Characterization and PCR-based detection of benzimidazole-resistant isolates of Monilinia laxa in California

Monilinia laxa is a pathogen of brown rot of stone fruit and almond in California, causing blossom blights and fruit rots. In this study, low-level resistance to the benzimidazole fungicides benomyl and thiophanate-methyl was detected in field isolates of M laxa collected from stone fruits and almonds in California. Low-resistant (LR) isolates grew in potato dextrose agar (PDA) plates amended with benomyl and thiophanate-methyl at 1 and 5 microg ml(-1), respectively, but not in plates amended with benomyl at 5 microg ml(-1) or thiophanate-methyl at 50 microg ml(-1). The benzimidazole LR isolates were characterized by temperature sensitivity and the DNA sequence of the beta-tubulin gene. The LR isolates showed high-temperature sensitivity, being sensitive to 1 microg ml(-1) of benomyl at 28 degrees C but resistant at 8-24 degrees C. Analysis of the DNA sequence of the beta-tubulin gene showed that the LR isolates had a point mutation at the amino-acid position 240, causing substitution of leucine by phenylalanine. Based on the point mutation, a pair of allele-specific PCR primers was developed for rapid detection of LR isolates of M laxa. In addition, a pair of PCR primers specific to M laxa was developed on the basis of the differences in the DNA sequence of the intron 6 of beta-tubulin gene from M laxa, M fructicola and other fungal species. The primer pair amplified the expected 376-bp DNA fragment from all M laxa isolates tested, but not from 14 other fungal species isolated from stone fruit and almond crops. The restriction endonuclease BsmA I recognized the sequence GTCTCC in the PCR products from sensitive (S) isolates only, but not the GTTTCC sequence in the PCR products from LR isolates. The endonuclease digested the 376-bp PCR products from S isolates to produce two bands (111 and 265 bp) on agarose gels. Thus, both allele-specific PCR and the PCR-restriction fragment length polymorphism (PCR-RFLP) methods could be useful for rapidly detecting benzimidazole-resistant isolates of M laxa from stone fruit and almond crops in California.     

Quantification of airborne spores of <Emphasis Type="Italic">Monilinia fructicola</Emphasis> in stone fruit orchards of California using real-time PCR

The fungal pathogen Monilinia fructicola causes blossom blight and fruit brown rot of stone fruits in California. In this study, spore densities in the air were monitored in six orchard/year combinations with Burkard spore traps. A real-time PCR assay was developed to efficiently quantify the dynamics of spore density in these orchards during the growing season. Different patterns of dynamics of spore density were observed in these orchards. A linear relationship between numbers of spores counted with a compound microscope and those determined with the real-time PCR assay was obtained, using the same samples of spore traps. Spore density in five of six orchard/year combinations ranged from 0.0 to 0.05 spores l⁻¹, except for that in orchard 4, which showed much higher values of spore density in the air, as well as higher values and wider range of incidences of blossom infection and fruit rot than those in the other orchards. The results demonstrated a potential method to quantitatively determine spore inoculum potential in orchards by using a real-time PCR assay.     

Using real-time PCR to survey frequency of azoxystrobin-resistant allele G143A in Alternaria populations from almond and pistachio orchards in California

Alternaria spp. cause leaf spot of almond and Alternaria late blight of pistachio in California, and azoxystrobin is a strobilurin fungicide that has been registered for the control of these diseases. To date, only a single point mutation of G143A in cytochrome b resulting to azoxystrobin resistance in Alternaria spp. was found in California. Based on this single point mutation, a real-time PCR assay was developed to quantify the frequency of the resistant allele G143A (FA) in pathogen samples taken from orchards. Forty-one almond and pistachio orchards were arbitrarily selected in eight counties of California. Fifty leaf lesions caused by Alternaria spp. per orchard were cut to extract the fungal DNA for a real-time PCR assay to determine the FA. About 88% of 41 surveyed orchards had Alternaria spp. with FA > 0.90, while six pistachio orchards showed a FA < 0.90. Therefore, azoxystrobin-resistant Alternaria populations are predominant in almond and pistachio orchards in California, and sprays of azoxystrobin to control Alternaria diseases are not recommended in these orchards. This study shows a potential use of a real-time PCR assay to efficiently quantify the frequency of azoxystrobin-resistant Alternaria spp. from large number of samples.     

