GB1573850A

GB1573850A - Mycofungicidal products

Info

Publication number: GB1573850A
Application number: GB37967/77A
Authority: GB
Original assignee: Individual
Current assignee: Individual
Priority date: 1976-09-13
Filing date: 1977-09-12
Publication date: 1980-08-28
Also published as: ATA650577A; ES462550A1; NL7710042A; AU2869777A; CA1106201A; DE2740052A1; TR19524A; AT360274B; NZ185151A; GR63078B; JPS5341424A; AU514726B2; SE7709935L

Description

(54) IMPROVEMENTS IN OR RELATING TO MYCOFUNGICIDAL PRODUCTS (71) I, JACQUES RICARD, a citizen of the United States of America of 26 Herrhagsvagen, S 190 30 Sigtuna, Sweden, do hereby declare the invention, for which I pray that a patent may be granted to me, and the method by which it is to be performed, to be particularly described in and by the following statement:- The present invention relates to a product having mycofungicidal activity containing propagules or spores of immunizing commensals and cereal flour.

The product according to the invention is characterized in that it contains propagules or spores of Scvtalidium lignicola ATCC 16,675, of Trichoderma polysporum Rifai ATCC 20,475, of Trichoderma viride sensu Bisby ATCC 20,476 or of Trichoderma viride sensu Bisby strain CG/BINAB-INRA I 030 (Institut Pasteur, Paris-France).

According to one embodiment of the invention, the product is preferably in the form of a powder concentrate containing from 20x 10' to 30x 109 spores.

According to another embodiment of the invention, said concentrate is conveniently prepared in the form of a wettable powder, granules or pellets, the form being adapted to the contemplated use.

The size of the particles may vary for example from 3 to 300 microns according to the distribution method considered e.g. atomizer or ordinary sprayer.

The invention is concerned also with a manufacturing process of the mycofungicidal product above containing spores or propagules of the immunizing commensals mentioned earlier, which comprises inoculating cereal kernels, such as those of barley or other plants, which have first been ground, moistened, acidified and then sterilized, with the commensals defined above after which the inoculated kernels are incubated, dried and powdered.

This process may be easily applied industrially as illustrated, by way of example, as follows: A plastic bag of 0.5 to 50 kg or more in capacity is filled with grain. Water acidified with lactic, acetic, sulfuric, etc. acid to a pH below 4.5 and a small amount of copper sulfate is added to the bag contents. The bag is heat sealed under vacuum and placed in an autoclave.

After autoclaving, the bags are placed horizontally on a moving belt for cooling by a water shower or by ventilation, they are then inoculated with a needle puncturing the bags as they go by. The puncture is preceded by pressing an alcohol soaked plug onto the bag and followed by sealing with plastic tape. The injection of an inoculating propagule suspension is followed by an air discharge into the bag to lightly blow up the bag. The bags are then placed on the shelves of trucks for temporary storage in an illuminated incubation room. As soon as spores appear, the bags are placed again on a moving belt for the following operations: 1) gas removal by aspiring needle; 2) light compression under a roll intended to break the blocks of kernels interwoven with mycelium in chunks having a volume of about 3 to 5 cm3; 3) blowing the bags again with fresh air.

These operations are repeated twice at 2 to 3 days intervals during incubation according to the saturation of the chunks with spores. The second time the roll is lowered so as to break the chunks in pieces of about 1 to 2 cm3 volume. The third roll is intended to break up the pieces completely back to the original bag contents of separated kernels. However, at the end of the process the kernels are green, black or yellow depending on the immunizing commensal whose spores are saturating them. The bag contents is then emptied over a belt having about a 1 mm2 mesh for warm air draft or vacuum drying. When dried to less than 10% moisture, the propagule saturated kernels are ground to a particle size of 3 to 300 microns and packaged.

If kernels, saturated with immunizing commensal, especially Trichoderma, are dried and ground, as indicated above, the resulting flour usually contains up to sox 109 spores/g. Although such a concentration is sufficient for many uses, it has been observed that, in some cases, and in particular for the treatment of grapevines against Botrytis, it is preferable to use concentrates with 15x 109 spores/g or more.

