CROP TREATMENT

Abstract

The presence of microorganisms in or on food can lead to, for example, disease, damage (loss of food), a shortening of shelf life, premature ripening, reduced yield, spoilage and/or rotting. Microbial contamination of food also represents a potential health risk, increasing the likelihood of food poisoning and the like. This disclosure provides methods, uses and compositions which may be exploited as a means to reduce or eliminate the presence of microorganisms on or in food and in turn, the various problems associated therewith. Provided is a method of decontaminating food or a food growing medium, substrate or compost of one or more microorganisms, said method comprising contacting the food and/or the food growing medium, substrate or compost with one or more phage.

Claims

1. A method of decontaminating food or a food growing medium, substrate or compost of one or more microorganisms, said method comprising contacting the food and/or the food growing medium, substrate or compost with one or more phages.

2-3. (canceled)

4. The method of claim 1, wherein the food is selected from the group consisting of: (i) any food, food stuff and/or food product for human or animal consumption; (ii) manufactured, harvested, farmed or processed food, food stuff, produce or product; (iii) crops; (iv) cereals; (v) dairy produce; (vi) meat; (vii) vegetables and root vegetables; (viii) edible leaves, herbs or salad; (ix) fruit; (x) seeds; (xi) tubers; (xii) fungi and/or mushrooms; (xiii) bagged salad; and (ix) potatoes.

5. The method of claim 1, wherein the food is mushrooms.

6. The method of claim 1, wherein the food growing medium, substrate or compost is a medium, substrate or compost for the growing or cultivation of mushrooms.

7. The method of claim 1, wherein the one or more microorganisms are part of the microbial flora of the food and/or food growing medium, substrate or compost.

8. The method of claim 1, wherein the one or more microorganisms are a microbial contaminant or pathogen of the food and/or food growing medium, substrate or compost.

9. The method of claim 1, wherein the one or more microorganisms are associated with spoilage, premature ripening and/or rotting of the food.

10. (canceled)

11. The method of claim 1, wherein the method comprises the phage deposited as NCIMB 42864.

12. The method of claim 1, wherein the one or more phages are selected from the group consisting of: (i) Phage deposited as NCIMB 42864; (ii) Phage deposited as NCIMB 42866; (iii) Phage deposited as NCIMB 42868; (iv) Phage deposited as NCIMB 43014; (v) Phage deposited as NCIMB 43016; (vi) Phage deposited as NCIMB 43018; (vii) Phage deposited as NCIMB 43467; (viii) Phage deposited as NCIMB 43469; (ix) Phage deposited as NCIMB 43471; (x) Phage deposited as NCIMB 43473; and (xi) Phage deposited as NCIMB 43475.

13. The method of claim 1, wherein the one or more phages comprise, consist essentially of or consist of: (i) Phage deposited as NCIMB 42864; (ii) Phage deposited as NCIMB 42866; (iii) Phage deposited as NCIMB 42868; (iv) Phage deposited as NCIMB 43014; (v) Phage deposited as NCIMB 43018; (vii) Phage deposited as NCIMB 43467; (viii) Phage deposited as NCIMB 43469; (ix) Phage deposited as NCIMB 43471; (x) Phage deposited as NCIMB 43473; and (xi) Phage deposited as NCIMB 43475.

14. Food and/or stored or packed food treated with the method of claim 1.

15.-17. (canceled)

18. The composition of claim 33, wherein the excipient, the diluent and/or the buffer comprises tris, NaCl and MgSO4.

19. The composition of claim 33, wherein the excipient, the diluent and/or the buffer comprises: (i) 50 mM Tris (pH 7.5); (ii) 100 mM NaCl; and (iii) 8 mM MgSo4.

