Identification and Characterization of Benzimidazole Resistance in Monilinia fructicola from Stone Fruit Orchards in California

Zhonghua Ma; Michael A Yoshimura; Themis J Michailides

doi:10.1128/AEM.69.12.7145-7152.2003

. 2003 Dec;69(12):7145–7152. doi: 10.1128/AEM.69.12.7145-7152.2003

Identification and Characterization of Benzimidazole Resistance in Monilinia fructicola from Stone Fruit Orchards in California

Zhonghua Ma ¹, Michael A Yoshimura ¹, Themis J Michailides ^1,^*

PMCID: PMC309941 PMID: 14660360

Abstract

Low and high levels of resistance to the benzimidazole fungicides benomyl and thiophanate-methyl were observed in field isolates of Monilinia fructicola, which is the causative agent of brown rot of stone fruit. Isolates that had low levels of resistance (hereafter referred to as LR isolates) and high levels of resistance (hereafter referred to as HR isolates) were also cold and heat sensitive, respectively. Results from microsatellite DNA fingerprints showed that genetic identities among the populations of sensitive (S), LR, and HR isolates were very high (>0.96). Analysis of DNA sequences of the β-tubulin gene showed that the LR isolates had a point mutation at codon 6, causing a replacement of the amino acid histidine by tyrosine. Codon 198, which encodes a glutamic acid in S and LR isolates, was converted to a codon for alanine in HR isolates. Based on these point mutations in the β-tubulin gene, allele-specific PCR assays were developed for rapid detection of benzimidazole-resistant isolates of M. fructicola from stone fruit.

Brown rot, caused by the fungal pathogen Monilinia fructicola (G. Wint.) Honey, is one of the most destructive diseases of stone fruits. Chemical control of brown rot is an important strategy for the management of this disease. The benzimidazole fungicides thiophanate-methyl and benomyl have been used widely for controlling the disease. In California, benomyl was used almost exclusively for controlling blossom blight after its registration in 1972. Since 1977, benomyl-resistant populations of M. fructicola have become widespread throughout California (24, 25, 28). Levels of resistance to benomyl have been reported to be relatively low in California, with 50% effective concentration (EC₅₀) values between 0.5 and 4 μg/ml (24, 25), when compared with resistance levels at 50 μg/ml or higher reported from other states (10, 33). However, in a preliminary study, we also observed high resistance to the benzimidazoles in field isolates of M. fructicola collected from stone fruit orchards in California. A genetic study on benzimidazole resistance revealed that the high resistance of M. fructicola to carbendazim was conferred by a single gene affected by gene conversion mechanisms (26).

Resistance to benzimidazole fungicides has been detected in many fungal species. In most cases, resistance has been shown to be associated with point mutations in the β-tubulin gene which result in altered amino acid sequences at the benzimidazole binding site (13). Studies with laboratory mutants of Aspergillus nidulans, Neurospora crassa, and Saccharomyces cerevisiae showed that changes at codons 6, 50, 134, 165, 198, 200, and 241 in the β-tubulin gene were responsible for benzimidazole resistance (13). In contrast to laboratory mutants, most field isolates of plant-pathogenic fungi exhibit codon changes that, strikingly, seem to be restricted to positions 50 (22), 198, 200 (1, 13), and 240 (1). Only a few exceptions of mutations at these positions have been reported for isolates of Venturia inaequalis, Penicillium expansum, and Penicillium aurantiogriseum with low levels of resistance to benomyl (13) and thiabendazole-resistant isolates of Gibberella pulicaris (12), but the exact molecular mechanisms for their resistance have not yet been determined.

A possible reason for the low variation in mutations found in field isolates compared to that of laboratory-induced mutants is that mutations in codons other than codons 198 and 200 might interfere with the fitness of mutants and impose a selective disadvantage on these mutants under field conditions (13). For example, a laboratory-induced benomyl-resistant mutant of Fusarium moniliforme with a mutation at codon 50 (31), resistant strains of A. nidulans with mutations at codon 50, 134, or 257 (13), and resistant mutants of S. cerevisiae (31) were also sensitive to heat or cold. However, such temperature sensitivity has not been reported from field isolates of benzimidazole-resistant plant pathogens.

In a preliminary study, we observed two levels of resistance (low and high) to the benzimidazole fungicides, benomyl and thiophanate-methyl, in field isolates of M. fructicola collected from stone fruit orchards in California. The objectives of this study were to (i) determine the sensitivity of M. fructicola from stone fruit to benzimidazole fungicides, (ii) determine the temperature sensitivity in benomyl-sensitive and -resistant isolates of M. fructicola, (iii) determine the genetic relationships among sensitive and resistant isolates, (iv) investigate molecular mechanisms of benzimidazole resistance in field isolates of M. fructicola, and (v) develop rapid molecular techniques to detect benzimidazole resistance in M. fructicola from stone fruits in California.

MATERIALS AND METHODS

Sensitivity of M. fructicola to benzimidazole fungicides.

Thirty-nine single-spore isolates of M. fructicola collected from 1994 to 2002 from nectarine, peach, plum, and prune throughout California were used in this study (Table 1). Isolates were routinely stored on grade 40 silica gel (Davison Chemical, Baltimore, Md.) at 4°C.

TABLE 1.

Sensitivity to benomyl and thiophanate-methyl in isolates of M. fructicola from California stone fruit

