AU640578B2 - Method of preparing va mycorrhizae inoculant - Google Patents

Description

AUSTRALIA

Patents Act COMPLETE SPECIFICATION

(ORIGINAL)

4 0 m/" Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: 4* 4 0 0@ ac Name of Applicant: Agricultural Genetics Company Limited Actual Inventor(s): Ingrid Arias de Williams Jonathan Day Address for Service: 0* S *9a 4* *9 S. S PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA 04 *r f> *5o* 9* 9 0*

SO

Invention Title: METHOD OF PREPARING VA MYCORRHIZAE INOCULANT Our Ref 311393 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 various field of agriculture and horticulture and, more precisely, to a method of preparing it by cultivating VA mycorrhizaeinfected plants in the presence of a carrier with applying a particular compound to the carrier so as to protect the VA mycorrhizae inoculant from phytopathogenic fungi of Pythium, Phytophthora and Peronosporaceae. Due to application of such a particular compound to the carrier, propagation of VA mycorrhizae and formation of spores of them are promoted so that a VA mycorrhizae inoculant having a high spore density and an elevated activity can be prepared by the present invention.

VA mycorrhizae (Vesicular Arbuscular mycorrhizae) are known to infect various plants to live together with them by symbiosis thereby to promote the growth of the plants as infected therewith and to improve the disease resistance of the infected plants (Agriculture and Horticulture, Vol. 62, No. 8, pages 930 to 937, 1987; Prevention of Plant Diseases, Vol. 42, No. 5, pages 259 to 266, 1988).

However, practical VA mycorrhizae inoculant which could be used for artificially propagating VA mycorrhizae so as to infect plants with them are not almost known up to these days. Hitherto, as VA mycorrhizae inoculant there have been known one as prepared by applying separated VA micorrhizal spores to a carrier along with a clay powder and one as prepared by using a porous carrier such as a foamed clay, However, the activity of the VA mycorrhizae in them is not sufficient and the VA mycorrhizae inoculant is high-priced. Therefore, the known VA mycorrhizae inoculant could not be said to be practical.

Known methods of prepari-i VA mycorrhizae inoculant could be grouped into the following two groups.

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 i ii: carboxymethyl cellulose, and granulating the resulting blend (JP-A 1-165369); and a method of using charcoal as a carrier (JP-A 3-103124). (The term "JP-A" as used herein means an "unexamined published Japanese patent application".) (ii) A method of using a soil as a medium (JP-A 3-58715, 3-76572); a method of using a porous'substance such as a foamed clay or pumice as a medium (JP-A 60-237987, 55-118390); and a method of using an ion exchanger as a medium S (JP-A 63-87973). In these methods, roots of plants and VA mycorrhizae are made S to live together by symbiosis so as to propagate the VA mycorrhizae, and the VA mycorrhizae as adhered to the medium are directly used as they are as a VA o. mycorrhizaa inoculant.

In accordance with the methods of the group 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 blend 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 quality by the methods.

On the other hand, in accordance with the methods of the group (ii), where a natural soil is used, there would occur a problem of contamination with pathogenic microbes. In the other method where a fine granular clay is used as a carrier for VA mycorrhizae, the carrier is filled in the interface to a coarse granular clay in the direction of the depth and plants are cultivated under the condition (JP-A 3-76572), VA mycorrhizae as propagated in the fine granular clay having a grain size of from 0.1 to 1 mm are recovered and used.

The case, however, often causes a problem that the fine granular clay is further broken during the preparation step (generally, 2 to 4 months) to cause clogging. Under the clogged condition, supply of oxygen to the growing VA r S mycorrhizae could not be effected sufficiently. Therefore, the method could not 8 be said to be favorable for propagation of VA mycorrhizae. In addition, the method has another risk that the VA mycorrhizae as grown would be contaminated with microbes.

In addition, as mentioned above, there is known a method of using a porous ampho-ion exchanger to propagate VA mycorrhizae with host plants of potato and the like. However, the method is not practical since the host plants usable therein are limited to only potato and the like plants and the carrier itself is expensive.

