BIOFUMIGANT

Abstract

A method of treating soil, the method comprising providing the soil with a glucosinolate-secreting plant or a source thereof and contacting said soil with a myrosinase-secreting microorganism or a source thereof.

Claims

1. A method of treating soil, the method comprising providing the soil with a glucosinolate-secreting plant or a source thereof and contacting said soil with a myrosinase-secreting microorganism or a source thereof.

2. A method according to claim 1 which is a biofumigation method.

3. A method according to claim 1 wherein the glucosinolate and myrosinase interact to form an isothiocyanate.

4. A method according to claim 3 wherein the isothiocyanate is selected from a group consisting of: benzyl isothiocyanate, isopropyl isothiocyanate, methyl isothiocyanate, 1-napthyl isothiocyanate, 2-phenylethyl isothiocyanate, phenyl isothiocyanate, and propyl isothiocyanate.

5. A method according to claim 1 wherein the glucosinolate-secreting plant is of the order Brassicales.

6. A method according to claim 1 which is a method of combating a pathogenic infection in the soil.

7. A method according to claim 6 which is a method of combatting pathogenic nematodes.

8. (canceled)

9. A kit for treating soil, the kit comprising a glucosinolate-secreting plant or a source thereof, and a myrosinase-secreting microorganism or a source thereof.

10. (canceled)

11. (canceled)

12. A seed of a glucosinolate-secreting plant comprising a coating, wherein the coating comprises a myrosinase-secreting microorganism or a source thereof.

13. A method of claim 1, wherein the myrosinase-secreting microorganism is a myrosinase-secreting bacterium.

14. A method of claim 1, wherein the myrosinase-secreting microorganism is a myrosinase-secreting fungus.

15. A method of claim 13, wherein the myrosinase-secreting bacterium is Bacillus sphaericus.

16. The kit of claim 9, wherein the myrosinase-secreting microorganism is a myrosinase-secreting bacterium.

17. The kit of claim 16, wherein the myrosinase-secreting bacterium is Bacillus sphaericus.

18. The kit of claim 9, wherein the myrosinase-secreting microorganism is a myrosinase-secreting fungus.

19. The seed of claim 12, wherein the myrosinase-secreting microorganism is a myrosinase-secreting bacterium.

20. The seed of claim 19, wherein the myrosinase-secreting bacterium is Bacillus sphaericus.

21. The seed of claim 12, wherein the myrosinase-secreting microorganism is a myrosinase-secreting fungus.

Description

[0059] Any of the aspects contained herein may be combined with any of the preferred embodiments contained herein in any workable combination.

[0060] The invention will now be further explained by way of example only with reference to the accompanying figures in which:

[0061] FIG. 1 is a graph of a replicated field trial of the level of potato cyst nematode eggs in plots of seven different biofumigant crops;

[0062] FIG. 2a is a representation of the level of potato cyst nematode eggs in a field plot before the growth of biofumigant crops;

[0063] FIG. 2b is a representation of the level of potato cyst nematode eggs in a field plot after the growth of biofumigant crops; and

[0064] FIG. 3 shows the number of potato cyst nematodes (PCN) hatched over a nine week period under various conditions.

EXAMPLE 1

[0065] A field trial was constructed to test the levels of pathogens in soil when traditional biofumigant crops are typically grown, especially the level of the pathogen PCN or ‘potato cyst nematodes’. A field was separated into blocks and sampled for nematode eggs using DGPS to map the field in 1/4 hectare blocks. Each block was then sampled by taking 40 soil cores at predetermined spacings using DGPS. The cores were then amalgamated into a single soil sample.

[0066] Samples were taken in each block as described in January 2011, before the growth of biofumigant crops (FIG. 2a), and again in December 2012 after the growth of biofumigant crops but before maceration of the crops (FIG. 2b).

[0067] The samples were processed and analysed using a standard method as described in ‘MAFF Reference book 402. Laboratory Methods for Work with Plant and Soil Nematodes’ Ed. J F Southey. Sixth edition 1986. The results given in eggs/g of soil.

[0068] The difference in the pathogen level in the soil before the biofumigant crops are grown and after the biofumigant crops are grown can be seen by comparing FIGS. 2a and FIG. 2b. Surprisingly, a reduction in pathogen level, indicated by a reduction in the level of potato cyst nematode eggs, is demonstrated in all blocks of the field except two. Some blocks showed an overall reduction of over 80% of eggs/g of soil and a reduction in the level of potato cyst nematodes to below 30 eggs/g of soil, and in some cases below 20 eggs/g of soil.

[0069] It is clear that the level of potato cyst nematode eggs is considerably reduced with the growth of some of the species of biofumigant plants in the field. However, surprisingly, this reduction in pathogens in the soil was achieved before the typical maceration of the biofumigant crops used in biofumigation processes.

[0070] Accordingly, the inventors surprisingly discovered that these plants secrete glucosinolates into the soil which are converted to isothiocyanates by bacteria present in the soil, found by the inventors to be myrosinase-secreting bacteria. On the basis of this data, the inventors proposed the method according to the invention in which glucosinolate secreting plants and myrosinase secreting bacteria can be used to generate the biofumigant isothiocyanates in the soil without the need to macerate or destroy the biofumigant crop. This method is tested in example 2.

