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Characterization of iprodione-resistant Alternaria isolates from pistachio in California
PESTICIDE Biochemistry & Physiology
Pesticide Biochemistry and Physiology 80 (2004) 75–84 www.elsevier.com/locate/ypest
Characterization of iprodione-resistant Alternaria isolates from pistachio in California Zhonghua Ma*, Themis J. Michailides* Department of Plant Pathology, Kearney Agricultural Center, University of California Davis, 9240 South Riverbend Ave., Parlier, CA 93648, USA Received 13 February 2004; accepted 30 June 2004 Available online 7 August 2004
Abstract 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. In this study, a field iprodione-resistant (FIR) isolate of the arborescens species-group and a laboratory-induced iprodione-resistant (LIIR) isolate of the alternata species-group were characterized by fungicide and osmotic sensitivity, virulence on detached pistachio leaflets, and sequence of the coiled-coil region (six repeats of approximately 90-amino-acid domain) of the two-component histidine kinase (HK) gene. Both FIR and LIIR isolates were sensitive to azoxystrobin and tebuconazole, and azoxystrobin-resistant isolates were sensitive to iprodione and tebuconazole. The LIIR isolate showed more sensitivity to osmotic stress than its wildtype parent. However, the FIR isolate did not show higher osmotic sensitivity compared to field iprodione-sensitive (FIS) isolates. Laboratory inoculation tests showed that both FIR and LIIR isolates remained highly virulent on pistachio. Analysis of DNA sequences of the HK coiled-coil region showed that there were no differences in deduced amino acid sequence of this region from the LIIR, FIR, and FIS Alternaria isolates from pistachio in California. 2004 Elsevier Inc. All rights reserved. Keywords: Alternaria late blight; Dicarboximide resistance; The two-component histidine kinase; Pistacia vera
1. Introduction Alternaria late blight caused by Alternaria spp. in the alternata, tenuissima, and arborescens species-groups is one of the most common fungal dis*
Corresponding authors. Fax: 1 559 646 6593. E-mail addresses: [email protected] (Z. Ma), themis@ uckac.edu (T.J. Michailides).
eases of pistachio, and affects foliage and fruit in California [1]. Alternaria late blight is difficult to be controlled and chemical control is a major strategy to manage this disease. Previous studies [2,3] showed that the strobilurin fungicide azoxystrobin (Abound), dicarboximide fungicide iprodione (Rovral), and the sterol demethylation inhibition tebuconazole (Elite) are very effective in controlling this disease. However, azoxystrobin-resistant
0048-3575/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2004.06.007
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fungal populations have been developed in California pistachio orchards after 2–3 sprays of azoxystrobin per season for only 3–4 seasons [4]. Additionally, the development of resistance to dicarboximides has been observed in field populations of several fungi species, including Alternaria alternata pv. citri [5], A. alternata [6,7], Alternaria brassicicola [8], and Alternaria panax [9] in other places. Iprodione highly resistant A. alternata pv. citri population had become dominant in an orchard in Makha, Israel, after this compound was applied for three years based on a standard spray schedule [5]. In Wisconsin, after applications of iprodione in an experimental plot for two continuous seasons at 1134 g/ha, the compound was not effective any longer against Alternaria leaf blight of ginseng caused by A. panax in the third season because all isolates collected from this experimental plot were resistant to iprodione [9]. The results suggest that Alternaria spp. may have a high risk to develop resistance to iprodione. Presently, iprodione has been shown to be very effective in controlling Alternaria late blight and there are plans to get it registered for California pistachio in the near future. Thus, it is necessary to incorporate effective anti-resistance strategies when iprodione is used extensively to control Alternaria late blight of pistachio in California. Information on fitness of resistant mutants is useful in developing effective anti-resistant strategies. If fitness costs are associated with fungicide resistance, the frequency of resistant pathogen population will decline when the fungicide selection pressure is discontinued in a field. Biggs [10] reported that the laboratory-induced iprodione-resistant A. alternata from apple exhibited no discernible differences in cultural characteristics or virulence from the sensitive isolates. Isolates of A. brassicicola resistant to iprodione in the field exhibited similar sporulation capacities or aggressiveness towards host plants compared to those of the sensitive isolates [8]. However, in another study, laboratory-induced iprodione-resistant isolates of A. brassicicola exhibited a greater osmotic sensitivity than sensitive isolates, and this high osmotic sensitivity was considered to be responsible for a lower pathogenicity and lower fitness of the resistant isolates than those of
their sensitive counterparts [11]. Thus, the fitness of dicarboximide-resistant fungal isolates varies among species and also among different mutants within species [12]. Although iprodione has been used for many years to control various fungal pathogens causing plant diseases, the mode of action and the resistance mechanisms are not well known yet. Genetic analysis of field and laboratory-induced dicarboximide-resistant isolates of Botrytis cinerea has demonstrated that the resistance is conferred by a single locus designated Daf1 [13]. The Daf1 has recently been confirmed to be a synonym of a two-component histidine kinase (HK) gene (Bos1) containing six approximately 90-amino-acid repeats, which putatively form a coiled-coil region, followed by a kinase domain [14]. When compared DNA sequences of Bos1 genes from laboratory-induced dicarboximide-resistant and -sensitive isolates of B. cinerea, Cui et al. [14] found that dicarboximide resistance resulted from the presence of mutations in second to fifth aminoacid repeats of the HK coiled-coil region. A point mutation causing a substitution of asparagine, arginine, or serine for isoleucine at the codon 86 in the second amino-acid repeat was reported to confer field resistance in this pathogen [15,16]. More recently, the HK gene (AaHK1) was isolated from A. alternata [17]. After compared DNA sequences of the AaHK1 genes from each two field iprodione-resistant and -sensitive isolates, Dry et al. [17] found that mutations within the first and fifth amino-acid repeats of the coiled-coil region resulting in premature termination of the open reading frame conferring A. alternata resistance to iprodione. Additionally, mutations in the coiled-coil region of the HK gene conferring dicarboximide resistance were also observed from laboratory-induced dicarboximide-resistant isolates of Neurospora crassa [18]. In light of recent studies on dicarboximide resistance, it appears that fungal resistance to dicarboximide fungicide may be associated with mutations in the coiled-coil region of the HK gene although other mechanisms were not excluded [19,20]. In a preliminary study, a field iprodione-resistant isolate was detected from screening 60 Alternaria isolates collected from California pistachios
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[21]. The objectives of this study were to: (i) compare the fitness of iprodione-resistant and -sensitive Alternaria isolates from California pistachio, and (ii) investigate the possible mechanism of iprodione resistance in Alternaria by comparing DNA sequences of the coiled-coil region of HK genes from sensitive and resistant isolates.
2. Materials and methods 2.1. Fungal isolates Single spore Alternaria isolates used in this study were obtained from different pistachio orchards in California (Table 1). Identification and assignment of these isolates into Alternaria species-group was performed according to a previously established protocol [1]. Isolates were maintained on grade 40 silica gels (Davison Chemical, Baltimore) at 4 C. To obtain a laboratory-induced iprodione-resistant (LIIR) isolate, a mycelial plug (5 mm in diameter) of a wild-type isolate 37E5S was placed on a potato dextrose agar (PDA) plate amended with iprodione (Rovral 41.6% a.i., BASF, Mount Olive, NJ) at 50 mg a.i./ L. After incubation at 22 C for 2 weeks in the dark, a growing (resistant) sector developed on the plate. A mycelial tip from this sector was transferred to a fresh PDA plate and the isolate originated from the resistant mycelial tip was designated 37E5R.
