Fungicide Resistance Profiles for 13 Botrytis cinerea Isolates from Strawberry in Southeastern Louisiana

Figures & data

FIGURE 1 Experimental set up for 96-well plate microtiter assay in which columns contain 0, 0.3, 3.0, and 30.0 concentrations of test compounds. Rows (A–H) contain fungicides and internal standards for comparison and validation. Sixteen positive growth control wells are used to establish the fungal growth mean and eight negative control wells without inoculum and test compounds serve as sterility checks. Blank reagent well indicated with stippling are used to negate the effects that colored molecules have on the turbidity measurements at 620 nM.

TABLE 1  Chemical and Trade Names, Chemical Class, Mode of Action, FRAC Group, Systemic Activity, Range of Controlled Pathogens, and Commercial Rate Range of Fungicides Used in This Study Based on Fungicide Labels and Fungicide Resistance Action Committee (FRAC) Website (http://www.frac.info)^z

Fungicide common and trade names	Chemical class	Mode of action	FRAC group	Systemic activity	Range of controlled pathogens	Commercial rate range per applications^y
Azoxystrobin Abound	Quinone outside inhibitor (QoI)	QoI complex 3 respiration inhibitor	11	Non-systemic	Broad spectrum	105.0–260.0 ml ai/ha
Benomyl Benlate 50 WP	Methyl benzimidazole carbamate	Inhibition of microtubule assembly	1	Systemic	Broad spectrum	No longer commercially available; 567 g ai/ha
Butrizol Enable	Triazole	C14-demethylation inhibitor	3	Non-systemic	Broad spectrum	Not registered for use in U.S.; 106.5–141.8 g ai/ha
Captan Captan	Multisite phthalamide	Multisite inhibitor	M4	Non-systemic	Broad spectrum	1.68–4.48 kg ai/ha
Chlorothalonil Bravo	Chloronitrile	Multisite inhibitor	M5	Non-systemic	Broad spectrum	1.23 kg ai/ha
Cyprodinil component of Switch	Anilinopyrimidine	Proposed methionine biosynthesis inhibitor	9	Systemic	Monilinia, Cladosporium, and Venturia	292.3–372.0 g ai/ha
Dodine Syllit	Anthraquinone aliphatic nitrogen fungicides	Multisite inhibitor	M9	Non-systemic	Broad spectrum	1.14–1.46 kg ai/ha
Fenhexamid Elevate 50	Hydroxyanilide	Ketoreductase inhibitor (sterol biosynthesis)	17	Non-systemic	Botrytis and Monilinia	560–840 g ai/ha
Fosetyl-Al Aliette	Phosphonate	Multisite inhibitor	33	Systemic	Broad spectrum	4.54 kg ai/ha
Iprodione Roval 50	Dicarboxymide	Inhibition of lipid and membrane synthesis	2	Non-systemic	Broad spectrum	560–1120 g ai/ha
Kresoxim-methyl Sovran	Quinone outside inhibitor (QoI)	QoI complex 3 respiration inhibitor	11	Non-systemic	Broad spectrum	112–280 g ai/ha
Metalaxyl Ridomil 2E	Phenylamide	RNA polymerase inhibitor	4	Systemic	Broad spectrum	1 liter ai/ha
Thiabendazole Mertect	Methyl benzimidazole carbamate	Inhibition of microtubule assembly	1	Systemic	Broad spectrum mold, rot, blight, and stain	479.8 g ai/ha
Thiophanate-methyl Topsin-M	Methyl benzimidazole carbamate	Inhibition of microtubule assembly	1	Systemic	Broad spectrum	850.5 g ai/ha
Thiram Thiram 65WP	Dithiocarbamate	Multisite inhibitor	M3	Non-systemic	Broad spectrum	2.95–3.69 kg ai/ha
Vinclozolin Ronilan 50DF	Dicarboxymide	Inhibition of lipid and membrane synthesis	2	Non-systemic	Broad spectrum	850.5 g ai/ha
^zTable adapted from Wedge et al., 2005.
^yRates based on US–EPA Section 3 registrations for use on fruit or vegetable crop.