20-21. (canceled)

22. A method of treating or preventing mushroom blotch, said method comprising contacting mushrooms at risk of mushroom blotch and/or a medium or substrate for growing or cultivating mushroom with one or more phages selected from the group consisting of: (i) Phage deposited as NCIMB 42864; (ii) Phage deposited as NCIMB 42866; (iii) Phage deposited as NCIMB 42868; (iv) Phage deposited as NCIMB 43014; (v) Phage deposited as NCIMB 43016; (vi) Phage deposited as NCIMB 43018; (vii) Phage deposited as NCIMB 43467; (viii) Phage deposited as NCIMB 43469; (ix) Phage deposited as NCIMB 43471; (x) Phage deposited as NCIMB 43473; and (xi) Phage deposited as NCIMB 43475.

23. A composition for use in treating or preventing mushroom blotch, said composition comprising one or more phages selected from the group consisting of: (i) Phage deposited as NCIMB 42864; (ii) Phage deposited as NCIMB 42866; (iii) Phage deposited as NCIMB 42868; (iv) Phage deposited as NCIMB 43014; (v) Phage deposited as NCIMB 43016; (vi) Phage deposited as NCIMB 43018; (vii) Phage deposited as NCIMB 43467; (viii) Phage deposited as NCIMB 43469; (ix) Phage deposited as NCIMB 43471; (x) Phage deposited as NCIMB 43473; and (xi) Phage deposited as NCIMB 43475.

24. The method of claim 1, wherein the one ore more microorganisms cause or contribute to mushroom blotch and/or Pseudomonas tolaasii.

25. A composition for the decontamination of food and/or a food growing medium, substrate or compost of one or more microorganisms, said composition comprising one or more phages.

26. The composition of claim 25, wherein the food is mushrooms.

27. The composition of claims 25, wherein the food growing medium, substrate or compost is a medium, substrate or compost for the growing or cultivation of mushrooms.

28. The composition of claim 25, wherein the one or more microorganisms are part of or a pathogen/contaminant of the microbial flora of the food and/or food growing medium, substrate or compost.

29. The composition of claim 25, wherein the one or more microorganisms cause or contribute to mushroom blotch and/or Pseudomonas tolaasii.

30. The composition of claim 25, wherein the composition comprises the phage deposited as NCIMB 42864.

31. The composition of claim 25, wherein the one or more phages are selected from the group consisting of: (i) Phage deposited as NCIMB 42864; (ii) Phage deposited as NCIMB 42866; (iii) Phage deposited as NCIMB 42868; (iv) Phage deposited as NCIMB 43014; (v) Phage deposited as NCIMB 43016; (vi) Phage deposited as NCIMB 43018; (vii) Phage deposited as NCIMB 43467; (viii) Phage deposited as NCIMB 43469; (ix) Phage deposited as NCIMB 43471; (x) Phage deposited as NCIMB 43473; and (xi) Phage deposited as NCIMB 43475.

32. The composition of claim 25, wherein the one or more phages comprise, consist essentially of or consist of: (i) Phage deposited as NCIMB 42864; (ii) Phage deposited as NCIMB 42866; (iii) Phage deposited as NCIMB 42868; (iv) Phage deposited as NCIMB 43014; (v) Phage deposited as NCIMB 43018; (vii) Phage deposited as NCIMB 43467; (viii) Phage deposited as NCIMB 43469; (ix) Phage deposited as NCIMB 43471; (x) Phage deposited as NCIMB 43473; and (xi) Phage deposited as NCIMB 43475.

33. The composition of claim 25, wherein the composition comprises an excipient, diluent and/or buffer.

Description

DETAILED DESCRIPTION

[0608] The present invention will now be described in detail with reference to the following Figures which show:

[0609] FIG. 1A, B and C: bacteriophage (1A: Podoviridae; 1B Myoviridae and 1C Podoviridae) analysis by transmission electron microscopy.

