BACTERIOPHAGES FOR FOOD DECONTAMINATION

Abstract

The disclosure provides phage-based methods, compositions and uses which may be applied to the prevention, management and/or control of microorganisms that cause damage, disease, spoilage and/or or loss of food, including vegetable, fruit and other fresh produce. The disclosure exploits bacteriophage (or phage) as a means to inactivate, kill or destroy microorganisms present on food and which are associated with, for example, disease, damage and spoilage (rotting).

Claims

1. A method of decontaminating food of one or more microorganisms, said method comprising contacting the food with one or more phage.

2. (canceled)

3. A composition for the decontamination of food of one or more microorganisms, said composition comprising one or more phage.

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) bagged salad; and (xiii) potatoes.

5. The method of claim 1, wherein the microorganism(s) is/are part of the microbial flora of the food.

6. The method claim 1, wherein the microorganism(s) is/are a microbial contaminant or pathogens of the food.

7. The method of claim 1, wherein the microorganism(s) is/are associated with spoilage, premature ripening and/or rotting of the food.

8. The method of claim 1, wherein the method comprises the use of one or more phage types specific for one or more microorganisms.

9. The method of claim 1, wherein the phage are selected from the group consisting of: (i) Phage deposited as NCIMB 42290; (ii) Phage deposited as NCIMB 42292; (iii) Phage deposited as NCIMB 42294; (iv) Phage deposited as NCIMB 42296; (v) Phage deposited as NCIMB 42298; (vi) Phage deposited as NCIMB 42300; (vii) Phage deposited as NCIMB 42302; (viii) Phage deposited as NCIMB 42304; (ix) Phage deposited as NCIMB 42520; (x) Phage deposited as NCIMB 42522; (xi) Phage deposited as NCIMB 42524; (xii) Phage deposited as NCIMB 42526; and (xiii) Phage deposited as NCIMB 42528;

10. The method of claim 1, wherein the phage comprise: (i) Phage deposited as NCIMB 42290; (ii) Phage deposited as NCIMB 42292; (iii) Phage deposited as NCIMB 42294; (iv) Phage deposited as NCIMB 42296; (v) Phage deposited as NCIMB 42298; (vi) Phage deposited as NCIMB 42300; (vii) Phage deposited as NCIMB 42302; and (viii) Phage deposited as NCIMB 42304.

11. The method of claim 1, wherein the phage are selected from the group consisting of: (i) Phage deposited as NCIMB 43321; (ii) Phage deposited as NCIMB 43323; (iii) Phage deposited as NCIMB 43325; and (iv) Phage deposited as NCIMB 43327.

12. The method of claim 11, wherein the phage comprise: (i) Phage deposited as NCIMB 43321; (ii) Phage deposited as NCIMB 43323; (iii) Phage deposited as NCIMB 43325; and (iv) Phage deposited as NCIMB 43327.

13. The method of claim 11, wherein the phage further comprises the phage deposited as NCIMB 42294 and/or the phage deposited as Accession No: NCIMB 42302.

14. Food and/or stored or packed food treated with a method of decontaminating food of one or more microorganisms, said method comprising contacting the food with one or more phage.

15. A food and/or stored or packed food of claim 14, wherein the one or more phage are selected from the group consisting of: (i) Phage deposited as NCIMB 42290; (ii) Phage deposited as NCIMB 42292; (iii) Phage deposited as NCIMB 42294; (iv) Phage deposited as NCIMB 42296; (v) Phage deposited as NCIMB 42298; (vi) Phage deposited as NCIMB 42300; (vii) Phage deposited as NCIMB 42302; (viii) Phage deposited as NCIMB 42304; (ix) Phage deposited as NCIMB 42520; (x) Phage deposited as NCIMB 42522; (xi) Phage deposited as NCIMB 42524; (xii) Phage deposited as NCIMB 42526; and (xiii) Phage deposited as NCIMB 42528.

16. A food and/or stored or packed food of claim 15, wherein the phage comprise: (i) Phage deposited as NCIMB 43321; (ii) Phage deposited as NCIMB 43323; (iii) Phage deposited as NCIMB 43325; and (iv) Phage deposited as NCIMB 43327.