TABLE 2  In Vitro Sensitivity of 13 Botrytis cinerea Isolates to 16 Antifungal Chemicals Was Determined Using a Standardized 96-Well Microtiter Assay. Fungal Growth Is Reported as Mean Percent Inhibition (-) or Stimulation (+) for Each Fungicide at 0.3 μM Concentration after 72 h of Growth Compared to the Growth of Each Isolate in Unamended RPMI Broth

	Antifungal chemical
Fungal	Cypro-	Fenhex-	Kresoxim	Azoxy-		Chloro-			Thiabend-			Vinclo-		Fosetyl-	Thiophanate
isolate	dinil	amide	methyl	strobin	Butrizol	thalonil	Benomyl	Captan	azole	Iprodione	Thiram	zolin	Dodine	Al	methyl	Metalaxyl	LSD
FP	−98g^z, B^y	−92fg, B	−94fg, B	−72ef, ABC	−63de, CDEF	−60de, C	−26bc, ABC	−42cd, B	−28bc, CD	−14ab, A	−39cd, C	−26bc, B	−22bc, B	−22bc, B	−11ab, BC	9a, ABC	24
GR	−98h, B	−99h, B	−96h, B	−84gh, C	−75gh, EF	−44ef, ABC	−62fg, DE	−39def, B	−33cde, D	−21bcde, A	−14abcd, BC	−12abc, AB	−20bcde, B	3ab, AB	−7ab, BC	10a, ABC	25
DW	−99e, B	−97e, B	−95e, B	−84de, C	−66cde, DEF	−50bcd, BC	−49bc, BCDE	−20ab, B	5a, ABCD	−12a, A	−1a, ABC	−4a, AB	−6a, AB	3a, AB	0a, BC	−19ab, C	35
HR	−96e, B	−97e, B	−91e, B	−72de, ABC	−79e, EF	−46cd, ABC	−7ab, ABC	−18abc, B	−8ab, D	−3ab, A	−9ab, BC	−8ab, AB	−26bc, B	2a, AB	−7ab, BC	−11ab, BC	28
LB	−96e, B	−83de, AB	−97e, B	−74de, BC	−74de, EF	−61d, C	1abc, ABC	−13bc, B	7ab, ABC	12a, A	8ab	0abc, AB	13a, AB	−11abc, AB	−22c, C	7ab, ABC	26
GH	−99e, B	−97e, B	−84de, AB	−70d, ABC	−39c, BC	−35c, ABC	−79de, E	−22bc, B	3a, ABCD	2a, A	16a, AB	4a, AB	8a, AB	6a, AB	1ab, BC	8a, ABC	24
P1	−92f, B	−95f, B	−90ef, B	−71def, ABC	−46cd,BCD	−25bc, AB	−52cde, CDE	−7ab, B	−1ab, ABCD	5ab, A	14a	23a, A	−1ab, AB	14a, AB	1ab, BC	−14abc, BC	39
BH	−98h, B	−98h, B	−85gh, B	−71g, ABC	−68g, DEF	−15ef, A	−85gh, E	−20f, B	−10def, BCD	5bcde, A	2cdef, AB	16bc, A	27ab, A	9bcd, AB	26ab, AB	45a, A	23
GC	−86d, B	−83d, AB	−87d, B	−68d, ABC	−59cd, CDEF	−46bcd, ABC	−3ab, ABC	−13abc, B	−13bc, BCD	20a, A	8a, AB	10a, AB	14a, AB	3a, AB	−2ab,BC	0ab, BC	49
DD	−72c, A	−71c, A	−62c, A	−54c, A	−51c, BCD	−41bc, ABC	0a, AB	−14ab, B	−9ab, BCD	−12ab, A	−7ab, BC	−6ab, AB	0a, AB	15a, AB	−3a, BC	−5a, BC	36
TG	−98h, B	−98h, B	−84gh, AB	−71fg, ABC	−31e, AB	−58f, BC	9bc, A	−16de, B	−4cd, ABCD	13bc, A	−2bcd	7bc,AB	6bc, AB	7bc, AB	19b, AB	45a, A	22
NW	−97f, B	−95f, B	−87ef, B	−64de, AB	−10c, A	−58d, BC	−90ef, E	33ab, A	18ab, AB	16abc, A	24ab	19ab, A	33ab, A	5bc, AB	41a, A	21ab, ABC	28
BB	−97e, B	−84de, AB	−91e, B	−77de, BC	−65cd, DEF	−41c, ABC	19a, A	−13b, B	32a, A	11ab, A	33a, A	22a, A	24a, A	22a, A	27a, AB	26a, AB	26
LSD	20	19	22	18	25	34	50	38	40	46	40	37	42	38	40	41
^zMean values followed by different lower case letters in the same row within a fungal isolate are significantly different as determined by Fisher's protected least significant difference (LSD) at α = 0.05.
^yMean values followed by different upper case letters in the same column within a fungicide are significantly different as determined by Fisher's protected least significant difference (LSD) at α = 0.05.