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2.2. Determination of sensitivity of Alternaria isolates to iprodione, tebuconazole, and azoxystrobin Sensitivities of Alternaria isolates to iprodione and tebuconazole were determined using mycelial growth assays. Iprodione (Rovral 41.6%) and tebuconazole (Elite 45% WP, Bayer Ag. Division, Kansas City, MO) were dissolved in sterile water, respectively, and added to PDA to produce the following concentrations: iprodione at 0, 0.02, 0.08, 0.32, 1.28, 5.12, 20.48, 81.92, and 327.68 mg a.i./ L medium, or tebuconazole at 0, 0.01, 0.04, 0.16, 0.64, and 2.56 mg a.i./L. Three plates of each fungicide concentration were used for each isolate. A mycelial plug (5 mm in diameter) was taken from the edge of a 5-day-old colony of each isolate and transferred onto a PDA plate amended with each concentration of the above fungicides. After the plates were incubated at 22 C in the dark for 5 days, the radial growth (colony diameter) on each plate was measure in millimeters with the original mycelial plug diameter (5 mm) subtracted from this measurement. For each isolate, a linear regression of the percent inhibition related to the control of the mycelial growth versus the log10 transformation of each concentration of iprodione or tebuconazole was obtained. The 50% effective concentration (EC50), which is the fungicide concentration that results in 50% mycelial growth inhibition, was calculated for each isolate to each fungicide using the linear equation. The experiment was performed twice and FisherÕs LSD test
Table 1 Sensitivity of Alternaria isolates from California pistachio to iprodione and tebuconazole Isolate
Species-group
EC50a Iprodione
Tebuconazole
25C2R KA48S 37E5Rb 37E5S 37E8S 37C11S 37D5S
Arborescens Arborescens Alternata Alternata Tenuissima Alternata Alternata
> 100 0.2729 ± 0.0029 > 100 0.3653 ± 0.0326 0.5641 ± 0.0100 0.4727 ± 0.0106 0.2210 ± 0.0046
0.2049 ± l0.0037 0.3759 ± 0.0233 0.9637 ± 0.0360 0.8521 ± 0.0449 0.9568 ± 0.0153 0.4185 ± 0.0427 0.2646 ± 0.0381
a
Results are means ± SD of two experiments since there were no significant (P > 0.05, FisherÕs LSD test) differences between the two experiments. b This was a laboratory-induced iprodione-resistant (LIIR) mutant derived from the wild-type isolate 37E5S.
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was used to determine significant differences in colony diameter of each isolate between the two experiments. The ANOVA procedure of SAS (Version 8.0, SAS Institute, Cary, NC) was used to determine significant differences in sensitivity among isolates to each fungicide. Sensitivity of Alternaria isolates to azoxystrobin was assayed by using an allele-specific PCR method as described by Ma and Michailides [22]. In a previous study, we found that Alternaria resistance to azoxystrobin correlated with a single point mutation resulting in the replacement of a glycine by an alanine at codon 143 (G143A) in the mitochondrial cytochrome b gene. Based on this point mutation, a pair of allele-specific PCR primers ARF4 (5 0 -ATG AGA GAT GTA AAT AAT GGG TGA T-3 0 ) and ARR4 (5 0 -AAG GTT AGT AAT AAC TGT TGC AG-3 0 ) was developed to detect this point mutation [22]. The primer pair can amplify a 246 bp DNA fragment from azoxystrobin-resistant isolates, but not from-sensitive isolates. PCR amplifications were performed in a 50 ll volume containing 2 ll DNA solution extracted by FastDNA kit (QbioGene, Carlsbad, CA) as described below, 0.2 lM of each primer, 0.2 mM of each dNTP, 2.5 mM MgCl2, 1· Promega Taq polymerase buffer, and 2 U of Promega Taq polymerase. The PCR amplifications were performed in a Eppendorf Mastercycler (Eppendorf AG, Hamburg, Germany) using the following parameters: an initial pre-heat for 3 min at 95 C, followed by 40 cycles of denaturation at 94 C for 40 s, annealing at 65.5 C for 40 s, extension at 72 C for 1 min, and a final extension at 72 C for 10 min. PCR products were examined by electrophoresis in a 1.5% agarose gel in Tris–acetate (TAE) buffer. 2.3. Determination of osmotic sensitivity For each isolate, a mycelial plug (5 mm in diameter) was taken from the edge of a 5-day-old colony and transferred onto a PDA plate amended with 1, 2, 4, 6, or 8% (w/v) NaCl, respectively. Plates without NaCl were used as a non-treatment control. Three plates of each NaCl concentration were used for each isolate. After plates were incubated at 22 C for 5 days, the diameter of the fungal colony was
measured in millimeters for each plate. The percentage of the mycelial radial growth inhibition (RGI) was calculated using the formula RGI% = ((C–N)/ (C–5)) · 100, where, C is colony diameter of nonNaCl control, and N is that of a NaCl treatment. The experiment was repeated twice and FisherÕs LSD test was used to determine significant differences in colony diameter of each isolate between the two experiments. The ANOVA procedure of SAS was used to determine significant differences in RGI among isolates at each NaCl concentration. 2.4. Efficacy of iprodione in controlling the disease caused by iprodione-sensitive or -resistant Alternaria isolates Fully expanded pistachio leaves taken from three-year-old potted trees were surface sterilized in 0.5% sodium hypochlorite (10% commercial bleach; Western Family Foods, Portland, OR) for 3 min and then washed three times in sterilized water. After drying the excess water for 30 min on a bench at a laboratory, the surface sterilized leaves were sprayed with iprodione (Rovral 41.6% a.i., BASF) at 500 mg a.i./L (a manufacturerÕs recommended dosage applied in the field) with a hand-held sprayer (ACE Hardware, Oak Brook, IL) until numerous droplets were deposited onto the leaves. Leaves sprayed with water were used as non-treatment control. After droplets on leaves air-dried for 30 min, each leaflet was slightly wounded on the midrib with a sterile needle. Each wounded leaflet was inoculated with a mycelial plug (5 mm in diameter) taken from the edge of a 5-day-old colony of each isolate. Twenty leaflets were used for each isolate. Leaflets inoculated with agar plugs were used as a negative control. Ten inoculated leaflets were placed on a plastic rack resting in a plastic container (32 · 23.5 · 10 cm) with 500 ml of water in the bottom, and petioles of inoculated leaflets were submerged in the water. After incubated at 25 ± 2 C for nine days, the lesion length on each leaflet was measured in centimeters. Efficacy of iprodione (EI) in controlling the disease was calculated using the following formula: EI = (LC–LT)/LC, where LC is the average lesion length (cm) from 10 non-treated control leaflets in each plastic container and LT is that
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from 10 iprodione-treated leaflets. The experiment was repeated twice and data were analyzed using ANOVA of SAS to determine the significant differences in disease lesion length and EI between sensitive and resistant Alternaria isolates. 2.5. Sequence analysis of the coiled-coil region of HK genes from iprodione-sensitive and -resistant Alternaria isolates To extract DNA, approximately 100 mg fresh mycelia of each isolate was harvested by gently scraping the surface of cultures on PDA plates with a sterilized loop. DNA from fungal mycelia was extracted by using the FastDNA kit, and final DNA from 100 mg mycelia of each isolate was dissolved in 50 ll water. A 2 ll aliquot of each DNA solution was used for each PCR amplification. A pair of PCR primers AaHKF (5 0 -GCA CTG CGG GAA ATC GGC-3 0 ) and AaHKR (5 0 -GCT TCC CTC GCA GCC GTG-3 0 ) has been developed to amplify the coiled-coil region of HK gene from A. alternata [17]. In this study, this pair of primers was used to amplify the coiled-coil region of HK genes from Alternaria isolates (Table 1) collected from California pistachios. The PCR amplifications were performed as described above except an annealing temperature 65 C was used. PCR products were size verified in 1.5% agarose gels in TAE buffer. The PCR products from each isolate were purified using the QIAquick Gel Extraction kit (Qiagen, Valencia, CA). The purified fragments from each isolate were ligated into the pGEM-T Easy vector (Promega, Madison, WI), and transformed into Escherichia coli (strain JM109) cells. Plasmids were purified using the QIAprep Spin Miniprep kit (Qiagen, Valencia, CA). PCR and restriction endonuclease analyses of the plasmids confirmed that the PCR products had been cloned into the pGEM-T Easy vector. Three independent PCR cloning processes were done for each isolate to distinguish original mutation from possible misinsertion during PCR amplification. The cloned fragments were sequenced by DBS Sequencing (Division of Biological Sciences, University of California at Davis) using the primers T7, SP6, and an internal primer AaHK3 (5 0 -CAT GGC TCA AAA CCT CAC CA-3 0 ).