Besides, when the mycofungicide is to be used for atomizing, the maximum particle size must be about 10 microns. Trichoderma spores usually have average diameters around 3 microns, but in the flour mentioned above particles exceeding 30 microns may be found.

It has been discovered, according to another embodiment of the invention that it is possible to obtain mycofungicidal products suitable for the uses described above by extraction of the spores on the kernels to eliminate most of the supporting material and obtain a powder much more concentrated in spores, as well as a residue of amino-acid enriched kernels.

As indicated above, the extraction may conveniently be achieved by two different methods, the first preferred method consists in the dry extraction of the spores coating the kernels after incubation by shaking or rubbing, followed by sieving, the concentrated spore powder passing through the sieve and the kernels staying on top of it.

Another method (wet extraction) consists in placing the incubated kernels in water acidified (for example with lactic, acetic, sulfuric, etc. acid) to a pH below 4.5 to avoid any significant bacterial contamination. After agitation in the acidified water for some time, the mixture is strained resulting in a fine suspension of spores and kernels permeated with mycelium. For actual use, the spore suspension is centrifuged, dried and ground to obtain the desired particle size.

The dry extraction method is preferred as it avoids practically all bacterial contamination while that risk exists in the wet extraction method.

By the extraction method mentioned above, it is possible to obtain a powder generally containing from lox109 to 30x109 spores/g and usually between 25x109 and -30x109 spores.

The mycofungicidal products of the invention may be provided in the following forms:- A/ Concentrates for spraying fruit trees, the casing soil of cultivated mushrooms, and grapevines; or for the dipping of flower, vine or potato cuttings; B/ Pellets for flowers, greenhouse plants, nursery stock, vegetable crops, grape vine pots, mature trees, and wooden telephone or electric poles; C/ Granules for composts, soils treated with steam or fumigants and various seed beds; D/ Powders for seed coating.

The various mycofungicidal products of the invention can be used especially for: a) the dipping of carnation cuttings against Fusarium: b) the treatment of cucumbers against Phomopsis sclerotioides; c) the treatmentnf Norway spruce and Scots pine seedlings against Armillaria mellea. Fomes annosus and damping off; d) the treatment of grapevines and fruit trees against Armillaria: for the treatment of grapevines seedlings, 100 g of Trichoderma pellets are mixed in the potting substrate used to hold the seedlings. These potted seedlings may then be placed in production vineyards; e) the treatment of elms against Graphium ulmi; for this treatment it is convenient to insert 5 to 30 pellets containing the mycofungicidal product in each tree, each pellet containing at least 50x 108 spores/g; f) the treatment of pine poles against Lentinus lepideus; g) the treatment of onion seedlings against smut; h) the coating of monogerm sugar beet seeds for protection against Phoma betae and Pvthium ultimum (damping off); i) the treatment of tomatoes in greenhouses; j) the treatment of casing soil for cultivated mushrooms, for this treatment, a suspension containing 5 gIl of a concentrate mentioned above (20x 108 to 20x 109 spores/g) at the rate of about 1 liter per m2 of soil is conveniently used.

Thus, in another aspect the invention provides a process for the treatment of plant materials against fungal diseases wherein an effective quantity of mycofungicidal product according to the invention is applied in or around said plant material.

The following non-limiting examples are given as illustrations of the invention.

EXAMPLE 1 About 250 g cracked barley and 250 g rice, 14 ml of 0.4 CuSO4. 5H2O solution, 400 ml of 0.5M CH3COOH and 0.5M sodium acetate solution are introduced in a thermostable plastic bag. The bag is then closed with a cotton plug and autoclaved for 2 hours at 1210C. After cooling, the bag is inoculated with T.

viride ATCC 20,476. It is then incubated at 20--220C for 10 days under ordinary light. The contents are kneaded gently, as needed, to obtain a uniform sporulation (as indicated by the occurrence of green conidia) throughout the substrate.

The contents of the bag can then be treated by either one of two distinctly different methods: A-Prepartion of Trichoderma powder.