TABLE 3  In vitro sensitivity of 13 Botrytis cinerea isolates to 16 antifungal chemicals was determined using a standardized 96-well microtiter assay. Fungal growth is reported as mean percent inhibition (-) or stimulation (+) for each fungicide at 3 μM concentration after 72 h of growth compared to the growth of each isolate in unamended RPMI broth

	Antifungal chemical
Fungal		Fenhex-		Cypro-	Chloro-	Kresoxim-	Azoxy-		Vincloz-	Thiabend-			Thiophanate		Fosetyl-
Isolate	Dodine	amide	Butrizol	dinil	thalonil	methyl	strobin	Captan	olin	azole	Iprodione	Benomyl	methyl	Thiram	Al	Metalaxyl	LSD
GH	−96g^z, B^y	−95g, B	−96g, B	−95g, B	−91fg, ABC	−88efg, B	−84defg, AB	−66d, BC	−96g, E	−75def, D	−41c, BCD	−71de, DE	−72de, CD	−2b, ABCDE	−5b, AB	19a, AB	18
GR	−97g, B	−96fg, B	−97g, AB	−97g, B	−91efg, ABC	−95fg, B	−91efg, B	−74defg, C	−59cde, CDE	−68cdefg, CD	−52cd, BCD	−60cdef, ABC	−41bcd, BC	−32bc, E	−7ab, AB	25a, AB	36
BH	−96e, B	−93e, B	−96e, B	−87e, AB	−85e, ABC	−93e, B	−88e, B	−61cd, BC	−81de, DE	−76cde, D	−56c, CD	−77cde, E	−84e, D	−7b, BCDE	7b, AB	46a, A	20
DW	−96c, B	−91c, AB	−94c, B	−96c, B	−77bc, A	−92c, B	−85bc, AB	−58bc, BC	−61bc, CDE	−56bc, BCD	−47b, BCD	−47b, CDE	−48b, BCD	1a, ABCDE	1a, AB	−4a, AB	41
NW	−96d, B	−91d, AB	−90d, AB	−94d, B	−79d, AB	−90d, B	−80d, AB	−28b, A	−90d, E	−81d, D	−33bc, ABCD	−82d, E	−66cd, CD	32a, A	5a, AB	21a, AB	33
P1	−97f, B	−97f, B	−95f, B	−96f, B	−86def, ABC	−92ef, B	−88def, B	−61cdef, BC	−48bcde, BCD	−55cdef, BCD	−44bcd, BCD	−39bc, BCDE	−53cdef, BCD	9a, ABC	21a, A	−4ab, AB	45
GC	−96d, B	−95d, B	−94d, B	−92d, AB	−95d, BC	−90d, B	−84d, AB	−53bcd, ABC	−71cd, DE	−16ab, AB	−60bcd, D	−4a, ABC	−5a, AB	−26abc, CDE	−22ab, B	4a, AB	47