Isolate	EC₅₀ (μg/ml) of		Sensitivity to benzimidazoles	Host	Location^a	Date of isolation (mo/yr)
Isolate	Benomyl	Thiophanate-methyl	Sensitivity to benzimidazoles	Host	Location^a	Date of isolation (mo/yr)
R2-12	0.03923	0.3689	S	Nectarine	Reedley, Fresno Co.	9/2002
R1-37	0.02531	0.2644	S	Nectarine	Reedley, Fresno Co.	9/2002
P1-32	0.03907	0.4164	S	Nectarine	Parlier, Fresno Co.	9/2002
K4-16	0.02633	0.4560	S	Nectarine	KAC, Fresno Co.	9/2002
LVN8	0.04511	0.5436	S	Nectarine	Merced Co.	9/2002
K3-4	0.04217	0.7629	S	Peach	KAC, Fresno Co.	9/2002
MS22	0.04827	0.4552	S	Peach	Madera Co.	9/2002
AG10	0.04227	0.4133	S	Peach	Merced Co.	9/2002
ATF16	0.02633	0.3133	S	Peach	Merced Co.	9/2002
ATC5	0.04192	0.4608	S	Peach	Merced Co.	9/2002
SP1-1	0.04551	0.5539	S	Plum	Sanger, Fresno Co.	9/2002
SP3-17	0.04529	0.7327	S	Nectarine	Sanger, Fresno Co.	9/2002
R2-9	0.6452	4.9714	LR	Nectarine	Reedley, Fresno Co.	9/2002
K2-4	0.7403	5.1612	LR	Nectarine	KAC, Fresno Co.	9/2002
P1-5	0.7419	4.8704	LR	Nectarine	Parlier, Fresno Co.	9/2002
P1-15	0.7361	2.3844	LR	Nectarine	Parlier, Fresno Co.	9/2002
P1-26	0.6097	4.1047	LR	Nectarine	Parlier, Fresno Co.	9/2002
LVN9	0.7347	3.1166	LR	Nectarine	Merced Co.	9/2002
22E5	0.7188	4.8493	LR	Peach	Parlier, Fresno Co.	3/1994
22E8	0.7883	4.1047	LR	Peach	Sanger, Fresno Co.	3/1994
MS15	0.6707	5.9524	LR	Peach	Madera Co.	9/2002
ATC27	0.7456	6.4212	LR	Peach	Merced Co.	9/2002
ATC22	0.6741	6.0752	LR	Peach	Merced Co.	9/2002
LVF32	0.8217	2.5215	LR	Peach	Merced Co.	9/2002
K8-14	0.7347	2.4095	LR	Plum	KAC, Fresno Co.	9/2002
K8-15	0.9579	2.8628	LR	Plum	KAC, Fresno Co.	9/2002
SP1-20	0.7807	2.5991	LR	Plum	Sanger, Fresno Co.	9/2002
23B2	0.7521	5.4009	LR	Prune	Parlier, Fresno Co.	11/1995
33C7	0.7456	4.1904	LR	Plum	Dinuba, Fresno Co.	5/1998
R2-3	>50	>50	HR	Nectarine	Reedley, Fresno Co.	9/2002
R2-11	>50	>50	HR	Nectarine	Reedley, Fresno Co.	9/2002
SP3-42	>50	>50	HR	Nectarine	Sanger, Fresno Co.	9/2002
MS1	>50	>50	HR	Nectarine	Madera Co.	9/2002
MS3	>50	>50	HR	Nectarine	Madera Co.	9/2002
MS9	>50	>50	HR	Nectarine	Madera Co.	9/2002
MS16	>50	>50	HR	Nectarine	Madera Co.	9/2002
MP4	>50	>50	HR	Plum	Madera Co.	9/2002
32F5	>50	>50	HR	Plum	Dinuba, Fresno Co.	5/1998
33D7	>50	>50	HR	Plum	Dinuba, Fresno Co.	5/1998

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KAC, Kearney Agricultural Center; Co., County.

To test the sensitivity of M. fructicola to benomyl (Benlate 50% WP; Du Pont de Nemours and Company, Wilmington, Del.) and thiophanate-methyl (70% WP; Elf Atochem North America, Inc., Philadelphia, Pa.), potato dextrose agar (PDA) (Microtech Scientific, Orange, Calif.) media were amended with benomyl at 0, 0.0125, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, 1, 5, 50, 100, or 500 μg of active ingredient (a.i.)/ml or thiophanate-methyl at 0, 0.0625, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 50, 100, or 500 μg of a.i./ml. Two replicates of each fungicide concentration were used for each isolate. Five-millimeter-diameter mycelial plugs taken from the edge of a 3-day-old colony of each isolate were placed onto PDA plates amended with each concentration of the above fungicides. After the plates were incubated at 24°C for 3 days in the dark, the radial growth (colony diameter) of each isolate was measured, with the original mycelial plug subtracted from each measurement. For each isolate, the average of the colony diameters measured in two perpendicular directions was used for calculating the EC₅₀, the fungicide concentration that results in 50% mycelial growth inhibition. For each isolate, a linear regression of the percent inhibition related to the mycelial growth of the control versus the log₁₀ of the fungicide concentration was obtained. The experiment was performed twice. Because the two experiments did not differ significantly (P > 0.05), the average EC₅₀ values from two experiments for each isolate were used in the data analysis.

Temperature sensitivity of M. fructicola isolates.

To determine the temperature sensitivity for isolates of M. fructicola that were benzimidazole sensitive (hereafter referred to as S isolates), isolates that had low resistance to benzimidazole (hereafter referred to as LR isolates), and isolates that had high resistance to benzimidazole (hereafter referred to as HR isolates), each of the 39 single-spore isolates (Table 1) was analyzed for its ability to grow at various temperatures on PDA with or without benomyl. A 5-mm mycelial plug taken from the edge of a 3-day-old colony of each isolate was transferred onto the PDA plate amended with benomyl at 0, 1, or 500 μg of a.i./ml. Three replicates of each fungicide concentration were used for each isolate. After the plates were incubated separately at 10, 15, 24, 31, or 35°C for 8 days in the dark, the mycelial growth was recorded for each plate. The experiment was performed twice.

Genetic relationships among the S, LR, and HR isolates.

To select representative isolates for analysis of DNA sequences of the β-tubulin gene, genetic relationships among S, LR, and HR isolates of M. fructicola were analyzed by using microsatellite-primed PCR (MP-PCR) (18, 19, 29). The 39 isolates of M. fructicola were grown in petri dishes containing 20 ml of potato dextrose broth (Difco Laboratories, Detroit, Mich.) at 24°C for 4 days. Mycelia were harvested and washed in sterile water, snap-frozen in liquid nitrogen, and lyophilized. Fungal genomic DNA from each isolate was extracted by using the FastDNAKit (Biogene Inc., Carlsbad, Calif.).