Inoculum dynamics, fruit infection, and development of brown rot in prune orchards in california

ABSTRACT Brown rot, caused by Monilinia fructicola, is a destructive disease of stone fruit in California. Disease management requires information on inoculum dynamics and development of latent and visible fruit infections during the season to help make decisions on timing of fungicide treatments and choice of cultural practices. In this study, the daily spore concentration (ascospores and conidia) of M. fructicola in the air was monitored with spore traps in two prune orchards during the growing seasons in 2001 and 2002. The spore concentrations were low to moderate at early bloom, increased at full bloom, and decreased to the lowest level at the end of bloom. Improper timing of fruit thinning and irrigation in midseason increased spore concentration in the air and fruit infections late in the season. Artificial fruit inoculations were conducted periodically in 10 prune orchards in 2002 and 2004, and incidence of fruit rot at different inoculation dates was assessed. Fruit rot development rate increased linearly with inoculation date during the growing season. Natural blossom and fruit infections were monitored periodically in 10 prune orchards, and incidence of latent fruit infection was determined by using the overnight freezing-incubation technique. Incidence of fruit rot also was assessed 2 weeks before harvest in these orchards. The incidence of latent fruit infection at the pit hardening stage significantly correlated with that at the late stages and with the incidence of fruit rot at harvest.     

Species-Specific Detection of Monilinia fructicola from California Stone Fruits and Flowers

ABSTRACT A set of molecular diagnostics was developed for Monilinia fructicola, causal agent of brown rot of stone fruits, capable of sensitive detection of the pathogen in planta. Species-specific repetitive sequences were identified from a partial library of 312 recombinant clones hybridized with total DNA, followed by subsequent screening for specificity. One hundred isolates, comprising 12 fungal species common to California stone fruits, were surveyed for specificity. Three clones hybridized to 60 geographically diverse M. fructicola isolates (California, Michigan, Georgia, Oregon, and Australia) to the exclusion of all other fungi surveyed, including the closely related M. laxa (n = 12). Two clones were identical and of extrachromosomal origin (pMF73 and pMF150), whereas the third (pMF210) migrated with uncut DNA. The sensitivity of all three was comparable and capable of detecting 50 pg of fungal DNA in dot blot hybridizations. Six species-specific primer pair sets were designed. They maintained the same specificity patterns observed in the initial hybridization surveys and were sensitive enough to detect 50 fg of fungal DNA template, approximately equivalent to 10 spores. The species-specific clones were capable of detecting the pathogen in planta, specifically from infected plum flowers and nectarine fruit tissue, using both hybridization- and polymerase chain reaction-based methodologies.     

Early brown rot infections in sweet cherry fruit are detected by monilinia-specific DNA primers

ABSTRACT Visible and nonvisible quiescent infections of immature and mature fruit are an integral component of the disease cycle of brown rot of sweet cherry in California. Detection of these infections is critical for developing efficient and efficacious fungicide management programs. The previously published DNA amplification primers mfs3 and NS5 for the identification of Monilinia fructicola were very specific in amplifying DNA of M. fructicola only and not M. laxa. This primer set, however, only detected DNA from some of the California isolates of M. fructicola. This genetic diversity was supported by random amplified polymorphic DNA (RAPD) analysis. Using eight 10-mer primers, seven M. fructicola isolates from California were all identified as genetically distinct. Using the same primers, only one polymorphism was detected among seven isolates of M. laxa. The multiple genotypes identified within the small population sample of M. fructicola, but not of M. laxa, using RAPD analysis could be indicative of genetic recombination within M. fructicola but not within M. laxa. To detect early brown rot infections in fruit, two primer sets that were developed from DNA sequences of either ribosomal DNA (MF5/ITS4/ITS3) or a RAPD fragment (X-09intF3/X-09R) specifically amplified DNA from isolates of M. fructicola and Monilinia species, respectively. No amplification products were present when using DNA from Botrytis cinerea or from other fungi commonly found on sweet cherry fruit. Primers X-09intF3 and X-09R were more sensitive and reliable for detecting small amounts of target DNA either extracted from conidia or from laboratory-inoculated cherry fruit with early brown rot infections that showed no visual symptoms or with visible quiescent infections. Furthermore, these primers also were effective for detecting visible quiescent infections in cherry fruit that were collected in the field.     