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To compare the deduced amino acid sequences of the HK coiled-coil region from resistant and sensitive isolates, the DNA sequences from each isolate were translated into amino acid sequences using standard code by the computer program EMBOSS Transeq (http://www.ebi.ac.uk/emboss/ transeq/). The sequences of DNA and deduced amino acid were aligned, respectively, using the computer program ClustalW 1.82 (http://www. ebi.ac.uk/clustalw/) (European Bioinformatics Institute, Cambridge, UK). The deduced amino acid sequences from Alternaria isolates collected from pistachio were also compared with those from other phytopathogenic fungal species using BLAST of NCBI/GenBank (http://www.ncbi.nlm. nih.gov/BLAST/).
3. Results 3.1. Sensitivity of Alternaria isolates to iprodione, tebuconazole, and azoxystrobin The field isolates 37C11S, 37D5S, 37E5S, 37E8S, and KA48S were resistant to azoxystrobin because the PCR primers ARF4 and ARR4 amplified a 246 bp DNA fragment from each of these isolates (Fig. 1), but they were sensitive to both iprodione and tebuconazole (Table 1). There were no significant (P = 0.156, ANOVA of SAS) differences in sensitivity to iprodione and tebuconazole
Fig. 1. An allele-specific PCR assay for detecting azoxystrobinresistant Alternaria spp. from pistachio in California. The allele-specific primer pair ARF4 + ARR4 amplified a 246 bp DNA fragment from azoxystrobin-resistant but not fromsensitive Alternaria isolates.
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among these five isolates. The EC50 to iprodione for LIIR isolate 37E5R, which was a laboratoryinduced iprodione-resistant isolate derived from the wild-type isolate 37E5S, was significantly (P < 0.01, ANOVA of SAS) higher than that of its original parent 37E5S. The field isolate 25C2R had a very high EC50 value to iprodione (Table 1), thus, it was designated field iprodioneresistant (FIR) isolate. This FIR isolate, however, was sensitive to tebuconazole (Table 1), and also sensitive to azoxystrobin because the primers ARF4 and ARR4 could not amplify the 246 bp DNA fragment from this isolate (Fig. 1). These results indicate that there is no cross-resistance among iprodione, tebuconazole, and azoxystrobin in these Alternaria isolates from pistachios. 3.2. Osmotic sensitivity The LIIR isolate 37E5R grew significantly (P < 0.03, ANOVA of SAS) slower than its wildtype sensitive parent 37E5S in PDA plates
amended with NaCl at each of the five concentrations (Fig. 2A). However, there were no significant differences (P > 0.21, ANOVA of SAS) in RGI between the FIR isolate 25C2R and the field iprodione-sensitive (FIS) isolates 37C11S, 37D5S, 37E5S, 37E8S, and KA48S at each NaCl concentration, which indicated that the FIR isolate 25C2R did not show higher sensitivity to osmotic stress (Fig. 2B). 3.3. Efficacy of iprodione against the disease caused by LIIR, FIR, and FIS Alternaria isolates Laboratory inoculation tests demonstrated that when iprodione was not applied, the LIIR isolate 37E5R and its original parent 37E5S caused similar lesion lengths (no statistical difference) on detached pistachio leaves (Fig. 3A). Although the FIR isolate 25C2R showed lower virulence than the FIS isolates 37E5S and 37E8S on detached pistachio leaves, it still caused longer disease lesions than the FIS isolates 37C11S and 37D5S (Fig. 3A).
Fig. 2. Comparisons in osmotic sensitivity between the laboratory-induced iprodione-resistant (LIIR) isolate 37E5R and its sensitive parent 37E5S (A), and among field iprodione-resistant (FIR) isolate 25C2R and -sensitive (FIS) Alternaria isolates collected from pistachios (B). The data represent means ± SD of the two experiments since there were no significant (P > 0.05, FisherÕs LSD test) differences between the two experiments.
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Fig. 3. Comparisons in disease lesion length among Alternaria isolates on detached pistachio leaflets treated with or without iprodione (A), and comparisons in iprodione efficacy among these isolates (B). The data represent means ± SD of the two experiments; gray or blank bars in each graph with the same letter are not significantly different at P = 0.05 (ANOVA of SAS).
When iprodione at 500 mg/L was applied, all five FIS isolates caused significantly smaller lesions than the FIR and LIIR isolates (Fig. 3A). The efficacy of iprodione in controlling disease caused by the FIR and LIIR isolates was significantly lower than that of the FIS isolates (Fig. 3B). 3.4. Sequences of the coiled-coil region of HK genes from Alternaria isolates A single DNA fragment (1605 bp) was amplified with the primers AaHKF and AaHKR from each of seven Alternaria isolates tested (Table 1). GenBank Accession Nos. of these sequences are AY559302–AY559308 for the isolate 25C2R, 37E5R, 37C11S, KA48S, 37D5S, 37E5S, and 37E8S, respectively. The coiled-coil region of HK gene from each isolate contained an intron within the second 90-amino-acid repeat. Deduced amino acid sequences of this section of HK gene from either LIIR, FIR, or FIS isolates were 518 residues in length. The sequenced section included all posi-
tions known to affect the sensitivity to dicarboximides in A. alternata and other fungi [14–18]. However, there were no differences in deduced amino acid sequence among the LIIR, FIR, FIS Alternaria isolates collected from pistachio, although there were 5–20 silent substitutions in the DNA sequences among these isolates. Deduced amino acid sequence of the coiled-coil region of HK gene from Alternaria isolates in this study showed identity of 100% to that from A. alternata (GenBank Accession No. AAO48475), 98% to Cochliobolus heterostrophus (BAC78684), 79% to Botryotinia fuckeliana (AAL30826), 78% to N. crassa (AAB01979), 77% to Magnaporthe grisea (EAA55993), and 75% to Fusarium solani f. sp. pisi (AAD09491).
