Isolation and characterization of eight bacteriophages infecting Erwinia amylovora and their potential as biological control agents in British Columbia, Canada

Figures & data

Table 1.  Origin and sources of Erwinia amylovora (Ea), Pantoea agglomerans^a (Eh, Pa) and Pseudomonas fluorescens (Pf) isolates used in this study

Isolate no.^b	Host Plant	Location
Ea1032	Apple ‘Gala’	Winfield, BC
Ea1033	Apple ‘Gala’	Winfield, BC
Ea1034	Apple ‘Gala’	Winfield, BC
Ea1280	Apple	Summerland, BC
Ea1337	Apple ‘Gala’	Kelowna, BC
Ea1583	Apple ‘Gala’	Summerland, BC
Ea1584	Apple	Oliver, BC
Ea1585	Apple	Oliver, BC
Ea1586	Apple	Winfield, BC
Ea1588	Apple ‘Gala’	Winfield, BC
Ea1593	Crab apple	Grand Forks, BC
Ea1594	unknown	Grand Forks, BC
Ea1595	Apple	Midway, BC
Ea1598	Pear	Summerland, BC
Ea1602	unknown	Creston, BC
Ea1611	Pear	Kelowna, BC
Ea1617	Apple ‘Gala’	Oyama, BC
Ea1618	Apple ‘Gala’	Oyama, BC
Ea1619	Apple ‘McIntosh’	Cawston, BC
Ea2345	Apple	Summerland, BC
Ea110^c	Apple	East Lansing, MI
Eh21-5^d	Pear	Vineland, ON
Pa2092	Grape ‘Pinot Noir’	Oliver, BC
Pf1100-6	M9 rootstock	Summerland, BC
Notes: ^a Pantoea agglomerans formerly Erwinia herbicola.
^b All E. amylovora were isolated from vegetative shoot except Ea2345 from a fruit.
^c Ea110 isolated by A.L. Jones, Department of Botany and Plant Pathology, Michigan State University, East Lansing, MI obtained by A. M. Svircev, Agriculture and Agri-Food Canada, Southern Crop Protection and Food Research Centre, Vineland Station, ON.
^d Pantoea agglomerans Eh21-5 isolated by A. M. Svircev, AAFC, Vineland, ON.

Table 2.  RFLP grouping, origin, morphotype with dimensions, PCR characterization and estimated genome size of the eight BC bacteriophages and phage ΦEa21-4 from Vineland, ON

						PCR ^e
RFLP Group ^a	Isolate no. ^b	Location of origin	Morphotype ^c	Head width ^d (nm)	Tail length (nm)	PEa1	Pun45	PCR ^e Genome size^f (kbp)
1	ΦEa2345-6	Agassiz, BC	A1	54	77	–	+	74
1	ΦEa2345-19	Kelowna, BC	A1	65	113	–	+	75
1	ΦEa21-4 ^g	Vineland, ON	A1	50	74	–	+	74
5	ΦEa1598-6	Agassiz, BC	A1	80	136	–	+	89
7	ΦEa1594-24	Oliver, BC	A1	48	74	+	+	n/a
7	ΦEa1594-26	Summerland, BC	A1	72	97	(+)	+	n/a
8	ΦEa1337-26	Summerland, BC	C1	53	14	+	+	36
8	ΦEa1598-19	Kelowna, BC	C1	60	20	–	+	41
-	ΦEa1615-26	Summerland, BC	A1	62	105	–	+	n/a
Notes: ^a RFLP Group designation based on restriction patterns created by four restriction enzymes EcoRI, BglII, BamHI and MvnI: RFLP group 1: phage DNA produced unique patterns when digested by EcoRI and MvnI; group 5: by EcoRI, BglII and MvnI; group 7: by MvnI only; and group 8: by EcoRI, BglII, BamHI and MvnI. None of the four enzymes digested ΦEa1615-26 consistently, therefore it remained ungrouped.
^b Designation of BC bacteriophages is ‘ΦEa’ followed by the four digit E. amylovora bacterial host number and the location number. Bacteriophages were isolated from soil beneath symptomatic apple trees cv. Gala, except for ΦEa2345-6 and ΦEa1598-6 isolated from soil beneath symptomatic apple seedlings, and for ΦEa21-4 (bacterial host Ea6-4) isolated from soil beneath a symptomatic pear tree.
^c Morphotype: tailed phages of the order Caudovirales, belonging to the families Myoviridae with contractile tails consisting of a sheath and a central tube designated A1, and Podoviridae with short tails designated C1.
^d Measurements of the icosahedral head width and uncontracted tail length (nm) are averages obtained by measuring an average of 20 phages per isolate from digitized negatives.
^e PCR amplification of a 304-bp fragment with PEa1 primers or a 140-bp fragment with Pun45 primers; positive amplification: +, weak positive: (+) or no amplification: -.
^f Average size (kbp) of each phage genome was estimated by adding DNA fragments generated by EcoRI; n/a: no estimation available.
^g Description of ΦEa21-4 in this study compares with its description by Lehman et al. (2009): Myoviridae with an icosahedral head of 60 nm across and an uncontracted tail measuring 90 nm. DNA sequencing results for ΦEa21-4 identified the genome size as 84 576 bp.