Monitoring of Venturia inaequalis harbouring the QoI resistance G143A mutation in French orchards as revealed by PCR assays

BACKGROUND: Genetic resistance to QoI fungicides may account for recent failures to control Venturia inaequalis (Cooke) Winter in French orchards. Two PCR-based assays were developed to detect the G143A point mutation in the fungal mitochondrial cytochrome b gene. The mutation is known to confer a high level of resistance to QoI fungicides. Occurrence of the G143A mutation in French field isolates collected from 2004 to 2007 was monitored. RESULTS: The QoI-resistant cytochrome b allele was specifically detected either following the cleavage of the amplified marker by a restriction endonuclease (CAPS assay) or its amplification using an allele-specific PCR primer. Using either method, the G143A mutation was found in 42% of the 291 field samples originating from French orchards in which apple scab proved difficult to be controlled. Monitoring of the G143A mutation in orchards located in 15 French administrative regions indicated that the mutation was detected at least once in nine of the regions, and its presence ranged from 33% to 64% of the orchards analysed in 2004 and in 2007 respectively. CONCLUSION: The PCR-based methods developed in this study efficiently reveal the presence of the G143A mutation in French V. inaequalis field populations.     

Genotyping Common Bean for the Potyvirus Resistance Alleles I and bc-1(2) with a Multiplex Real-Time Polymerase Chain Reaction Assay

ABSTRACT A multiplex real-time polymerase chain reaction (PCR) assay was developed to simultaneously genotype plants for the I and bc-1(2) alleles, which condition resistance in beans to Bean common mosaic virus and Bean common mosaic necrosis virus. A segregating F(2) population was derived from the cross between pinto bean breeding line P94207-189A (bc-1 bc-1 I I) and Olathe (bc-12 bc-1(2) i i). Real-time PCR assays were developed that were specific for each allele, and a multiplex PCR reaction could unambiguously assign F(2) plants to one of nine genotypes. Remnant F(1) plants were used as a comparative reference sample. PCR results among this sample fit a normal distribution for both real-time PCR assays, and 99% probability distributions were determined for heterozygotes. F(2) plants were genotyped based on results relative to the probability distributions for heterozygotes. F(2) plants also were genotyped for the I and bc-1(2) alleles by performing F(3) family progeny tests for virus resistance. Agreement between the two methods was 100% (198/198) for the bc-1(2) allele, and 92.4% (183/198) for the I allele. Erroneous genotyping was due to recombination between the amplicon and the I allele. Realtime PCR assays provide a robust method for genotyping seedlings and, in some cases, may eliminate the need for progeny testing.     

High‐throughput detection of highly benzimidazole‐resistant allele E198A with mismatch primers in allele‐specific real‐time polymerase chain reaction

BACKGROUND: A point mutation often confers resistance of organisms against medical drugs and agricultural pesticides. Allele‐specific nucleotide polymerase chain reaction (ASPCR) and allele‐specific quantitative real‐time PCR using SYBR Green (ASQPCR) are widely and effectively applied to detect and monitor this type of resistance. However, the former is unsuitable for high‐throughput detection, and the latter often reduces the accuracy of detection. RESULTS: In order to decrease background amplification, a rapid and high‐throughput genotyping method with mismatch primers was developed (ASQPCR‐MP) and applied specifically to survey the frequency of the highly benzimidazole‐resistant MBC^HR mutation (E198A) in the β‐tubulin gene of Sclerotinia sclerotiorum (Lib.) de Bary populations. Genomic DNA from 223 sclerotia was analysed. Similar genotype results were also obtained using ASPCR with mismatch primers and a mycelial growth inhibition assay. It was found that ASQPCR‐MP clearly differentiated MBC^HR and benzimidazole‐sensitive MBC^S phenotypes. Moreover, ASQPCR‐MP took less than 6 h to complete. CONCLUSION: ASQPCR‐MP appears suitable for large epidemiological studies involving resistant genotypes and requiring high‐throughout formats. Copyright © 2009 Society of Chemical Industry     

The biological characteristics of dicarboximide-resistant isolates of Monilinia fructicola from New Zealand stone-fruit orchards

Dicarboximide-resistant isolates of Monilinia fructicola were detected in New Zealand stone fruit orchards in 1985. The EC₅₀ values of resistant isolates ranged from 3 to 217 mg a.i./l iprodione, compared with 0-3 to 0-7 mg a.i./l for sensitive isolates. The degree of dicarboximide resistance was maintained over nine generations in fruit tissues in most isolates, but in four isolates there was a significant decline. Three resistant and two sensitive isolates were selected for further study on agar and host tissues. Resistant isolates caused disease, grew and sporulated as well as sensitive isolates on flowers not amended with dicarboximide fungicides, but some were less fit on fruit. The pathogenicity and spore production of a resistant strain on flowers was reduced significantly by dicarboximide applied prior to pathogen inoculation. Fitness of isolates on flowers and on fruit was poorly correlated. Resistant isolates were significantly less competitive than sensitive isolates when mixed inocula were applied to flowers and fruit. The relative performance on flowers and fruit was not a reliable indicator of competitive ability.     