FP	−96e, B	−93e, B	−94e, B	−94e, B	−94e, BC	−91e, B	−87de, B	−68d, BC	−27c, ABC	−29c, ABC	−27c, ABCD	−21bc, ABCD	−6b, AB	−33c, E	−16bc, AB	21a, AB	19
HR	−96e, B	−95e, B	−92e, AB	−92e, B	−92e, ABC	−92e, B	−88e, B	−63d, BC	−16bc, AB	−26c, AB	−13bc, AB	−5ab, ABC	−16bc, ABC	−21bc, BCDE	−8abc, AB	10a, AB	20
BB	−96d, B	−92d, B	−95d, B	−89d, AB	−96d, C	−89d, B	−90d, B	−49c, ABC	−91d, E	8b, A	−60c, D	16ab, A	36a, A	10ab, AB	−6b, AB	12ab, AB	26
DD	−76d, A	−75d, A	−71d, A	−78d, A	−95d, BC	−63cd, A	−67cd, A	−58bcd, ABC	−19ab, ABC	−16ab, AB	−19ab, ABC	0a, ABC	−5a, AB	−29abc, DE	−11a, AB	−21ab, B	41
TG	−95e, B	−95e, B	−94e, B	−93e, B	−95e, BC	−86e, AB	−79de, AB	−56d, ABC	1bc, A	0bc, A	4bc, A	12abc, AB	17ab, A	−9c, BCDE	5bc, AB	34a, A	23
LB	−89e, AB	−92e, B	−89e, AB	−90e, AB	−91e, ABC	−90e, B	−79e, AB	−44d, AB	0bc, A	7abc, A	−11c, AB	25a, A	0bc, AB	5abc, ABCD	2abc, AB	12ab, AB	23
LSD	16	17	21	13	16	23	18	29	41	39	40	53	57	37	40	51
^zMean values followed by different lower case letters in the same row within a fungal isolate are significantly different as determined by Fisher's protected least significant difference (LSD) at α = 0.05.
^yMean values followed by different upper case letters in the same column within a fungicide are significantly different as determined by Fisher's protected least significant difference (LSD) at α = 0.05.

TABLE 4  In Vitro Sensitivity of 13 Botrytis cinerea Isolates to 16 Antifungal Chemicals Was Determined Using a Standardized 96-Well Microtiter Assay. Fungal Growth Is Reported as Mean Percent Inhibition (-) or Stimulation (+) for Each Fungicide at 30 μM Concentration after 72 h of Growth Compared to the Growth of Each Isolate in Unamended RPMI Broth

