Shifts in Xanthomonas spp. causing bacterial spot in processing tomato in the Midwest of the United States

Figures & data

Fig. 1 Map of the counties surveyed for bacterial spot in processing tomato, 2010–2020. Northwest Ohio: Ottawa, Erie, Sandusky, Fulton, Wood, Henry, Putnam and Hancock; Southeast Michigan: Lenawee; and Central Indiana: Madison, Tipton counties.

Table 1. Xanthomonas strains isolated from processing tomatoes in the U. S. Midwest, by species and geographical location, 2010–2020. Xanthomonas spp. identification is based on the qPCR outcome.

				Xanthomonas species
Year	County	State	No. of fields	euvesicatoria	hortorum pv. gardneri	perforans
2010	Ottawa	OH	7	2	15	1
	Sandusky	OH	4	2	14	0
	Fulton	OH	2	1	6	0
	Unknown	OH	11	3	14	4
OH total (%)				8 (13)	49 (79)	5 (8)
	Lenawee	MI	6	2	13	4
Total (%)			30	10 (12)	62 (77)	9 (11)
2011	Ottawa	OH	1	0	0	1
	Sandusky	OH	1	0	0	1
	Henry	OH	2	0	4	0
	Wood	OH	2	0	5	1
	Putnam	OH	14	0	29	12
	Unknown	OH	3	0	2	1
Total (%)			23	0	40 (71)	16 (29)
2012	Ottawa	OH	1	0	1	0
	Sandusky	OH	2	0	2	1
	Wood	OH	1	0	2	1
	Putnam	OH	10	0	7	6
	Unknown	OH	2	0	2
Total (%)			16	0	14 (64)	8 (36)
2013	Erie	OH	2	0	1	1
	Sandusky	OH	3	0	2	1
	Wood	OH	4	1	3	0
Total (%)			9	1(11)	6 (67)	2 (22)
2017	Ottawa	OH		0	87	17
	Wood	OH		0	19	12
	Hancock	OH		1	12	60
	Putnam	OH		0	0	24
OH total (%)				1 (<1)	118 (51)	113 (49)
	Tipton	IN		1	1	22
Total (%)				2 (0.8)	119 (47)	135 (53)
2018	Ottawa	OH		0	16	6
	Wood	OH		0	81	0
	Hancock	OH		0	0	91
	Putnam	OH		0	0	139
OH total (%)				0 (0)	97 (29)	236 (71)
	Madison	IN		0	0	48
Total (%)				0	97 (25)	284 (75)
2019	Erie	OH		0	0	5
	Wood	OH		0	14	20
	Hancock	OH		0	11	23
	Putnam	OH		0	0	6
OH total (%)				0 (0)	25 (32)	54 (68)
	Tipton	IN		0	0	42
Total (%)				0	25 (21)	96 (79)
2020	Ottawa	OH		0	23	11
OH total (%)				0	23 (68)	11(32)
	Madison	IN		0	0	50
Total (%)				0	23 (27)	61 (73)
2010–2020 Total (%)				13 (1)	385 (38)	611 (61)

Fig. 2 BOX-PCR fingerprints of Xanthomonas spp. strains isolated from processing tomato in the U.S. Midwest. L) ladder 2 kb; 1 to 12) X. hortorum pv. gardneri; 13 and 14) putative X. perforans; 15 and 16) X. perforans; reference strains 17) X. hortorum pv. gardneri XcgA2; 18) X. euvesicatoria 110C; 19) X. perforans 1220.

Fig. 3 Xanthomonas species distribution (percent) in processing tomatoes in Ohio, Indiana and Michigan, 2010–2020. Data for years 2012 and 2013 are not reported in the figure because of their small sample size, and no sampling was done in 2014–2016.

Table 2. Association between Xanthomonas species isolated from processing tomatoes in the U.S. Midwest and tomato seed sources.

Year	Seed source	X. hortorum pv. gardneri	E^x	X. perforans	E^x	DF^y	X^2	p-value
	A	9	(0.5)	2	(0.5)	-	-	-
2010	B	50	(0.5)	6	(0.5)		34.5714	0
	A	9	(.13)	2	(.03)	-	-	-
	B	50	(.75)	6	(.09)
	A	2	(0.5)	3	(0.5)	-	-	-
2011	B	15	(0.5)	0	(0.5)		-	-
	E	11	(0.5)	2	(0.5)		6.230	0.013
	A	0	(0.5)	1	(0.5)	-	-	-
2012	B	7	(0.5)	5	(0.5)	-	0.33	0.564
	F	1	(0.5)	1	(0.5)	-	-	-
	C	0	(0.5)	1	(0.5)	-	-	-
	A	1	(0.5)	-	(0.5)	-	-	-
2013	B	3	(0.5)	-	(0.5)	-	-	-
	C	0	(0.5)	-	(0.5)	-	-	-
	B	72	(0.5)	96	(0.5)	1	3.428	0.034
	F	17	(0.5)	12	(0.5)	1	0.86	0.35
	E	8	(0.5)	8	(0.5)	1	0	1
	A	0	(0.5)	10	(0.5)	-	-	-
2017	D	14	(0.5)	0	(0.5)	-	-	-
	B	72	(.32)	96	(.47)	2	3.67	0.597
	F	17	(.10)	12	(.04)
	E	8	(.04)	8	(.03)
	A	17	(0.5)	52	(0.5)	1	17.75	<0.0001
	B	46	(0.5)	166	(0.5)	1	67.93	<0.0001
	G	14	(0.5)	40	(0.5)	1	12.51	<0.0001
	F	0	(0.5)	22	(0.5)	-	-	-
2018	E	0	(0.5)	4	(0.5)	-	-	-
	A	17	(0.05)	52	(0.16)	2	0.569	0.7525
	B	46	(0.14)	166	(0.5)
	G	14	(0.04)	40	(0.12)
	A	10	(0.05)	7	(0.05)	1	0.529	0.467
2019	B	0	-	38	-	-	-	-
	E	14	-	0	-	-	-	-
	D	1	-	9	-	-	-	-
2020	A	0	(0.5)	10	(0.5)	-		-
	B	23	(0.5)	31	(0.5)	1	1.185	0.276
	F	0	(0.5)	10	(0.5)	-	-	-
	E	0	(0.5)	10	(0.5)	-	-	-