Table 3.  Host range of eight BC bacteriophages and ΦEa21-4 from Vineland, ON on 20 BC E. amylovora isolates, one E. amylovora from Michigan Ea110, Pantoea agglomerans ^a Eh21-5 and Pa2092 and Pseudomonas fluorescens Pf1100-6 by using the soft agar overlay method

	RFLP Group 1			Group 5	Group 7		Group 8		Ungrouped
	ΦEa2345	ΦEa2345	ΦEa21	ΦEa1598	ΦEa1594	ΦEa1594	ΦEa1598	ΦEa1337	ΦEa1615
	−6	−19	−4	−6	−24	−26	−19	−26	−26
Ea1032	+ ^b	+	+	+	−^c	–	+	+	+
Ea1033	+	+	+	+	–	–	+	+	–
Ea1034	+	+	+	+	–	(+)^d	+	–	(+)
Ea1280	–	(+)	+	+	–	–	–	+	+
Ea1337	+	–	–	+	–	–	+	+	+
Ea1583	+	+	+	+	–	–	–	–	+
Ea1584	+	+	(+)	–	(+)	+	+	–	+
Ea1585	+	+	+	+	+	–	+	–	+
Ea1586	+	+	+	+	–	–	–	–	+
Ea1588	+	(+)	+	+	–	–	+	–	(+)
Ea1593	+	+	+	+	–	–	+	+	+
Ea1594	+	+	–	–	+	+	–	–	+
Ea1595	+	+	+	–	+	–	–	+	+
Ea1598	+	+	+	+	–	–	(+)	–	+
Ea1602	+	+	–	–	–	–	–	–	–
Ea1611	+	–	+	+	+	–	+	+	+
Ea1617	+	+	(+)	+	–	(+)	–	–	–
Ea1618	+	+	+	+	–	–	–	–	+
Ea1619	+	+	+	+	(+)	–	(+)	(+)	+
Ea2345	+	+	+	+	+	+	+	–	(+)
Ea110	+	+	+	–	–	+	–	+	+
Eh21-5	+	+	+	+	+	+	+	+	(+)
Pa2092	–	+	nt ^e	–	nt	–	–	–	–
Pf1100-6	–	–	–	–	–	–	–	–	–
Notes: ^a Pantoea agglomerans formerly Erwinia herbicola.
^b ‘+’ indicates that a bacteriophage effectively infected the host by producing clearly visible plaques (host sensitive).
^c ‘–’ no infection (host not sensitive).
^d ‘(+)’ indicates pale plaques (host slightly sensitive).
^e ‘nt’ not tested.

Fig. 1. Electron micrographs of representative phages belonging to a, Myoviridae in a contracted and uncontracted state (Φ1598-6); and b, Podoviridae (Φ1337-26).

Fig. 2. Agarose gel electrophoresis of restriction patterns representative of those exhibited by four phage groups: RFLP groups 1, 5, 7 and 8: a, digestion with EcoRI; b, digestion with BglII; c, digestion with BamHI; and d, digestion with MvnI. Individual lanes are labelled as follows: M: 1 kb Plus DNA ladder (Qiagen), labels 1, 5, 7 and 8 indicating the phage group the pattern represents.

Fig. 3. Evaluation of phage control of fire blight infection on detached pear blossoms. The rating scale of 0 to 5 was used where 0: no symptoms and 5: infection spread on entire flower surface. In experiment 1, phage ΦEa1337-26 was tested against Erwinia amylovora Ea1337; and in experiment 2, ΦEa2345-6, ΦEa2345-19 and ΦEa21-4 were tested against E. amylovora Ea2345. In both experiments, phages were applied with the carrier Pantoea agglomerans Eh21-5. Each phage carrier combination was compared with streptomycin, Eh21-5 in phosphate buffer (PB), and PB alone (negative control). The severity index of three replications of five detached flowers was averaged and the data from three trials (experiment 1) and two trials (experiment 2) were pooled. Bars of each experiment with the same letters are not significantly different (P ≤ 0.05) according to Waller–Duncan k-ratio (k = 100) t test.

Fig. 4. Per cent incidence of infected flower clusters and vegetative shoots on potted apple trees in a screenhouse treated with ΦEa2345-6 and ΦEa2345-19 and Pantoea agglomerans Eh21-5. The trees were inoculated with a mixture of E. amylovora Ea1337 and Ea2345. Clusters displaying symptoms of fire blight indicated by blackening and wilting were recorded 8 d (flowers) and 20 d (shoots) following inoculation. The data were arcsine transformed for analysis. Bars of the same data set with the same letters are not significantly different (P ≤ 0.05) according to Waller–Duncan k-ratio (k = 100) t test.

Lehman , S.M. , Kropinski , A.M , Castle , A.J. and Svircev , A.M. 2009 . Complete genome of the broad-host-range Erwinia amylovora phage ΦEa21-4 and its relationship to Salmonella phage Felix O1 . Appl. Environ. Microbiol. , 75 : 2139 – 2147 .

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