4. Discussion In a previous study, we showed that most Alternaria isolates (59 of 60) collected from pistachios
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in California were sensitive to iprodione; only one isolate (25C2R) was highly resistant to this compound [21]. In this study, we found that this field iprodione resistant (FIR) isolate did not show higher osmotic sensitivity than field iprodione sensitive (FIS) isolates, and it still remained high virulence on pistachio. These results suggest that the FIR isolate may have sufficient parasitic fitness to compete with FIS isolates in the field. Thus, under selection pressure of multiple applications of iprodione, the FIR isolate may spread in pistachio orchards. Since both LIIR and FIR isolates were sensitive to azoxytrobin and tebuconazole, and azoxystrobin-resistant isolates were sensitive to iprodione and tebuconazole, alternative applications of iprodione, azoxystrobin, and tebuconazole could delay the development of iprodione- and azoxystrobin-resistant Alternaria populations in California pistachio orchards. The alternative applications of these fungicides are necessary since azoxystrobin-resistant Alternaria populations have been developed in some pistachio orchards. Under laboratory conditions, dicarboximide-resistant mutants could be induced easily in many fungal species, including A. alternata [10], A. brassicicola [10], B. cinerea [14,15], Botryosphaeria dothidea [23], and N. crassa [18]. Generally, most laboratory-induced mutants had high levels of resistance to dicarboximides and also showed higher osmotic sensitivity compared to their original sensitive parents. In this study, we also found the LIIR isolate 37E5R was more sensitive to osmotic stress than its sensitive parent 37E5S. However, the FIR isolate 25C2R did not show higher osmotic sensitivity compared to the FIS isolates. These observations suggest that the resistance
mechanism of the FIR Alternaria isolate differs from that of the LIIR isolate. In N. crassa, mutations in several osmotic sensitivity genes (os-1, os-2, os-4, os-5, smco-8, and smco-9) were found to confer resistance to dicarboximide, aromatic hydrocarbon, and phenylpyrrole fungicides [20]. The os-1 gene has been cloned and shown to encode a histidine kinase, which plays an important role in the regulation of cell-wall assembly and other cell responses to changes in external osmolarity [24]. Recently, several studies on N. crassa, B. cinerea, and A. alternata suggested that the potential target site of dicarboximides could be the coiled-coil region of the two-component histidine kinase (HK) [14– 18]. In those previous studies, amino acid changes in the HK coiled-coil region were found to be associated with dicarboximide resistance (Table 2). In A. alternata, a duplication of an 11-base nucleotide sequence was found in the first amino-acid repeat of the coiled-coil region of the HK gene from a FIR isolate Aa-8495, which was collected from passion fruit in Australia. This duplication of 11base nucleotide sequence resulted in a premature stop codon at amino acid position 95 [17]. In another FIR isolate Aa-8501, a four-base nucleotide deletion was detected within the fifth amino-acid repeat which caused termination of the open reading frame at amino acid position 434 [17]. In our study, however, LIIR, FIR, and FIS Alternaria isolates from California pistachio had identical amino acid sequences of the HK coiled-coil region, which indicates that an alternative resistance mechanism other than mutation in the coiled-coil region of HK gene is responsible for dicarboximide resistance in Alternaria isolates from pistachio.
Table 2 Summary of mutations at two-component histidine kinase genes from dicarboximide-resistant mutants Fungal species
Origina
Mutation position in coiled-coil regionb
Reference
A. alternata species-group A. alternata species-group A. arborescens species-group B. cinerea B. cinerea N. crassa
Field Lab Field Field Lab Lab
1, 5 No mutations observed No mutations observed 2 2, 3, 4, 5 2, 5
[17] This study This study [15,16] [14] [18]
a b
Origin from where the resistant mutant was obtained. The 90-amino-acid repeat in which mutation was observed.
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Alternative resistance mechanisms other than mutation in the HK coiled-coil region have been observed in other laboratory induced dicarboximide-resistant fungi, e.g., B. cinerea and Ustilago maydis. In B. cinerea, genetic analysis of LIIR mutants showed that dicarboximide resistance in some resistant mutants was conferred by another locus, Daf2 [25], which was not linked to the HK gene. Since the Daf2 gene has not been cloned yet, a comparison of the Daf2 sequences from the resistant mutant and wild-type sensitive parent has not been reported. In the basidiomycete U. maydis, Ramesh et al. [20] reported that mutants disrupted in the ubc1 gene, which encodes the regulatory subunit of a cAMP-dependent protein kinase (PKA), were resistant to dicarboximides. The authors speculated that the resistance of ubc1 mutants is due to the absence of the regulatory subunit and the resulting increase in the PKA activity. Currently, it is under investigation if such alternative mechanisms are responsible for iprodione resistance in Alternaria spp. from pistachio. In light of recent studies on dicarboximide resistance and the results from this study, we conclude that resistance mechanisms to dicarboximides in Alternaria spp. are complex. Subsequently, applications of molecular methods for detections of resistant Alternaria populations should be difficult. Acknowledgments We thank Tae H. Lim for testing fungicide sensitivity in Alternaria isolates, David P. Morgan for collecting Alternaria isolates from pistachio, and Dr. Yong Luo for helping with statistical analysis.
References [1] B.M. Pryor, T.J. Michailides, Morphological pathogenic, molecular characterization of Alternaria isolates associated with Alternaria late blight of pistachio, Phytopathology 92 (2002) 406–416. [2] T.J. Michailides, D.P. Morgan, D. Felts, Chemical control of Alternaria late blight of California pistachio in 1998, in: California Pistachio Industry Annual Report Crop Year 1998–1999, Fresno, CA, 1999, p. 82.
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[3] T.J. Michailides, D.P. Morgan, D. Felts, Chemical control of Alternaria late blight of California pistachio in 1999, in: California Pistachio Industry Annual Report Crop Year 1999–2000, Fresno, CA, 2000, p. 87. [4] Z. Ma, D. Felts, T.J. Michailides, Resistance to azoxystrobin in Alternaria isolates from pistachio in California, Pestic. Biochem. Physiol. 77 (2003) 66–74. [5] Z. Sole, L. Timmer, M. Kimchi, Iprodione resistance of Alternaria alternata pv. citri from Minneola tangelo in Israel and Florida, Plant Dis. 80 (1996) 291–293. [6] D.G. Hutton, The appearance of dicarboximide resistance in Alternaria alternata in passion fruit in south-east Queenland, Aust. Plant Path. 17 (1988) 34–36. [7] W.J. McPhee, Some characteristics of Alternaria alternata strains resistant to iprodione, Plant. Dis. 64 (1980) 847– 849. [8] B. Iacomi-Vasilescu, H. Avebot, N. Bataille-Simoneau, E. Laurent, M. Guenard, P. Simoneau, In vitro fungicide sensitivity of Alternaria species pathogenic to crucifers and identification of Alternaria brassicicola field isolates highly resistant to both dicarboximides and phenylpyrroles, Crop Protection 23 (2004) 481–488. [9] M.K. Rahimian, Resistance of Alternaria panax to iprodione under field conditions, Phytopathology 77 (1987) 1747. [10] A.R. Biggs, Mycelial growth, sporulation, and virulence to apple fruit of Alternaria alternata isolates resistant to iprodione, Plant Dis. 78 (1994) 732–735. [11] R. Huang, Y. Levy, Characterization of iprodione-resistant isolates of Alternaria brassicicola, Plant Dis. 79 (1995) 828–833. [12] R. Raposo, V. Gomez, T. Urrutia, P. Melgarejo, Fitness of Botrytis cinerea associated with dicarboximide resistance, Phytopathology 90 (2000) 1246–1249. [13] F. Faretra, S. Pollastro, Genetic basis of resistance to benzimidazole and dicarboximide fungicides in Botryotinia fuckeliana (Botrytis cinerea), Mycol. Res. 95 (1991) 457– 464. [14] W. Cui, R.E. Beever, S.L. Parkes, P.L. Weeds, M.D. Templeton, An osmosensing histidine kinase mediates dicarboximide fungicide resistance in Botryotinia fuckeliana (Botrytis cinerea), Fungal Genet. Biol. 36 (2002) 187– 198. [15] M. Oshima, M. Fujimura, S. Banno, C. Hashimoto, T. Motoyama, A. Ichiishi, I. Yamaguchi, A point mutation in the two-component histidine BcOS-1 gene confers dicarboximide resistance in field isolates of Botrytis cinerea, Phytopathology 92 (2002) 75–80. [16] P. Leroux, R. Fritz, D. Debieu, C. Albertini, C. Lanen, J. Bach, M. Gredt, F. Chapeland, Mechanisms of resistance to fungicides in field strains of Botrytis cinerea, Pest Manag. Sci. 58 (2002) 876–888. [17] I.B. Dry, K.H. Yuan, D.G. Hutton, Dicarboximide resistance in field isolates of Alternaria alternata is mediated by a mutation in a two-component histidine kinase gene, Fungal Genet. Biol. 41 (2004) 102– 108.