Four microsatellite primers, i.e., M13 (5′-GAGGGTGGCGGTTCT-3′), AAG₈, (AG)₈C, and (GACA)₄, which were found to be informative in a preliminary study, were used for MP-PCR. The primers were synthesized by Invitrogen Life Technologies (Grand Island, N.Y.). PCR was performed with an Eppendorf AG (Hamburg, Germany) Mastercycler in a 50-μl volume containing 50 ng of fungal genomic template, a 1.0 μM concentration of each microsatellite primer, a 0.2 mM concentration of each deoxynucleoside triphosphate (Promega, Madison, Wis.), 2.0 mM MgCl₂, 1× Promega Taq polymerase buffer (10 mM Tris-Cl [pH 9.0], 50 mM KCl, 0.1% Triton X-100), and 1.5 U of Promega Taq polymerase. The following PCR run parameters were used: an initial preheating for 3 min at 95°C, followed by 40 cycles of denaturation at 94°C for 1 min, annealing at 50°C for 1 min, and extension at 72°C for 1.5 min, with a final extension at 72°C for 10 min. PCR was performed twice for each isolate. Amplicons were separated on 1.5% agarose gels in Tris-acetate (TAE) buffer and photographed after being stained with ethidium bromide.

For analysis of the MP-PCR data set, each isolate was scored for the presence or absence of each amplicon by using Kodak Digital Science ID image analysis software (Eastman Kodak Co., Rochester, N.Y.). MP-PCR markers are considered dominant markers, since homozygotes could not be differentiated from heterozygotes without a progeny test. However, dominance of MP-PCR markers is not an issue for haploid fungi, e.g., M. fructicola (21). Genetic similarities (S) were calculated by the following formula: S = 2N_xy/(N_x + N_y), where N_xy is the number of bands in common and N_x and N_y are the numbers of bands found in isolates x and y, respectively (17). A phenogram was constructed by using the unweighted pair-group method with arithmetic average and the program SAHN (sequential, agglomerative, hierarchical, and nested clustering methods) of the software package NTSYS-pc 2.1 (Department of Ecology and Evolution, State University of New York). The analyses of Nei's unbiased genetic identity among the three populations of S, LR, and HR isolates were performed by using the computer software POPGENE (version 1.32; University of Alberta, Edmonton, Canada).

Isolation of the β-tubulin gene from M. fructicola.

Based on the MP-PCR data, 10 isolates (2 S isolates [LVN8 and ATF16], 4 LR isolates [33C7, 23B2, P1-5, and R2-9], and 4 HR isolates [SP3-42, R2-3, 32F5, and MS9]), which represented various MP-PCR profiles, were selected for analysis of the DNA sequence of the β-tubulin gene.

The conserved PCR primers TubA and TubR1 (Table 2) (22) were used to amplify the β-tubulin gene fragment from M. fructicola. The PCR was performed in a 50-μl volume containing 50 ng of fungal DNA, a 0.2 μM concentration of each primer, a 0.2 mM concentration of each deoxynucleoside triphosphate, 2.0 mM MgCl₂, 1× Promega Taq polymerase buffer, and 1.5 U of Promega Taq polymerase. The PCR was performed with the following parameters: an initial preheating for 3 min at 95°C, followed by 40 cycles of denaturation at 94°C for 40 s, annealing at 50°C for 40 s, and extension at 72°C for 1.5 min, with a final extension at 72°C for 10 min. PCR products were separated by electrophoresis in a 1.5% agarose gel in Tris-acetate (TAE) buffer.

TABLE 2.

PCR primers used to isolate the β-tubulin gene of M. fructicola

Primer	Sequence (5′-3′)	Location (5′-3′) in β-tubulin gene (bp)
TubA	AAA TGC GTG AGA TTG TA	−2→15
TubR1	TGT ACC AAT GCA AGA AAG CCT T	1610→1631
MfTF	TCG ACA ATA CAA AAT CAC TCC GC	495→517
MfTR3	GAC AGG TGG TAA CAC CGG ACA T	1068→1098
LRF	ACC AAG GGA CTT TTC AAT CTA CAG GTT T	124→151
LRR2	GTT CAT ACG CTC AAG TTG GAG GTC	440→463
HRF	TAA CAA CTG GAA GGC TTC CGG	518→538
HRR	CTC GTT ATC GAT ACA GAA GGT TG	964→986

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The PCR products from each isolate were purified by using the QIAquick gel extraction kit (Qiagen Inc., Valencia, Calif.). The purified fragments from each isolate were ligated into the pGEM-T Easy vector (Promega) and transformed into Escherichia coli (strain JM109) cells. Recombinant plasmids were purified by using the QIAprep Spin Miniprep kit (Qiagen Inc.). PCR and restriction endonuclease analyses of the recombinant plasmids confirmed that the PCR products from M. fructicola isolates had been cloned into the recombinant plasmids. The primers TubA and TubR1 were used in PCR analysis of the recombinant plasmids, and EcoRI (Promega) was used in restriction endonuclease analysis. The cloned fragments were sequenced by DBS Sequencing Inc. (Division of Biological Sciences, University of California at Davis) by using the primers T7 and SP6 and two internal primers, MfTF and MfTR3 (Table 2).

To compare the amino acid sequences of the β-tubulin genes from S, LR, and HR isolates, the DNA sequences of the β-tubulin genes from the representative 10 isolates of M. fructicola were translated into amino acid sequences by using standard code with the computer program EMBOSS Transeq (http://www.ebi.ac.uk/emboss/transeq/). The deduced amino acid sequences were aligned by using the computer program Clustal W 1.82 (http://www.ebi.ac.uk/clustalw/) (European Bioinformatics Institute, Cambridge, United Kingdom). The deduced amino acid sequences from two S isolates of M. fructicola were also compared with those from other phytopathogenic fungal species by using BLAST of NCBI/GenBank (http://www.ncbi.nlm.nih.gov/BLAST/).

Development of specific primers for detection of benzimidazole-resistant isolates of M. fructicola.

Based on the mutations in the β-tubulin gene conferring the LR and HR levels to benzimidazole in M. fructicola isolates, the primer sets LRF and LRR2 and HRF and HRR (Table 2) were designed to prime the β-tubulin gene from LR and HR isolates only. The forward primer LRF and the reverse primer HRR were designed to target the single point mutations at codons 6 and 198, respectively. The forward primer HRF and the reverse primer LRR2 were designed based on the sequences of two regions of β-tubulin from M. fructicola which were identified as unique from published β-tubulin sequences of other fungal species by using BLAST of NCBI/GenBank. Thus, the primers LRR2 and HRF might be specific to the β-tubulin gene from M. fructicola but not from those of other fungal species.

