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

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

Characterization of iprodione-resistant Alternaria isolates from pistachio in California

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 wild-type 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.     

Resistance to pyraclostrobin, boscalid and multiple resistance to Pristine® (pyraclostrobin + boscalid) fungicide in Alternaria alternata causing alternaria late blight of pistachios in California

Pristine® (pyraclostrobin + boscalid) is a fungicide registered for the control of alternaria late blight in pistachio. A total of 95 isolates of Alternaria alternata collected from orchards with and without a prior history of Pristine® sprays were tested for their sensitivity towards pyraclostrobin, boscalid and Pristine® in conidial germination assays. The EC₅₀ values for 35 isolates from orchards without Pristine® sprays ranged from 0·09 to 3·14 µg mL^?1 and < 0·01 to 2·04 µg mL^?1 for boscalid and Pristine®, respectively. For pyraclostrobin, 27 isolates had EC₅₀ < 0·01 µg mL^?1 and six had low resistance (mean EC₅₀ value = 4·71 µg mL^?1). Only one isolate was resistant to all three fungicides tested, with EC₅₀ > 100 µg mL^?1. Among 59 isolates from the orchard with a history of Pristine® sprays, 56 were resistant to pyraclostrobin; only two were sensitive (EC₅₀ < 0·01 µg mL^?1) and one was weakly resistant (EC₅₀ = 10 µg mL^?1). For the majority of these isolates EC₅₀ values ranged from 0·06 to 4·22 µg mL^?1 for boscalid and from 0·22 to 7·74 µg mL^?1 for Pristine®. However, seven isolates resistant to pyraclostrobin were also highly resistant to boscalid and Pristine® and remained pathogenic on pistachio treated with Pristine®. Whereas strobilurin resistance is a common occurrence in Alternaria of pistachio, this is the first report of resistance to boscalid in field isolates of phytopathogenic fungi. No cross resistance between pyraclostrobin and boscalid was detected, suggesting that Pristine® resistance appears as a case of multiple resistance.     

Pathogenicity,Morpho-Species and Mating Types of <Emphasis Type="Italic">Alternaria</Emphasis> spp. causing Alternaria blight in <Emphasis Type="Italic">Pistacia</Emphasis> spp. in Turkey

Alternaria genus includes many plant pathogens on numerous hosts, causing leaf spots, rots and blights. Alternaria blight has been observed as one of the important fungal diseases of pistachio (Pistacia vera L.) as well as its wild relatives (P. terebinthus, P. lentiscus, P. khinjuk, P. atlantica, P. mutica) in Turkey. Alternaria species were sampled from Pistacia spp. hosts from different geographic regions in Turkey during field trips in late spring to early fall of 2013. Alternaria blight symptoms were observed mainly on fruits and rarely on leaves. Four hundred and twenty two of the isolates were morphologically defined as A. alternata, A. tenuissima, A. arborescens and also intermediate morpho-species between A. alternata/A. arborescens. Pathogenicity of the isolates was confirmed with host inoculations on detached fruits. Mating types of 270 isolates of Alternaria spp. from the collection were identified using a PCR-based mating type assay that amplifies either a MAT1-1 or a MAT1-2 fragment from the mating locus. Although a strongly clonal population structure was expected due to the putative asexual reproduction of these fungi, both idiomorphs were detected at equal frequencies at several different spatial scales. The distribution of mating types within each geographic region, within host species as well as in overall collection was not significantly different from 1:1. Amplified fragments of partial idiomorph sequences were obtained for representative isolates. Parsimony trees were depicted based on sequence data of mating type genes for these representative isolates as well as some other Alternaria species obtained by Genebank. Several point mutations presented a few clusters which are supported by high bootsrapped values. The Alternaria blight disease agents both from cultivated and wild hosts were pathogenic on pistachio which may cause difficulties to control the disease because of extensity of pathogen sources. Besides, equal mating type distribution of the pathogen at both geographic and host species levels suggests a potential for sexual reproduction of Alternaria spp. in Turkey.     