The contents of the bag are dried at 450C in a warm air draft. When the moisture content is below 20% by weight, the product is ground in a hammer mill equipped with a 0.6 mm mesh-sieve. The powder obtained can contain up to 10x 109 spores/g and more particularly from 50x 106 to 10x 109 spores/g. This powder can be mixed with surfactants and used to spray fruit trees and cultivated mushrooms, against fungal diseases, as well as in other spraying uses. The spores are counted under the microscope according to techniques described by Gindrat & Ricard ("Counting techniques for Trichoderma viride conidia dispersed in barley flour based inoculants", Plant Disease Reporter 1976--60 p. 321-325). For a second type of use, the powder can be pelleted, without additives, for the inoculation of trees or poles susceptible to infection by Stereum, Ceratocystis, Lentinus or Poria spp.

For a third type of use, the separated Trichoderma saturated kernels can be mixed with fresh or composted bark, with or without peat, and used as a substrate for growth of plants seeded in greenhouses, in nurseries, or potted plants sensitive to infection by plant pathogens such as Phomopsis, Pythium, Verticillium, Fusarium spp.

For a fourth type of use, the powder is added to standard ingredients for seed coatings to protect seeds, for example, against damping-off of monogerm seeds of sugar beets (Phoma betae).

B-Preparation of "pure" Trichoderma spores and an animal feed To each bag, 1.5 liter of water acidified with lactic, acetic, sulfuric acid, etc.

and buffered at about pH 4.0 but definitely below 4.5, in order to avoid significant bacterial contamination, particularly from Enterobacteriaceae, is added. Then the bag is shaken mechanically for about 30 minutes. The contents are then sieved yielding a dark green water suspension of spores, and kernels permeated with mycelium.

(a) the kernels are dried, yielding an animal feed enriched in amino acids as a result of the fungal growth converting low cost nitrogen, ammonium salts for example, into hyphal cellular components, including amino acids.

(b) the spore suspension is centrifuged, dried and ground, yielding a spore powder containing more than lox 108 spores and up to 30x109 spores, often between 25 and 30x109 spores/g. These spores exhibit the usual Van der Waals forces of microparticles and form an excellent mycofungicidal product for spraying without any adjuvant. When special circumstances make it necessary, the preparations can be sterilized with gas or by heating, because killed spores, in sufficient concentrations, can still exert a mycofungicidal action probably because of the enzymes immobilized on their surface.

EXAMPLE 2 One hundred and forty six elms attacked by the Dutch elm disease were selected. These trees had a diameter ranging from less than 20 cm to 80 cm. The extent of the disease was measured by the proportion of damaged foliage.

Seventy eight trees inoculated in various ways were compared with 47 control trees.

The inoculations were performed with a preparation derived from Trichoderma viride ATCC 20,476 grown on cracked barley kernels. The kernels were ground into a flour containing at least 50x 108 viable spores per gram dry weight. The flour was milled into pellets with a diameter of about 3 mm and with a length of 20 mm weighing an average of 0.5 g. each. By drilling or puncturing, holes of suitable size were made to introduce 5 to 20 pellets per tree, spaced evenly in a circle or spiral pattern. The inoculations were made in March or July. The results were recorded in July the following year according to the changes shown by the leaves.

Twenty one trees were treated with bark pellets instead of Trichoderma pellets to determine the possible effects of mechanical binders.

The damaged foliage increased in 40.4% of the control trees. A proportion nearly equal was observed in the-trees inoculated in March. Among those inoculated in July, the percentage of trees showing in increase in damage was 12.520/,. Twelve of the trees "inoculated" with bark pellets showed an increase in damage, that is 57.1% of that group.

The selection of timing seems quite important to control that disease with Trichoderma propagules.

This would be expected if the Trichoderma inoculants act by release of their metabolites in the vascular system of the tree. When the tree reacts quickly to the mechanical break caused by the introduction of the Trichoderma pellets, there is only little or no distribution of metabolites coming from the introduced fungus.

Whether the beneficial effect of Trichoderma pellets is caused directly. by extraction of water soluble metabolites or indirectly by the growth of hyphae, or by translocation of conidia away from the pellets, a certain distribution of the pellet contents must take place within the tree. An immediate plugging of the vessels in places where the pellets are introduced would prevent the Trichoderma inoculant from performing as intended. It is known that such reaction rates vary at different times during the growing season.