17. A composition according to claim 3, wherein the one or more phage are selected from the group consisting of: (i) Phage deposited as NCIMB 42290; (ii) Phage deposited as NCIMB 42292; (iii) Phage deposited as NCIMB 42294; (iv) Phage deposited as NCIMB 42296; (v) Phage deposited as NCIMB 42298; (vi) Phage deposited as NCIMB 42300; (vii) Phage deposited as NCIMB 42302; (viii) Phage deposited as NCIMB 42304; (ix) Phage deposited as NCIMB 42520; (x) Phage deposited as NCIMB 42522; (xi) Phage deposited as NCIMB 42524; (xii) Phage deposited as NCIMB 42526; and (xiii) Phage deposited as NCIMB 42528.

18. A composition according to claim 3, wherein the phage comprise: (i) Phage deposited as NCIMB 43321; (ii) Phage deposited as NCIMB 43323; (iii) Phage deposited as NCIMB 43325; and (iv) Phage deposited as NCIMB 43327.

Description

DETAILED DESCRIPTION

[0103] The present invention will now be described in detail with reference to the following figures which show:

[0104] FIG. 1: Factory success data: Pectobacterium spp. contamination of tubers is reduced following bacteriophage treatment.

[0105] FIG. 2: Application of a bacteriophage mix to tubers prior to planting prevents blackleg formation during growth.

[0106] FIG. 3: reducing seed-derived blackleg with bacteriophage. *: significant reduction compared with positive control (F.sub.4,25=39.12 P<0.001,n=6).

[0107] FIG. 4: Pectorbacterium loading in stolon end tissue of individual and combined FG.sub.2 Rudolph samples by Q-PCR (copy number/g). Rudolf PB2 seed treated with the eight phage mix of this disclosure at planting. Bacteriophage applied with a hand mister & allowed to dry before planting. Randomised block- 4 blocks, 1 plot of each treatment per block (98 tubers/plot). 3 trials completed. Seed contamination levels measured pre-planting

[0108] FIG. 5: % Blackleg plants/plot (n=4). Bacteriophage treatment cannot make completely reverse the effects of contaminated seed but can reduce the expression of disease if environmental conditions are suitable. *: significant reduction compared with untreated control (F.sub.3,12=4,68 P=0.02,n=4)

[0109] FIG. 6: PB2 seed (Rudolf) (0% Pba). 4 bacteriophage treatments (control, in furrow (3 L product/ha, in 80 L, Foliar (3 L product/ha, in 200 L, applied for 9 weeks from rosette stage) and in furrow & foliar). Randomised block- 4 blocks, 1 plot of each treatment per block with 98 tubers per plot. Infector plants to increase the likelihood of disease symptoms. qPCR analysis on 3 tubers/each of 10 plants/plot. *: significant reduction compared with untreated control (F.sub.2,42=6.35, P<0.001,n=40).

[0110] FIG. 7: Replanted material from 2017 trial. Data represents observations onlyno treatments. 50 tubers were planted per treatment. qPCR analysis on 20 groups of 15 tubers per treatment. *: significant reduction compared with untreated control (Mann Whitney tests, P<0.05).

[0111] FIG. 8: Species distribution of the 248 Pectobacterium species isolated from potato material/potato wash water from The Netherlands and assessed for phage susceptibility in FIG. 9: Pcc: Pectobacterium carotovorum subsp. carotovorum, Pcbr: Pectobacterium carotovorum subsp. brasiliense and Ppar. Pectobacterium parmentieri. Lytic activity of the eight phage mix and a new 6-phage mix against the isolated Dutch Pectobacterium spp. FIG. 10: current UK Bacteriophage mix (the 8 phage mix) vs new European mix: % coverage of Pectobacterium spp.

[0112] Selective Bacteriophage Combinations for managing Spoilage Microorganisms of Fresh Produce

[0113] Bacteriophage compositions may contain the core bacteriophage composition APSPb1, APSPb2, APSDs1, APSPs1. Some bacteriophage compositions can contain additional bacteriophages to increase the efficacy of the core bacteriophage composition in certain samples, for example in samples from a range of geographic areas, farms and processing factories as well as samples of different types and/or varieties of food.

[0114] Phage (for example bacteriophage) mixtures for use in this invention are selected by their ability to lyse (kill) bacteria isolated from samples (soil, plants, tubers, water) which exhibit rot symptoms.

[0115] Bacteriophage compositions may be used to target potato soft rots caused by Pectobacterium & Dickeya spp., and/or in potato processing factories (post-harvest). The bacteriophage compositions could also be used in the field or in potato stores to prevent blackleg/bacterial rots.