Pathogenicity and toxigenicity of <Emphasis Type="Italic">Fusarium verticillioides</Emphasis> isolates collected from maize roots,stems and ears in South Africa

Sunflower (Helianthus annuus L.) is an important oilseed crop in South Africa, and is grown in rotation with maize in some parts of North West, Limpopo, Free State, Mpumalanga and Gauteng provinces. Alternaria leaf blight is currently one of the major potential disease threats of sunflower and is capable of causing yield losses in all production regions. Alternaria helianthi was reported as the main cause of Alternaria leaf blight of sunflower in South Africa; however small-spored Alternaria species have been consistently isolated from leaf blight symptoms during recent surveys. The aim of this study was to use morphological and molecular techniques to identify the causal agent(s) of Alternaria blight isolated from South African sunflower production areas. Alternaria helianthi was not recovered from any of the sunflower lesions or seeds, with only Alternaria alternata retrieved from the symptomatic tissue. Molecular identification based on a combined phylogenetic dataset using the partial internal transcribed spacer regions, RNA polymerase second largest subunit, glyceraldehyde-3-phosphate dehydrogenase, translation elongation factor and Alternaria allergen gene regions was done to support the morphological identification based on the three-dimensional sporulation patterns of Alternaria. Furthermore, this study aimed at evaluating the pathogenicity of the recovered Alternaria isolates and their potential as causal agents of Alternaria leaf blight of sunflower. Pathogenicity tests showed that all the Alternaria alternata isolates tested were capable of causing Alternaria leaf blight of sunflower as seen in the field. This is the first report of A. alternata causing leaf blight of sunflower in South Africa.     

Characterization of Alternaria species associated with leaf blight of sunflower in China

Nine Alternaria species have been reported to be associated with sunflower leaf blight worldwide, and A. helianthi has been recognized as the most prevalent and damaging species. However, the population structure of Alternaria species causing leaf blight of sunflower in China had not been examined thoroughly prior to this study. During 2010 to 2013, a total of 272 Alternaria isolates were obtained from infected sunflower leaves in 11 provinces, autonomous regions, and municipalities of China. Based on morphological traits and sequence analyses of the internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA) and the partial coding sequences of the histone 3 gene, 227 (83.5 %) isolates were identified as belonging to Alternaria tenuissima, the remainder 45 isolates were grouped to A. alternata (16.5 %). Compared with the ITS regions of rDNA, sequence analyses of the partial coding sequences of histone 3 gene displayed a critical role in discrimination of the small-spored Alternaria species. Phylogenetic analysis of the partial coding sequence of histone 3 gene clearly divided the representative Alternaria isolates into two main clades, A. tenuissima and A. alternata. The pathogenicity of A. tenuissima and A. alternata on detached leaves of sunflower cv. Gankui No.2 did not significantly differ between the two species or among isolates from different geographical origins. Our results indicate that the population structure of Alternaria species associated with sunflower leaf blight differed from that reported previously in China since A. helianthi was not found in this study. In addition, this is the first report about A. tenuissima causing leaf blight on sunflower in China.     

Occurrence and molecular characterization of azoxystrobin resistance in cucumber downy mildew in Shandong province of China

Cucumber downy mildew caused byPseudoperonospora cubensis (Berk. and Curt.) Rostov. limits crop production in Shandong Province of China. Since management of downy mildew is strongly dependent on fungicides, a rational design of control programs requires a good understanding of the fungicide resistance phenomenon in field populations of the pathogen. A total of 106 and 97 isolates ofP. cubensis were obtained in 2006 and 2007, respectively. The EC₅₀ values for the growth of all the 106 isolates collected in 2006 were 0.0063–0.0688μg ml⁻¹ (average: 0.0196±0.0048μg ml⁻¹) azoxystrobin and these were therefore considered sensitive isolates. However, 57 field isolates ofP. cubensis of the 97 collected in 2007 with EC₅₀ values that ranged from 0.609 to >51.2μg ml⁻¹ were considered resistant to azoxystrobin. Fragments of the fungicide-targeted mitochondrial cytochromeb gene from total pathogen DNA were amplified using polymerase chain reaction and their sequences analyzed to elucidate the molecular mechanism of resistance. A single point mutation (GGT to GCT) in the cytochromeb gene, resulting in substitution of glycine by alanine at position 143, was found in the three selected azoxystrobin-resistant isolates of downy mildew. This substitution in cytochromeb exhibited different resistance levels, with the resistance factor from 21.15 to greater than 2618.9. In addition, the different resistance levels seemed to appear within 1 year (between 2006 and 2007). Therefore, growers of Shandong Province in China now are faced with a challenge in managing the azoxystrobin resistance in cucumber downy mildew. http://www.phytoparasitica.org posting March 10, 2008.     

