- Email: [email protected]
A real-time PCR assay for the detection of azoxystrobin-resistant Alternaria populations from pistachio orchards in California
ARTICLE IN PRESS
Crop Protection 23 (2004) 1259–1263 www.elsevier.com/locate/cropro
Short communication
A real-time PCR assay for the detection of azoxystrobin-resistant Alternaria populations from pistachio orchards in California Zhonghua Ma1, T.J. Michailides Department of Plant Pathology, University of California, Davis, Kearney Agricultural Center, 9240 South Riverbend Avenue, Parlier, CA 93648, USA Received 11 March 2004; received in revised form 28 May 2004; accepted 28 May 2004
Abstract Azoxystrobin-resistant populations of Alternaria spp. in the alternata, tenuissima, and arborescens species–groups, the causal agents of Alternaria late blight of pistachio, have been selected in pistachio orchards in California. The azoxystrobin resistance in Alternaria spp. was found to be correlative to a single point mutation resulting in the replacement of a glycine by an alanine at codon 143 (G143A) in the mitochondrial cytochrome b (cyt b) gene. Based on this point mutation, a real-time PCR method was developed to rapidly detect azoxystrobin-resistant Alternaria populations in field leaf samples collected from pistachio orchards in California. r 2004 Elsevier Ltd. All rights reserved. Keywords: Alternaria late blight of pistachio; QoI fungicides; Real-time PCR
1. Introduction Alternaria late blight caused by Alternaria spp. in the alternata, tenuissima, and arborescens species–groups is one of the most common fungal diseases of pistachio in California, and affects foliage and fruit (Pryor and Michailides, 2002). The strobilurin fungicide azoxystrobin (Abounds ) provided excellent control of this disease. However, azoxystrobin-resistant Alternaria populations had been selected in some pistachio orchards after multiple sprays of azoxystrobin for only a few years (Ma et al., 2003). In a previous study, we found a single point mutation resulting in the replacement of a glycine by an alanine at codon 143 (G143A) in the mitochondrial cytochrome b (cyt b) from all azoxystrobin resistant Alternaria isolates (Ma et al., 2003). Based on this point mutation, we developed PCR-restriction fragment length polymorphism (PCR-RFLP) (Ma et al., 2003) and allele-specific PCR methods (Ma and
Michailides, 2004) to rapidly detect azoxystrobin resistant Alternaria isolates. Compared to the classical spore germination assay, the PCR-RFLP and allelespecific PCR assays can rapidly detect azoxystrobin resistance for individual isolates/disease lesions. However, these methods are not quantitative and are time consuming if large numbers of samples need to be tested. Recently, a real-time PCR method has been used successfully to quantify azoxystrobin-resistant Blumeria graminis on wheat (Fraaije et al., 2002). Thus, the objective of this study was to develop a real-time PCR method to quantitatively detect azoxystrobin resistant Alternaria spp. from pistachios in California. Rapid quantitative detection of azoxystrobin resistance in Alternaria populations will be valuable to pistachio growers by enabling timely decisions on management of azoxystrobin resistance in commercial orchards.
2. Materials and methods Corresponding author. Tel.: +1-559-646-6546; fax: +1-559-6466593. E-mail address: [email protected] (T.J. Michailides). 1 Present address: Department of Plant Pathology, Michigan State University, East Lansing, Michigan 48824.
