Species specific primers for identification of Alternaria solani, in combination with analysis of the F129L substitution associated with loss of sensitivity toward strobilurins. / Edin, E

Year published: 2012

Journal: Crop Protection 38, 72-73

DOI:  10.1016/j.cropro.2012.03.021

Abstract: A species specific forward primer was designed in order to identify the phytopathogenic fungus Alternaria solani, which causes early blight on potato. The reverse primer came from another survey of the American population of A. solani. The combination of these two primers can be used both as molecular identification of A. solani as well as sending the PCR product for sequencing in search for the F129L substitution associated with loss of sensitivity toward fungicides based on strobilurins. The diagnostic method has been successfully used when identifying the causal agent of lesions on potato leaflets during large field assays.

Differentiation of the closely related species, Alternaria solani and A. tomatophila, by molecular, morphological and physiological features. /Gannibal Ph.B., Orina A.S., Mironenko N.V., Levitin M.M.

Year published: 2014

Journal: European Journal of Plant Pathology. DOI 10.1007/s10658-014-0417-6

Abstract: 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 primedpolymerase 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.

Characterization of a tomato pathogen, Alternaria tomatophila, previously unidentified in Russia. / Gannibal Ph.B., Orina A.S.

Year published: 2013

Journal: Mikologiya i Fitopatologiya. 47 (1): 51-55 [in Russian]

Abstract: Anamorphic fungus Alternaria solani Sorauer is traditionally considered to be the causal agent of Alternaria leaf spot of tomato in Russia. However long ago different research teams revealed sufficient intraspecific diversity in this pathogen. A little more than 10 years ago large-spored Alternaria species from potato and tomato were established as different species basing on thorough morphological observations. The causal agent of the early blight of tomato in USA was described as a new species, A. tomatophila E. G. Simmons. Re-identification of some Russian A. solani-like isolates revealed their strong similarity with A. tomatophila. The present paper contains detailed description of A. tomatophila and the identification key for all large-spored Alternaria species that ever have been found on tomato. Some findings of A. tomatophila in Russia in 2006-2011 are listed. According to that list this pathogen is distributed in all main tomato-growing regions of the country.

Shift in Sensitivity of Alternaria solani in Response to QoI Fungicides. / Pasche, Julie S., Wharam, C. and Gudmestad, Neil C

Year published: 2004

Journal: Plant Disease 88:181-187

Abstract: Isolates of Alternaria solani, cause of potato early blight, collected in 1998 through 2001 from various potato growing areas across the midwestern United States, were tested for sensitivity to azoxystrobin. Isolates collected in 1998, prior to the introduction of azoxystrobin, were tested to establish the baseline sensitivity of the fungus to this fungicide. Isolates collected in subsequent years, not necessarily from the same sites as baseline isolates, were tested to determine if populations of A. solani had become less sensitive to azoxystrobin. Azoxystrobin sensitivity was determined utilizing an in vitro spore germination assay. The effective fungicide concentration that inhibited spore germination by 50% (EC50) was determined for each isolate. There was no significant difference in mean EC50 values between baseline isolates and all other isolates collected through 1999. Mean azoxystrobin EC50 values of A. solani isolates collected in 2000 and 2001 were significantly higher compared with means from previous years, and mean azoxystrobin EC50 values from 2001 were significantly higher than means from isolates collected in 2000. A subset of 54 A. solani isolates was evaluated in vitro for cross-sensitivity to pyraclostrobin and trifloxystrobin. A highly significant and strong correlation among the isolates tested for fungicide cross-sensitivity was detected between azoxystrobin and pyraclostrobin; however, the correlation between azoxystrobin and trifloxystrobin, and between trifloxystrobin and pyraclostrobin, was significant but weak. A second subset of five isolates was chosen for in vivo assessment of azoxystrobin, pyraclostrobin, and trifloxystrobin sensitivity. Disease severity on plants treated with azoxystrobin and pyraclostrobin was significantly greater with reduced-sensitive A. solani isolates compared with sensitive isolates. Disease severity was not statistically different between azoxystrobin reduced-sensitive and sensitive A. solani isolates on plants treated with trifloxystrobin. This is the first report of a shift in sensitivity to QoI fungicides in a fungus possessing only an anamorphic stage.