Sensitivity to azoxystrobin in Didymella bryoniae isolates collected before and after field use of strobilurin fungicides

BACKGROUND: Isolates of Didymella bryoniae (Auersw.) Rehm, causal agent of gummy stem blight on cucurbits, developed insensitivity to azoxystrobin in the eastern United States 2 years after first commercial use in 1998. Baseline sensitivity of this fungus to azoxystrobin has never been reported. The objectives were to compare baseline sensitivities of D. bryoniae from South Carolina and other locations to sensitivities of isolates exposed to azoxystrobin for one or more seasons, and to compare sensitivity in vitro and in vivo. RESULTS: Sixty‐one isolates of D. bryoniae collected before 1998 were sensitive. Median EC₅₀ was 0.055 mg L^?1 azoxystrobin (range 0.005 to 0.81). Forty isolates collected after exposure during 1998 also were sensitive. Fifty‐three of 64 isolates collected in South and North Carolina between 2000 and 2006 were insensitive to 10 mg L^?1 azoxystrobin. Sensitive and insensitive isolates were distinguished by disease severity on Cucumis melo L. seedlings treated with azoxystrobin (20 or 200 mg L^?1). CONCLUSIONS: An azoxystrobin baseline sensitivity distribution was established in vitro for isolates of D. bryoniae never exposed to strobilurins. Baseline values were comparable with those of other ascomycetes. Insensitive isolates were found in fields with a history of strobilurin applications. An in vivo method distinguished sensitive and insensitive isolates. Copyright © 2009 Society of Chemical Industry     

Assessing the Belgian potato <Emphasis Type="Italic">Alternaria</Emphasis> population for sensitivity to fungicides with diverse modes of action

Early blight and brown spot, caused by respectively Alternaria solani and Alternaria alternata, can lead to severe yield losses in potato-growing areas. To date, fungicide application is the most effective measure to control the disease. However, in recent years, a reduced sensitivity towards several active ingredients has been reported. To shed light on this issue, Alternaria isolates were collected from different potato fields in Belgium during two growing seasons. Subsequently, the sensitivity of these isolates was assessed using four widely used fungicides with different modes of action. Demethylation inhibitors, quinone outside inhibitors, a dithiocarbamate and a carboxylic acid amide were included in this study. Although all fungicides reduced spore germination and vegetative growth of Alternaria species to some extent, the interspecies sensitivity was very variable. In general, A. solani was more suppressed by the fungicides compared to A. alternata. The effectiveness of the dithiocarbamate mancozeb was high, whereas the quinone outside inhibitor azoxystrobin showed a limited activity, especially towards A. alternata. Therefore, a subset of the A. alternata and A. solani isolates was tested for the presence of, respectively, the G143A substitution and the F129L substitution in the cytochrome b. The frequency of A. alternata isolates bearing the resistant G143A allele (approximately 65%) was comparable in both sampling years, although sensitivity of isolates decreased during the growing season. This finding points to a shift of the population towards resistant isolates. Both the European genotype I and American genotype II were present in the A. solani population, with genotype I being the most prevalent. None of the genotype I isolates carried the F129L substitution, whereas in 83% of the genotype II isolates this substitution was present. Our results demonstrate for the first time that the Belgian Alternaria population on potato comprises a considerable broad spectrum of isolates with different sensitivity to fungicides.     