0261-2194/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.cropro.2004.05.012
2.1. Sample collection and DNA extraction In 2003, 50 disease lesions (0.5 cm in diameter) were collected from each of six pistachio orchards with
ARTICLE IN PRESS Z. Ma, T.J. Michailides / Crop Protection 23 (2004) 1259–1263
1260
AGA GAT GTA AAT AAT GGG TGA T-30 ) + ARR4 (50 -AAG GTT AGT AAT AAC TGT TGC AG-30 ), which is specific to resistant genotype (Ma and Michailides, 2004), was used to detect resistant allele only. The reverse primer ARR4 was designed to match the point mutation A143 at the 30 end of the primer. Real-time PCR amplifications were performed with the DNA Engine Opticons 2 System (MJ Research, Waltham, MA, USA) using the SYRB Green I fluorescent dye detection. Amplifications were conducted in 25-ml volumes containing 12:5 ml DyNAmos HS master mix (Finnzymes Oy, Finland), 2-ml template DNA extracted from disease lesions, and 2 ml of both forward and reverse primers (4 mM each). To create a standard curve, 10-fold serial dilutions of resistant genotype fungal DNA (ranging from 0.001 to 100 ng), which was extracted from the resistant isolate 37E3, spiked with 10 ng pistachio leaf DNA, were used for each experiment. Additionally, each experiment also included a positive control (template DNA from the resistant isolate 37B8) and negative controls (template DNA from the sensitive isolates 3A2 and A039, and no template DNA). There were two replicates for each sample, and the experiment was performed twice. The PCR amplifications were performed using the following parameters: an initial pre-heat for 15 min at 95 C, followed by 40 cycles at 94 C for 15 s, 55 C for the primer pair ARF4+ARR4, or 58 C for the primer pair AF+AR for 25 s, 72 C for 30 s, and 73 C for 1 s in
different histories of azoxystrobin application (Table 1). DNA from each 50 disease lesions was extracted by using the FastDNAs kit (Qbiogene, Carlsbad, CA, USA) with the buffer Cell Lysis/DNA Solubilizing Solution for Vegetation (CLS-VF) according to the manufacturer’s protocol. The DNA was suspended in 200 ml H2 O, and further purified with an addition of 200 ml of the Binding Matrix Solution of FastDNAs kit. The final DNA was resuspended in 50 ml H2 O, and 2-ml aliquots of 1:10 DNA dilutions were used for realtime PCR amplifications. DNA from pure cultures of azoxystrobin-resistant Alternaria isolates 37E3 and 37B8, and sensitive isolates A039 and 3A2, and from healthy pistachio leaves was also extracted by the FastDNAs kit and quantified using the Hoefers DyNA Quant 200 Fluorometer (Hoefer Pharmacia Biotech. Inc., San Francisco, CA, USA). 2.2. Real-time PCR amplification Since there are no differences in the partial DNA sequence of the cyt b genes from A. alternata, A. tenuissima, and A. arborescens, the primer pair AF (50 -ACA CTG CTT CAG CAT TTT TCT TCA TAG30 )+AR (50 -TTG TCC AAT TCA TGG TAT AGC ACT CA-30 ), which is specific to these Alternaria spp. from pistachio (Ma et al., 2003), was used to detect the Alternaria spp. [including both resistant (A143) and sensitive (G143) alleles]. The primer pair ARF4 (50 -ATG
Table 1 Quantification of azoxystrobin-resistant genotype of Alternaria spp. in the alternata, tenuissima, and arborescens species–groups in California pistachio orchards Sample
Amount of resistant (A143) and sensitive (G143) alleles SE (ng)a
Amount of resistant allele (A143) SE (ng)a
Percent of resistant genotype SE (%)a
Negative control 1 (without template DNA) Negative control 2 (template DNA extracted from the sensitive isolate A039) Negative control 3 (template DNA extracted from the sensitive isolate 3A2) Positive control (template DNA extracted from the resistant isolate 37B8) Fresno Co. (KAC1)b
0
0
0
9.520.42
0.150.05
1.570.55
61.121.52
0.190.01
0.300.03
9.770.08
9.710.07
99.361.50
1.330.04
0.100.01
7.110.32
0.750.01 0.640.05 1.88 0.09 1.76 0.11 1.530.11
0.010.00 0.010.00 1.780.01 1.62 0.04 1.130.02
1.810.39 1.620.51 94.703.87 92.78.00 74.576.65
Fresno Co. (KAC2)c Fresno Co. (KAC3)c Kern Co. Glenn Co. Sacramento Co. a
History of azoxystrobin application
Two applications on some trees in this orchards No application No application Multiple applications Multiple applications Multiple applications