The 39 isolates of M. fructicola (Table 1) were used in specificity tests for primer pairs LRF and LRR2 and HRF and HRR. Twenty other fungal species were used as well (isolate collection numbers are in parentheses): Alternaria alternata (CH1), Aspergillus ochraceus (2568), Bipolaris spicifera (CH2), Botryosphaeria dothidea (MP1), Botryosphaeria rhodina (Br30), Botrytis cinerea (549), Cladosporium cladosporioides (1887), Curvularia inaequalis (CH3), Drechslera biseptata (CH6), Epicoccum purpurascens (CH7), Exserohilum parlierensis (CH26), Fusarium moniliforme (CH8), Humicola grisea (CH12), Monilinia laxa (515), Paecilomyces lilacinus (CH14), Penicillium expansum (CH15), Rhizomucor sp. (1886), Stemphylium botryosum (CH18), Trichoderma harzianum (CH20), and Ulocladium atrum (CH21). PCRs were performed as described above by using 50 ng of fungal DNA and a 0.2 μM concentration of each primer. Additionally, the internal transcribed spacer primers ITS1 and ITS4 (30) were used to test the efficacy of the DNA template. The PCR amplification parameters were an initial preheating for 3 min at 95°C, 35 cycles of denaturation at 94°C for 40 s, annealing at 56°C for the primers ITS1 and ITS4, 67°C for the primers HRF and HRR, or 69°C for the primers LRF and LRR2 for 40 s, extension at 72°C for 1 min, and a final extension at 72°C for 10 min. A 15-μl aliquot of PCR product from each sample was analyzed in a 1.5% agarose gel in TAE buffer.

Detection of benzimidazole-resistant isolates of M. fructicola from stone fruit flowers by PCR assays.

Flowers of peach and nectarine suspected of being infected by M. fructicola were collected from stone fruit orchards at Kearney Agricultural Center (Parlier, Calif.) and placed in humidified plastic containers to encourage sporulation and mycelial growth. Genomic DNA from M. fructicola on each flower sample was extracted by a modification of a previously published protocol (20). Briefly, after incubation of the flower samples at 24°C for 3 days, the resultant mycelia and conidia were removed from each flower and placed in a 1.5-ml microtube containing 30 μl of 1 M Tris-HCl (pH 8.0) amended with 0.05% Tween 20 (Mallinckrodt, Hazelwood, Mo.) and overlaid with 2 drops of mineral oil. The samples were boiled at 98 to 100°C for 15 min and immediately placed on ice for 5 min. After centrifugation at 10,000 × g for 2 min, a 2-μl aliquot of supernatant was used for PCR amplification. PCR primer pairs LRF and LRR2 and HRF and HRR were used to detect LR and HR isolates, respectively. The PCR amplifications were performed as described above.

To verify the PCR methods in assessing M. fructicola resistance to benzimidazoles, after conidia and mycelia were removed from each flower sample for DNA extraction the flowers were surface disinfected with 0.5% sodium hypochlorite for 3 min (10% commercial bleach; Western Family Foods, Inc., Portland, Oreg.), rinsed with sterile water, and placed on acidified (2.5 ml of 25% [vol/vol] lactic acid per liter) Difco PDA. After incubation at 24°C for 4 days, the resultant cultures of M. fructicola were used to determine their sensitivities to benomyl on PDA plates amended with benomyl as described above. The results from this conventional bioassay method were compared with those from the PCR assays for detecting benzimidazole resistance in M. fructicola.

Nucleotide sequence accession numbers.

The GenBank accession numbers of the 1,631-bp DNA fragment sequences of the β-tubulin gene from M. fructicola determined in this study are AY283676 through AY283685.

RESULTS

Sensitivity to benomyl and thiophanate-methyl in the isolates of M. fructicola from stone fruit.

Two different resistance levels (low and high resistance) were observed for 39 field isolates of M. fructicola based on their sensitivity to benomyl and thiophanate-methyl. The EC₅₀s of 12 S isolates to benomyl ranged from 0.02531 to 0.04827 μg/ml. The EC₅₀s of 17 LR isolates, with a range from 0.6097 to 0.9579 μg/ml, were significantly (P < 0.001) higher than those of the S isolates. The S and LR isolates had EC₅₀s to thiophanate-methyl ranging from 0.2644 to 0.7362 μg/ml and 2.3844 to 6.4212 μg/ml, respectively (Table 1). EC₅₀s of HR isolates to either benomyl or thiophanate-methyl were greater than 50 μg of a.i./ml.

After incubation at 24°C for 3 days, each of the 10 HR isolates was growing aggressively on PDA medium amended with benomyl at 500 μg of a.i./ml. The S isolates were unable to grow on the plates amended with benomyl at 1 μg of a.i./ml; however, the LR isolates were able to grow at this concentration but not at 5 μg of a.i. of benomyl/ml of medium. Thus, the 1 μg of a.i. of benomyl/ml was used to differentiate S from LR isolates and 500 μg of a.i. of benomyl/ml was used as the threshold concentration for monitoring HR isolates.

Sensitivity of the S, LR, and HR isolates of M. fructicola to low and high temperatures.

After incubation at various temperatures for 8 days, the LR and HR isolates of M. fructicola showed different temperature-sensitive phenotypes on PDA amended with benomyl. The LR isolates were unable to grow at 10 and 15°C on PDA amended with benomyl at 1 μg of a.i./ml but were able to grow at 24 and 31°C. The HR isolates failed to grow at 31°C on PDA amended with benomyl at 500 μg of a.i./ml but did grow at 10, 15, and 24°C (Table 3).

TABLE 3.