In vitro inhibition of Sclerotinia sclerotiorum by mixtures of azoxystrobin, SHAM, and thiram

The necrotrophic fungal phytopathogen Sclerotinia sclerotiorum (Lib.) de Bary has a broad host range and frequently causes destructive diseases. The extensive use of common fungicides to control these diseases has selected for resistance in populations of S. sclerotiorum. In this study, 105 isolates of S. sclerotiorum from different geographical regions in Jiangsu Province of China were characterized for baseline sensitivity to azoxystrobin, and the average EC₅₀ value was 0.2932 μg/mL for mycelial growth. Of the mixtures of the fungicides thiram and azoxystrobin that were tested using an in vitro mycelial growth assay, the 1:4 ratio provided the greatest inhibition of S. sclerotiorum. When tested against nine isolates, the 1:4 mixture resulted in a mean synergy ratio of 2.31, indicating synergistic inhibition. Mycelial respiration was inhibited for about 2 h by azoxystrobin alone but for 48 h by the mixture of thiram and azoxystrobin. Salicylhydroxamic acid (SHAM, a known inhibitor of alternative respiration) also increased the inhibition of mycelial growth and respiration caused by azoxystrobin. These results suggest the need for further study of effects of combinations of azoxystrobin with thiram or SHAM in planta to evaluate their potential for management of diseases caused by S. sclerotiorum.     

Occurrence of the F129L mutation in Alternaria solani populations in Germany in response to QoI application,and its effect on sensitivity

Early blight caused by Alternaria solani is a highly destructive disease of potatoes. Control of early blight mainly relies on the use of preventive fungicide treatments. Because of their high efficacy, azoxystrobin and other quinone outside inhibitors (QoIs) are commonly used to manage early blight. However, loss of sensitivity to QoIs has previously been reported for A. solani in the United States. Two hundred and three A. solani field isolates collected from 81 locations in Germany between 2005 and 2011 were screened for the presence of the F129L mutation in the cytochrome b gene; of these, 74 contained the F129L mutation. Sequence analysis revealed the occurrence of two structurally different cytb genes, which differed in the presence (genotype I) or absence (genotype II) of an intron, with genotype I being the most prevalent (63% of isolates). The F129L mutation was detected only in genotype II isolates, where it occurred in 97%. Sensitivity to azoxystrobin and pyraclostrobin was determined in conidial germination assays. All isolates possessing the F129L mutation had reduced sensitivity to azoxystrobin and, to a lesser extent, to pyraclostrobin. Early blight disease severity on plants treated with azoxystrobin was significantly higher for A. solani isolates with reduced fungicide sensitivity in the conidial germination assay compared with sensitive isolates. Data suggest an accumulation of F129L isolates in the German A. solani population over the years 2009–2011. It is assumed that the application of QoIs has selected for the occurrence of F129L mutations, which may contribute to loss of fungicide efficacy.     

广东省辣椒疫霉菌对嘧菌酯的敏感性

从广东省各地采集了54个辣椒疫霉菌株,分别测定了菌丝生长和游动孢子囊形成对嘧菌酯的敏感性。结果表明,嘧菌酯对辣椒疫霉的菌丝生长具有很强的抑制作用,EC₅₀范围为0.1428～1.9509μg/mL,平均为0.9649μg/mL;对游动孢子囊形成的抑制作用更强,EC₅₀范围为0.0010～0.0844μg/mL平均为0.0239μg/mL。正态分析表明,辣椒疫病菌54个菌株菌丝生长对嘧菌酯的敏感性符合正态分布,而孢子萌发则在高浓度区具有拖尾现象,表明自然界中已存在个别具有一定耐药性的辣椒疫霉种群。     

5种链格孢属植物病原真菌对10种杀菌剂的敏感性比较

离体条件下测定并比较了10种杀菌剂对番茄早疫病菌、苹果斑点落叶病菌、梨黑斑病菌、烟草赤星病菌和甘蓝黑斑病菌等5种链格孢属植物病原真菌的抗菌活性。结果表明,不同种的链格孢属病菌对同一药剂的敏感性相近;咪鲜胺、啶菌恶唑、苯醚甲环唑、异菌脲、腐霉利等5种杀菌剂对供试病菌菌丝生长的抑制活性最好,其EC₅₀在0.01～1.28 μg/mL之间;百菌清对病菌分生孢子萌发的抑制活性最高,其次是醚菌酯和嘧菌酯。     

抑制番茄早疫病菌活性化合物的 高通量筛选方法研究

以OD 630 值作为评价指标,在高通量筛选设备上建立了番茄早疫病菌Alternaria solania的高通量定量筛选方法,并对嘧菌酯、百菌清、 NFDA1 醚唑、霜脲氰、甲霜灵、代森锰锌、福美双和咪鲜胺8种常用杀菌剂对番茄早疫病菌孢子萌发和菌丝生长的抑制效果进行了评价,结果与孢子萌发法和目测筛选法所得结论基本一致。     