Values are the mean of two experiments SE. Samples from experimental orchards 1, 2, and 3 at Kearney Agricultural Center, University of California, Parlier, CA. c Samples from experimental orchards 1, 2, and 3 at Kearney Agricultural Center, University of California, Parlier, CA. b
ARTICLE IN PRESS Z. Ma, T.J. Michailides / Crop Protection 23 (2004) 1259–1263
order to detect and quantify the fluorescence at a temperature above the denaturation of primer-dimers. Once amplifications were completed, melting curves were obtained based on a standard protocol (refer to manual) and used to identify PCR products. After the amplifications were completed, data were analyzed by using the DNA Engine Opticons 2 software (version 2.02). Because there were no differences in the DNA fragments of the cyt b genes amplified by the primer pair AF+AR from azoxystrobin-sensitive and -resistant isolates except for the point mutation at G143A, this primer pair amplified an expected 226-bp fragment from both sensitive and resistant isolates. Standard curves in the amplifications with this primer pair were used to
1261
detect the amount of both A143 and G143 alleles in each sample. The threshold cycle (Ct) was recorded as the PCR cycle, in which the gain in fluorescence generated by the accumulating amplicon exceeds 10 standard deviations of the mean baseline fluorescence, using data taken from cycles 1 to 10. For detection of azoxystrobin-resistant genotype, the primer pair ARF4+ARR4 was used (Ma and Michailides, 2004). In amplifications with the primers ARF4+ARR4, standard curves based on threshold cycles (Ct) for 10-fold dilution series of resistant genotype template DNA were used to detect the amount of resistant allele A143 in each sample. Subsequently, a frequency of resistant allele was calculated by dividing the amount of resistant allele (A143) by the amount of both resistant (A143) and sensitive (G143) alleles for each sample. To confirm that expected PCR products were amplified, amplicons of each sample were also analyzed in 2% agarose gels in 1TAE buffer.
3. Results In real-time PCR amplifications with the primer pair AF+AR, different SYBR Green I signal intensities
Y = -0.24x + 7.64; R 2 = 0.994 Log Quantity
5 4 3 2 1 0 10
15
20
25
30
35
C(T) Cycle
(A)
Y = -0.24x + 8.31; R 2 = 0.991 Log Quantity
5 4 3 2 1 0 15 Fig. 1. Quantification of both A143 (resistant) and G143 (sensitive) alleles of Alternaria by real-time PCR with the primer pair AF+AR, which is specific to Alternaria spp. in the alternata, tenuissima, and arborescens species–groups in California pistachio orchards: (A) Kinetics of SYBR Green I fluorescence signal at different concentrations of template DNA extracted from the azoxystrobin-resistant isolate 37E3; (B) Agarose gel (2%) electrophoresis of real-time PCR products from different concentrations of template DNA; (C) The melting curve of the real-time PCR product.
(B)
20
25
30
35
C (T) Cycle
Fig. 2. Standard curve obtained by plotting the amount of A143 allele of Alternaria by real-time PCR with: (A) the primer pair AF+AR, which is specific to Alternaria spp. in the alternata, tenuissima, and arborescens species–groups, and (B) primer pair ARF4+ARR4, which is specific to azoxystrobin-resistant Alternaria spp. in the alternata, tenuissima, and arborescens species–groups in California pistachio orchards, versus the threshold cycle.
ARTICLE IN PRESS 1262
Z. Ma, T.J. Michailides / Crop Protection 23 (2004) 1259–1263
For quantification of azoxystrobin-resistant allele (A143), the primer pair ARF4+ARR4 amplified the expected 246-bp DNA fragment from serial dilutions of resistant genotype DNA spiked with 10 ng pistachio leaf DNA (Figs. 3A–C). Using the standard curve (Fig. 2B), the amounts of resistant allele in the tested samples were calculated, subsequently, the percent of resistant allele in each sample was obtained (Table 1). The results showed that the three orchards with multiple applications of azoxystrobin had high frequencies of resistant genotype. The frequencies of resistant genotype in the two orchards without a history of azoxystrobin application were close to negative controls with the template DNA from the sensitive isolates (Table 1).