Effect of the F129L mutation in Alternaria solani on fungicides affecting mitochondrial respiration. / Pasche, J.S., Piche, L.M., and Gudmestad, N. C.

Year published: 2005

Journal: Plant Disease 89:269-278

Abstract: Isolates of Alternaria solani previously collected from throughout the Midwestern United States and characterized as being azoxystrobin sensitive or reduced sensitive were tested for sensitivity to the Quinone outside inhibitor (QoI) fungicides famoxadone and fenamidone and the carboxamide fungicide boscalid. All three fungicides affect mitochondrial respiration: famoxadone and fenamidone at complex III, and boscalid at complex II. A. solani isolates possessing reduced-sensitivity to azoxystrobin also were less sensitive in vitro to famoxadone and fenamidone compared with azoxystrobin-sensitive isolates, but the shift in sensitivity was of lower magnitude, approximately 2- to 3-fold versus approximately 12-fold for azoxystrobin. The in vitro EC50 values, the concentration that effectively reduces germination by 50% relative to the untreated control, for sensitive A. solani isolates were significantly lower for famoxadone and azoxystrobin than for fenamidone and boscalid; whereas, for reduced-sensitive isolates, famoxadone EC50 values were significantly lower than all other fungicides. Isolates of A. solani with reduced-sensitivity to azoxystrobin were twofold more sensitive in vitro to boscalid than were azoxystrobin- sensitive wild-type isolates, displaying negative cross-sensitivity. All isolates determined to have reduced-sensitivity to azoxystrobin also were determined to possess the amino acid substitution of phenylalanine with leucine at position 129 (F129L mutation) using real-time polymerase chain reaction. In vivo studies were performed to determine the effects of in vitro sensitivity shifts on early blight disease control provided by each fungicide over a range of concentrations. Reduced-sensitivity to azoxystrobin did not significantly affect disease control provided by famoxadone, regardless of the wide range of in vitro famoxadone EC50 values. Efficacy of fenamidone was affected by some azoxystrobin reduced-sensitive A. solani isolates, but not others. Boscalid controlled azoxystrobin-sensitive and reduced-sensitive isolates with equal effectiveness. These results suggest that the F129L mutation present in A. solani does not convey cross-sensitivity in vivo among all QoI or related fungicides, and that two- to threefold shifts in in vitro sensitivity among A. solani isolates does not appreciably affect disease control.

Prevalence, competitive fitness and impact of the F129L mutation in Alternariasolani in the United States. / Pasche, J.S. and Gudmestad, Neil C.

Year published: 2008

Journal: Crop Protection 27:427-435.

Abstract: Reduced sensitivity to quinone outside inhibitor (QoI) fungicides conferred by the presence of the F129L mutation was identified in Alternaria solani isolates collected in Nebraska in 2000 and in isolates collected from the Midwestern states of Minnesota and North Dakota in 2001. Over 4200 isolates of A. solani collected over a 5-year period from 2002 to 2006 from 11 potato-producing states were evaluated for the presence of the F129L mutation utilizing azoxystrobin spore germination assays and real-time PCR. From this population, 96.5% of isolates were determined to have reduced sensitivity to QoI fungicides and/or to contain the F129L mutation. In addition to Midwestern states of Minnesota, Nebraska and North Dakota, A. solani isolates containing the F129L mutation were collected from Colorado, Michigan and Texas in 2003, Wisconsin in 2005, as well as from the Western United States including Idaho, Washington and Wyoming in 2005 and Oregon in 2006. The detection of these isolates in areas outside the Midwest suggests that the F129L mutation is stable and present under conditions that are less conducive for the pathogen and where it is under less selection pressure by QoI fungicides. Competitive fitness tests confirm that, while isolates of A. solani with the F129L mutation had a decrease in in vitro percentage germination compared with wild-type isolates, F129L mutant isolates produced higher disease severity in greenhouse trials than wild-type isolates. Field trials were performed in central Minnesota and central North Dakota in 2000 and 2001 when the A. solani population was dominated by wild-type isolates as well as in 2002, 2003, 2005 and 2006 when F129L mutant isolates dominated. These trials support in vitro and greenhouse results indicating that the F129L mutation has affected the field performance of all QoI fungicides. Field trial results suggest that these fungicides do not provide improved disease control over standard protectant fungicides such as chlorothalonil and mancozeb.