[0116] Bacteriophage compositions may be used to decontaminate salad produce (including washed and bagged salad leaves) of microorganisms which induce rot, including, for example, rots (such as blackleg) caused by Pseudomonas spp. and Pectobacterium spp.

[0117] Indeed, the various phage compositions described herein may be used to decontaminate a wide range of fresh produce of microorganisms which are associated with rot and spoilage. The compositions of this invention which comprise bacteriophage mixtures may have increased activity, when compared to individual bacteriophage. Some bacteriophage may act synergistically.

[0118] The compositions of this invention may be provided as liquid formulations comprising buffer(s) and/or salts. The compositions may be diluted for use.

TABLE-US-00002 TABLE 2 Composition of exemplar bacteriophage mixtures Target bacterial Core Supplementary Supplementary Supplementary Supplementary Supplementary genus bacteriophage bacteriophage 1 bacteriophage 2 bacteriophage 3 bacteriophage 4 bacteriophage 5 Pectobacterium APSPb1 APSPb3 APSPb4 APSPb5 APSPb6 APSPb7 Pectobacterium APSPb2 Dickeya APSDs1 APSDs2 Pseudomonas APSPs1 APSPs2 APSPs3 APSPs4 APSPs5

TABLE-US-00003 TABLE 3a Species targeted by each bacteriophage and microbiological deposit information for bacteriophage (and their hosts) employed in the examples and in compositions of this invention. Bacterial Isolate Details Species Accession Numbers Sample Targeted Phage code Phage Host Location type Pectobacterium APSPb1 NCIMB NCIMB Scotland Potato atrosepticum (APS Ph 1) 42290* 42291* stem Pectobacterium APSPb5 NCIMB NCIMB N. Ireland Soil atrosepticum (APS Ph 2) 42292* 42293* Pectobacterium APSPb2 NCIMB NCIMB Scotland water atrosepticum (APS Ph 3) 42294* 42295* Pectobacterium APSPb6 NCIMB NCIMB Scotland water atrosepticum (APS Ph 4) 42296* 42297* Pectobacterium APSPb3 NCIMB NCIMB Scotland Potato atrosepticum (APS Ph 5) 42298* 42299* Tuber Pectobacterium APSPb4 NCIMB NCIMB Scotland water atrosepticum (APS Ph 6) 42300* 42301* Pectobacterium APSPb7 NCIMB NCIMB UK water sp. (APS Ph 7) 42302* 42303* Dickeya solani APSDs1 NCIMB NCIMB Non-UK Potato (APS Ph 8) 42304* 42305* stem Pseudomonas APS Ps1 NCIMB NCIMB UK Bagged fluorescens (APS PF1) 42520.sup.y 42519.sup.y salad leaves Pseudomonas APS Ps2 NCIMB NCIMB UK Bagged fluorescens (APS PF2) 42522.sup.y 42521.sup.y salad leaves Pseudomonas APS Ps3 NCIMB NCIMB UK Bagged fluorescens (APS PF3) 42524.sup.y 42523.sup.y salad leaves Pseudomonas APS Ps4 NCIMB NCIMB UK Bagged fluorescens (APS PF4) 42526.sup.y 42525.sup.y salad leaves Pseudomonas APS Ps5 NCIMB NCIMB UK Bagged fluorescens (APS PF5) 42528.sup.y 42527.sup.y salad leaves *All deposits made with the National Collection of Industrial Food and Marine Bacteria (NCIMB) Ltd., of Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA. All deposits (NCIMB 42290, NCIMB 42291, NCIM 42292, NCIMB 42293, NCIMB 42294, NCIMB 42295, NCIMB 42296, NCIMB 42297, NCIMB 42298, NCIMB 42299, NCIMB 42300, NCIMB 42301, NCIMB 42302, NCIMB 42303, NCIMB 42304 and NCIMB 42305) made 24 Sep. 2014. .sup.yAll deposits made with the National Collection of Industrial Food and Marine Bacteria (NCIMB) Ltd., of Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA. All deposits (NCIMB 42519, NCIMB 42520, NCIMB 42521, NCIM 42522, NCIMB 42523, NCIMB 42524, NCIMB 42525, NCIMB 42526, NCIMB 42527 and NCIMB 42528) made 27 Jan. 2016.