Effects of temperature on growth of S, LR, and HR isolates of M. fructicola on PDA plates amended with benomyl

Temp (°C)	Benomyl concn (μg a.i./ml)	Mycelial growth of M. fructicola isolates^a
Temp (°C)	Benomyl concn (μg a.i./ml)	S (n = 12)	LR (n = 17)	HR (n = 10)
10	0	+	+	+
	1	−	−	+
	500	−	−	+
15	0	+	+	+
	1	−	−	+
	500	−	−	+
24	0	+	+	+
	1	−	+	+
	500	−	−	+
31	0	+	+	+
	1	−	+	+
	500	−	−	−
35	0	−	−	−
	1	−	−	−
	500	−	−	−

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+, the isolates grew on the plates; −, the isolates did not grow.

Genetic relationships among the S, LR, and HR isolates of M. fructicola.

Four microsatellite primers generated 20 polymorphic bands from 39 isolates of M. fructicola. Examples of MP-PCR using the primer M13 are presented in Fig. 1. Analysis of MP-PCR data showed that genetic identities among the populations of S, LR, and HR isolates were very high. Nei's unbiased genetic identities between the populations of S and LR, S and HR, and LR and HR isolates were 0.9914, 0.9644, and 0.9971, respectively. The LR and HR isolates did not cluster independently from the S isolates (Fig. 2). MP-PCR profiles of some LR and HR isolates were the same as those of some S isolates (Fig. 2). Based on the MP-PCR data, four groups of isolates were identified (Fig. 2). One LR and one HR isolate from each group were selected for analyzing the sequences of the β-tubulin gene.

FIG. 1. — Electrophoretic separation of PCR amplicons of *Monilinia fructicola* isolates obtained from the primer M13.

FIG. 2. — Phenogram generated by the unweighted pair-group method with average cluster analysis of microsatellite-primed PCR data sets from S, LR, and HR isolates of *M. fructicola* collected from stone fruit in California.

Partial sequences of the β-tubulin gene from M. fructicola.

A single DNA fragment (1,631 bp) was amplified with the primer pair TubA and TubR1 (Table 2) from each of the 10 (2 S, 4 LR, and 4 HR) representative isolates tested. The section of the β-tubulin gene amplified by the primer pair TubA and TubR1 contained six introns and seven extrons. The two S isolates LVN8 and ATF16 had an identical sequence of 398 deduced amino acids. The sequenced section of the β-tubulin gene includes all positions in other fungi known to affect the sensitivity to benzimidazoles (1, 13). The deduced amino acid sequence of the β-tubulin gene from sensitive M. fructicola isolates showed homologies of 96% to that of Botryotinia fuckeliana (GenBank accession number P53373), 95% to Blumeria graminis f. sp. hordei (P16040) and Leptosphaeria maculans (AAF66992), 94% to Rhynchosporium secalis (P53376), and 93% to Phaeosphaeria nodorum (S30254).

All four HR and two S isolates sequenced, which represented various MP-PCR profiles, had the sequence CAT (histidine) at codon position 6 in the β-tubulin gene. But all four LR isolates were identified as having codon 6 converted from CAT (histidine) to TAT (tyrosine). As expected with other filamentous fungi, the S and LR isolates of M. fructicola had the sequence GAA (glutamic acid) at codon 198 in the β-tubulin gene. All four HR isolates had a punctual allelic change at codon 198, GCA (alanine) instead of GAA (glutamic acid). Apart from these mutations at codon 6s and 198, all 10 isolates analyzed had identical deduced amino acid sequences of the partial β-tubulin gene.

Specific primers for detection of benzimidazole resistance in M. fructicola.

The primer pair HRF and HRR amplified a 469-bp DNA fragment only from each of the 10 HR isolates tested and not from the 29 S and LR isolates or the 20 other fungal species listed in Materials and Methods (Fig. 3A and Table 1). The primer pair LRF and LRR2 amplified a 340-bp DNA fragment only from each of the 16 LR isolates tested and not from the 23 S and HR isolates or 20 other fungal species (Fig. 3B and Table 1).

FIG. 3. — Specificity of the primer pair HRF + HRR (A) and the primer pair LRF + LRR2 (B) for detection of isolates of *Monilinia fructicola* with high resistance (HR) and low resistance (LR), respectively, to benzimidazole.

Detection of benzimidazole-resistant isolates of M. fructicola from naturally infected flower samples.

DNA extracted from boiled conidia and mycelia, which were collected from flower samples, was suitable for PCR detection of HR isolates but not of LR isolates. The results from the PCR assay with the primer pair HRF and HRR detecting HR isolates were in agreement with those from the bioassay tests. Using the PCR assay with primer pair HRF and HRR, we observed that 5 of 32 flower samples were infected with HR isolates. The isolates recovered from these five flower samples were also identified as HR isolates by the conventional bioassay on PDA plates amended with benomyl at 500 μg of a.i./ml. Among the remaining 27 isolates, 2 and 25 were identified as LR and S isolates, respectively, by the conventional bioassay using the threshold concentration of benomyl at 1 μg of a.i./ml. In contrast to the detection of HR isolates, the two LR isolates were detected by PCR assay with the primers LRF and LRR2 only when DNAs extracted from the cultures of these two isolates with a FastDNA kit were used. The PCR amplification of DNA from boiled mycelia and conidia with the primer pair LRF and LRR2 was unable to detect the two LR isolates.

DISCUSSION

One of the objectives of this study was to characterize benzimidazole-resistant isolates of M. fructicola from stone fruit in California. Two distinct resistance levels, low and high resistance to benomyl and thiophanate-methyl, were observed for field isolates of M. fructicola. Analysis of the sequence of the β-tubulin gene showed that a single base pair mutation at codon 6 was responsible for the low resistance level to benzimidazoles in all the LR isolates of M. fructicola examined. The mutation resulted in a change at codon 6 from CAT (histidine) to TAT (tyrosine). The LR isolates were collected from different locations in different years and were also differentiated by MP-PCR, which indicated that this point mutation at codon 6 occurred independently in the LR isolates. To date, we have not found a report of a mutation at codon 6 conferring benzimidazole resistance in any field isolates from more than 15 phytopathogenic fungi (1, 13, 22), although the mutation at this locus was reported to occur in laboratory-induced benomyl-resistant mutants of A. nidulans (11), Septoria nodorum (2), and Trichoderma viride (8). Therefore, this study appears to be the first report of a mutation at codon 6 in the β-tubulin gene from field isolates of a benzimidazole-resistant phytopathogenic fungus. Binding studies have shown that the β-tubulin from an A. nidulans mutant with a change at codon 6 from histidine to leucine had a binding affinity for carbendazim (a benzimidazole fungicide) lower than that of wild-type tubulin (4, 11). These studies suggested that the resistance conferred by the mutation at codon 6 in the β-tubulin gene resulted from a direct effect on benzimidazole binding.