	Antifungal compound
Fungal				Fenhex-	Kresoxim-		Cypro-	Azoxy-	Chloro-			Thiaben-		Thiophanate-	Fosetyl-
isolate	Dodine	Captan	Thiram	amide	methyl	Butrizol	dinil	strobin	thalonil	Iprodione	Vinclozolin	dazole	Benomyl	methyl	Al	Metalaxyl	LSD
GH	−99c^z, A^y	−99c, B	−96c, AB	−97c, B	−94c, B	−98c, B	−98c, B	−88bc, ABC	−86c, AB	−98c, C	−99c, G	−74b, EF	−77b, D	−75b, C	2a, ABC	12a, AB	14
NW	−96cd, A	−99d, B	−94cd, AB	−96cd, B	−96cd, B	−95cd, B	−93cd, AB	−92cd, ABC	−73bc, A	−97d, C	−99d, G	−91cd, F	−91cd, D	−73c, C	16a, A	31a, AB	23
BH	−99d, A	−95cd, AB	−93cd, AB	−97d, B	−98d, B	−96d, B	−72c, A	−96d, C	−100d, B	−98d, C	−82cd, EFG	−84cd, F	−83cd, D	−84cd, C	5b, AB	40a, A	23
GR	−98d, A	−95cd, AB	−99d, B	−98d, B	−100d, B	−96d, B	−99d, B	−97d, C	−94cd, AB	−93cd, BC	−55b, CDE	−65bcd, DEF	−61bc, CD	−50b, BC	2a, ABC	18a, AB	35
DW	−97e, A	−99e, B	−97e, AB	−94e, B	−98e, B	−97e, B	−91de, AB	−97e, C	−85cde, AB	−91de, BC	−68bcde, DEFG	−61bcd, DEF	−54bc, BCD	−45b, ABC	2a, ABC	6a, AB	32
P1	−100d,A	−97cd, AB	−96cd, AB	−98cd, B	−97cd, B	−98cd, B	−98cd, B	−83bcd, A	−84bcd, AB	−86bcd, ABC	−58bc, CDEF	−58bcd, CDEF	−54b, BCD	−47b, BC	17a, A	9a, AB	41
GC	−97c, A	−98c, AB	−96c, AB	−94c, B	−96c, B	−92c, AB	−87c, AB	−91c, ABC	−93c, AB	−97c, C	−74bc, EFG	−21a, ABC	5a, A	−13a, AB	−33ab, C	4a, AB	44
HR	−98c, A	−91c, A	−98c, B	−97c, B	−99c, B	−98c, B	−98c, B	−96c, C	−85c, AB	−82c, ABC	−34b, BCD	−32b, ABCD	−20b, ABC	−18b, AB	−23b, BC	15a, AB	17
FP	−100e, A	−99e, B	−97e, AB	−98e, B	−98e, B	−97e, B	−99e, B	−98e, C	−86de, AB	−70d, AB	−43c, BCDE	−40c, BCDE	−25bc, ABC	−15b, AB	−16b, ABC	26a, AB	21
BB	−95c, A	−95c, AB	−94c, AB	−98c, B	−94c, B	−94c, B	−97c, B	−95c, BC	−95c, AB	−89c, ABC	−96c, FG	5ab, A	5ab, A	6ab, A	−11b, ABC	12a, AB	19
DD	−98b, A	−96b, AB	−94b, AB	−77b, A	−73b, A	−72b, A	−93b, AB	−94b, ABC	−99b, B	−86b, ABC	−24a, ABC	−27a, ABCD	−9a, AB	−4a, AB	−17a, ABC	−11a, B	38
LB	−98e, A	−96e, AB	−93e, A	−91e, AB	−100e, B	−95e, B	−97e, B	−88de, ABC	−92e, AB	−67d, A	−15bc, AB	1ab, A	−6ab, A	1ab, AB	−28c, BC	11a, AB	21
TG	−96ef,A	−98f, AB	−99f, B	−96ef, B	−89ef, AB	−97f, B	−99f, B	−84ef, AB	−90ef, AB	−77e, ABC	4bc, A	−16d, AB	−9cd, AB	−2bcd, AB	14ab, A	31a, AB	19
LSD (2)	5	7	5	16	18	21	22	11	22	23	39	40	46	53	36	47
^zMean values followed by different lower case letters in the same row within a fungal isolate are significantly different as determined by Fisher's protected least significant difference (LSD) at α = 0.05.
^yMean values followed by different upper case letters in the same column within a fungicide are significantly different as determined by Fisher's protected least significant difference (LSD) at α = 0.05.
Bold values indicate growth inhibition levels of greater than 70%.