4. Discussion
Fig. 3. Quantification of azoxystrobin-resistant allele (A143) of Alternaria by real-time PCR with the primer pair ARF4+ARR4, which is specific to azoxystrobin-resistant Alternaria spp. in the alternata, tenuissima, and arborescens species–groups in California pistachio orchards: (A) Kinetics of SYBR Green I fluorescence signal at different concentrations of template DNA extracted from the azoxystrobin-resistant isolate 37E3; (B) Agarose gel (2%) electrophoresis of real-time PCR products from different amounts of template DNA extracted from the azoxystrobin-resistant isolate 37E3; and (C) The melting curve of the real-time PCR product.
were detected from serial dilutions of template DNA from a resistant isolate spiked with 10 ng pistachio leaf DNA (Fig. 1A). An expected 226-bp DNA fragment amplified by this primer pair was determined by both the electrophoresis of PCR product (Fig. 1B) and melting curve analysis (Fig. 1C). Using the DNA Engine Opticon 2 software, a standard curve (Fig. 2A) was generated, and the amounts of both A143 and G143 alleles in the tested samples (Table 1) were calculated by comparing Ct values to the crossing point values of the linear regression line of the standard curve.
Determination of fungicide sensitivity in pathogen populations is the first important step in management of fungicide resistance. The real-time PCR assay described in this paper is rapid and applicable for high-throughput routine resting. This method can be used to rapidly evaluate the efficacy of different anti-resistant programs. Using this real-time PCR method, frequencies of resistant Alternaria populations from at least 60 orchards can be detected within two workdays. Obviously, this assay will enable pistachio growers to rapidly adjust fungicide applications based on the test results. The strobilurin fungicides have a single-site mode of action. They inhibit mitochondrial respiration by binding to the Qo site of the cytochrome bc1 enzyme complex, thus blocking electron transfer in the respiration pathway (Bartlett et al., 2002). It is well known that single-site fungicides generally possess a high risk of resistance development if resistant mutants are not impaired in their ability to survive and reproduce in the agricultural environment. In a previous study, a single point mutation resulting in the replacement of a glycine by an alanine at codon 143 (G143A) in the mitochondrial cytochrome b (cyt b) has been found in all azoxystrobin-resistant isolates of Alternaria from California pistachio (Ma et al., 2003). Results from this study also showed that azoxystrobin-resistant Alternaria populations have become predominant in all three sampled commercial pistachio orchards where azoxystrobin was sprayed for several times. However, also in California pistachio orchards, after multiple applications of azoxystrobin for more than 5 years with three to five applications/season, azoxystrobin resistance has not been detected in another pistachio pathogen Botryosphaeria dothidea, the causal agent of panicle and shoot blight of pistachio (Michailides et al., unpublished data). Currently, azoxystrobin and other strobilurin fungicides are considered the most effective compounds
ARTICLE IN PRESS Z. Ma, T.J. Michailides / Crop Protection 23 (2004) 1259–1263
for controlling panicle and shoot blight of pistachio. Because both Botryosphaeria panicle and shoot blight and Alternaria late blight have been frequently found together in some pistachio orchards, when growers spray azoxystrobin to control panicle and shoot blight, they should be concerned with the selection pressure of azoxystrobin on Alternaria populations since Alternaria populations could develop resistance rapidly to this compound.
Acknowledgements This work was partially supported by grants to T. J. Michailides from the California Pistachio Commission. We thank D. Morgan and D. Felts for assistance with the figures of this manuscript.