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[18] N. Ochiai, M. Fujimura, T. Motoyama, A. Ichiishi, R. Usami, K. Horijoshi, I. Yamaguchi, Characterization of mutations in the two-component histidine kinase gene that confer fludioxonil resistance and osmotic sensitivity in the os-1 mutants of Neurospora crassa, Pest Manag. Sci. 57 (2002) 437–442. [19] A.B. Orth, M. Rzhetskaya, E.J. Pell, M. Tien, A serine (threonine) protein kinase confers fungicide resistance in the phytopathogenic fungus Ustilago maydis, Appl. Environ. Microbiol. 61 (1995) 2341–2345. [20] M.A. Ramesh, R.D. Laidlaw, F. Durrenberger, A.B. Orth, J.W. Kronstad, The cAMP signal transduction pathway mediates resistance to dicarboximide and aromatic hydrocarbon fungicides in Ustilago maydis, Fungal Genet. Biol. 32 (2001) 183–193. [21] T.H. Lim, B.M. Pryor, D.P. Morgan, T.J. Michailides, Sensitivity to iprodione, vinclozolin, and tebuconazole and
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characters of iprodione-resistant isolates of Alternaria spp. from pistachio, Phytopathology 93 (2003) S128. Z. Ma, T.J. Michailides, An allele-specific PCR assay for detecting azoxystrobin-resistant Alternaria isolates from pistachio in California, J. Phytopathology 152 (2004) 118– 121. Z. Ma, Y. Luo, T.J. Michailides, Resistance of Botryosphaeria dothidea from pistachio to iprodione, Plant Dis. 85 (2001) 183–188. M.M. Schumacher, C.S. Enderlin, C.P. Selitrennikoff, The osmotic-1 locus of Neurospora crassa encodes a putative histidine kinase similar to osmosensors of bacteria and yeast, Curr. Microbiol. 34 (1997) 340–347. F. Faretra, S. Pollastro, Isolation, characterization and genetic-analysis of laboratory mutants of Botryotiniafuckeliana resistant to the phenylpyrrole fungicide CGA173506, Mycol. Res. 97 (1993) 620–624.
Pesticide Biochemistry and Physiology 80 (2004) 75–84 www.elsevier.com/locate/ypest
Characterization of iprodione-resistant Alternaria isolates from pistachio in California Zhonghua Ma*, Themis J. Michailides* Department of Plant Pathology, Kearney Agricultural Center, University of California Davis, 9240 South Riverbend Ave., Parlier, CA 93648, USA Received 13 February 2004; accepted 30 June 2004 Available online 7 August 2004
Abstract 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. In this study, a field iprodione-resistant (FIR) isolate of the arborescens species-group and a laboratory-induced iprodione-resistant (LIIR) isolate of the alternata species-group were characterized by fungicide and osmotic sensitivity, virulence on detached pistachio leaflets, and sequence of the coiled-coil region (six repeats of approximately 90-amino-acid domain) of the two-component histidine kinase (HK) gene. Both FIR and LIIR isolates were sensitive to azoxystrobin and tebuconazole, and azoxystrobin-resistant isolates were sensitive to iprodione and tebuconazole. The LIIR isolate showed more sensitivity to osmotic stress than its wildtype parent. However, the FIR isolate did not show higher osmotic sensitivity compared to field iprodione-sensitive (FIS) isolates. Laboratory inoculation tests showed that both FIR and LIIR isolates remained highly virulent on pistachio. Analysis of DNA sequences of the HK coiled-coil region showed that there were no differences in deduced amino acid sequence of this region from the LIIR, FIR, and FIS Alternaria isolates from pistachio in California. 2004 Elsevier Inc. All rights reserved. Keywords: Alternaria late blight; Dicarboximide resistance; The two-component histidine kinase; Pistacia vera
1. Introduction Alternaria late blight caused by Alternaria spp. in the alternata, tenuissima, and arborescens species-groups is one of the most common fungal dis*
Corresponding authors. Fax: 1 559 646 6593. E-mail addresses: [email protected] (Z. Ma), themis@ uckac.edu (T.J. Michailides).
eases of pistachio, and affects foliage and fruit in California [1]. Alternaria late blight is difficult to be controlled and chemical control is a major strategy to manage this disease. Previous studies [2,3] showed that the strobilurin fungicide azoxystrobin (Abound), dicarboximide fungicide iprodione (Rovral), and the sterol demethylation inhibition tebuconazole (Elite) are very effective in controlling this disease. However, azoxystrobin-resistant
0048-3575/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2004.06.007
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fungal populations have been developed in California pistachio orchards after 2–3 sprays of azoxystrobin per season for only 3–4 seasons [4]. Additionally, the development of resistance to dicarboximides has been observed in field populations of several fungi species, including Alternaria alternata pv. citri [5], A. alternata [6,7], Alternaria brassicicola [8], and Alternaria panax [9] in other places. Iprodione highly resistant A. alternata pv. citri population had become dominant in an orchard in Makha, Israel, after this compound was applied for three years based on a standard spray schedule [5]. In Wisconsin, after applications of iprodione in an experimental plot for two continuous seasons at 1134 g/ha, the compound was not effective any longer against Alternaria leaf blight of ginseng caused by A. panax in the third season because all isolates collected from this experimental plot were resistant to iprodione [9]. The results suggest that Alternaria spp. may have a high risk to develop resistance to iprodione. Presently, iprodione has been shown to be very effective in controlling Alternaria late blight and there are plans to get it registered for California pistachio in the near future. Thus, it is necessary to incorporate effective anti-resistance strategies when iprodione is used extensively to control Alternaria late blight of pistachio in California. Information on fitness of resistant mutants is useful in developing effective anti-resistant strategies. If fitness costs are associated with fungicide resistance, the frequency of resistant pathogen population will decline when the fungicide selection pressure is discontinued in a field. Biggs [10] reported that the laboratory-induced iprodione-resistant A. alternata from apple exhibited no discernible differences in cultural characteristics or virulence from the sensitive isolates. Isolates of A. brassicicola resistant to iprodione in the field exhibited similar sporulation capacities or aggressiveness towards host plants compared to those of the sensitive isolates [8]. However, in another study, laboratory-induced iprodione-resistant isolates of A. brassicicola exhibited a greater osmotic sensitivity than sensitive isolates, and this high osmotic sensitivity was considered to be responsible for a lower pathogenicity and lower fitness of the resistant isolates than those of
their sensitive counterparts [11]. Thus, the fitness of dicarboximide-resistant fungal isolates varies among species and also among different mutants within species [12]. Although iprodione has been used for many years to control various fungal pathogens causing plant diseases, the mode of action and the resistance mechanisms are not well known yet. Genetic analysis of field and laboratory-induced dicarboximide-resistant isolates of Botrytis cinerea has demonstrated that the resistance is conferred by a single locus designated Daf1 [13]. The Daf1 has recently been confirmed to be a synonym of a two-component histidine kinase (HK) gene (Bos1) containing six approximately 90-amino-acid repeats, which putatively form a coiled-coil region, followed by a kinase domain [14]. When compared DNA sequences of Bos1 genes from laboratory-induced dicarboximide-resistant and -sensitive isolates of B. cinerea, Cui et al. [14] found that dicarboximide resistance resulted from the presence of mutations in second to fifth aminoacid repeats of the HK coiled-coil region. A point mutation causing a substitution of asparagine, arginine, or serine for isoleucine at the codon 86 in the second amino-acid repeat was reported to confer field resistance in this pathogen [15,16]. More recently, the HK gene (AaHK1) was isolated from A. alternata [17]. After compared DNA sequences of the AaHK1 genes from each two field iprodione-resistant and -sensitive isolates, Dry et al. [17] found that mutations within the first and fifth amino-acid repeats of the coiled-coil region resulting in premature termination of the open reading frame conferring A. alternata resistance to iprodione. Additionally, mutations in the coiled-coil region of the HK gene conferring dicarboximide resistance were also observed from laboratory-induced dicarboximide-resistant isolates of Neurospora crassa [18]. In light of recent studies on dicarboximide resistance, it appears that fungal resistance to dicarboximide fungicide may be associated with mutations in the coiled-coil region of the HK gene although other mechanisms were not excluded [19,20]. In a preliminary study, a field iprodione-resistant isolate was detected from screening 60 Alternaria isolates collected from California pistachios
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[21]. The objectives of this study were to: (i) compare the fitness of iprodione-resistant and -sensitive Alternaria isolates from California pistachio, and (ii) investigate the possible mechanism of iprodione resistance in Alternaria by comparing DNA sequences of the coiled-coil region of HK genes from sensitive and resistant isolates.
