivated by tissue culture (JP-B 62-49037 the term "JP-B" as used herein means an "examined Japanese patent publication"). Any of them can be used in the present invention.

In the case that the medium containing calcined montomorillonite is usd as the medium, other VA Mycorrhizae than VA Mycorrhizae belonging to the genus Glomus may be used.

As other VA Mycorrhizae than VA Mycorrhizae belonging to the genus Glomus, there are various kinds of VA mycorrhizae.

For example, those are VA Mycorrhizae belonging to the genera Gigaspora, Acaulospora, Entrophospora, Sclerocystis and Scutellospora.

More concretely, those are Gigaspora margarita, Acaulospora laevis, S Entrophospora infrequens, Sclerocystis dussii and Scutellospora gregaria.

Plants to be infected with VA mycorrhizae or host plants for propagation of VA mycorrhizae for use in the present invention are not specifically defined provided that they may grow rapidly to have well grown roots and may easily be 'i infected with VA mycorrhizae. For instance, seeds are sowed in a field and cultivated there to plants or the seedlings from them are then transplanted in a different field and are further cultivated there; or plants are cultivated and propagated by vegetable propagation or by cutting or grafting or from bulbs.

*Concretely, as host plants for propagation of VA mycorrhizae, there are mentioned true grasses of Gramineae such as corn, crabgrass, sorgo (sweet sorghum or sugar sorghum), wheat, barley and lawn grass; solanous plants of Solanaceae such as eggplant, tomato, pimento (Spanish paprika) and green pepper; leguminous plants of Leguminosae such as soy bean and tare; and liliaceous plants of Liliaceae such as Welsh onion and common onion.

VA mycorrhizae may be applied to the medium before or after rooting of the above-mentioned host plants. Infection of the host plants with VA mycorrhizae may be effected by any known method. For instance, the conditions are such that the temperature is from 5 to 600 C, preferably from 10 to 450 C, and the pH of the soil is from 4 to 9.5, preferably from 4.5 to In accordance with the present invention, the above-mentioned host plants as infected with VA mycorrhizae of the genus Glomus are characteristical ly cultivated in a medium containing calcined attapulgite or calcined montomorillonite.

In the present invention, the host plants as infected with VA mycorrhizae of the genus Glomus may well be cultivated in at least a medium containing calcined attapulgite or calcined montomorillonite. If desired, any other medium may be used for infection of host plants with VA mycorrhizae of S. the genus Glomus.

The calcined attapulgite to be used in the present invention is one as obtained by calcining attapulgite at a temperature of from 200 to 13000 C, S preferably from 300 to 10000 C. If desired, calcined attapulgite granules as obtained by granulating a powdery attapulgite along with a binder of alumina or boehmite may also be used. In the case, the calcined attapulgite granules are desired to be adjusted to have pH of from 4.5 to 7.5. The grain size of them S may be from 0.25 to 10 mm, preferably from 1 to 5 mm.

On the other hand, the calcined montmorillonite to be used in the present invention is one as obtained by calcining montmorillonite at a temperature of from 200 to 1300 C, preferably from 300 to 10000 C. If desired, calcined montmorillonite granules as obtained by granulating a powdery montmorillonite along with a binder of alumina or boehmite may also be used.

In the case, the calcined montmorillonite granules are desired to be adjusted to have pH of from 4,5 to 7.5. The grain size of them may be from 0.25 to mm, preferably from 1 to 5 mm.

In the present invention, the above-mentioned calcined attapulgite or calcined montmorillonite may be used singly as the medium, or as the case may be, it may be combined with one or urore of any other medium components such as zeolite, pearlite, vermiculite, (calcined) diatomaceous earth and/or pumice.

The proportion of the calcined attapulgite to additional medium components, if any, may be from 1:0 to 1:1 preferably from 1:0.1 to 1:1 more preferably from 1:1/6 to 1:1/2 As the additional medium component, preferred is pumice having a grain size of from 0.5 to 10 mm, preferably from 1 to 5 mm.