In this study, all 10 HR isolates of M. fructicola collected from different locations of California in different years had the same single base pair mutation at codon 198 of the β-tubulin gene. The mutation resulted in an alanine (GCA) replacing the glutamic acid (GAA). This mutation in HR isolates of M. fructicola collected from stone fruit orchards in California was different from a previously described mutation at this locus in a benomyl-resistant isolate of M. fructicola, which resulted in a lysine (AAA) replacing the glutamic acid (GAA) at codon 198 (13). The replacement of glutamic acid with either alanine or lysine at codon 198 had been detected benzimidazole-resistant field isolates of several other phytopathogenic fungi including Botrytis cinerea (15, 32) (a relative of M. fructicola), Helminthosporium solani (23), Penicillium spp., Sclerotinia homoeocarpa, V. inaequalis, Venturia pirina (13), Tapesia yallundae, and Tapesia acutormis (1). The direct involvement of these mutations at codon 198 in conferring resistance to benzimidazole has been confirmed by site-directed mutagenesis (7). Recently, using a gel filtration assay, Hollomon et al. (9) observed that benzimidazole fungicides indeed bound to β-tubulin and this binding was reduced by the mutation at codon 198 from glutamic acid to glycine.

Several benomyl-resistant mutants of model fungi, including A. nidulans and S. cerevisiae, exhibited temperature-sensitive phenotypes (3). A. nidulans benomyl-resistant strains with mutations at codon 50, 134, or 257 were temperature sensitive, and these mutants were expected to have reduced fitness under field conditions (13). Such effects of temperature sensitivity have never been reported for a plant-pathogenic fungus. In this study, we observed that the HR isolates were heat sensitive and did not show high resistance to benomyl at 31°C, while the LR isolates of M. fructicola were cold sensitive and did not show resistance to benomyl at temperatures below 15°C. Interestingly, the frequencies of LR isolates decreased from 84.6% in 1994 to 63.6% in 1995, 55.8% in 1998, and 25.4% in 2002 in California stone fruit orchards (our unpublished data). The decrease in frequency of LR isolates indicates that the LR isolates might indeed have a reduced fitness in the field, which had been proposed for the laboratory mutants of A. nidulans (13).

Brown rot causes significant losses in stone fruit production. Fungicide spray in the early bloom, such as at the green tip or popcorn stage, is conventionally applied in commercial orchards, and it is an important control strategy to reduce the risk of blossom blight, latent infection, and fruit rot in California (16). With the withdrawal of benomyl from the market, more and more growers will use thiophanate-methyl instead. This study showed that the LR isolates were not resistant to benomyl at low temperatures (below 15°C). Generally, the temperatures in the Central Valley of California during early bloom season are approximately 6 to 15°C. Thus, the benzimidazoles could still be effective in controlling the blossom blight caused by LR isolates at such low temperatures. This finding is in agreement with previous reports that in California stone fruit orchards with high populations of isolates resistant at 1 to 4 μg of a.i. of benomyl/ml, disease control with benomyl or carbendazim has been effective (25, 28). However, the benzimidazoles cannot control the blossom blight caused by HR isolates of M. fructicola. Laboratory inoculation tests also showed that a single application of thiophanate-methyl could select HR isolates and increase the frequency of an HR subpopulation by 50% (M. A. Yoshimura and T. J. Michailides, unpublished data). Since HR isolates have been detected in some stone fruit orchards in the Central Valley, the detection of HR isolates is critical before application of benzimidazole fungicides for control of blossom blight of stone fruit.

The current method for assessing benzimidazole resistance in M. fructicola requires the isolation of the pathogen as a pure culture and subsequent plating to a medium containing the fungicide. This procedure is labor-intensive and time-consuming if large numbers of isolates are to be tested. Advances in molecular biology, particularly the PCR, have provided new opportunities for rapidly detecting fungicide-resistant isolates of plant pathogens. The techniques of PCR linked with allele-specific probes (14), PCR-restriction fragment length polymorphism (15), and allele-specific PCR (5) have been used successfully to detect fungicide-resistant isolates of various plant pathogens. In this study, we developed an accurate, simple, and rapid method for the detection of benzimidazole HR isolates of M. fructicola without isolation of the fungus in a pure culture. However, the PCR amplification of DNA from boiled mycelia and conidia with the primer pair LRF and LRR2 was unable to detect the LR isolates on flower samples, although the LR isolates were identified by PCR using the DNA template extracted, with a FastDNA kit, from the cultures of those isolates recovered from flower samples. This was probably because the primer pair LRF and LRR2 required a very high annealing temperature (69°C) and the boiling method for DNA extraction from a small amount of mycelia and conidia produced on a blighted flower could not generate enough high-quality DNA template for the PCR analysis of LR isolates. However, the detection of HR isolates of M. fructicola is more important than the detection of LR isolates for chemical control of blossom blight of stone fruit, because the LR isolates did not show resistance at low temperatures during the early bloom season. Thus, the PCR method developed in this study for detection of HR isolates is expected to produce results in a timely fashion so that growers can select the appropriate registered fungicides against blossom blight of stone fruit.

The PCR assays developed in this study were not quantitative and detected only the presence of benzimidazole-resistant isolates of M. fructicola from stone fruits. However, the recently developed real-time PCR technique can be used to directly determine the amount of target DNA in a sample (27) and to rapidly detect frequencies of fungicide-resistant isolates in field pathogen populations (6). The PCR assays described in this study will be the basis for developing real-time PCR to determine the presence of M. fructicola resistance to benzimidazole at the population level in orchards. The real-time PCR technique should rapidly provide more-accurate information with which to evaluate the proficiency of strategies designed for the management of fungicide resistance.