[0610] FIG. 2: Genome detail of P1

[0611] FIG. 3 Genome detail of P51

[0612] FIG. 4: Genome detail of P2

[0613] FIG. 5: Individual Phage: % P. tolaasii isolates lysed (n=484)

[0614] FIG. 6: Combined Phage: % P. tolaasii isolates lysed (n=484)

[0615] FIG. 7: Comparison between P1 and P51 genome sequences

EXAMPLE 1

[0616] Genome and morphology analysis of mushroom phage P1, P2 and P51

[0617] The suspensions of each bacteriophage were analysed by transmission electron microscopy (TEM): P1 and P51 were classified as Podoviridae due to the presence of a very short tail and P2 was identified as a member of the Myoviridae due to the presence of a contractile tail and a clearly visible neck.

[0618] The capsid sizes of all phages and the tail length of P2 were measured from the TEM images (FIGS. 1A, B and C).

TABLE-US-00002 TABLE 2 Capsid sizes and tail length of bacteriophage Mean head Mean head Mean tail diameter volume length Bacteriophage Tail Classification (nm) (nm.sup.3) (nm) P1 Absent Podoviridae 43 2.25 × 10.sup.4 — P2 Contractile Myoviridae 70 1.09 × 10.sup.5 119 P51 Absent Podoviridae 48 3.51 × 10.sup.4 —

[0619] Genome Sequence Analysis

[0620] The phage genomic DNA was purified from pure phage suspension by phenol-chloroform method and submitted to BaseClear BV, The Netherlands, for full genome sequencing, assembly and annotation.

[0621] P1 and P51 (see FIG. 7)

[0622] The complete genome sizes of both phage are about 40.4 kB in size. The phage share overall 84.8% sequence identity. The average sequence identity of the identified ORFs is above 92%, with the notable exception of 17CDS in each of the genomes annotated as Tail Fibre Protein, which share 23% DNA sequence identity and 38.4% amino acid sequence identity and 50.9% sequence similarity. This sequence is responsible for a big mismatch of the aligned sequences and suggest significant differences in the affinity of P1 and P51 to cell surface receptors.

[0623] The genome of P1 (top) shows rearrangement of the end block of the sequence (highlighted in green) in comparison to the genome sequence of P51 (bottom). The colour boxes highlight the blocks of each sequence sharing homology. The height of the peaks within each box represent sequence identity within the blocks. The area with the lowest sequence identity corresponds to the coding sequence of the tail fibre protein 17CDS.

[0624] Analysis of the genome sequences was performed by BaseClear using Prokka v1.12. Overall there were 51 ORFs and 50 ORFs, respectively recognised in P1 and P51, out of which products of 24 ORFs in each genome had a function predictable based on SwissProt analysis:

TABLE-US-00003 TABLE 3 ORFs with predicted function in the genome of P1 ORF name annotation ID Predicted function 1.3 CDS Prokka_00004 DNA ligase 1 CDS Prokka_00009 DNA-directed RNA polymerase 19 CDS Prokka_00014 Terminase, large subunit gp19 18.5 CDS Prokka_00015 Spanin, inner membrane subunit 18 CDS Prokka_00016 DNA maturase A 17.5 CDS Prokka_00017 Holin 17 CDS Prokka_00018 Tail fiber protein 16 CDS Prokka_00019 Peptidoglycan hydrolase gp16 15 CDS Prokka_00020 Internal virion protein gp15 14 CDS Prokka_00021 Internal virion protein gp14 12 CDS Prokka_00023 Tail tubular protein gp12 11 CDS Prokka_00024 Tail tubular protein gp11 Ig-like virion Prokka_00025 Ig-like virion protein protein CDS 10 CDS Prokka_00026 Major capsid protein 10A 9 CDS Prokka_00027 Capsid assembly scaffolding protein 8 CDS Prokka_00028 Portal protein 6.5 CDS Prokka_00031 putative gene 6.5 protein 6 CDS Prokka_00032 Exonuclease 5.7 CDS Prokka_00033 Gene 5.7 protein 5 CDS Prokka_00035 DNA-directed DNA polymerase 4 CDS Prokka_00037 DNA primase/helicase 3.5 CDS Prokka_00041 Endolysin 3 CDS Prokka_00042 Endonuclease I 2.5 CDS Prokka_00043 Single-stranded DNA-binding protein gp2.5