Characterization of the cytochrome b gene fragment of Puccinia species responsible for the binding site of QoI fungicides

The fragment of the cytochrome b (cyt b) gene responsible for the binding site of QoI fungicides was sequenced for different Puccinia species by using DNA and RNA as template for PCR and RT-PCR, respectively. Degenerated primers for the cyt b gene amplified in P. recondita f.sp. tritici a 450 bp fragment, which was cloned and sequenced. At cDNA level, several Thermal Asymmetric InterLaced (TAIL)-PCR cycles were needed to produce a 996 bp long fragment, which corresponded to almost the whole cyt b gene (about 1160-1180 bp, without introns). This fragment was sequenced and specific primers were designed. Amplification with cyt b specific primers using genomic DNA as template revealed the presence of an intron of about 1500 bp length after the codon for glycine at amino acid position 143. By using the same primer pair, the cyt b gene fragment was amplified and sequenced both at cDNA and genomic DNA level also for other rust species, including P. graminis f.sp. tritici (length: 506 bp), P. striiformis f.sp. tritici (755 bp), P. coronata f.sp. avenae (644 bp), P. hordei (660 bp), P. recondita f.sp. secalis (687 bp), P. sorghi (709 bp), and P. horiana (478 bp). At the same position as for P. recondita f.sp. tritici, an intron of about 1500-1600 bp length was detected also in all other Puccinia species. High homologies were observed among all Puccinia species for both the exonic and intronic fragments of the cyt b gene. Specific primers for the cyt b gene of all eight Puccinia species were developed, which easily amplified the fragment of the gene including all possible mutations known to confer resistance to QoIs in several plant pathogens. However, in all tested isolates of the Puccinia species included in this study, the sequence of cyt b gene fragment did not contain any point mutations.     

Thiophanate-methyl sensitivity and fitness in Lasiodiplodia theobromae populations from papaya in Brazil

Stem-end rot, caused by Lasiodiplodia theobromae, is an important postharvest disease of papaya in Brazil. The use of fungicides is one of the main disease management measures. However, there are no data available on the sensitivity of L. theobromae to thiophanate methyl (methyl benzimidazole carbamate), the most common fungicide used in papaya orchards in northeastern Brazil. Thus, the effective concentration that results in 50 % of mycelial growth inhibition (EC₅₀) of 109 isolates, representing five populations of the pathogen was estimated in vitro. Seven components of fitness were measured for the 10 isolates with lower and high values of EC₅₀. Of the 109 isolates, 20.2 % were resistant to the fungicide with EC₅₀ values greater than 300 μg ml^?1, whereas the remaining 79.8 % were sensitive with an average EC₅₀ of 1.87 μg ml^?1. The EC₅₀ values for the resistant isolates were significantly (P?≤?0.05) higher than those for the sensitive isolates. When the fitness components were evaluated, only in relation to the spore production was significant difference among sensitive and resistant isolates, and resistant isolates showed sporulation capacity significantly lower than the S isolates, indicating a fitness cost.     

2013年湖北省小麦赤霉病菌对多菌灵和戊唑醇的敏感性

2013年从湖北省7个小麦主产区分离获得106株禾谷镰刀菌,测定了其对戊唑醇和多菌灵的敏感性。结果表明:戊唑醇和多菌灵对所有供试菌株的EC50值分别为0.064~0.778和0.090~0.858 mg/L。采用SAS软件的W法对EC50分布进行了正态性检验,表明106株菌株对戊唑醇和多菌灵敏感性的频率分布符合正态分布,其EC50平均值分别为(0.383±0.129)和(0.526±0.151)mg/L。不同地区来源的菌株对两种药剂的敏感性存在显著差异,其中襄阳的菌株对两种药剂的敏感性显著低于其他6个地区的。研究结果显示:湖北省小麦赤霉病菌未出现对戊唑醇和多菌灵抗性菌群,两种药剂用于防治小麦赤霉病仍具有使用价值。     

Multiple Alternaria species groups are associated with leaf blotch and fruit spot diseases of apple in Australia