In order to overcome the problems in the above related arts, the present inventors earnestly studied and, as a result, have found that application of a particular compound to plants as inoculated with VA mycorrhizae during cultivation of them is effective for protecting the plants from diseases to be caused by phytopathogenic fungi of Pythium or Peronosporaceae. In addition, propagation of mycelium of the VA mycorrhizae is thereby accelerated and formation of spores of them is also promoted. Accordingly, they have found that a low-priced and practical VA mycorrhizae inoculant can thereby be obtained.

On the basis of the findings, the inventors have achieved the present invention.

SUMMARY OF THE INVENTION Specifically, the present invention provides a method of preparing a VA mycorrhizae inoculant by cultivating VA mycorrhizae-infected plants in the presence of a carrier to be inoculated with VA mycorrhizae, in which a compound of a general formula (I a N-1 CH, n -NH-C-R' (HC (I)

II

0 where R' represents a hydrogen atom or a methyl group; R' represents an oxygen atom or a sulfur atom; R' represents an alkyl group having from 2 to 4 carbon atoms, a 1-methyl-2-propionyl group or an allyl group; n represents from 2 to 5; and m represents 0 or 1, 0 is applied to the medium to be inoculated with VA mycorrhizae.

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

There are various kinds of VA mycorrhizae usable in the present invention, including, for example, those belonging to the genera Gigaspora, Acaulospora, Entrophospora, Sclerocystis, Scutellospora and Glomus.

More precisely, there are mentioned, for example, Gigaspora margarita, Acaulospora laevis, Entrophospora infrequens, Sclerocystis dussii, Scutellospora gregaria, Glomus mosseae, Glomus intraradicies, Glomus caledonium, and Glomus fasciculatum.

For collecting these VA mycorrhizae, various methods may be employed.

For instance, there are mentioned a method of collecting them from a natural world by the use of a sieve (Suzuki, Problems on VA Mycorrhizae Agriculture and Horticulture, Vol. 62, No. 3, pp. 28 to 33, 1987); and a centrifugal method of also collecting them from a natural world (JP-A 63-309178) e SIn addition, also usable are a nutrient thin film culture method (JP-A 55-118390) and 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"), in both of which VA mycorrhizae are propagated under a germ-free condition to form spores of them.

*9 Where the spores thus formed are separated from the mycelium of the cells and are handled singly, they are desired to be stored under a wet condition. This is because if they are dried, the activity of them would lower.

In addition, for the purpose of preventing germination of the stored spores, maintaining the activity of them and preventing them from being contaminated with microbes, the spores are desired to be stored at a low temperature.

Host plants which are to live together with VA mycorrhizae by symbiosis in the present invention are not specifically defined provided that they may grow rapidly to have well grown roots and may easily be infected with VA mycorrhizae. For instance, preferred are true grasses of Gramineae such as corn, crabgrass, sorgo 'sweet sorghum or sugar sorghum), wheat, barley, lawn grass and Sudan grass; solanous plants of Solanaceae such as eggplant, tomato, pimento (Spanish paprika) and green pepper; leguminous plants of Leguminosae such as soy bean, tare, mung bean, peanut, alfalfa and clover; composite plants of Cormpositae such as marigold, sunflower, cineraria and chrysanthemum; rose plants of Rosaceae such as strawberry; and liliaceous plants of Liliaceae such as Welsh onion and common onion. These plants are cultivated and propagated in various ways and are used in the present invention. For instance, seeds are sowed in a field and cultivated there to plants or the seedlings from them are :li: 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.

S"The medium (which may also be called a support material) to be inoculated with VA mycorrhizae and to be used for cultivation of plants in the present invention is not specifically defined provided that it is an inorganic 9e material and is hardly disintegrated after it has absorbed water. In view of prevention of introduction of aboriginal microbes into the medium (support Smaterial) the medium (support material)is desired to be a sterilized (or calcined) one. For instance, especially preferred are calcined attapulgite, calcined montmorillonite, calcined diatomaceous earth, zeolite, calcined amber clay and pumice. These may be used singly or in combination of two or more of them.

As calcined attapulgite or calcined montmorillonite to be used, preferred is one to be obtained by calcining attapulgite or montmorillonite having a grain size of from 0.5 to 10 mm, preferably from 1 to 3 mm, at a temperature of from 200 to 1300 C, preferably from 300 to 10000 C. If desired, they may be in the form of granules as granulated with a binder such as alumina.