EXAMPLE 3 Spraying Trichoderma viride sensu Bisby ATCC 20,476 on casing soil at the rate of 100x 108 spores per liter and 1 liter per m2 is effective for the control of Verticillium malthousei in cultivated mushrooms.

A tolerance to benomyl (methyl l-(butylcarbamoyl)-2-benzimidazole carbamate) by Verticillium malthousei, cause of the dry bubble disease, has been observed by commercial mushroom growers, hence there is a need for substitutes.

Commercial production of the cultivated mushroom, Agaricus bisporus, offers a system in which the effect of biological control agents is obvious and where the cycles are short, in contrast with the forest trees from which a mycoparasitic Trichoderma isolate was obtained earlier. Propagules of Trichoderma isolate were tested on the casing soil in production trays and compared with benomyl containing commercial preparations.

Propagules of Trichoderma viride sensu Bisby ATCC 20,476 cultivated on cracked barley kernels with a concentration of conidia of about 20x 108 per gram dry weight were mixed with a wetting agent and an homogenizer. The powder was suspended in water and applied in the usual manner for spraying of fungicides against the dry bubble disease of cultivated mushrooms under commercial production conditions. The effectiveness of the spraying of Trichoderma at two concentrations (2.5 gIl and 5 girl) was measured by the yield in healthy carpophores per tray and also by picking diseased caps. Similar observations were made on trays sprayed with (1) benomyl, (2) surfactants and barley flour without Trichoderma propagules, (3) surfactants only and (4) controls without any spraying.

The observations were reported for an average 2.2 m2 tray based on 4 replicates. The trays were placed under commercial conditions. The caps of the mushrooms were picked daily.

The yield of caps damaged by the disease up till 52 days after casing is shown in Fig. 1 of the accompanying drawings starting with the results from the first flush after 24 days. The trend remains constant, but with all parameters except one, there are greater differences with time. The exception is the spraying with surfactants and the barley flour but without propagules of Trichoderma. That treatment resulted in a roughly median yield after 24 days, but showed the second highest yield in diseased mushrooms at the end of the experiment, just below that of the control trays. All the other parameters gave consistent relative yields, the trays sprayed with benomyl at 2 g/m2 showing the lowest yield in diseased caps (1.878 g per tray). The highest level was recorded in the control with no spraying, that is 3.458 g per tray. The trays with a spraying with Trichoderma concentrate followed closely those receiving benomyl spray, but with a yield slightly higher (1.976 g per tray) in an application of 5 git2. The trays with a spraying of only 2.5 g/m2 of Trichoderma concentrate, or only with inert ingredients yielded all about the same quantities of caps damaged by the disease.

In Fig. 1, the various curves show: 1. Control (no spraying).

2. Spraying with 2.5 g/m2 surfactants and flour.

3. Spraying with 2.5 g/m2 surfactants.

4. Spraying with 2.5 g/m2 of preparation complete with Trichoderma.

5. Spraying with 5.0 g/m2 of preparation complete with Trichoderma.

6. Spraying with 2.0 g/m2 benomyl.

The Fig. 2 of the accompanying drawings shows the yield in healthy mushrooms: in the trays sprayed with Trichoderma at 5 g/m2 gradually takes the lead. The cumulative total is 24.37 kg per tray, while the benomyl treatment yields 21.98 kg. It is the control with no spraying that shows the lowest yield (20.43 kg/tray). The spraying with the surfactants only has given nearly the same yield with 20.49 kg/tray. When it is applied at the rate of 2.5 g/m2 the Trichoderma concentrate is more effective than benomyl.

concentrate is more effective than benomyl.

Since the Trichoderma spray concentrate is available at prices competitive with benomyl, it offers an effective and economic biological substitute for synthetic chemical fungicides. The LDso of Trichoderma flour is non-existent until 4,000 mglkg body weight. At least until that level, this flour does not cause distress symptoms in any of the animals tested.

In Fig. 2, the different curves represent: 1. Control (no spraying).

2. Spraying with 2.5 g/m2 surfactants and flour.

3. Spraying with 2.5 g/m2 surfactants.

4. Spraying with 2.5 g/m2 of preparation complete with Trichoderma.

5. Spraying with 5.0 g/m2 of preparation complete with Trichoderma.

6. Spraying with 2.0 g/m2 benomyl.

In Figs. 1 and 2, the ordinate axes indicate the yields of diseased and healthy mushrooms respectively, and the abscissa axes show the time in days.