The proportion of the calcined montmorillonite to additional medium components, if any, may be from 1:0 to 1:1 preferably from 1:0.1 to 1:1 more preferably from 1:1/8 to 1:1/3 As the additional medium *e* component, preferred is pumice having a grain size of from 0.5 to 5 mm, preferably from 1 to 3 mm.

With growth of the host plants, VA mycorrhizae propagate. In general, after the host plants have well grown in 2 to 5 months, irrigation and others thereto is stopped and the thus grown plants are allowed to stand as they are for a while, whereupon VA mycorrhizae form spores. Cultivation of the host plants may be effected under ordinary conditions. For instance, the temperature may be generally from 5 to 60 C. If desired, water and fertilizer may be applied to the host plants being cultivated.

Then, the medium to which the thus formed VA mycorrhizal spores have adhered (VA mycorrhizae inoculant) may be recovered. Thus, the intended VA mycorrhizae inoculant is obtained.

In accordance with the method of the present invention, a VA mycorrhizae inoculant having a high VA mycorrhizal spore density and having a stabilized VA mycorrhizal activity can be prepared at low cost.

Accordingly, the method of the present invention can be utilized effectively in various fields of agriculture, horticulture, gardening and nursery.

EXAMPLE

Next, the present invention will be explained in more detail by way of the following examples.

Example 1: A plastic pot No. 8 (diameter 240 mmx height 169 mm) was filled with calcined attapulgite passing through a 3 mm-mesh sieve but not through a 1 mm- S mesh sieve (grain size, 1 to 3 mm; firing temperature 540 In the center of the pot, 320 spores of VA mycorrhiza (Glomus fasciculatum) were embedded at the depth of 3 cm, they being wrapped with tissue paper so that they would not run out downwards along with water. Applicant has a mind to furnish a sample of Glomus fasciculatum to any interested party in response to request for the furnishing of the sample.

Next, two grains of corn (a variety of Golden Dent DK649, by Kaneko Nursery Co.) were put 1 cm above the spores.

Next, the calcined attapulgite montmorillonite, VA mycorrhizae and corn seeds in the plastic pot were well wetted with water, and was put in a glass green house.

The temperature in the inside of the glass green house in which the pots were put was kept to be from 20 to 25° C, and the plants were cultivated for one week with applying sufficient water thereto. Among the grown seedlings, others except healthy ones were removed, Then, the remaining seedlings were cultivated further in the glass greenr house with sprinkling water over them every day.

After one month from the beginning of the cultivation, a 1/1000 diluted solution of Peter's liquid fertilizer (N/P/K 20/10/20) was sprinkled over the plants being cultivated once a week.

S• The cycle was repeated, and the plants were cultivated for further o months. Afterwards, sprinkling of water and the liquid fertilizer were *o C stopped, and the plants were allowed to be as they were for 30 days.

Next, the medium pot was turned over a polyvinyl resin sheet and the roots of the corn plants and the calcined attapulgite containing VA mycorrhiza were spread over the sheet. The thick roots of the corn plants were removed and the others were dried as they were at 15° C in the dark.

-Five samples, each weighing one gram, of the thus processed medium (calcined attapulgite containing roots of corn plants and VA mycorrhiza) were taken out at random, and the number of the adhered spores was counted in each sample. An average number was obtained from the counted data and was shown in Table 1 below as the number of spores adhered to the medium.

Example 2: The same process as in Example 1 was repeated except that a mixture comprising 6 parts of calcined attapulgite and one part of pumice was used as the medium in place of calcined attapulgite only. The number of the spores as adhered to the medium was counted, and an average number was obtained from the counted data and was shown in Table 1 as the number of spores adhered to the medium.

Comparative Example 1: The same process as in Example 1 was repeated except that a foamed clay (Lecadan as registered trade mark by Lecadan Deutscheland) as pulverized to have a grain size of from 1 to 3 mm was used as the medium in place of calcined attapulgite. The number of the spores as adhered to the medium was counted, and an average number was obtained from the counted data and was shown in Table 1 as the number of spores adhered to the medium.