TABLE-US-00004 TABLE 4 ORFs with predicted function in the genome of P51 ORF name annotation ID Predicted function 1.3 CDS Prokka_00004 DNA ligase 1 CDS Prokka_00009 DNA-directed RNA polymerase 19 CDS Prokka_00014 Terminase, large subunit gp19 18.5 CDS Prokka_00015 Spanin, inner membrane subunit 18 CDS Prokka_00016 DNA maturase A 17.5 CDS Prokka_00017 Holin 17 CDS Prokka_00018 Tail fiber protein 16 CDS Prokka_00019 Peptidoglycan hydrolase gp16 15 CDS Prokka_00020 Internal virion protein gp15 14 CDS Prokka_00021 Internal virion protein gp14 12 CDS Prokka_00023 Tail tubular protein gp12 11 CDS Prokka_00024 Tail tubular protein gp11 Ig-like virion Prokka_00025 Ig-like virion protein protein CDS 10 CDS Prokka_00026 Major capsid protein 10A 9 CDS Prokka_00027 Capsid assembly scaffolding protein 8 CDS Prokka_00028 Portal protein 6.5 CDS Prokka_00031 putative gene 6.5 protein 6 CDS Prokka_00032 Exonuclease 5.7 CDS Prokka_00033 Gene 5.7 protein 5 CDS Prokka_00035 DNA-directed DNA polymerase 4 CDS Prokka_00037 DNA primase/helicase 3.5 CDS Prokka_00041 Endolysin 3 CDS Prokka_00042 Endonuclease I 2.5 CDS Prokka_00043 Single-stranded DNA-binding protein gp2.5

[0625] P2

[0626] The complete genome sequence of P2 is 94.9 kB and does not align with the other two sequences, which is to be expected from an unrelated bacteriophage. A total of 177 ORF was recognised by Prokka out of which 35 had function predictable by SwissProt.

TABLE-US-00005 TABLE 5 ORFs with predicted function in the genome of P2 ORF name annotation ID Predicted function prs CDS Prokka_00001 Ribose-phosphate pyrophosphokinase 66 CDS Prokka_00012 Gene 66 protein hegA CDS Prokka_00013 H-N-H endonuclease F-TfIIV LIG CDS Prokka_00017 DNA ligase fbh1 CDS Prokka_00019 F-box DNA helicase protein 1 comEB CDS Prokka_00022 ComE operon protein 2 cIpP CDS Prokka_00025 3.4.21.92 cIpP ATP-dependent Clp protease proteolytic subunit tRNA-GIn Prokka_00030 tRNA-Gln tRNA-Trp Prokka_00032 tRNA-Trp tRNA-Arg Prokka_00033 tRNA-Arg tRNA-Asp Prokka_00036 tRNA-Asp tRNA-Thr Prokka_00037 tRNA-Thr tRNA-Ile Prokka_00038 tRNA-Ile tRNA-Asn Prokka_00040 tRNA-Asn tRNA-Pro Prokka_00043 tRNA-Pro tRNA-Ala Prokka_00044 tRNA-Ala tRNA-His Prokka_00045 tRNA-His tRNA-Leu Prokka_00046 tRNA-Leu tRNA-Gly Prokka_00047 tRNA-Gly xkdF CDS Prokka_00051 Phage-like element PBSX protein XkdF ORF4 CDS Prokka_00054 Major capsid protein Structural Prokka_00061 Structural protein ORF10 protein ORF10 CDS Structural Prokka_00062 Structural protein ORF10 protein ORF10 CDS Structural Prokka_00063 Structural protein ORF10 protein ORF10 CDS segD CDS Prokka_00064 Putative endonuclease segD putative tape Prokka_00066 putative tape measure protein measure protein CDS 4 CDS Prokka_00093 DNA primase/helicase 59 1 CDS Prokka_00095 Gene 59 protein 59 2 CDS Prokka_00097 Gene 59 protein 59 3 CDS Prokka_00111 Gene 59 protein thyX CDS Prokka_00112 Flavin-dependent thymidylate synthase FV3-038R CDS Prokka_00114 Putative ribonucleoside-diphosphate reductase large subunit FV3-067L CDS Prokka_00116 Putative ribonucleoside-diphosphate reductase small subunit 067L nhaP CDS Prokka_00171 Na(+)/H(+) antiporter NhaP y06D CDS Prokka_00172 putative 17.5 kDa protein in tk-vs intergenic region