EXAMPLE 2

[0071] A replicated field trial was constructed to test various typical biofumigant crops with myrosinase-secreting bacteria in accordance with the method of the invention.

[0072] Plots of seven biofumigant plant varieties plus a fallow (empty) plot as a control were laid out. Some of the plant varieties include commercial biofumigant crops such as SpudGuard and variety 199. Each ‘treatment’ consisted of 5 plots/replicate. Each plot measured 10 m×4 m. The trial was laid out in a randomised block design. Soil samples from each plot were taken at drilling of the seeds of each crop, and again just before the crop was destroyed. Each soil sample consisted of 20 soil cores to a depth of 25 cm taken in a grid pattern across each plot. The duration between drilling the crop and the second sampling was 56 days.

[0073] The soil samples were processed and analysed using standard methods as described in ‘MAFF Reference book 402. Laboratory Methods for Work with Plant and Soil Nematodes’ Ed. J F Southey. Sixth edition 1986. The results are given in potato cyst nematode eggs/g of soil.

[0074] Data analysis was undertaken using ARMS. (Bartletts followed by ANOVA).

[0075] Referring to FIG. 1, for each plant, the left hand blue column shows the level of potato cyst nematode eggs per gram of soil at drilling of the seeds of the biofumigant crops, the right hand red column shows the level of potato cyst nematodes after the crops have been grown for 56 days. The soil the plants were grown in contained myrosinase-secreting bacteria of Bacillus sphaericus.

[0076] From FIG. 1, it is clear that the level of potato cyst nematode eggs is considerably reduced with the growth of some of the species of biofumigant plants, in particular, Keva and Loti, both species of Brassica in soil containing myrosinase-secreting bacteria. Again, this reduction in pathogens in the soil was achieved before the typical maceration of the biofumigant crops used in traditional biofumigation processes.

[0077] Accordingly, example 2 demonstrates that certain plants of the Brassica genus secrete glucosinolates into the soil which are converted to isothiocyanates by bacteria present in the soil, found by the inventors to be myrosinase-secreting bacteria, in particular bacteria of the Bacillus genus. Example 2 furthermore replicates the situation shown in the field in example 1 under a controlled environment.

[0078] Both examples show that the methods of the invention give a significant reduction in pathogen load within the soil of fields without the need to destroy the biofumigant crop, and with a sustained release of isothiocyanate biofumigant into the soil starting from only 56 days after planting and up to at least 1 year.

EXAMPLE 3

[0079] Globodera paffida cysts which had been stored at 4° C. for 4 weeks were sealed in silk teabags and placed into soil. Keva seedlings were placed into 1 L pots containing a 50:50 mix of loam and sand. Cereal was planted into the control pots, with all others growing mustard. The test pots either contained the mustard only, mustard and myrosinase-secreting bacteria (B. sphaericus and Lactobaccilus), or mustard and a mixture of fungal isolates of Fusarium.

[0080] Fungi 1A, 4A, 4D were grown on PDA plates. Six confluent 90 mm petri dishes of each (freshly cultured) fungi were harvested using a pipette tip, added to 40 ml SDW, mixed, resulting in 45 ml of a fungal mix. 7 ml of mix was delivered to the root of the Keva plants using a syringe.

[0081] Bacteria E7 and E10 were grown in separate 50 ml falcon tubes containing 35 ml MRS Broth, shaken 48 hours at RT. OD600 of 1.000 and 0.400 were obtained respectively, mixed, then 5 ml added per pot.

[0082] After the plants were grown for 10 weeks and mustards were flowered 50%, the cysts were removed for further processing. Cysts were rehydrated in water for 7 days at 15° C. and transferred to potato root diffusate (PRD) for incubation at 15° C. PRD was obtained from Estima plants 4 weeks post emergence. The cysts were removed to fresh PRD weekly for 9 weeks and the number of hatched juveniles was counted. At the end of the hatching experiment, the remaining eggs were counted. The percentage of hatched juveniles was calculated using the total count.

[0083] Table 1 and FIG. 3 show the mean cumulative hatching of PCN juveniles from treated pots over 9 weeks.

TABLE-US-00001 TABLE 1 Treatment 1 2 3 4 5 6 7 8 9 Cereal 1967 3511 3682.5 3789.5 3828.5 3882 3893.5 3925 3926 Mustard 1729 3132 3366 3489.5 3524 3589 3601.5 3616 3620 Mustard + Bacteria 1718 2986 3138.5 3250.5 3275.5 3308.5 3316 3328 3330.5 Mustard + Fungi 1385 2346 2552.5 2671.5 2717 2759 2769.5 2800 2807

[0084] The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

[0085] All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.

[0086] Each feature disclosed in this specification (including any accompanying claims, abstract and drawings) may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features. The invention is not restricted to the details of the foregoing embodiment(s). The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.