应用real-time PCR定量检测小麦条锈菌潜伏侵染量方法的建立

 小麦条锈病是我国小麦主要病害之一。快速、及时地诊断与定量监测处于潜育状态下的病叶,对准确估计越冬、越夏后的病情,制定正确的防治方案具有重要的意义。根据小麦条锈菌Puccinia striiformis的β-tubilin基因序列设计对该病原菌的种具有特异性的引物betaf/betar,并分别在普通PCR和real-time PCR扩增时对该引物的特异性和灵敏性进行了测定。结果表明该引物对小麦条锈菌特异性高,可稳定扩增出243 bp的目标条带。Real-time PCR的灵敏度为普通PCR的100倍。应用此特异性引物,建立了real-time PCR测定系统,定量测定了条锈菌在小麦叶片接种后组织内的DNA随时间的变化。结果表明,在接种后12 h,可在小麦叶片内检测到条锈菌,且条锈菌在小麦叶片内潜育期间随时间呈指数增长。接种第6 d后叶片内的菌量有明显的增加。建立的小麦条锈菌的real-time PCR早期定量测定方法,为及时、快速监测小麦条锈病在潜育期间的发病规律以及为该病的预测、防治提供依据。     

Using allele-specific oligonucleotide probes to characterize benzimidazole resistance in Rhynchosporium secalis

Benzimidazole fungicides are important mixture components in strategies to combat fungicide resistance in Rhynchosporium secalis Davis. To monitor the performance of these strategies, a rapid, accurate assay has been developed to detect point mutations in the β-tubulin gene which confers resistance of benzimidazoles. The β-tubulin gene of a benzimidazole-resistant strain of R. secalis has been cloned and sequenced. Except for the difference in the position of one of its six introns, this gene showed a strong homology with other β-tubulin genes from filamentous fungi. Resistance was related to a point mutation in codon 198 which caused a glutamic acid to glycine change in resistant field strains, but glutamic acid to lysine in a laboratory mutant. A DNA fragment surrounding codon 198 was amplified directly from diseased lesions using a ‘nested’ set of PCR primers. Combining PCR amplificiation of a target DNA sequence with hybridization of Allele-Specific Oligonucleotide probes (ASO_s, 15-mers) allowed accurate detection of benzimidazole resistance. Only two probes, one sensitive and one resistant, were sufficient to monitor current field populations. Detection was achieved using either ³²P-labelled probe, or non-radioactively using a biotin-labelled probe coupled to streptavidin/alkaline phosphatase. This rapid method using ASOS can detect benzimidazole resistance within 48 h compared with 6–8 weeks by conventional assay procedures.     

A real‐time (TaqMan) PCR assay to differentiate Monilinia fructicola from other brown rot fungi of fruit crops

To prevent the entry and spread of the brown rot fungus Monilinia fructicola in Europe, a fast and reliable method for detection of this organism is essential. In this study, an automated DNA extraction method combined with a multiplex real‐time PCR based on TaqMan chemistry was developed for fast, convenient and reliable detection of both the EU quarantine organism Monilinia fructicola and the three other brown rot fungi M. fructigena, M. laxa and Monilia polystroma. Using the internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene repeat, a Monilinia genus‐specific primer pair and two differently labelled fluorogenic probes specific for M. fructicola and the group M. fructigena/M. laxa/Monilia polystroma were developed. The analytical specificity of the assay was assessed by testing 33 isolates of the four brown rot fungi and 13 isolates of related fungal species or other fungal species that can be present on stone and pome fruit. No cross‐reactions were observed. The assay was found to have a detection limit of 0·6 pg of DNA, corresponding to 27 haploid genomes or four conidia. Comparison of a manual DNA isolation followed by a conventional PCR with an automated DNA isolation combined with the presently developed real‐time PCR showed that the latter method gave improved results when tested with 72 naturally infected stone fruit samples. The detection rate increased from 65 to 97%.     