Leaf blotch and fruit spot of apple caused by Alternaria species occur in apple orchards in Australia. However, there is no information on the identity of the pathogens and whether one or more Alternaria species cause both diseases in Australia. Using DNA sequencing and morphological and cultural characteristics of 51 isolates obtained from apple leaves and fruit with symptoms in Australia, Alternaria species groups associated with leaf blotch and fruit spot of apples were identified. Sequences of Alternaria allergen a1 and endopolygalacturonase gene regions revealed that multiple Alternaria species groups are associated with both diseases. Phylogenetic analysis of concatenated sequences of the two genes resulted in four clades representing A. arborescens and A. arborescens‐like isolates in clade 1, A. tenuissima/A. mali isolates in clade 2, A. alternata/A. tenuissima intermediate isolates in clade 3 and A. longipes and A. longipes‐like isolates in clade 4. The clades formed using sequence information were supported by colony characteristics and sporulation patterns. The source of the isolates in each clade included both the leaf blotch variant and the fruit spot variant of the disease. Alternaria arborescens‐like isolates were the most prevalent (47%) and occurred in all six states of Australia, while A. alternata/A. tenuissima intermediate isolates (14%) and A. tenuissima/A. mali isolates (6%) occurred mostly in Queensland and New South Wales, respectively. Implications of multiple Alternaria species groups on apples in Australia are discussed.     

Shift of sensitivity of <Emphasis Type="Italic">Botrytis cinerea</Emphasis> to azoxystrobin in greenhouse vegetables before and after exposure to the fungicide

From 2004 to 2006, 213 isolates of Botrytis cinerea never exposed to Q_O center inhibitors (Q_OIs) were collected to determine the baseline sensitivity to azoxystrobin. In the absence of salicylhydroxamic acid (SHAM), the mean EC₅₀ values were 10.49 ± 13.12 and 0.36 ± 0.48 mg l⁻¹ for inhibiting mycelial growth and conidium germination, respectively. In the presence of SHAM, the mean EC₅₀ values were 2.24 ± 1.29 and 0.22 ± 0.11 mg l⁻¹. In 2010, five azoxystrobin-resistant isolates were detected with the resistance frequency of 2.25% in greenhouse tomatoes after 4 years of continuous exposure. These resistant isolates showed cross-resistance to other Q_OIs but not to boscalid. In addition, these resistant isolates had comparable growth, sporulation and pathogenicity ability as sensitive isolates and maintained resistance in plants and the presence of SHAM. The G143A point mutation predicted to cause a change from glycine to alanine at codon 143 of cyt b gene was found in all resistant isolates.     

Differentiation of the closely related species,Alternaria solani and A. tomatophila,by molecular and morphological features and aggressiveness

The main causal agent of early blight, the noxious disease of solanaceous crops, is generally considered to be Alternaria solani Sorauer (in a broad sense). However, heterogeneity in many morphological features of this pathogen has been noted suggesting that the disease may not be caused by a single species. Recent research has revealed that several large-spored Alternaria species may cause disease of potato and tomato including A. solani sensu stricto and A. tomatophila. The goal of our research was to compare Russian large-spored Alternaria isolates from tomato and potato to test the hypothesis that early blight of tomato and potato are caused by different species. Cluster analysis of genetic distances estimated from 12 polymorphic molecular markers (universally primed-polymerase chain reaction and randomly amplified polymorphic DNA) revealed two groups of isolates accepted here as A. solani and A. tomatophila that were supported by morphology and host plant association. Differentiation of species was supported by phylogeny derived from the DNA sequences of a portion of the Alt a1, gpd and calmodulin genes. Species-specific primers based on the Alt a1 and calmodulin gene sequences for both species were designed. Under laboratory conditions, A. solani isolates were equally aggressive on both tomato and potato, whereas A. tomatophila was highly aggressive to tomato but only weakly aggressive to potato. In the field, A. solani was isolated from potato, tomato and from several wild potato species including S. schickii, S. papita and S. kurtzianum. The majority (90 %) of A. solani isolates carried the mating type locus 1 (MAT1) idiomorph MAT1-1 while the majority (88 %) of A. tomatophila isolates carried the MAT1-2 idiomorph.