As calcined diatomaceous earth to be used, preferred is one as obtained by calcining diatomaceous earth having grain size of from 0.5 to 5 mm, preferably from 1 to 3 mm, at a temperature of from 200 to 13000 C, preferably from 300 to 1000° C. Clay of montmorillonite or attapulgite can be used as a binder. Alumina may also be used as a binder, but in the case, the pH value is desired to be adjusted to from 5.5 to :'As zeolite to be used, preferred is such that the surface is spherical 0 but is not smooth and it partly has plane structures and is rough. Desirably, *Of0 it has a grain size of from 0.5 to 5 om, preferably from 1 to 3 mm.

As calcined amber clay to be used, preferred is one to be obtained by calcining amber clay having a grain size of from 0.5 to 5 mm, preferably from 1 to 3 mm, at a temperature of from 200 to 13000 C, preferably from 200 to 10000 00

C.

A mixture comprising from 1 to 10 parts of the above-mentioned medium and one part of pumice is also used preferably. In the case, the pumice to be used is desired to have a grain size of from 0.5 to 5 mm.

The process of infecting host plants with VA mycorrhizae will be mentioned below. Regarding the time, the spores may be applied to host plants at any time of before and after shooting of roots from the seeds. Preferably, they are applied during pre-treatment for sowing or cutting or at the same time with sowing or cutting or during transplantation of seedlings. Regarding the means for application, preferably, the spores may be blended with a medium (support material) for host plants or may be applied the seeds or buds of host plants underneath or may also be applied to the holes of a medium into which the seedlings of host plants are transplanted permanently from a seed bed. The number of VA mycorrhizae spores to be applied to host plants is not specifically defined, but in general, it may be approximately from 1 to 100,000 c! Is per one plant.

Infection of host plants with VA mycorrhizae as well as cultivation of the infected host plants may be effected by any known method. For instance, the conditions are such that the temperature is from 5 to 60° C, preferably from 10 to 450 C, and the pH of the soil isfrom 3 to 9.5, preferably from 4 to After cultivating the host plants in this way, infection of the plants with VA mycorrhizae is completed along with growth of the plants so that the VA mycorrhizae propagates with the plants.

In accordance with the present invention, a compound of the following 44** 4e S general formula is applied to a medium to be inoculated with VA *.a6 mycorrhizae, in cultivation of plants as infected with VA mycorrhizae in the Sa presence of the above-mentioned medium.

N- CCH, n -NH-C-R' (HCl)m (I) S"

II

where R' represents a hydrogen atom or a methyl group; R' represents an oxygen atom or a sulfur atom; R' represents an alkyl group having from 2 to 4 carbon atoms, a 1-mehtyl-2-propionyl group or an allyl group; n represents from 2 to and m represents 0 or 1.

The alkyl group having from 2 to 4 carbon atoms as referred to herein includes an ethyl group, a propyl group, a butyl group, a t-butyl group and an iso-butyl group.

Examples of the compound are mentioned below, in which Me indicates a methyl group, Et indicates an ethyl group, Pr indicates a propyl group, and Bu indicates a butyl group.

Me, N- (H 2 -NH-C-0C-rS 0 CH

OHS

H, N- (OH, 4 -NH-C-C-C-OHs o OH 3

OHS

H, N- (OH, 5 NHC0COHs

OH

He, N- 3 -NH-C-OCH 3

C-C

Me, N- (OH, 3-NH-C-S-P rHC Me, N- [CH, Me, N- [CH, Me, N- [CH, Me, N- (CH, Me, N- (CH, Me, N- Me, N- (CH, -NH-C-O-Et

II

0 -NH-C-O-CH, -C-CH, II I 0 CH,

-NH-C-O-CH-CH=CH,

11

O

-NH-C-0-Bu

II

O

-NH-C-0-Pr

II

O

*0 *r 6 000 4 00 @0 *6 00 S S 00 0000 6 0 06 0 0@ 0 S S

-NH-C-S-E

II

0 t HCI -NH-C-S-E t

II

0 0 5

S

es. o 05 0 05 Application of the above-mentioned compounds to the medium (support material) is not specifically defined but may be effected by any ordinary way.