2. Materials and methods 2.1. Fungal isolates Single spore Alternaria isolates used in this study were obtained from different pistachio orchards in California (Table 1). Identification and assignment of these isolates into Alternaria species-group was performed according to a previously established protocol [1]. Isolates were maintained on grade 40 silica gels (Davison Chemical, Baltimore) at 4 C. To obtain a laboratory-induced iprodione-resistant (LIIR) isolate, a mycelial plug (5 mm in diameter) of a wild-type isolate 37E5S was placed on a potato dextrose agar (PDA) plate amended with iprodione (Rovral 41.6% a.i., BASF, Mount Olive, NJ) at 50 mg a.i./ L. After incubation at 22 C for 2 weeks in the dark, a growing (resistant) sector developed on the plate. A mycelial tip from this sector was transferred to a fresh PDA plate and the isolate originated from the resistant mycelial tip was designated 37E5R.
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2.2. Determination of sensitivity of Alternaria isolates to iprodione, tebuconazole, and azoxystrobin Sensitivities of Alternaria isolates to iprodione and tebuconazole were determined using mycelial growth assays. Iprodione (Rovral 41.6%) and tebuconazole (Elite 45% WP, Bayer Ag. Division, Kansas City, MO) were dissolved in sterile water, respectively, and added to PDA to produce the following concentrations: iprodione at 0, 0.02, 0.08, 0.32, 1.28, 5.12, 20.48, 81.92, and 327.68 mg a.i./ L medium, or tebuconazole at 0, 0.01, 0.04, 0.16, 0.64, and 2.56 mg a.i./L. Three plates of each fungicide concentration were used for each isolate. A mycelial plug (5 mm in diameter) was taken from the edge of a 5-day-old colony of each isolate and transferred onto a PDA plate amended with each concentration of the above fungicides. After the plates were incubated at 22 C in the dark for 5 days, the radial growth (colony diameter) on each plate was measure in millimeters with the original mycelial plug diameter (5 mm) subtracted from this measurement. For each isolate, a linear regression of the percent inhibition related to the control of the mycelial growth versus the log10 transformation of each concentration of iprodione or tebuconazole was obtained. The 50% effective concentration (EC50), which is the fungicide concentration that results in 50% mycelial growth inhibition, was calculated for each isolate to each fungicide using the linear equation. The experiment was performed twice and FisherÕs LSD test
Table 1 Sensitivity of Alternaria isolates from California pistachio to iprodione and tebuconazole Isolate
Species-group
EC50a Iprodione
Tebuconazole
25C2R KA48S 37E5Rb 37E5S 37E8S 37C11S 37D5S
Arborescens Arborescens Alternata Alternata Tenuissima Alternata Alternata
> 100 0.2729 ± 0.0029 > 100 0.3653 ± 0.0326 0.5641 ± 0.0100 0.4727 ± 0.0106 0.2210 ± 0.0046
0.2049 ± l0.0037 0.3759 ± 0.0233 0.9637 ± 0.0360 0.8521 ± 0.0449 0.9568 ± 0.0153 0.4185 ± 0.0427 0.2646 ± 0.0381
a
Results are means ± SD of two experiments since there were no significant (P > 0.05, FisherÕs LSD test) differences between the two experiments. b This was a laboratory-induced iprodione-resistant (LIIR) mutant derived from the wild-type isolate 37E5S.