TABLE-US-00004 TABLE 3b Bacterial Species Phage Accession numbers Targeted code Phage Host Pectobacterium sp. APS PH9 NCIMB 43321* NCIMB 43322* Pectobacterium APS PH10 NCIMB 43323* NCIMB 43324* brasiliense Pectobacterium APS PH11 NCIMB 43325* NCIMB 43326* parmentieri Pectobacterium APS PH12 NCIMB 43327* NCIMB 43328* carotovorum *All deposits made with the National Collection of Industrial Food and Marine Bacteria (NCIMB) Ltd., of Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA. All deposits (NCIMB 43321, NCIM 43323, NCIMB 43325 and NCIMB 43327) made 19 Dec. 2018.

[0119] Methods

[0120] Bacterial Isolation:

[0121] Samples (typically wash water from processing factories or rotten tubers/plant material) are processed by producing a homogenous suspension of bacteria in buffer, serially diluting and plating on selective microbiological media (crystal violet pectate medium for Pectobacterium spp. and King's B medium for Pseudomonas spp.). Liquid samples are diluted directly and solid samples are pulsified in buffer (MRD) before dilution. Bacteria are identified using standard microbiological tests (growth or appearance on selective agar, Gram staining etc.) and confirmed by PCR. Selected bacterial colonies are purified and used for subsequent bacteriophage screening.

[0122] Bacteriophaqe Isolation:

[0123] A range of environmental samples (inc. soil, water, plant material, digestate from waste processing, sewage, animal stomach contents etc.) were screened for the presence of bacteriophage by enrichment of the environmental samples with the target bacteria in liquid microbiological media. Following enrichment, samples are centrifuged or filtered to remove solid components and bacteria before being spotted against the target bacterial strains. A classic over-lay technique, where the target bacteria are added to a layer of top agar overlaying an agar plate, provides a lawn on which bacteriophage enrichment mixes are spotted and instances of lysis (clearing of the bacteria) can be observed following overnight incubation at room temperature. Enrichment mixes which lyse the target bacteria are selected and individual bacteriophage can be isolated, picking individual plaques in top agar.

EXAMPLES

[0124] Example of Tubers of a Single Variety, from Multiple Growers

TABLE-US-00005 TABLE 4 Testing of Bacteriophage mixes against Pectobacterium spp. isolated from tubers supplied from multiple growers % bacteria % bacteria % bacteria % bacteria % bacteria % bacteria lysed by lysed by lysed by lysed by lysed by Location lysed by core phage + core phage + core phage + core phage + core phage + of growers Variety core phage APSPb3 APSPb4 APSPb5 APSPb6 APSPb7 Scotland Purple 85% 85% 100% 85% 85% 85% majesty Scotland Desiree 77% 77% nd 77% 85% 85% E. Maris 60% 60% nd 60% 90% 80% England Piper Scotland King 0% 29% nd 0% 43% 100% and SW. Edward England

[0125] Example of Different Varieties in the Same Factory

TABLE-US-00006 TABLE 5 Testing of Bacteriophage mixes against Pectobacterium spp. isolated from tubers supplied from a single processing factory and tested against the core or supplemented core bacteriophage mixes % bacteria % bacteria % bacteria % bacteria % bacteria lysed by core lysed by core lysed by core lysed by core lysed by core % bacteria bacteriophage bacteriophage bacteriophage bacteriophage bacteriophage lysed by core composition + composition + composition + composition + composition + Location Maris bacteriophage supplementary supplementary supplementary supplementary supplementary of growers Piper composition APSPb3 APSPb4 APSPb5 APSPb6 APSPb7 Scotland Purple 60% 60% nd 60% 60% 60% majesty Scotland Desiree 77% 77% nd 77% 85% 85%

[0126] Example of Same Variety in Different Locations

TABLE-US-00007 TABLE 6 Testing of Bacteriophage mixes against Pectobacterium spp. isolated from a single variety of tubers supplied from two potato processing factories % bacteria % bacteria % bacteria % bacteria % bacteria lysed by core lysed by core lysed by core lysed by core lysed by core % bacteria bacteriophage bacteriophage bacteriophage bacteriophage bacteriophage lysed by core composition + composition + composition + composition + composition + Location bacteriophage supplementary supplementary supplementary supplementary supplementary of growers Variety composition APSPb3 APSPb4 APSPb5 APSPb6 APSPb7 Scotland Maris 70% 70% nd 70% 70% 70% Piper E. Maris 60% 60% nd 60% 90% 80% England Piper

[0127] Factory Success DataFIG. 1

[0128] Tubers were washed in water and either left with no treatment (wash barrel) or sprayed with a bacteriophage composition APS90 containing core bacteriophage+APSPb3 & APSPb5 at 10.sup.6 pfu/ml. Numbers of Pectobacterium spp. were enumerated from tubers after 5 days storage at room temperature.