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References Bartlett, D.W., Clough, J.M., Godwin, J.R., Hall, A.A., Hamer, M., Parr-Dobrzanski, B., 2002. The strobilurin fungicides. Pest Manage. Sci. 58, 649–662. Fraaije, B.A., Butters, J.A., Coelho, J.M., Jones, D.R., Hollomon, D.W., 2002. Following the dynamics of strobilurin resistance in Blumeria graminis f. sp. tritici using quantitative allele-specific realtime PCR measurements with the fluorescent dye SYBR green I. Plant Pathol. 51, 45–54. Ma, Z., Michailides, T.J., 2004. An allele-specific PCR assay for detecting azoxystrobin-resistant Alternaria isolates from pistachio in California. J. Phytopathol. 152, 118–121. Ma, Z., Felts, D., Michailides, T.J., 2003. Resistance to azoxystrobin in Alternaria isolates from pistachio in California. Pesticide Biochem. Physiol. 77, 66–74. Pryor, B.M., Michailides, T.J., 2002. Morphological, pathogenic, and molecular characterization of Alternaria isolates associated with Alternaria late blight of pistachio. Phytopathology 92, 406–416.
Crop Protection 23 (2004) 1259–1263 www.elsevier.com/locate/cropro
Short communication
A real-time PCR assay for the detection of azoxystrobin-resistant Alternaria populations from pistachio orchards in California Zhonghua Ma1, T.J. Michailides Department of Plant Pathology, University of California, Davis, Kearney Agricultural Center, 9240 South Riverbend Avenue, Parlier, CA 93648, USA Received 11 March 2004; received in revised form 28 May 2004; accepted 28 May 2004
Abstract Azoxystrobin-resistant populations of Alternaria spp. in the alternata, tenuissima, and arborescens species–groups, the causal agents of Alternaria late blight of pistachio, have been selected in pistachio orchards in California. The azoxystrobin resistance in Alternaria spp. was found to be correlative to a single point mutation resulting in the replacement of a glycine by an alanine at codon 143 (G143A) in the mitochondrial cytochrome b (cyt b) gene. Based on this point mutation, a real-time PCR method was developed to rapidly detect azoxystrobin-resistant Alternaria populations in field leaf samples collected from pistachio orchards in California. r 2004 Elsevier Ltd. All rights reserved. Keywords: Alternaria late blight of pistachio; QoI fungicides; Real-time PCR
1. Introduction Alternaria late blight caused by Alternaria spp. in the alternata, tenuissima, and arborescens species–groups is one of the most common fungal diseases of pistachio in California, and affects foliage and fruit (Pryor and Michailides, 2002). The strobilurin fungicide azoxystrobin (Abounds ) provided excellent control of this disease. However, azoxystrobin-resistant Alternaria populations had been selected in some pistachio orchards after multiple sprays of azoxystrobin for only a few years (Ma et al., 2003). In a previous study, we found a single point mutation resulting in the replacement of a glycine by an alanine at codon 143 (G143A) in the mitochondrial cytochrome b (cyt b) from all azoxystrobin resistant Alternaria isolates (Ma et al., 2003). Based on this point mutation, we developed PCR-restriction fragment length polymorphism (PCR-RFLP) (Ma et al., 2003) and allele-specific PCR methods (Ma and
Michailides, 2004) to rapidly detect azoxystrobin resistant Alternaria isolates. Compared to the classical spore germination assay, the PCR-RFLP and allelespecific PCR assays can rapidly detect azoxystrobin resistance for individual isolates/disease lesions. However, these methods are not quantitative and are time consuming if large numbers of samples need to be tested. Recently, a real-time PCR method has been used successfully to quantify azoxystrobin-resistant Blumeria graminis on wheat (Fraaije et al., 2002). Thus, the objective of this study was to develop a real-time PCR method to quantitatively detect azoxystrobin resistant Alternaria spp. from pistachios in California. Rapid quantitative detection of azoxystrobin resistance in Alternaria populations will be valuable to pistachio growers by enabling timely decisions on management of azoxystrobin resistance in commercial orchards.
2. Materials and methods Corresponding author. Tel.: +1-559-646-6546; fax: +1-559-6466593. E-mail address: [email protected] (T.J. Michailides). 1 Present address: Department of Plant Pathology, Michigan State University, East Lansing, Michigan 48824.