TABLE 5  In vitro growth response of 13 Botrytis cinerea isolates^z to 6 fungicides. All isolates were obtained from strawberry except isolate GR, which was obtained from grape, and isolate BB, which was obtained from blueberry. Fungal growth to each chemical using an in vitro 96-well microtiter assay is reported as sensitive^y (S), intermediate^y (I), or resistant^y (R). The 72 h growth response to antifungal compounds at 3 μM concentrations identified chemically sensitive B. cinerea isolates and the growth response at 30 μM concentrations identified chemically resistant B. cinerea isolates

			Botrytis cinerea isolate
Fungicide class	Antifungal compound	Level	GH	GR	BH	DW	NW	P1	GC	FP	HR	BB	DD	TG	LB
Benzimidazole	Benomyl	0.3 μM	S	S	S	I	S	I	R	I	R	R	R	I	R
Benzimidazole	Thiabendazole	0.3 μM	R	I	I	R	R	R	I	I	R	R	R	I	R
Benzimidazole	Thiophanate methyl	0.3 μM	R	R	R	R	I	R	R	R	R	R	R	I	S
Dicarboxymide	Iprodione	0.3 μM	R	I	I	R	R	R	R	R	R	R	R	I	R
Dicarboxymide	Vinclozolin	0.3 μM	R	R	I	R	R	R	R	I	R	R	R	I	R
Dithiocarbamate	Thiram	0.3 μM	R	R	I	R	R	R	R	I	R	R	R	I	R
Benzimidazole	Benomyl	3 μM	S	I	S	I	S	R	R	I	R	R	R	R	R
Benzimidazole	Thiabendazole	3 μM	S	I	S	I	S	I	R	I	I	R	R	I	R
Benzimidazole	Thiophanate methyl	3 μM	S	I	S	I	S	I	R	I	I	R	R	R	R
Dicarboxymide	Iprodione	3 μM	I	I	I	I	S	R	S	I	I	S	R	I	I
Dicarboxymide	Vinclozolin	3 μM	S	I	S	S	S	R	S	I	I	S	R	I	R
Dithiocarbamate	Thiram	3 μM	I	I	I	R	R	R	R	R	I	R	R	I	R
Benzimidazole	Benomyl	30 μM	I	S	S	I	S	I	R	I	I	R	R	I	R
Benzimidazole	Thiabendazole	30 μM	I	S	S	I	S	I	R	I	I	R	R	I	R
Benzimidazole	Thiophanate methyl	30 μM	I	I	S	I	I	I	R	I	I	R	R	I	R
Dicarboxymide	Iprodione	30 μM	S	S	S	S	S	S	S	I	S	S	S	I	I
Dicarboxymide	Vinclozolin	30 μM	S	I	S	S	S	I	S	I	I	S	R	I	I
Dithiocarbamate	Thiram	30 μM	S	S	S	S	S	S	S	S	S	S	S	S	S
^zAll 13 isolates were sensitive at 3 and 30 μM to dodine, fenhexamide, cyprodinil, butrizol, and azoxystrobin; therefore, these fungicides are not shown on this table.
^yThese categories were defined as follows within each concentration: R = chemical performed the same as the metalaxyl (noneffective control) by the LSD (α = 0.05); I = chemical caused more inhibition than metalaxyl but less than captan by the LSD (α = 0.05); S = chemical caused the same or more inhibition than captan (effective control) by the LSD (α = 0.05).

TABLE 6  Benomyl Resistance Profile and the Identity of Codon Position 198 in the β-Tubulin Gene for the 11 Strawberry Isolates of Botrytis cinerea

Isolate	Benomyl profile	β-Tubulin
HR	Resistant	GCG^z
LB	Resistant	GCG
FP	Resistant	GCG
TG	Resistant	GCG
DD	Resistant	GCG
GC	Resistant	GCG
P1	Intermediate	GAG
DW	Intermediate	GAG
BH	Sensitive	GAG
NW	Sensitive	GAG
GH^y	Sensitive	GCG
^zGCG = alanine; GAG = glutamic acid.
^yGH is the only benomyl sensitive isolate in which the β-tubulin amino acid substitution corresponding to the resistance allele occurred.