[0129] Data presented in FIG. 1 shows a 654-fold reduction in bacterial numbers in tubers treated with the bacteriophage composition APS90.

[0130] Storage Trial Data

[0131] (a) Prevention of Rots During Storage

[0132] Maris Piper tubers were treated with either no treatment, a water or bacteriophage spray (core+APSPb5 at 10.sup.6pfu/m1). The tubers were assessed for rot development after 1, 3 and 6 months cold storage at 2.8 C.

[0133] At each assessment tubers were visually inspected for the presence of rots and when found, rotten tubers were removed and planted in glasshouses to determine blackleg levels in daughter plants (see part b). Numbers represent cumulative number of rots identified across all assessment points.

TABLE-US-00008 TABLE 7 Prevention of rots in tubers treated with bacteriophage prior to cold storage. Month 1 Month 3 Month 6 Treatment rots rots rots 1 No Treatment 9 13 13 2 Water spray 10 11 17 3 Bacteriophage spray 9 10 11

[0134] The number of rotten tubers treated with bacteriophage spray was lower than that of tubers stored without treatment or treated with a water spray. The difference in the number of rots is greater at longer storage times. This indicates that adding bacteriophage to tubers can increase the shelf life of said tubers when they are stored over a period of time.

[0135] (b) Prevention of Blackleg Once Planted

[0136] Maris Piper tubers were treated with either no treatment, water or bacteriophage (core +APSPb5) spray at 10.sup.6 pfu/ml and stored for 1, 3 or 6 months at 2.8 C. Tubers from part a) of the assessment were planted in glasshouses to determine blackleg levels in daughter plants. No effect of the bacteriophage was observed on emergence of the plants but stem number increased with phage treatment.

TABLE-US-00009 TABLE 8 Prevention of blackleg by application of bacteriophage mixes to mother tuber prior to cold storage (1, 3 or 6 months pre-planting) Emergence Total number of stems Duration between No Water Bacteriophage No Water Bacteriophage treatment and planting treatment Spray spray treatment Spray spray After 1 month of storage 23 24 24 32 24 27 After 3 months of storage 24 24 24 55 50 65 After 6 months of storage 24 21 24 110 105 115

[0137] Treatment of Seed Immediately Before Planting

[0138] Pectobacterium spp. Example on Tubers Variety: Desiree were damaged and inoculated with water (negative control) or Pectobacterium spp. (10.sup.6cfu/ml, all other treatments) to contaminate the seed potatoes. Immediately prior to planting bacteriophage mixes (core+APSPb3, APSPb4 & APSPb5 at 10.sup.6 (Dose 1), 10.sup.7 (Dose 2) or 10.sup.8 (Dose 3) pfu/ml) were sprayed onto tubers and planted.

[0139] The number of plants which developed blackleg was assessed following emergence.

[0140] Dickeya spp. Example on Tubers

[0141] Tubers (Variety: Desiree) were inoculated with water (negative control) or Dickeya spp. (107 cfu/ml, all other treatments) to contaminate the seed potatoes. Immediately prior to planting, bacteriophage (core APSDs1 at 107 cfu/ml) was sprayed onto tubers and the treated tubers were planted. The number of plants which successfully emerged was assessed as a measure of disease prevention.

[0142] Selection of a Bacteriophage Composition Suitable for Salad Processing

[0143] Pseudomonas ExampleSalad Leaves

[0144] Pseudomonas spp. bacteria were isolated from bagged salad samples from a UK salad processor. Bacteriophage were then isolated and tested against these isolates and the proportion of lysis calculated.