For instance, approximately from 100 to 500 ml, per one plant or seedling, of an aqueous solution of the compound having a concentration of from 0.1 to mg/liter, preferably from 0.3 to 1.5 mg/liter, may be applied to the vicinity of plants or seedlings. In cultivating plants, if desired, water and/or nutrient components may be applied thereto.

Host plants well grow generally in about 2 to 5 months after the application of the above-mentioned compound thereto, whereupon application of water and/or nutrient components thereto is stopped and the plants are allowed to stand as they are. Then, VA mycorrhizae as being penetrated into root and soil form spores. The medium (support material) to which the VA mycorrhizal spores thus formed have adhered is recovered to obtain an intended VA mycorrhizae inoculant (preparation).

The thus obtained VA mycorrhizae inoculant may be applied to plants capable of being infected with VA mycorrhizae and are utilized for cultivation of various plants.

In accordance with the method of the present invention, propagation of mycelium of VA mycorrhizae is rapid and formation of spores of them is promoted. In addition, due to application of a particular compound of formula e a0 to plants being cultivated, contamination of the plants with microbes may be prevented.

0 Therefore, a VA mycorrhizal preparation having a high VA mycorrhizal spore density and an exceilent VA mycorrhizal activity can be prepared at low cost. Accordingly, the VA mycorrhizal preparation to be obtained by the o present invention can be utilized effectively as a practical material.

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

Examples 1 to 3, and Comparative Examples 1 and 2: 15 flower pots No. 6 (each having a size of 187 X 146 fam H; hereinafter referred to as a pot) were filled with calcined montmorillonite passing through a 3 mm-mesh sieve but not through a 1 mm-mesh sieve. In each pot, 190 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 watering. 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 .den Tent DK 649, by Kaneko Nursery Co.) were put 1 cm above the spores. The calcined montmorillonite, VA mwcorrhiza and corn grains in each pot were well wetted with water. These pots were put in a glass green house kept to have an inside temperature of from 20 to C, and the corn plants were cultivated therein with applying sufficient water thereto for one week.

Among the grown seedlings, others except healthy ones were removed.

Then, the remaining seedlings were cultivated further in the glass green house 0* Swith sprinkling water over them every day. In one month after sowing, 300 ml of S an aqueous solution of 700 mg/liter of propyl-3-(dimethylamino)propyl carbamate hydrochloride (hereinafter referred to as chemical compound) was applied to five of 15 pots (Example while water only was applied to the other 10 pots.

•Afterwards, 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, and cultivation of the plants was continued for further one month.

At the time, the calcined montmorillonite in the position separated 5 cm from the roots of the plants being cultivated was sampled by the use of a cork borer, and the number of the samples of the calcined montmorillonite to which VA mycorrhizal mycelium adhered were counted. The number with VA mycorrhiza adhering thereto per the total number of the samples of the calcined montmorillonite is called a mycelium-adhered percentage, and the mean value of five tested pots is shown in Table 1 below.

Five of 10 control pots to which the chemical compound was not applied were selected at random, and sampling of the calcined montmorillonite and counting of the number of the VA mycorrhizal mycelium adhered samples, if any, were effected in the same manner as above (Comparative Example The mycelium-adhered percentage per 5 pots was obtained and shown in Table 1.

Further, cultivation of the plants was continued for one more month with applying the above-mentioned liquid fertilizer thereto once a week.

Afterwards, application of water and liquid fertilizer was stopped, whereupon 300 ml of the same chemical compound solution having the same concentration as above was additionally applied to five pots to which the chemical compound solution was previously applied (Example and 300 ml of the same chemical compound solution having the same concentration as above was newly applied to the other five pots of the remaining ten pots (to which the chemical compound solution was not applied in the previous one month cultivation) (Example 3).

To the last five pots, no chemical compound solution was applied as a control l group (Comparative Example 2).