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was used to determine significant differences in colony diameter of each isolate between the two experiments. The ANOVA procedure of SAS (Version 8.0, SAS Institute, Cary, NC) was used to determine significant differences in sensitivity among isolates to each fungicide. Sensitivity of Alternaria isolates to azoxystrobin was assayed by using an allele-specific PCR method as described by Ma and Michailides [22]. In a previous study, we found that Alternaria resistance to azoxystrobin correlated with a single point mutation resulting in the replacement of a glycine by an alanine at codon 143 (G143A) in the mitochondrial cytochrome b gene. Based on this point mutation, a pair of allele-specific PCR primers ARF4 (5 0 -ATG AGA GAT GTA AAT AAT GGG TGA T-3 0 ) and ARR4 (5 0 -AAG GTT AGT AAT AAC TGT TGC AG-3 0 ) was developed to detect this point mutation [22]. The primer pair can amplify a 246 bp DNA fragment from azoxystrobin-resistant isolates, but not from-sensitive isolates. PCR amplifications were performed in a 50 ll volume containing 2 ll DNA solution extracted by FastDNA kit (QbioGene, Carlsbad, CA) as described below, 0.2 lM of each primer, 0.2 mM of each dNTP, 2.5 mM MgCl2, 1· Promega Taq polymerase buffer, and 2 U of Promega Taq polymerase. The PCR amplifications were performed in a Eppendorf Mastercycler (Eppendorf AG, Hamburg, Germany) using the following parameters: an initial pre-heat for 3 min at 95 C, followed by 40 cycles of denaturation at 94 C for 40 s, annealing at 65.5 C for 40 s, extension at 72 C for 1 min, and a final extension at 72 C for 10 min. PCR products were examined by electrophoresis in a 1.5% agarose gel in Tris–acetate (TAE) buffer. 2.3. Determination of osmotic sensitivity For each isolate, a mycelial plug (5 mm in diameter) was taken from the edge of a 5-day-old colony and transferred onto a PDA plate amended with 1, 2, 4, 6, or 8% (w/v) NaCl, respectively. Plates without NaCl were used as a non-treatment control. Three plates of each NaCl concentration were used for each isolate. After plates were incubated at 22 C for 5 days, the diameter of the fungal colony was
measured in millimeters for each plate. The percentage of the mycelial radial growth inhibition (RGI) was calculated using the formula RGI% = ((C–N)/ (C–5)) · 100, where, C is colony diameter of nonNaCl control, and N is that of a NaCl treatment. The experiment was repeated twice and FisherÕs LSD test was used to determine significant differences in colony diameter of each isolate between the two experiments. The ANOVA procedure of SAS was used to determine significant differences in RGI among isolates at each NaCl concentration. 2.4. Efficacy of iprodione in controlling the disease caused by iprodione-sensitive or -resistant Alternaria isolates Fully expanded pistachio leaves taken from three-year-old potted trees were surface sterilized in 0.5% sodium hypochlorite (10% commercial bleach; Western Family Foods, Portland, OR) for 3 min and then washed three times in sterilized water. After drying the excess water for 30 min on a bench at a laboratory, the surface sterilized leaves were sprayed with iprodione (Rovral 41.6% a.i., BASF) at 500 mg a.i./L (a manufacturerÕs recommended dosage applied in the field) with a hand-held sprayer (ACE Hardware, Oak Brook, IL) until numerous droplets were deposited onto the leaves. Leaves sprayed with water were used as non-treatment control. After droplets on leaves air-dried for 30 min, each leaflet was slightly wounded on the midrib with a sterile needle. Each wounded leaflet was inoculated with a mycelial plug (5 mm in diameter) taken from the edge of a 5-day-old colony of each isolate. Twenty leaflets were used for each isolate. Leaflets inoculated with agar plugs were used as a negative control. Ten inoculated leaflets were placed on a plastic rack resting in a plastic container (32 · 23.5 · 10 cm) with 500 ml of water in the bottom, and petioles of inoculated leaflets were submerged in the water. After incubated at 25 ± 2 C for nine days, the lesion length on each leaflet was measured in centimeters. Efficacy of iprodione (EI) in controlling the disease was calculated using the following formula: EI = (LC–LT)/LC, where LC is the average lesion length (cm) from 10 non-treated control leaflets in each plastic container and LT is that
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from 10 iprodione-treated leaflets. The experiment was repeated twice and data were analyzed using ANOVA of SAS to determine the significant differences in disease lesion length and EI between sensitive and resistant Alternaria isolates. 2.5. Sequence analysis of the coiled-coil region of HK genes from iprodione-sensitive and -resistant Alternaria isolates To extract DNA, approximately 100 mg fresh mycelia of each isolate was harvested by gently scraping the surface of cultures on PDA plates with a sterilized loop. DNA from fungal mycelia was extracted by using the FastDNA kit, and final DNA from 100 mg mycelia of each isolate was dissolved in 50 ll water. A 2 ll aliquot of each DNA solution was used for each PCR amplification. A pair of PCR primers AaHKF (5 0 -GCA CTG CGG GAA ATC GGC-3 0 ) and AaHKR (5 0 -GCT TCC CTC GCA GCC GTG-3 0 ) has been developed to amplify the coiled-coil region of HK gene from A. alternata [17]. In this study, this pair of primers was used to amplify the coiled-coil region of HK genes from Alternaria isolates (Table 1) collected from California pistachios. The PCR amplifications were performed as described above except an annealing temperature 65 C was used. PCR products were size verified in 1.5% agarose gels in TAE buffer. The PCR products from each isolate were purified using the QIAquick Gel Extraction kit (Qiagen, Valencia, CA). The purified fragments from each isolate were ligated into the pGEM-T Easy vector (Promega, Madison, WI), and transformed into Escherichia coli (strain JM109) cells. Plasmids were purified using the QIAprep Spin Miniprep kit (Qiagen, Valencia, CA). PCR and restriction endonuclease analyses of the plasmids confirmed that the PCR products had been cloned into the pGEM-T Easy vector. Three independent PCR cloning processes were done for each isolate to distinguish original mutation from possible misinsertion during PCR amplification. The cloned fragments were sequenced by DBS Sequencing (Division of Biological Sciences, University of California at Davis) using the primers T7, SP6, and an internal primer AaHK3 (5 0 -CAT GGC TCA AAA CCT CAC CA-3 0 ).
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To compare the deduced amino acid sequences of the HK coiled-coil region from resistant and sensitive isolates, the DNA sequences from each isolate were translated into amino acid sequences using standard code by the computer program EMBOSS Transeq (http://www.ebi.ac.uk/emboss/ transeq/). The sequences of DNA and deduced amino acid were aligned, respectively, using the computer program ClustalW 1.82 (http://www. ebi.ac.uk/clustalw/) (European Bioinformatics Institute, Cambridge, UK). The deduced amino acid sequences from Alternaria isolates collected from pistachio were also compared with those from other phytopathogenic fungal species using BLAST of NCBI/GenBank (http://www.ncbi.nlm. nih.gov/BLAST/).
3. Results 3.1. Sensitivity of Alternaria isolates to iprodione, tebuconazole, and azoxystrobin The field isolates 37C11S, 37D5S, 37E5S, 37E8S, and KA48S were resistant to azoxystrobin because the PCR primers ARF4 and ARR4 amplified a 246 bp DNA fragment from each of these isolates (Fig. 1), but they were sensitive to both iprodione and tebuconazole (Table 1). There were no significant (P = 0.156, ANOVA of SAS) differences in sensitivity to iprodione and tebuconazole
Fig. 1. An allele-specific PCR assay for detecting azoxystrobinresistant Alternaria spp. from pistachio in California. The allele-specific primer pair ARF4 + ARR4 amplified a 246 bp DNA fragment from azoxystrobin-resistant but not fromsensitive Alternaria isolates.
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among these five isolates. The EC50 to iprodione for LIIR isolate 37E5R, which was a laboratoryinduced iprodione-resistant isolate derived from the wild-type isolate 37E5S, was significantly (P < 0.01, ANOVA of SAS) higher than that of its original parent 37E5S. The field isolate 25C2R had a very high EC50 value to iprodione (Table 1), thus, it was designated field iprodioneresistant (FIR) isolate. This FIR isolate, however, was sensitive to tebuconazole (Table 1), and also sensitive to azoxystrobin because the primers ARF4 and ARR4 could not amplify the 246 bp DNA fragment from this isolate (Fig. 1). These results indicate that there is no cross-resistance among iprodione, tebuconazole, and azoxystrobin in these Alternaria isolates from pistachios. 3.2. Osmotic sensitivity The LIIR isolate 37E5R grew significantly (P < 0.03, ANOVA of SAS) slower than its wildtype sensitive parent 37E5S in PDA plates
amended with NaCl at each of the five concentrations (Fig. 2A). However, there were no significant differences (P > 0.21, ANOVA of SAS) in RGI between the FIR isolate 25C2R and the field iprodione-sensitive (FIS) isolates 37C11S, 37D5S, 37E5S, 37E8S, and KA48S at each NaCl concentration, which indicated that the FIR isolate 25C2R did not show higher sensitivity to osmotic stress (Fig. 2B). 3.3. Efficacy of iprodione against the disease caused by LIIR, FIR, and FIS Alternaria isolates Laboratory inoculation tests demonstrated that when iprodione was not applied, the LIIR isolate 37E5R and its original parent 37E5S caused similar lesion lengths (no statistical difference) on detached pistachio leaves (Fig. 3A). Although the FIR isolate 25C2R showed lower virulence than the FIS isolates 37E5S and 37E8S on detached pistachio leaves, it still caused longer disease lesions than the FIS isolates 37C11S and 37D5S (Fig. 3A).