0 Comparative Example 2: The same process as in Example 1 was repeated except that amber clay as sterilized with methyl bromide and well degassed to have a grain size of from 2 to 4 mm was used as the medium in place of fired attapulgite. The number of the o spores as adhered to the medium was counted, and an average number was obtained from the counted data and was shown in Table 1 as the number of spores adheied to the medium.

Example 3: The same process as in Example 1 was repeated except that 40 spores of Glomus caledonium were used in place of 320 spores of Glomus fasciculatum as the VA mycorrhiza. The number of the spores as adhered to the medium was counted, and an average number was obtained from the counted data and was shown in Table 2 below as the number of spores adhered to the medium. Applicant has a mind to furnish a sample of Glomus caledonium to any interested party in response to request for the furnishing of the sample.

Example 4: The same process as in Example 1 was repeated except that 40 spores of Glomus mosseae were used in place of 320 spores of Glomus fasciculatum as the VA mycorrhiza. The number of the spores as adhered to the medium was counted, and an average number was obtained from the counted data and was shown in Table 2 as the number of spores adhered to the medium. Applicant has a mind to furnish a sample of Glomus mosseae to any interested party in response to request for the furnishing of the sample.

Comparative Example 3: The same process as in Example 1 was repeated except that 40 spores of Scutellospora gregaria were used in place of 320 spores of Glomus fasciculatum as the VA mycorrhiza. The number of the spores as adhered to the medium was counted, and an average number was obtained from the counted data and was shown in Table 2 as the number of spores adhered to the medium.

Table 1 Example 1 Example 2 Comparative Comparative Example 1 Example 2 Type of the Calcined Gaicined medium attapulgite attapulgite, Foamed clay Amber clay Pumice VA Plycorrhiza Glomus Glomus Glomus Glomus fasciculatum fasciculatum fasciculatum fasciculatum Number of spores adhered 87 108 36 44 to the medium 9. 9.

9 *9 9 *94* 9, 9. 9 9. 9 9 .9 9 9* .9 9099 9*9e9O 9 9 .9 ft 9 99** Table 2 Example 3 Example 4 Comparative Example 3 Type of the Calcined Calcined Calcined medium attapuloite attapulgite attapulgite VA mycorrhiza Glomus Glomus Scutello!;pora caledonium mosseae gregaria Number of spores adhered 61 85 8 to the medium Example F: A long pot No. 5 (diameter 150 mm x height 157 mm) was fillec with calcined montmorillonite passing through a 3 .i-mesh sieve but not through a 1 mm-mesh sieve (grain size, 1 to 3 mm; calcining temperature 6000 In the center of the pot, 250 spores of VA mycorrhiza (Glomus fasciculatum) were embedded at the depth of 3 cm, they being wrapped with tissue paper so that they would not run out downwards along with water. Next, two seeds of Sudan grass (a variety of Best Sudan, by Takii Nursery Co.) were put 1 cm above the spores.

Next, the calcined montmorillonite, VA mycorrhizae and Sudan grass seeds in the long pot were well wetted with water, and was put in a glass green house.

0 .The temperature in the inside of the glass green house in which the pots were put was kept to be from 20 to 250 C, and the plants were cultivated for one week with applying sufficient water thereto. Among the grown seedlings, others except healthy ones were removed. Then, the remaining seedlings were cultivated further in the glass green house with sprinkling water over them every day.

After one month from the beginning of the cultivation, a 1/1000 diluted solution of Peter's liquid fertilizer (N/P/K 20/10/20) was sprinkled over the plants being cultivated once a week.

The cycle was repeated, and the plants were cultivated for further months. Afterwards, sprinkling of water and the liquid fertilizer were stopped, and the plants were allowed to be as they were for 20 days.