After the process, application of water and liquid fertilizer was stopped, and all the pots were allowed to stand as they were for 20 days. Then, each pot was turned over and the corn roots and the calcined montmorillonite containing VA mycorrhiza were spread over a polyvinyl resin sheet. The thick roots of the corn plants were removed and the others were dried as they were at 150 C in the dark. Three samples, each weighing one gram, of the thus processed medium (calcined montmorillonite) were taken out at random from each pot, and the number of the adhered spores was counted in each sample. An average number was obtained from the counted data of the five pots of each group and was shown in Table 1 below as the number of VA mycorrhizal spores.

Table 1 VA mycorrhizal mycelium- number of adhered percentage VA mycorrhizal spores Example 1 47 Comparative Example 1 31 Example 2 -93 Example 3 Comparative Example 2 67 O* a :0 so a 0 *0 of ooS.SS too* 00 4 is 0 04 Examples 4 to 5, and Comparative Examples 3 to 4: Same process as in Example 2 was repeated except that the calcined attapulgite was used in place of the calcined montmorillonite and that Glomus caledonium (Example 4) or Glomus mosseae (Example 5) was used in place of Glomus fasciculatum as the mycorrhiza.

In the same manner as Example 2, the number of the adhered spores was counted. An average number was shown in Table 2 below.

And the same process as in Example 4 was repeated except that no chemical compound solution was applied (Comparative Example 3).

And further the same process as in Example 5 was repeated except that no chemical compound solution was applied (Comparative Example 4).

In the same manner as Example 2, the number of the adhered spores was counted. An average number was shown in Table 2 below.

Applicant has a mind to furnish a sample of Glomus caledonium or Glomus mosseae to any interested party in response to request for the furnishing of the sample, Table 2 Number of VA mycorrhizal spores Glomus caledonium Glomus mosseae Example 4 124 Comparative Example 3 64 Example 5 132 Comparative Example 4 79

Claims (5)

1. A method of preparing a VA mycorrhizae inoculant by cultivating VA mycorrhizae-infected plants in the presence of a medium to be inoculated with VA mycorrhizae, in which a compound of a general formula 0 (RI)2N-[CH2]n-NH- -R2-R 3 (HC1) m 1 2 where R represents a hydrogen atom or a methyl group; R represents an oxygen atom or a sulfur atom; R 3 represents an alkyl group having from 2 to 4 carbon atoms, a l-methyl-2- propionyl group or an allyl group; n represents from 2 to and m represents 0 or 1, is applied to the medium to be inoculated with VA mycorrhizae.

2. A method according to claim 1 wherein the VA mycorrhizae is at least one selected from the genera consisting of o. Gigaspora, Acaulospora, Entrophospora, Sclerocystis, Scutellospora and Glomus.

3. A method according to any one of claims 1 or 2 wherein the medium is at least one selected from the group consisting of calcined attapulgite, calcined montmorillonite, calcined S diatomaceous earth, zeolite, calcined amber clay and pumice.

4. A method according to any one of claims 1 to 3 wherein the compound of the general formula I is propyl-3- (dimethylamino)propyl carbamate hydrochloride.

5. A method according -to claim 1 substantially as hereinbefore described with reference to any one of examples 1 to 5 excluding the comparative examples. e DATED: 23rd November 1992 PHILLIPS ORMONDE FITZPATRICK Attorneys for: uap AGRICULTURAL GENETICS COMPANY LIMITED 39 -16- I 1 ABSTRACT OF THE DISCLOSURE The present invention provides a method of preparing a VA mycorrhizae inoculant by cultivating VA mycorrhizae-infected plants in the presence of a medium to be inoculated with VA mycorrhizae, in which a compound of a general formula N- (CH, n -NH-C-R' (HC1) (I) II 0 where R' represents a hydrogen atom or a methyl group; R' represents an oxygen atom or a sulfur atom; R' represents an alkyl group having from 2 to 4 carbon atoms, a 1-methyl-2-propionyl group or an allyl group; n represents from 2 to S 5; and m represents 0 or 1, is applied to the medium to be inoculated with VA mycorrhizae. In accordance with the method of the present invention, propagation of mycelium of VA mycorrhizae is rapid and formation of spores of them is promoted. In addition, due to application of a particular compound of formula I to plants being cultivated, contamination of the plants with microbes may be S prevented. Therefore, a VA mycorrhizal preparation having a high VA mycorrhizal ts spore density and an excellent VA mycorrhizal activity can be prepared at low cos cost.