TABLE-US-00010 TABLE 9 Selecting a bacteriophage mix suitable for salad processing. % bacteria % bacteria % bacteria % bacteria % bacteria lysed by core lysed by core lysed by core lysed by core lysed by core bacteriophage % bacteria bacteriophage bacteriophage bacteriophage bacteriophage composition + lysed by core composition + composition + composition + composition + supplementary bacteriophage supplementary supplementary supplementary supplementary APSPs2 and Sample composition APSPs2 APSPs3 APSPs4 APSPs5 APSPs3 Salad 30% 36% 36% 35% 33% 41% mix 1 Salad 16% 20% 18% 17% 17% 20% mix 2

[0145] A proportion of the Pseudomonas bacteria present in bagged salad leaves was lysed by the core bacteriophage APSPs1. However, adding one additional supplementary bacteriophage (APSPs2, APSPs3, APSPs4 or APSPs5) to the core bacteriophage composition leads to an increase in the efficacy of the treatment, measured in terms of increased % of lysed bacteria. Adding two supplementary bacteriophage to the core simultaneously leads to a greater increase in efficacy (at least for Salad Mix 1) compared to the treatment with core alone or core+a single supplementary phage. This indicates that there may be a synergistic effect in the efficacy of bacteriophage mixtures in lysing bacteria when multiple bacteriophage are used concurrently.

[0146] Reduction in Blackleg Symptoms

[0147] Reducing Seed-Derived Blackleg with Bacteriophage.

[0148] FIG. 3 shows Desiree seed treated damaged & inoculated with Pba (10 cfu/ml) and sprayed (until visibly damp) with bacteriophage the eight phage mix of this disclosure at final concentrations of 10.sup.6, 10.sup.7 & 10.sup.8 pfu/ml. Results show a significant reduction in seed-derived blackleg compared with positive control. Thus Blackleg may be reduced if seed is externally contaminated.

[0149] FIG. 4 shows Pectobacterium loading in stolon end tissue of individual and combined FG.sub.2 Rudolph samples by Q-PCR (copy number/g). Rudolf PB2 seed was treated with the eight phage mix at planting. Bacteriophage applied with a hand mister & allowed to dry before planting. Randomised block4 blocks, 1 plot of each treatment per block (98 tubers/plot). Seed contamination levels measured pre-planting.

[0150] FIG. 5 shows that there was a significant reduction in the % blackleg plants/plot and that while Bacteriophage treatment cannot completely reverse the effects of contaminated seed, it can reduce the expression of disease if environmental conditions are suitable.

[0151] The data presented in FIGS. 6 and 7 looks at whether disease counts accurately reflect harvest seed quality. The data shows examples of bacteriophage treatment of potato seed leading to reduced bacterial loading in the progeny tubers over 2 generations: keeping potato seed clean of Pectobacterium (blackleg-causing bacteria) is very important, since very small amounts of disease (<0.5%) means that the crop can be downgraded, which represents a financial loss to the grower. In FIG. 6, a potato crop was treated with bacteriophage either at planting (in-furrow) or with a weekly foliar spray or a combination of both treatments in 2016. qPCR on the harvested progeny tubers showed that the in-furrow & in-furrow & foliar treatment had significantly lower Pectobacterium than the control. These progeny tubers were replanted in 2017 (FIG. 7), with no further treatments and less blackleg was seen in all of the treatments using seed from the 2016 phage treatments and in addition, the progeny tubers of the 2017 trial also had lower bacterial loadingi.e. a carry-over effect from the 2016 treatments.

TABLE-US-00011 TABLE 10 Current UK (8 phage) Mix New European Mix Total 28% 40% Pectobacterium 36% 38% Pectobacterium atrosepticum 78% 78% Pectobacterium carotovorum 20% 34% Pectobacterium brasiliense 45% 65% Pectobacterium parmentieri 10% 41% Dickeya spp. 58% 0% (above): current UK bacteriophage mix vs new European mix: % coverage of Pectobacterium spp. UK mix = 8 UK phage mix (composition comprising, consisting essentially of or consisting of: APSPb1 (deposited as NCIMB 42290); APSPb5 (deposited as NCIMB 42292); APSPb2 (deposited as NCIMB 42294); APSPb6 (deposited as NCIMB 42296); APSPb3 (deposited as NCIMB 42298); APSPb4 (deposited as NCIMB 42300); APSPb7 (deposited as NCIMB 42302); and APSDs1 (deposited as NCIMB 42304)). New European mix = APS PH9 (Accession no: NCIMB 43321; APS PH10 (Accession no: NCIMB 43323; APS PH11 (Accession no: NCIMB 43325); APS PH12 (Accession no: NCIMB 43327; APS Ph3 (Accession No: NCIMB 42294); and APS Ph7 (Accession No: NCIMB 42302). Also see FIG. 10.