Next, the parts above the ground of the thus grown Sudan grass plants were cut and removed, and each long pot was turned over lyvinyl resin sheet and the roots of the Sudan grass plants and the calcined monimorillonite containing VA mycorrhiza were spread over the sheet. The thick roots of the Sudan grass plants were removed and the others were dried as they were at 150 C in the dark. Five samples, each weighing one gram, of the thus processed medium (calcined montmorillonite) were taken out at random, and the number of the adhered spores was counted in each sample. An average number was obtained from the counted data and was shown in Table 3 below as the number of spoies adhered to the medium of calcined montmorillonite.

S Example 6: 0*0Q The same process as in Example 5 was repeated except that a mixture comprising 6 parts of calcined montmorillonite and one part of pumice was used as the medium in place of calcined montmorillonite only. The number of the spores as adhered to the medium was counted, and an average number was obtained from the counted data and was shown in Table 3 as the number of spores adhered to the medium.

Comparative Example 4: The same process as in Example 5 was repeated except that a foamed clay (Lecadan as registered trade mark by Lecadan Deutscheland) as pulverized to have a grain size of from 1 to 3 mm was used as the medium in place of calcined montmorillonite. The number of the spores as adhered to the medium was counted, and an average number was obtained from the counted data and was shown in Table 3 as the number of spores adhered to the medium.

Comparative Example The same process as in Example 5 was repeated except that amber clay as sterilized with methyl bromide and well degassed to have a grain size of from 2 to 4 mm was used as the medium in place of calcined montmorillonite. The number of the spores as adhered to the medium was counted, and an average number was obtained from the counted data and was shown in Table 3 as the number of spores adhered to the medium.

Example 7: The same process as in Example 5 was repeated except that 40 spores of Glomus mosseae) were used in place of 250 spores of Glomus fasciculatum as the VA mycorrhiza. The number of the spores as adhered to the medium was counted, S and an average number was obtained from the counted data and was shown in Table '3 as the number of spores adhered to the medium.

o a a a a. f Table 3 Example 5 Example 6 Comparative Comparative Example 7 Example 4 Example Type of the Calcined Calcined Calcined montmori- montmori- Foamed clay Amber clay montmorimedium Ilonite llonite' I lonite Pumice VA Glomus Glomus Glomus Glomus Glomus mycorrhiza fascicu- fascicu- fasciculatum fasciculatum mosseae latum latum Number (/g of spores 76 88 41 48 61 adhered to the medium S S

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SS S 5* *55* S S *5 a. I.

Claims (5)

1. A method of preparing a VA mycorrhizae inoculant characterized by cultivating plants as infected with VA mycorrhizae of the genus Glomus in a medium containing calcined attapulgite or calcined montmorillonite so as to propagate the VA mycorrhizae.

2. A method according to claim 1 in which a mixture comprising calcined attapulgite and pumice in a proportion of from 1/0.1 to 1/1 is used as the medium.

3. A method according to claim 1 in which a mixture comprising calcined montmorillonite and pumice in a proportion of from 1/0.1 to 1/1 is used as the medium.

4. A method according to any one of claims 1 to 3 wherein the VA mycorrhiza is at least one selected from the group consisting of Glomus fasciculatum, Glomus mosseae, Glomus etunicatum, Glomus intraradicies, Glomus manifotis and Glomus Scaledonium. S

5. A method according to claim 1 substantially as hereinbefore described with reference to the examples excluding any of the comparative examples. DATED: 23rd November, 1992 PHILLIPS ORMONDE FITZPATRICK Attorneys for: AGRICULTURAL GENETICS COMPANY LIMITED 39 -16- ,l ABSTRACT OF THE DISCLOSURE The present invention provides a method of preparing a VA mycorrhizae inoculant characterized by cultivating plants as infected with VA mycorrhizae of the genus Glomus in a medium containing calcined attapulgite or calcined montmorillonite so as to propagate the VA mycorrhizae. In accordance with the method of the present invention, a VA mycorrhizae inoculant having a high VA mycorrhizal spore density and having a stabilized VA mycorrhizal activity can be prepared at low cost. Accordingly, the method of the present invention can be utilized effectively in various fields of agriculture, horticulture, gardening and nursery. e. a o e