^xE = Expected probability for each Chi-square analysis. The expected probability for Chi-square

analyses with degrees of freedom (DF) = 5 was calculated using the joint probability by considering sample size of seed sources.

^yDF = degrees of freedom comparing the distribution of X. hortorum pv. gardneri (Xhg) and X. perforans (Xp) on seed source.

Table 3. Association of Xanthomonas hortorum pv. gardneri and X. perforans with green and ripe processing tomato fruit, 2017–2020.

	Maturity	X. hortorum pv. gardneri	E^x	X. perforans	E^x	DF^y	χ^2	p-value
2017	Green	43	(0.5)	56	(0.5)	1	1.707	0.191
	Ripe	76	(0.5)	78	(0.5)	1	0.025	0.872
	Green	43	(0.18)	56	(0.21)	1	0.89	0.83
	Ripe	76	(0.29)	78	(0.32)
2018	Green	79	(0.5)	196	(0.5)	1	49.78	<0.0001
	Ripe	18	(0.5)	88	(0.5)	1	46.22	<0.0001
	Green	79	(0.18)	196	(0.54)	1	5.479	0.0192
	Ripe	18	(0.07)	88	(0.21)
2019	Green	2	(0.5)	23	(0.5)	-	-	-
	Ripe	23	(0.5)	72	(0.5)	1	25.27	<0.0001
	Green	2	(0.04)	23	(0.17)	-	-	-
	Ripe	23	(0.16)	72	(0.62)
2020	Green	11	(0.5)	43	(0.5)	1	18.96	<0.0001
	Ripe	12	(0.5)	18	(0.5)	1	1.2	0.273
	Green	11	(0.18)	40	(0.47)	1	3.385	0.0658
	Ripe	12	(0.1)	18	(0.26)

^xE = Expected probability for each Chi-square analysis. The expected probability for Chi-square

analyses with degrees of freedom (DF) = 1 was calculated using the joint probability by considering sample size of the fruit maturity category.

^yDF = degrees of freedom comparing the distribution of X. hortoruom pv. gardneri and X. perforans on fruit maturity category.

Table 4. Percentage of copper sulphate- and streptomycin sulphate-resistant Xanthomonas hortorum pv. gardneri and X. perforans strains isolated from processing tomatoes in the U.S. Midwest, 2010–2020.

		Copper sulphate 100 µg mL^−1			Copper sulphate 200 µg mL^−1			Streptomycin 20 µg mL^−1			Streptomycin 200 µg mL^−1
Year	Xanthomonas species	% Resistant	χ^2	P-value	% Resistant	χ^2	P-value	% Resistant	χ^2	P-value	% Resistant	χ^2	P-value
2010	Xhg^x	90	1.200	0.530	0			86	22.761	<.0001	86	22.761	<.0001
	Xp^y	100			0			13			13
2011	Xhg	39	12.600	0.004	5	0.886	0.347	31	0.617	0.4323	31	3.358	0.067
	Xp	93			13			20			7
2012	Xhg	77	0.359	0.549	0			31	0.777	0.3782	23	1.615	0.204
	Xp	87			7			50			50
2013	Xhg	NA^z			NA			NA			NA
	Xp	NA			NA			NA			NA
2017	Xhg	99			81	16.790	<.0001	66	9.21	0.01	50	27.401	<.0001
	Xp	99			84			80			80
2018	Xhg	100	1.376	0.241	85	22.478	<.0001	100	12.703	0.0004	59	33.492	<.0001
	Xp	99			58			88			86
2019	Xhg	100			20	63.313	<.0001	100			100	7.480	0.006
	Xp	100			93			100			76
2020	Xhg	100			100			100			100	18.250	<0.0001
	Xp	100			100			100			49

^xX. hortorum pv. gardneri.

^yX. perforans.

^zNA = Not available, N < 5 for each category.

Fig. 4 Mosaic charts showing the proportion of Xanthomonas spp. strains from processing tomatoes in the U.S. Midwest resistant to copper sulphate (200 µg mL⁻¹) for each surveyed year. Resistance is indicated in black, sensitivity in white. The width of each column is proportional to the relative abundance of each species.

Fig. 5 Mosaic charts showing the proportion of Xanthomonas spp. strains from processing tomatoes in the U.S. Midwest resistant to streptomycin sulphate (200 µg mL⁻¹) for each surveyed year. Resistance is indicated in black, sensitivity in white. The width of each column is proportional to the relative abundance of each species.

Table 5. Percentage of bacteriocin-producing Xanthomonas perforans strains isolated from processing tomatoes in the U.S. Midwest, 2010–2020 that inhibited the growth of X. euvesicatoria 91–106 K.

Year	Bacteriocin-Producing	Strains Tested
2010	50%	8
2011	53%	15
2012	63%	8
2013	50%	4
2017	45%	134
2018	64%	285
2019	58%	96
2020	66%	61