In summary, in this study, we observed low and high levels of resistance to the benzimidazole fungicides in field isolates of M. fructicola. LR and HR isolates were also cold and heat sensitive, respectively. The LR and HR isolates had single point mutations at codons 6 and 198, respectively. Based on these point mutations in the β-tubulin gene, allele-specific PCR assays were developed for rapid detection of benzimidazole-resistant isolates of M. fructicola from stone fruit in California.

Acknowledgments

We thank Y. Luo for collecting isolates of M. fructicola and D. P. Morgan for technical assistance.

This research was supported partially by grant no. 2002-S1100-01990.

REFERENCES

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16.Luo, Y., D. P. Morgan, and T. J. Michailides. 2001. Risk analysis of brown rot blossom blight of prune caused by Monilinia fructicola. Phytopathology 91:759-768. [DOI] [PubMed] [Google Scholar]
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23.McKay, G. J., and L. R. Cook. 1997. A PCR-based method to characterize and identify benzimidazole resistance in Helminthosporium solani. FEMS Microbiol. 152:371-378. [DOI] [PubMed] [Google Scholar]
24.Michailides, T. J., J. M. Ogawa, and D. C. Opgenorth. 1987. Shift of Monilinia spp. and distribution of isolates sensitive and resistant to benomyl in California prune and apricot orchards. Plant Dis. 71:893-896. [Google Scholar]
25.Osorio, J. M., J. E. Adaskaveg, and J. M. Ogawa. 1994. Inhibition of mycelial growth of Monilinia species and suppression and control of brown rot blossom blight of almond with iprodione and E-0858. Plant Dis. 78:712-716. [Google Scholar]
26.Sanoamuang, N., R. E. Gaunt, and A. G. Fautrier. 1995. The segregation of resistance to carbendazim in sexual progeny of Monilinia fructicola. Mycol. Res. 99:677-680. [Google Scholar]
27.Schaad, N. W., and R. D. Frederick. 2002. Real-time PCR and its application for rapid plant disease diagnostics. Can. J. Plant Pathol. 24:250-258. [Google Scholar]
28.Sonoda, R. M., J. M. Ogawa, B. T. Manji, E. Shabi, and D. Rough. 1983. Factors affecting control of blossom blight in a peach orchard with low level benomyl-resistant Monilinia fructicola. Plant Dis. 67:681-684. [Google Scholar]
29.Weising, K., R. G. Atkinson, and R. C. Gardner. 1995. Genomic fingerprinting by microsatellite-primed PCR: a critical evaluation. PCR Methods Appl. 4:249-255. [DOI] [PubMed] [Google Scholar]
30.White, T. J., T. Bruns, S. Lee, and J. Taylor. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics, p. 315-322. In M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White (ed.), PCR protocols: a guide to methods and applications. Academic Press, San Diego, Calif.
31.Yan, K., and M. B. Dickman. 1996. Isolation of a β-tubulin gene from Fusarium moniliforme that confers cold-sensitive benomyl resistance. Appl. Environ. Microbiol. 62:3053-3056. [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Yarden, O., and T. Katan. 1993. Mutations leading to substitutions at amino acid 198 and 200 of beta-tubulin that correlate with benomyl-resistant phenotypes of field strains of Botrytis cinerea. Phytopathology 83:1478-1483. [Google Scholar]
33.Zehr, E. I., J. E. Toler, and L. A. Luszcz. 1991. Spread and persistence of benomyl-resistant Monilinia fructicola in South Carolina peach orchards. Plant Dis. 75:590-593. [Google Scholar]

[r1] 1.Altertini, C., M. Gredt, and P. Leroux. 1999. Mutations of the β-tubulin gene associated with different phenotypes of benzimidazole resistance in the cereal eyespot fungi Tapesia yallundae and Tapesia acuformis. Pesticide Biochem. Physiol. 64:17-31. [Google Scholar]

[r2] 2.Cooley, R. N., and C. E. Caten. 1993. Molecular analysis of the Septoria nodorum beta-tubulin gene and characterization of a benomyl-resistant mutation. Mol. Gen. Genet. 237:58-64. [DOI] [PubMed] [Google Scholar]

[r3] 3.Davidse, L. C. 1986. Benzimidazole fungicides: mechanism of action and biological impact. Annu. Rev. Phytopathol. 24:43-65. [Google Scholar]

[r4] 4.Davidse, L. C., and W. Flach. 1977. Differential binding of methyl-benzimidazole-2-yl carbamate to fungal tubulin as a mechanism of resistance to this antimitotic agent in mutant strains of Aspergillus nidulans. J. Cell Biol. 72:174-193. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r5] 5.Délye, C., F. Laigret, and M. F. Corio-Costet. 1997. A mutation in the 14α-demethylase gene of Uncinula necator that correlates with resistance to a sterol biosynthesis inhibitor. Appl. Environ. Microbiol. 63:2966-2970. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r6] 6.Fraaije, B. A., J. A. Butters, J. M. Coelho, D. R. Jones, and D. W. Hollomon. 2002. Following the dynamics of strobilurin resistance in Blumeria graminis f. sp. tritici using quantitative allele-specific real-time PCR measurements with the fluorescent dye SYBR green I. Plant Pathol. 51:45-54. [Google Scholar]

[r7] 7.Fujimura, M., T. Kamakura, S. Inoue, and I. Yamaguchi. 1992. Sensitivity of Neurospora crassa to benzimidazoles and N-phenylcarbamates: effect of amino acid substitutions at position 198 in β-tubulin. Pesticide Biochem. Physiol. 44:165-167. [Google Scholar]

[r8] 8.Goldman, G. H., W. Temmerman, D. Jacobs, R. Contreras, M. Vanmontagu, and A. Herreraestrella. 1993. A nucleotide substitution in one of the beta-tubulin genes of Trichoderma viride confers resistance to the antimitotic drug methyl benzimidazole-2-yl carbamate. Mol. Gen. Genet. 240:73-80. [DOI] [PubMed] [Google Scholar]

[r9] 9.Hollomon, D. W., J. A. Butters, H. Barker, and L. Hall. 1998. Fungal β-tubulin, expressed as fusion protein, binds benzimidazole and phenylcarbamate fungicides. Antimicrob. Agents Chemother. 9:2171-2173. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r10] 10.Jones, A. L., and G. R. Ehret. 1976. Isolation and characterization of benomyl-tolerant strains of Monilinia fructicola. Plant Dis. Rep. 60:765-769. [Google Scholar]