0261-2194/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.cropro.2004.05.012
2.1. Sample collection and DNA extraction In 2003, 50 disease lesions (0.5 cm in diameter) were collected from each of six pistachio orchards with
ARTICLE IN PRESS Z. Ma, T.J. Michailides / Crop Protection 23 (2004) 1259–1263
1260
AGA GAT GTA AAT AAT GGG TGA T-30 ) + ARR4 (50 -AAG GTT AGT AAT AAC TGT TGC AG-30 ), which is specific to resistant genotype (Ma and Michailides, 2004), was used to detect resistant allele only. The reverse primer ARR4 was designed to match the point mutation A143 at the 30 end of the primer. Real-time PCR amplifications were performed with the DNA Engine Opticons 2 System (MJ Research, Waltham, MA, USA) using the SYRB Green I fluorescent dye detection. Amplifications were conducted in 25-ml volumes containing 12:5 ml DyNAmos HS master mix (Finnzymes Oy, Finland), 2-ml template DNA extracted from disease lesions, and 2 ml of both forward and reverse primers (4 mM each). To create a standard curve, 10-fold serial dilutions of resistant genotype fungal DNA (ranging from 0.001 to 100 ng), which was extracted from the resistant isolate 37E3, spiked with 10 ng pistachio leaf DNA, were used for each experiment. Additionally, each experiment also included a positive control (template DNA from the resistant isolate 37B8) and negative controls (template DNA from the sensitive isolates 3A2 and A039, and no template DNA). There were two replicates for each sample, and the experiment was performed twice. The PCR amplifications were performed using the following parameters: an initial pre-heat for 15 min at 95 C, followed by 40 cycles at 94 C for 15 s, 55 C for the primer pair ARF4+ARR4, or 58 C for the primer pair AF+AR for 25 s, 72 C for 30 s, and 73 C for 1 s in
different histories of azoxystrobin application (Table 1). DNA from each 50 disease lesions was extracted by using the FastDNAs kit (Qbiogene, Carlsbad, CA, USA) with the buffer Cell Lysis/DNA Solubilizing Solution for Vegetation (CLS-VF) according to the manufacturer’s protocol. The DNA was suspended in 200 ml H2 O, and further purified with an addition of 200 ml of the Binding Matrix Solution of FastDNAs kit. The final DNA was resuspended in 50 ml H2 O, and 2-ml aliquots of 1:10 DNA dilutions were used for realtime PCR amplifications. DNA from pure cultures of azoxystrobin-resistant Alternaria isolates 37E3 and 37B8, and sensitive isolates A039 and 3A2, and from healthy pistachio leaves was also extracted by the FastDNAs kit and quantified using the Hoefers DyNA Quant 200 Fluorometer (Hoefer Pharmacia Biotech. Inc., San Francisco, CA, USA). 2.2. Real-time PCR amplification Since there are no differences in the partial DNA sequence of the cyt b genes from A. alternata, A. tenuissima, and A. arborescens, the primer pair AF (50 -ACA CTG CTT CAG CAT TTT TCT TCA TAG30 )+AR (50 -TTG TCC AAT TCA TGG TAT AGC ACT CA-30 ), which is specific to these Alternaria spp. from pistachio (Ma et al., 2003), was used to detect the Alternaria spp. [including both resistant (A143) and sensitive (G143) alleles]. The primer pair ARF4 (50 -ATG
Table 1 Quantification of azoxystrobin-resistant genotype of Alternaria spp. in the alternata, tenuissima, and arborescens species–groups in California pistachio orchards Sample
Amount of resistant (A143) and sensitive (G143) alleles SE (ng)a
Amount of resistant allele (A143) SE (ng)a
Percent of resistant genotype SE (%)a
Negative control 1 (without template DNA) Negative control 2 (template DNA extracted from the sensitive isolate A039) Negative control 3 (template DNA extracted from the sensitive isolate 3A2) Positive control (template DNA extracted from the resistant isolate 37B8) Fresno Co. (KAC1)b
0
0
0
9.520.42
0.150.05
1.570.55
61.121.52
0.190.01
0.300.03
9.770.08
9.710.07