Molecular characterization of the two-component histidine kinase gene from Monilinia fructicola

Brown rot of stone fruit caused by Monilinia fructicola (G. Wint) Honey is one of the most common fungal diseases in California. In this study, two laboratory-induced iprodione-resistant (LIR) mutants of M. fructicola were characterized by osmotic sensitivity, virulence on prune and sequence of the two-component histidine kinase gene (Mfos-1). The LIR mutants showed more sensitivity to osmotic stress and lower virulence on prune than their wild-type parent. Analysis of deduced amino acid of Mfos-1 showed that this protein exhibited all the characteristic features of the two-component histidine kinase genes, including osmotic sensing domain, six 90-amino-acid repeat motifs (coiled coil region) and kinase core and response regulator domains. Comparison of DNA sequences of the Mfos-1 from LIR mutants and the wild-type sensitive (S) isolate showed that LIR mutants had single point mutations in the coiled coil region of Mfos-1.     

Pcr Assay for specific detection of European stone fruit yellows phytoplasmas and its use for epidemiological studies in France

DNA amplification by polymerase chain reaction was used to specifically detect phytoplasmas associated with severe decline diseases of European stone fruits. PCR primers were designed according to the partial sequence of a nonribosomal genomic fragment of European stone fruit yellows phytoplasmas obtained by direct sequencing of a specific PCR product. A PCR assay was developed which resulted in specific amplification of a 237 bp-DNA fragment from total DNA extracts derived from over 300 stone fruit samples. No PCR product was obtained with DNA from healthy controls or plants diseased with various other phytoplasmas, e.g. the closely related apple proliferation and pear decline phytoplasmas. Phytoplasma infection was checked in all samples by PCR amplification with universal ribosomal primers. Detection rate with specific and universal primers was correlated by 97%. European stone fruit yellows phytoplasmas were detected in samples of 114 out of 139 examined orchards which represent the major stone fruit growing regions of France. Typical symptoms like chlorotic leaf roll in summer and off-season growth in winter were correlated by 95% to the presence of phytoplasmas. However, phytoplasmas were also detected in 51% of samples derived from trees showing non-specific symptoms. A comparison study including 201 samples showed that 81% of the PCR-positive samples were also tested positive using fluorescence microscopy with DAPI staining.     

Factors Affecting Latent Infection of Prune Fruit by Monilinia fructicola

ABSTRACT Experiments were conducted in three prune orchards in California. In each orchard, inoculations with Monilinia fructicola, the causal agent of brown rot of stone fruits, were performed on branches of trees at bloom and fruit developmental stages. Five inoculum concentrations were used in each inoculation. Six and four wetness durations were created for each inoculum concentration at bloom and fruit developmental stages, respectively. Fruit were harvested 3 weeks before commercial harvest. The overnight freezing incubation technique was used to promote sporulation and to determine incidence of latent infection (ILI) of fruit brown rot. No differences in ILI among locations were found. A seasonal pattern of bloom and fruit susceptibility to latent infection was determined. Susceptibility to latent infection at bloom stage was at a moderate level and increased to reach the highest level at pit hardening stage. Subsequently, fruit susceptibility to latent infection decreased, reaching the lowest level in early June at embryo growth stage. Thereafter, the susceptibility increased again with fruit development and maturity until harvest. Linear relationships between ILI and inoculum concentration were obtained for most combinations of growth stage and wetness duration. Incidence of latent infection increased linearly with increased wetness duration at bloom stage and increased exponentially with increased wetness duration at early and late fruit developmental stages. The optimum temperatures for latent infection at pit hardening stage ranged from 14 to 18 degrees C, but the effect of temperature on latent infection was reduced at resistant stages. The temperature range favorable to latent infection varied for different wetness durations.     

不同类型Monilinia fructicola菌株的温度适应性及其对苯并咪唑类杀菌剂和乙霉威的交互抗性

褐腐病菌Monilinia fructicola是引起多种果树褐腐病的重要病原菌,前期研究发现该病原菌对甲基硫菌灵的抗性与Tub2蛋白的多个氨基酸变异有关.为明确不同类型菌株的温度适应性及乙霉威是否对所有抗性类型菌株均具有抑菌活性,本研究测定了敏感型菌株S及3种抗性类型包括R(E198A)、R(E198Q)及R(F20...     