Fig. 2. Comparisons in osmotic sensitivity between the laboratory-induced iprodione-resistant (LIIR) isolate 37E5R and its sensitive parent 37E5S (A), and among field iprodione-resistant (FIR) isolate 25C2R and -sensitive (FIS) Alternaria isolates collected from pistachios (B). The data represent means ± SD of the two experiments since there were no significant (P > 0.05, FisherÕs LSD test) differences between the two experiments.
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Fig. 3. Comparisons in disease lesion length among Alternaria isolates on detached pistachio leaflets treated with or without iprodione (A), and comparisons in iprodione efficacy among these isolates (B). The data represent means ± SD of the two experiments; gray or blank bars in each graph with the same letter are not significantly different at P = 0.05 (ANOVA of SAS).
When iprodione at 500 mg/L was applied, all five FIS isolates caused significantly smaller lesions than the FIR and LIIR isolates (Fig. 3A). The efficacy of iprodione in controlling disease caused by the FIR and LIIR isolates was significantly lower than that of the FIS isolates (Fig. 3B). 3.4. Sequences of the coiled-coil region of HK genes from Alternaria isolates A single DNA fragment (1605 bp) was amplified with the primers AaHKF and AaHKR from each of seven Alternaria isolates tested (Table 1). GenBank Accession Nos. of these sequences are AY559302–AY559308 for the isolate 25C2R, 37E5R, 37C11S, KA48S, 37D5S, 37E5S, and 37E8S, respectively. The coiled-coil region of HK gene from each isolate contained an intron within the second 90-amino-acid repeat. Deduced amino acid sequences of this section of HK gene from either LIIR, FIR, or FIS isolates were 518 residues in length. The sequenced section included all posi-
tions known to affect the sensitivity to dicarboximides in A. alternata and other fungi [14–18]. However, there were no differences in deduced amino acid sequence among the LIIR, FIR, FIS Alternaria isolates collected from pistachio, although there were 5–20 silent substitutions in the DNA sequences among these isolates. Deduced amino acid sequence of the coiled-coil region of HK gene from Alternaria isolates in this study showed identity of 100% to that from A. alternata (GenBank Accession No. AAO48475), 98% to Cochliobolus heterostrophus (BAC78684), 79% to Botryotinia fuckeliana (AAL30826), 78% to N. crassa (AAB01979), 77% to Magnaporthe grisea (EAA55993), and 75% to Fusarium solani f. sp. pisi (AAD09491).
4. Discussion In a previous study, we showed that most Alternaria isolates (59 of 60) collected from pistachios
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in California were sensitive to iprodione; only one isolate (25C2R) was highly resistant to this compound [21]. In this study, we found that this field iprodione resistant (FIR) isolate did not show higher osmotic sensitivity than field iprodione sensitive (FIS) isolates, and it still remained high virulence on pistachio. These results suggest that the FIR isolate may have sufficient parasitic fitness to compete with FIS isolates in the field. Thus, under selection pressure of multiple applications of iprodione, the FIR isolate may spread in pistachio orchards. Since both LIIR and FIR isolates were sensitive to azoxytrobin and tebuconazole, and azoxystrobin-resistant isolates were sensitive to iprodione and tebuconazole, alternative applications of iprodione, azoxystrobin, and tebuconazole could delay the development of iprodione- and azoxystrobin-resistant Alternaria populations in California pistachio orchards. The alternative applications of these fungicides are necessary since azoxystrobin-resistant Alternaria populations have been developed in some pistachio orchards. Under laboratory conditions, dicarboximide-resistant mutants could be induced easily in many fungal species, including A. alternata [10], A. brassicicola [10], B. cinerea [14,15], Botryosphaeria dothidea [23], and N. crassa [18]. Generally, most laboratory-induced mutants had high levels of resistance to dicarboximides and also showed higher osmotic sensitivity compared to their original sensitive parents. In this study, we also found the LIIR isolate 37E5R was more sensitive to osmotic stress than its sensitive parent 37E5S. However, the FIR isolate 25C2R did not show higher osmotic sensitivity compared to the FIS isolates. These observations suggest that the resistance
mechanism of the FIR Alternaria isolate differs from that of the LIIR isolate. In N. crassa, mutations in several osmotic sensitivity genes (os-1, os-2, os-4, os-5, smco-8, and smco-9) were found to confer resistance to dicarboximide, aromatic hydrocarbon, and phenylpyrrole fungicides [20]. The os-1 gene has been cloned and shown to encode a histidine kinase, which plays an important role in the regulation of cell-wall assembly and other cell responses to changes in external osmolarity [24]. Recently, several studies on N. crassa, B. cinerea, and A. alternata suggested that the potential target site of dicarboximides could be the coiled-coil region of the two-component histidine kinase (HK) [14– 18]. In those previous studies, amino acid changes in the HK coiled-coil region were found to be associated with dicarboximide resistance (Table 2). In A. alternata, a duplication of an 11-base nucleotide sequence was found in the first amino-acid repeat of the coiled-coil region of the HK gene from a FIR isolate Aa-8495, which was collected from passion fruit in Australia. This duplication of 11base nucleotide sequence resulted in a premature stop codon at amino acid position 95 [17]. In another FIR isolate Aa-8501, a four-base nucleotide deletion was detected within the fifth amino-acid repeat which caused termination of the open reading frame at amino acid position 434 [17]. In our study, however, LIIR, FIR, and FIS Alternaria isolates from California pistachio had identical amino acid sequences of the HK coiled-coil region, which indicates that an alternative resistance mechanism other than mutation in the coiled-coil region of HK gene is responsible for dicarboximide resistance in Alternaria isolates from pistachio.
Table 2 Summary of mutations at two-component histidine kinase genes from dicarboximide-resistant mutants Fungal species
Origina
Mutation position in coiled-coil regionb
Reference
A. alternata species-group A. alternata species-group A. arborescens species-group B. cinerea B. cinerea N. crassa
Field Lab Field Field Lab Lab
1, 5 No mutations observed No mutations observed 2 2, 3, 4, 5 2, 5
[17] This study This study [15,16] [14] [18]
a b
Origin from where the resistant mutant was obtained. The 90-amino-acid repeat in which mutation was observed.
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Alternative resistance mechanisms other than mutation in the HK coiled-coil region have been observed in other laboratory induced dicarboximide-resistant fungi, e.g., B. cinerea and Ustilago maydis. In B. cinerea, genetic analysis of LIIR mutants showed that dicarboximide resistance in some resistant mutants was conferred by another locus, Daf2 [25], which was not linked to the HK gene. Since the Daf2 gene has not been cloned yet, a comparison of the Daf2 sequences from the resistant mutant and wild-type sensitive parent has not been reported. In the basidiomycete U. maydis, Ramesh et al. [20] reported that mutants disrupted in the ubc1 gene, which encodes the regulatory subunit of a cAMP-dependent protein kinase (PKA), were resistant to dicarboximides. The authors speculated that the resistance of ubc1 mutants is due to the absence of the regulatory subunit and the resulting increase in the PKA activity. Currently, it is under investigation if such alternative mechanisms are responsible for iprodione resistance in Alternaria spp. from pistachio. In light of recent studies on dicarboximide resistance and the results from this study, we conclude that resistance mechanisms to dicarboximides in Alternaria spp. are complex. Subsequently, applications of molecular methods for detections of resistant Alternaria populations should be difficult. Acknowledgments We thank Tae H. Lim for testing fungicide sensitivity in Alternaria isolates, David P. Morgan for collecting Alternaria isolates from pistachio, and Dr. Yong Luo for helping with statistical analysis.
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