99.361.50
1.330.04
0.100.01
7.110.32
0.750.01 0.640.05 1.88 0.09 1.76 0.11 1.530.11
0.010.00 0.010.00 1.780.01 1.62 0.04 1.130.02
1.810.39 1.620.51 94.703.87 92.78.00 74.576.65
Fresno Co. (KAC2)c Fresno Co. (KAC3)c Kern Co. Glenn Co. Sacramento Co. a
History of azoxystrobin application
Two applications on some trees in this orchards No application No application Multiple applications Multiple applications Multiple applications
Values are the mean of two experiments SE. Samples from experimental orchards 1, 2, and 3 at Kearney Agricultural Center, University of California, Parlier, CA. c Samples from experimental orchards 1, 2, and 3 at Kearney Agricultural Center, University of California, Parlier, CA. b
ARTICLE IN PRESS Z. Ma, T.J. Michailides / Crop Protection 23 (2004) 1259–1263
order to detect and quantify the fluorescence at a temperature above the denaturation of primer-dimers. Once amplifications were completed, melting curves were obtained based on a standard protocol (refer to manual) and used to identify PCR products. After the amplifications were completed, data were analyzed by using the DNA Engine Opticons 2 software (version 2.02). Because there were no differences in the DNA fragments of the cyt b genes amplified by the primer pair AF+AR from azoxystrobin-sensitive and -resistant isolates except for the point mutation at G143A, this primer pair amplified an expected 226-bp fragment from both sensitive and resistant isolates. Standard curves in the amplifications with this primer pair were used to
1261
detect the amount of both A143 and G143 alleles in each sample. The threshold cycle (Ct) was recorded as the PCR cycle, in which the gain in fluorescence generated by the accumulating amplicon exceeds 10 standard deviations of the mean baseline fluorescence, using data taken from cycles 1 to 10. For detection of azoxystrobin-resistant genotype, the primer pair ARF4+ARR4 was used (Ma and Michailides, 2004). In amplifications with the primers ARF4+ARR4, standard curves based on threshold cycles (Ct) for 10-fold dilution series of resistant genotype template DNA were used to detect the amount of resistant allele A143 in each sample. Subsequently, a frequency of resistant allele was calculated by dividing the amount of resistant allele (A143) by the amount of both resistant (A143) and sensitive (G143) alleles for each sample. To confirm that expected PCR products were amplified, amplicons of each sample were also analyzed in 2% agarose gels in 1TAE buffer.
3. Results In real-time PCR amplifications with the primer pair AF+AR, different SYBR Green I signal intensities
Y = -0.24x + 7.64; R 2 = 0.994 Log Quantity
5 4 3 2 1 0 10
15
20
25
30
35
C(T) Cycle
(A)
Y = -0.24x + 8.31; R 2 = 0.991 Log Quantity
5 4 3 2 1 0 15 Fig. 1. Quantification of both A143 (resistant) and G143 (sensitive) alleles of Alternaria by real-time PCR with the primer pair AF+AR, which is specific to Alternaria spp. in the alternata, tenuissima, and arborescens species–groups in California pistachio orchards: (A) Kinetics of SYBR Green I fluorescence signal at different concentrations of template DNA extracted from the azoxystrobin-resistant isolate 37E3; (B) Agarose gel (2%) electrophoresis of real-time PCR products from different concentrations of template DNA; (C) The melting curve of the real-time PCR product.
(B)
20
25
30
35
C (T) Cycle
Fig. 2. Standard curve obtained by plotting the amount of A143 allele of Alternaria by real-time PCR with: (A) the primer pair AF+AR, which is specific to Alternaria spp. in the alternata, tenuissima, and arborescens species–groups, and (B) primer pair ARF4+ARR4, which is specific to azoxystrobin-resistant Alternaria spp. in the alternata, tenuissima, and arborescens species–groups in California pistachio orchards, versus the threshold cycle.