[r11] 11.Jung, M. K., I. B. Wilder, and B. R. Oakley. 1992. Amino-acid alterations in the BenA (beta-tubulin) gene of Aspergillus nidulans that confer benomyl resistance. Cell Motil. Cytoskelet. 22:170-174. [DOI] [PubMed] [Google Scholar]

[r12] 12.Kawchuk, L. M., L. J. Hutchison, C. A. Verhaeghe, D. R. Lynch, P. S. Bains, and J. D. Holley. 2002. Isolation of the β-tubulin gene and characterization of thiabendazole resistance in Gibberella pulicaris. Can. J. Plant Pathol. 24:233-238. [Google Scholar]

[r13] 13.Koenraadt, H., S. C. Somerville, and A. L. Jones. 1992. Characterization of mutations in the beta-tubulin gene of benomyl-resistant field strains of Venturia inaequalis and other plant pathogenic fungi. Phytopathology 82:1348-1354. [Google Scholar]

[r14] 14.Koenraadt, H., and A. L. Jones. 1992. The use of allele-specific oligonucleotide probes to characterize resistance to benomyl in field strains of Venturia inaequalis. Phytopathology 82:1354-1358. [Google Scholar]

[r15] 15.Luck, J. E., and M. R. Gillings. 1995. Rapid identification of benomyl resistant strains of Botrytis cinerea using the polymerase chain reaction. Mycol. Res. 99:1483-1488. [Google Scholar]

[r16] 16.Luo, Y., D. P. Morgan, and T. J. Michailides. 2001. Risk analysis of brown rot blossom blight of prune caused by Monilinia fructicola. Phytopathology 91:759-768. [DOI] [PubMed] [Google Scholar]

[r17] 17.Lynch, M. 1990. The similarity index and DNA fingerprinting. Mol. Biol. Evol. 7:478-484. [DOI] [PubMed] [Google Scholar]

[r18] 18.Ma, Z., E. W. Boehm, Y. Luo, and T. J. Michailides. 2001. Population structure of Botryosphaeria dothidea from pistachio and other hosts in California. Phytopathology 91:665-672. [DOI] [PubMed] [Google Scholar]

[r19] 19.Ma, Z., and T. J. Michailides. 2002. Characterization of Botryosphaeria dothidea isolates collected from pistachio and other plant hosts in California. Phytopathology 92:519-526. [DOI] [PubMed] [Google Scholar]

[r20] 20.Ma, Z., and T. J. Michailides. 2002. A PCR-based technique for identification of Fusicoccum sp. from pistachio and various other hosts in California. Plant Dis. 86:515-520. [DOI] [PubMed] [Google Scholar]

[r21] 21.McDonald, B. A. 1997. The population genetics of fungi: tools and techniques. Phytopathology 87:448-453. [DOI] [PubMed] [Google Scholar]

[r22] 22.McKay, G. J., D. Egan, E. Morris, and A. E. Brown. 1998. Identification of benzimidazole resistance in Cladobotryum dendroides using a PCR-based method. Mycol. Res. 102:671-676. [Google Scholar]

[r23] 23.McKay, G. J., and L. R. Cook. 1997. A PCR-based method to characterize and identify benzimidazole resistance in Helminthosporium solani. FEMS Microbiol. 152:371-378. [DOI] [PubMed] [Google Scholar]

[r24] 24.Michailides, T. J., J. M. Ogawa, and D. C. Opgenorth. 1987. Shift of Monilinia spp. and distribution of isolates sensitive and resistant to benomyl in California prune and apricot orchards. Plant Dis. 71:893-896. [Google Scholar]

[r25] 25.Osorio, J. M., J. E. Adaskaveg, and J. M. Ogawa. 1994. Inhibition of mycelial growth of Monilinia species and suppression and control of brown rot blossom blight of almond with iprodione and E-0858. Plant Dis. 78:712-716. [Google Scholar]

[r26] 26.Sanoamuang, N., R. E. Gaunt, and A. G. Fautrier. 1995. The segregation of resistance to carbendazim in sexual progeny of Monilinia fructicola. Mycol. Res. 99:677-680. [Google Scholar]

[r27] 27.Schaad, N. W., and R. D. Frederick. 2002. Real-time PCR and its application for rapid plant disease diagnostics. Can. J. Plant Pathol. 24:250-258. [Google Scholar]

[r28] 28.Sonoda, R. M., J. M. Ogawa, B. T. Manji, E. Shabi, and D. Rough. 1983. Factors affecting control of blossom blight in a peach orchard with low level benomyl-resistant Monilinia fructicola. Plant Dis. 67:681-684. [Google Scholar]

[r29] 29.Weising, K., R. G. Atkinson, and R. C. Gardner. 1995. Genomic fingerprinting by microsatellite-primed PCR: a critical evaluation. PCR Methods Appl. 4:249-255. [DOI] [PubMed] [Google Scholar]

[r30] 30.White, T. J., T. Bruns, S. Lee, and J. Taylor. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics, p. 315-322. In M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White (ed.), PCR protocols: a guide to methods and applications. Academic Press, San Diego, Calif.

[r31] 31.Yan, K., and M. B. Dickman. 1996. Isolation of a β-tubulin gene from Fusarium moniliforme that confers cold-sensitive benomyl resistance. Appl. Environ. Microbiol. 62:3053-3056. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r32] 32.Yarden, O., and T. Katan. 1993. Mutations leading to substitutions at amino acid 198 and 200 of beta-tubulin that correlate with benomyl-resistant phenotypes of field strains of Botrytis cinerea. Phytopathology 83:1478-1483. [Google Scholar]

[r33] 33.Zehr, E. I., J. E. Toler, and L. A. Luszcz. 1991. Spread and persistence of benomyl-resistant Monilinia fructicola in South Carolina peach orchards. Plant Dis. 75:590-593. [Google Scholar]

PERMALINK

Identification and Characterization of Benzimidazole Resistance in Monilinia fructicola from Stone Fruit Orchards in California

Zhonghua Ma

Michael A Yoshimura

Themis J Michailides

Abstract

MATERIALS AND METHODS