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 yeast of the x 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 yeast of the Cryptococcus genus 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 1 which is a method of combatting pathogenic nematodes.

8. A combination of a myrosinase-secreting Cryptococcus yeast or a source thereof, and a glucosinolate-secreting plant or a source thereof for treating soil.

9-12. (canceled)

13. A seed of a glucosinolate-secreting plant comprising a coating, wherein the coating comprises a myrosinase-secreting Cryptococcus yeast or a source thereof.

14. The method of claim 1, wherein the myrosinase-secreting yeast is selected from Cryptococcus macerans and Cryptococcus oierensis.

15. The method of claim 1, wherein the myrosinase-secreting yeast has the SEQUENCE ID 1 or the SEQUENCE ID 2.

Description

EXAMPLE 1

[0054] The effectiveness of various microorganisms at degrading sinigrin was tested. The tested organisms included different types of bacteria and fungi, including yeasts. Sinigrin is a glucosinolate expressed by some plants of the Brassicaceace family.

[0055] The tests were carried out using sinigrin-barium agar plates.

[0056] Microorganisms were tested in 5 mM sinigrin media, both with and without glucose. This was to investigate whether myrosinase secretion would be maintained in the presence of another carbon source.

[0057] The sinigrin concentration was measured after 1 day using a spectrophotometer @ 227.5 nm.

[0058] The most effective organisms were identified by DNA extraction and next generation sequencing using known techniques.

[0059] The two most promising species were found to be yeasts and their genomes are provided in the attached sequence listings. These are believed to be novel species and have been deposited with the National Collection of Yeast Cultures.

[0060] SEQUENCE ID 1 is believed to be a yeast of the Cryptococcus macerans species.

[0061] SEQUENCE ID 2 is believed to be a yeast of the Cryptococcus oierenis species.

EXAMPLE 2

[0062] The effectiveness at degrading sinigrin of the yeasts of Sequence ID 1 and Sequence ID 2 is shown in table 1, compared with the effectiveness of some bacteria and fungi.

TABLE-US-00001 TABLE 1 Microorganism Temperature Glucose Pseudomonas Paecilomyces SEQ ID 1 SEQ ID 2 ( C.) pH present? mM singrin degraded per day 4 7 Y 0.002119 0.0059233 0.068774 0.050274 10 7 Y 0.03737 0.0428455 0.069025 10 7 N 0.0366505 0.0782975 0.072742 20 7 Y 0.020295 0.021389 0.130728 0.309697 20 7 N 0.026905 0.041608 0.195898 0.141093 4 5.8 Y 0.026156 0.011059 0.075355 0.105971 10 5.8 Y 0.0383695 0.1166665 0.116347 10 5.8 N 0.013629 0.1508395 0.078537 20 5.8 Y 0.01432 0.0269786 0.21814 0.140887 20 5.8 N 0.036319 0.070273 0.253426 0.199889 4 8 Y 0.01219 0.1306555 0.0796165 10 8 Y 0.005955 0.1333335 0.126299 20 8 Y 0.02494 0.3205435 0.123701 20 8 N 0.056155 0.276512 0.155096

EXAMPLE 3

[0063] Tests were carried out to check that seeds coated with the Cryptococcus yeasts of the invention were able to germinate.

[0064] Seeds were sprayed twice with an inoculm comprising a yeast of SEQUENCE ID 1 or SEQUENCE ID 2.

[0065] Once dried seeds were sown in 50 ml falcon tubes containing 35 ml of John Innes No. 2 compost and stored at 20 C.

[0066] The percentage of seeds that had germinated was counted after 10 days. The results are shown in FIG. 1.

EXAMPLE 4

[0067] Molecular analysis experiments were carried out to determine the presence of yeasts of SEQ ID1 and SEQ ID2.

[0068] Roots from 4 seed treated Brassicas were extracted from soil and stored in ethanol.

[0069] The presence of the yeasts of SEQ ID1 and SEQ ID2 was confirmed using Real-Time PCR protocols.

EXAMPLE 5

[0070] Root-Microbe interactions were studied by the following method.

[0071] Roots of plants grown from seeds treated with yeasts of SEQ ID 1 and SEQ ID 2 were checked for presence of these yeasts after 10 days of growth.

[0072] The roots were plated onto agar plates to examine growth of microbes.

[0073] The presence or absence of microbes was determined and the microbes identified using colony morophologies and real time PCR for SEQ ID1 and SEQ ID2.

[0074] FIG. 2 shows the percentage of the roots of different types of Brassica seedlings which had the yeasts present.

[0075] Biological Deposits

[0076] The application refers to the following indications of deposited biological material: [0077] Name: National Collection of Yeast Cultures [0078] Address: National Collection of Yeast Cultures, [0079] Institute of Food Research, [0080] Norwich Research Park, [0081] Norwich, [0082] Norfolk, [0083] NR4 7UA, [0084] United Kingdom [0085] Date: 10 Aug. 2016 [0086] Accession Number: NCYC 4134, NCYC 4135 [0087] Depositor: Arcis Biotechnology