ARTICLE IN PRESS 1262
Z. Ma, T.J. Michailides / Crop Protection 23 (2004) 1259–1263
For quantification of azoxystrobin-resistant allele (A143), the primer pair ARF4+ARR4 amplified the expected 246-bp DNA fragment from serial dilutions of resistant genotype DNA spiked with 10 ng pistachio leaf DNA (Figs. 3A–C). Using the standard curve (Fig. 2B), the amounts of resistant allele in the tested samples were calculated, subsequently, the percent of resistant allele in each sample was obtained (Table 1). The results showed that the three orchards with multiple applications of azoxystrobin had high frequencies of resistant genotype. The frequencies of resistant genotype in the two orchards without a history of azoxystrobin application were close to negative controls with the template DNA from the sensitive isolates (Table 1).
4. Discussion
Fig. 3. Quantification of azoxystrobin-resistant allele (A143) of Alternaria by real-time PCR with the primer pair ARF4+ARR4, which is specific to azoxystrobin-resistant Alternaria spp. in the alternata, tenuissima, and arborescens species–groups in California pistachio orchards: (A) Kinetics of SYBR Green I fluorescence signal at different concentrations of template DNA extracted from the azoxystrobin-resistant isolate 37E3; (B) Agarose gel (2%) electrophoresis of real-time PCR products from different amounts of template DNA extracted from the azoxystrobin-resistant isolate 37E3; and (C) The melting curve of the real-time PCR product.
were detected from serial dilutions of template DNA from a resistant isolate spiked with 10 ng pistachio leaf DNA (Fig. 1A). An expected 226-bp DNA fragment amplified by this primer pair was determined by both the electrophoresis of PCR product (Fig. 1B) and melting curve analysis (Fig. 1C). Using the DNA Engine Opticon 2 software, a standard curve (Fig. 2A) was generated, and the amounts of both A143 and G143 alleles in the tested samples (Table 1) were calculated by comparing Ct values to the crossing point values of the linear regression line of the standard curve.
Determination of fungicide sensitivity in pathogen populations is the first important step in management of fungicide resistance. The real-time PCR assay described in this paper is rapid and applicable for high-throughput routine resting. This method can be used to rapidly evaluate the efficacy of different anti-resistant programs. Using this real-time PCR method, frequencies of resistant Alternaria populations from at least 60 orchards can be detected within two workdays. Obviously, this assay will enable pistachio growers to rapidly adjust fungicide applications based on the test results. The strobilurin fungicides have a single-site mode of action. They inhibit mitochondrial respiration by binding to the Qo site of the cytochrome bc1 enzyme complex, thus blocking electron transfer in the respiration pathway (Bartlett et al., 2002). It is well known that single-site fungicides generally possess a high risk of resistance development if resistant mutants are not impaired in their ability to survive and reproduce in the agricultural environment. In a previous study, a single point mutation resulting in the replacement of a glycine by an alanine at codon 143 (G143A) in the mitochondrial cytochrome b (cyt b) has been found in all azoxystrobin-resistant isolates of Alternaria from California pistachio (Ma et al., 2003). Results from this study also showed that azoxystrobin-resistant Alternaria populations have become predominant in all three sampled commercial pistachio orchards where azoxystrobin was sprayed for several times. However, also in California pistachio orchards, after multiple applications of azoxystrobin for more than 5 years with three to five applications/season, azoxystrobin resistance has not been detected in another pistachio pathogen Botryosphaeria dothidea, the causal agent of panicle and shoot blight of pistachio (Michailides et al., unpublished data). Currently, azoxystrobin and other strobilurin fungicides are considered the most effective compounds
ARTICLE IN PRESS Z. Ma, T.J. Michailides / Crop Protection 23 (2004) 1259–1263
for controlling panicle and shoot blight of pistachio. Because both Botryosphaeria panicle and shoot blight and Alternaria late blight have been frequently found together in some pistachio orchards, when growers spray azoxystrobin to control panicle and shoot blight, they should be concerned with the selection pressure of azoxystrobin on Alternaria populations since Alternaria populations could develop resistance rapidly to this compound.
Acknowledgements This work was partially supported by grants to T. J. Michailides from the California Pistachio Commission. We thank D. Morgan and D. Felts for assistance with the figures of this manuscript.
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