[0627] 12 of the recognised ORFs were identified as tRNA loci.

TABLE-US-00006 TABLE 6 Percentage of P. tolaasii isolates lysed with individual phage compositions 10{circumflex over ( )}7 10{circumflex over ( )}6 10{circumflex over ( )}5 pfu/ml pfu/ml pfu/ml MW141215_1.02 14%  14%  14%  P1 MW310715_1.08 7% 7% 7% P2 AS150616_1.06 10%  8% 6% P51 TW021116_1.20 13%  12%  11%  P3 MW141215 1_15 3% 3% 2% P3 MF171017_4.5 7% 7% 6% E1P1 MF171017_4.5 C 6% 6% 6%

TABLE-US-00007 TABLE 7 Percentage of P. tolaasii isolates lysed with combined phage compositions 10{circumflex over ( )}7 10{circumflex over ( )}6 10{circumflex over ( )}5 pfu/ml pfu/ml pfu/ml P1 + P2 + P51 26% 25% 25% P1 + P2 + P51 + 1.20 P3 36% 34% 32% P1 + P2 + P51 + 1_15 P3 28% 27% 27% P1 + P2 + P51 + E1P1 33% 32% 31% P1 + P2 + P51 + 4.5 C 32% 31% 31% P1 + P2 + P51 + 1.20 43% 41% 38% P3 + E1P1

EXAMPLE 2

[0628]

TABLE-US-00008 TABLE 8 Percentage of P. tolaasii isolates lysed with individual phage compositions 10{circumflex over ( )}7 10{circumflex over ( )}6 10{circumflex over ( )}5 pfu/ml pfu/ml pfu/ml MP1 12%  11%  11%  MP2 9 8 8 MP3 9% 7% 6% MP4 11%  9% 8% MP5 4% 4% 3% MP6 8% 8% 7% MP7 9% 8% 8% MP8 7% 6% 6% MP9 22%  22%  21%  MP10 11%  10%  10%  MP11 6% 5% 4%

TABLE-US-00009 TABLE 9 Percentage of P. tolaasii isolates lysed with combined phage compositions 10{circumflex over ( )}7 10{circumflex over ( )}6 10{circumflex over ( )}5 pfu/ml pfu/ml pfu/ml MP9 22% 20% 19% MP9 + MP1 34% 32% 31% MP9 + MP2 27% 25% 24% MP9 + MP3 30% 27% 25% MP9 + MP4 33% 30% 28% MP9 + MP7 30% 27% 26% MP9 + MP8 28% 26% 25% MP9 + MP10 30% 28% 27% MP9 + MP11 27% 25% 24% MP9 + MP1 + MP2 39% 37% 36% MP9 + MP1 + MP4 42% 39% 37% MP9 + MP1 + MP10 42% 40% 39% MP9 + MP1 + MP11 40% 36% 35% MP9 + MP1 + MP2 + MP4 47% 44% 42% MP9 + MP1 + MP2 + MP4 + MP10 56% 52% 50% MP9 + MP1 + MP2 + MP4 + 60% 57% 54% MP10 + MP11