EXAMPLE 4 The effectiveness of a mycofungicidal product derived from Trichoderma according to the invention has been tested for the treatment of grapevines against Botrytis Cinerea.

It has been established thus that spores of Trichoderma viride sensu Bisby strain CG/BINAB-INRA I 030 (Institut Pasteur, Paris) when used in a concentration of 108/ml have an effectiveness of about 70% according to the Abbot coefficient.

The results of these tests are reported in Table I below: TABLE I Effectiveness of Trichoderma viride I 030 pn the grey mold caused by Botrytis Cinerea % effectiveness of /n moldy ground up agar medium grapes in the coated with Trichoderma untreated Treatment viride spores control Natural infection 70 31 Artificial infection 71 61 Four treatments were made in each case, with the exception of the control.

Observations were recorded from berries (Desaymard Method).

The product was applied as a suspension of ground up agar medium overgrown with sporulated Trichoderma viride I 030 mycelium.

A second series of tests was made with plain ground up agar and with ground up agar plus a protecting cover.

It was found that when the concentrate of the invention containing spores of Trichoderma I 030 was sprayed under the conditions of classical application of insecticide, anti-mildew and anti-oidium agents, namely 4 times during the growing season, that is at the setting of the flower (A), at the closing of the berry cluster (B), at the turning (change in color) of the grapes (C) and 3 weeks before harvest (D), the results were amply sufficient to satisfy established effectiveness standards.

Indeed these standards state that: (a) the average decrease in rate of grey mold must be equal to at least 50 /n of that in the unsprayed control; (b) the maximum rate of grey mold allowed in the treated plots is 20%. The tests have yielded 61 of grey mold in the controls and 71% of effectiveness for the Trichoderma viride in the plots infected artificially with Botrytis. In the plots left for natural infection, the grey mold percentage was 31% in the controls with 70 X" effectiveness for Trichoderma viride.

The results obtained are shown in Table II below: TABLE II Effectiveness of Trichoderma viride I 030 on the grey mold caused by Botrvtis Cinerea at harvest % of Treatment effectiveness Ground up agar suspension without cover 54.8 Ground up agar suspension with cover 22.6 Concentrate of the invention without cover 63.5 Concentrate of the invention with cover 47.5 Control with cover (% grey mold) 16.5 Four treatments were made in each case, control excepted.

The cover consists in the simultaneous use of insecticide (methomyl), antioidium (S) and anti-mildew (Cu) fungicides and of Trichoderma.

EXAMPLE 5 The mycofungicidal product mentioned in Example 4 was tested also for the treatment of grape vines against the "excoriose" caused by Phomopsis viticola. It has been found that the product of the invention had the same effectiveness as sodium arsenite when the control showed 50 /" excoriose at the first internodal space.

The results obtained are shown in Table III below.

TABLE III Effectiveness of Trichoderma viride I 030 on "excoriose" caused by Phomopsis viticola Rating of damaged surfaces Treatment (Desaymard method) Ground up agar 82% effectiveness Culture filtrate 69% effectiveness Sodium arsenite l250g pflhl 91 /" effectiveness Untreated control 57% damaged surfaces WHAT I CLAIM IS: 1. A mycofungicidal product containing propagules or spores of immunizing commensals and cereal flour, characterized in that the immunizing commensal is Scvtalidium lignicola ATCC 16,675, Trichoderma polysporum Rifai ATCC 20,475, Trichoderma viride sensu Bisby ATCC 20,476 or Trichoderma viride sensu Bisby strain CG BINAB. INRA I 030 (Pasteur Institute, Paris).

2. A mycofungicidal product according to claim 1 in the form of a powder concentrate containing from 20x 108 to 30x 109 spores of microorganisms per gram.

3. A mycofungicidal product according to claim 2, containing more than 1x109 spores of microorganism per gram.

4. A mycofungicidal product according to claim 3 containing 25 to 30x109 spores of microorganisms per gram.

5. A mycofungicidal product according to claim 1 in the form of a concentrate according to any one of claims 2 to 4 in combination with a surfactant for spraying or atomizing.