Identification of a novel point mutation in the <Emphasis Type="Italic">β</Emphasis>-tubulin gene of <Emphasis Type="Italic">Botrytis cinerea</Emphasis> and detection of benzimidazole resistance by a diagnostic PCR-RFLP assay

The molecular basis of resistance to benzimidazole fungicides with laboratory and field mutant isolates of Botrytis cinerea was investigated. After chemical mutagenesis with N-methyl-N-nitrosogouanidine (NMNG) two different benzimidazole-resistant phenotypes were isolated on media containing carbendazim or a mixture of carbendazim and diethofencarb. The mutant isolates from the fungicide-mixture-containing medium were moderately resistant to carbendazim with wild-type tolerance to diethofencarb while mutant isolates from carbendazim-containing medium were highly resistant to carbendazim but sensitive to diethofencarb. The studied field isolates were highly resistant to benzimidazoles and sensitive to diethofencarb. Study of fitness characteristics of benzimidazole highly-resistant isolates showed that the resistance mutation(s) had no apparent effect on fitness-determining parameters. Contrary to this, the moderately benzimidazole-resistant strains, with no increased diethofencarb sensitivity, had a significant reduction in certain ecological fitness-determining characteristics. Analysis of the sequence of the β-tubulin gene revealed two amino acid replacements in the highly benzimidazole-resistant mutants compared to that of the wild-type parent strain. One was the glutamic acid (GAG) to alanine (GCG) change at position 198 (E198A), identified in both laboratory and field highly benzimidazole-resistant isolates, a mutation previously implicated in benzimidazole resistance. The second was a novel benzimidazole resistance mutation of glutamic acid (GAG) to glycine (GGG) substitution at the same position 198 (E198G), identified in a highly benzimidazole-resistant laboratory mutant strain. Molecular analysis of the moderately benzimidazole-resistant strains revealed no mutations at the β-tubulin gene. A novel diagnostic PCR-RFLP assay utilising a BsaI restriction site present in the benzimidazole-sensitive (E198) but absent in both resistant genotypes (E198G and E198A) was developed for the detection of both amino acid replacements at the β-tubulin gene.     

Detection and quantification of Rhizoctonia cerealis in soil using real-time PCR

Rhizoctonia cerealis E.P. van der Hoeven (anamorph of Ceratobasidium cereale D.I. Murray and Burpee), which causes sharp eyespot in wheat, is a major soilborne fungal pathogen that severely impairs yield and quality of winter wheat in China. Because the pathogen is difficult to identify and quantify in soil using conventional methods, a rapid and reliable method is needed to detect and quantify the fungus. In this study, we developed an SYBR Green-based quantitative real-time polymerase chain reaction assay for specific, sensitive detection and quantification of R. cerealis in soil samples. Using a specific primer pair based on the β-tubulin gene of the fungal DNA sequence, we could specifically detect R. cerealis at quantities as low as 100?fg of purified pathogen DNA. Using the real-time PCR assays, we were able to quantify R. cerealis in artificially and naturally infested soil samples. This new technique to quantify R. cerealis is rapid and accurate and will be a useful tool for future studies of pathogenic R. cerealis.     

Molecular characterization and a multiplex allele-specific PCR method for detection of thiabendazole resistance in Penicillium expansum from apple

Thiabendazole (TBZ) is commonly used as a postharvest treatment for control of blue mold in apples caused by Penicillium expansum. Different point mutations in the β-tubulin gene conferring benzimidazole resistance have been reported in plant pathogens, but molecular mechanisms of TBZ resistance in P. expansum from apple in Washington State are unknown. Determination of TBZ resistance level showed that all 102 TBZ-resistant (TBZ-R) isolates were highly resistant. Sequencing of the majority of the β-tubulin gene showed that 76 TBZ-R isolates harboured the E198V mutation and 26 harboured the F167Y mutation, and all the sensitive isolates did not possess any of the mutations, indicating that these two point mutations in the β-tubulin gene were correlated with TBZ resistance in P. expansum from apple in Washington State. There was no association between levels of TBZ resistance and types of point mutations (E198V or F167Y) in the β-tubulin gene. A multiplex allele-specific PCR assay was developed to detect these two mutations simultaneously. Microsatellite-primed PCR derived presence-absence matrix used to assess the genetic relationship among 56 isolates suggested that the resistance mutations originated several times independently and that there was no correlation between the types of point mutation and the genetic background of the isolates.