6. A process for producing a mycofungicidal product according to claim 1 which comprises inoculating cereal kernels which have first been ground, moistened, acidified and then sterilized, with a commensal defined in claim 1, after which the inoculated kernels are then incubated, dried and powdered.

7. A process according to claim 6 wherein the incubated and dried kernels are subject to dry or wet extraction in order to obtain concentrated spores of the

**WARNING** end of DESC field may overlap start of CLMS **.

Claims

**WARNING** start of CLMS field may overlap end of DESC **. the results were amply sufficient to satisfy established effectiveness standards. Indeed these standards state that: (a) the average decrease in rate of grey mold must be equal to at least 50 /n of that in the unsprayed control; (b) the maximum rate of grey mold allowed in the treated plots is 20%. The tests have yielded 61 of grey mold in the controls and 71% of effectiveness for the Trichoderma viride in the plots infected artificially with Botrytis. In the plots left for natural infection, the grey mold percentage was 31% in the controls with 70 X" effectiveness for Trichoderma viride. The results obtained are shown in Table II below: TABLE II Effectiveness of Trichoderma viride I 030 on the grey mold caused by Botrvtis Cinerea at harvest % of Treatment effectiveness Ground up agar suspension without cover 54.8 Ground up agar suspension with cover 22.6 Concentrate of the invention without cover 63.5 Concentrate of the invention with cover 47.5 Control with cover (% grey mold) 16.5 Four treatments were made in each case, control excepted. The cover consists in the simultaneous use of insecticide (methomyl), antioidium (S) and anti-mildew (Cu) fungicides and of Trichoderma. EXAMPLE 5 The mycofungicidal product mentioned in Example 4 was tested also for the treatment of grape vines against the "excoriose" caused by Phomopsis viticola. It has been found that the product of the invention had the same effectiveness as sodium arsenite when the control showed 50 /" excoriose at the first internodal space. The results obtained are shown in Table III below. TABLE III Effectiveness of Trichoderma viride I 030 on "excoriose" caused by Phomopsis viticola Rating of damaged surfaces Treatment (Desaymard method) Ground up agar 82% effectiveness Culture filtrate 69% effectiveness Sodium arsenite l250g pflhl 91 /" effectiveness Untreated control 57% damaged surfaces WHAT I CLAIM IS:

1. A mycofungicidal product containing propagules or spores of immunizing commensals and cereal flour, characterized in that the immunizing commensal is Scvtalidium lignicola ATCC 16,675, Trichoderma polysporum Rifai ATCC 20,475, Trichoderma viride sensu Bisby ATCC 20,476 or Trichoderma viride sensu Bisby strain CG BINAB. INRA I 030 (Pasteur Institute, Paris).

microorganisms involved together with a residue of kernels enriched in aminoacids.

8. A process according to claim 7 wherein the dry extraction is performed by shaking or rubbing followed by sieving to separate the incubated kernels from the mycofungicidal powder.

9. A process according to claim 7 wherein wet extraction is performed using water acidified to a pH lower than 4.5 followed by screening to separate out the kernels and centrifuging the spore suspension so obtained, the spores and kernels being then dried separately.

10. A process for the treatment of plant materials against fungal diseases wherein an effective quantity of mycofungicidal product according to any one of claims 1 to 4 is applied in or around said plant material.

Il. A process according to claim 10 wherein said mycofungicidal product is used in the treatment of cultivated mushrooms.

12. A process according to claim 10 wherein said mycofungicidal product is used in the treatment of vineyards.

13. A process according to claim 10 wherein said mycofungicidal product is used in the treatment of elms.

14. A process according to claim 10 wherein said mycofungicidal product is used for the treatment of greenhouse crops.

15. A process according to claim 10 wherein said mycofungicidal product is used for the treatment of seeds.

16. A process according to claim 10 wherein said mycofungicidal product is used in the treatment of wooden structures.

17. A mycofungicidal product as claimed in claim 1 substantially as herein described.

18. A process as claimed in claim 6 substantially as herein described in the examples.

19. A process as claimed in claim 10 substantially as herein described in the Examples.

20. A mycofungicidal product whenever produced by a process claimed in any of claims 6 to 9.