Prevalence and impact of SDHI resistance in Alternaria solani. / Gudmestad, N.C., Arabiat, S., Pasche, J.S., and Miller, J.S.

Year published: 2013

Journal: Plant Disease 97:952-960.

Abstract: Early blight, caused by Alternaria solani, is an important chronic foliar disease of potato (Solanum tuberosum) present every growing season in the Midwestern United States. Most currently grown potato cultivars lack resistance to early blight; therefore, foliar fungicides are relied upon for disease management. Foliar fungicides with high efficacy against the pathogen, such as boscalid, frequently are used under high disease pressure situations, such as potatoes grown under overhead irrigation. Boscalid is a member of the succinate dehydrogenase inhibiting (SDHI) fungicide group and was registered for use on potato in 2005. Baseline sensitivity of A. solani to the SDHI fungicides boscalid, penthiopyrad, and fluopyram using a spore germination assay demonstrated similar intrinsic activity against A. solani with mean EC50 values of 0.33, 0.38, and 0.31 μg/ml, respectively. However, isolates varied in their sensitivity to each of these fungicides, resulting in very low correlations (r) among isolate sensitivity to each fungicide. Resistance to boscalid in A. solani was detected in the states of North Dakota, Minnesota, Nebraska, Texas, Idaho, Wisconsin, and Florida from early blight samples collected in 2010 and 2011. Two phenotypes of boscalid resistance were detected. Approximately 80% of all A. solani assayed were found to have some level of resistance to boscalid with about 5 and 75% of the population moderately resistant (5 to 20 μg/ml) and highly resistant (>20 μg/ml), respectively, to the fungicide.

Nearly 99% of all boscalid resistant isolates possessed the F129L mutation in the cytochrome b gene, indicating that an A. solani population with dual fungicide resistance predominates in the states surveyed. However, A. solani isolates resistant to boscalid remained sensitive to fluopyram, and a large proportion of moderately resistant and resistant isolates were sensitive to penthiopyrad. Disease control data from in vivo trials demonstrated a significant loss of fungicide efficacy when boscalid and fluxapyroxad were used to control moderately and highly resistant isolates of A. solani relative to the control these fungicides provided wild-type isolates. Fluopyram, however, controlled boscalid resistant isolates as well as it controlled wild-type isolates of A. solani. These data will assist potato growers in regions where boscalid resistance is prevalent by assisting them in avoiding fungicides that do not effectively control early blight and in selecting SDHI fungicide molecules that remain efficacious.

Molecular characterization and detection of mutations associated with resistance to succinate dehydrogenase inhibiting (SDHI) fungicides in Alternaria solani. / Mallik, I., Arabiat, S., Pasche, J.S., Bolton, M.D., Patel, J., and Gudmestad, N.C.

Year published: 2014

Journal: Phytopathology104:40-49

Abstract: Early blight, caused by Alternaria solani, is an economically important foliar disease of potato in several production areas of the United States. Few potato cultivars possess resistance to early blight; therefore, the application of fungicides is the primary means of achieving disease control. Previous work in our laboratory reported resistance to the succinate dehydrogenase-inhibiting (SDHI) fungicide boscalid in this plant pathogen with a concomitant loss of disease control. Two phenotypes were detected, one in which A. solani isolates were moderately resistant to boscalid, the other in which isolates were highly resistant to the fungicide. Resistance in other fungal plant pathogens to SDHI fungicides is known to occur due to amino acid exchanges in the soluble subunit succinate dehydrogenase B (SdhB), C (SdhC), and D (SdhD) proteins. In this study, the AsSdhB, AsSdhC, and AsSdhD genes were analyzed and compared in sensitive (50% effective concentration [EC50] < 5 μg ml–1), moderately resistant (EC50 = 5.1 to 20 μg ml–1), highly resistant (EC50 = 20.1 to 100 μg ml–1), and very highly resistant (EC50 > 100 μg ml–1) A. solani isolates. In total, five mutations were detected, two in each of the AsSdhB and AsSdhD genes and one in the AsSdhC gene. The sequencing of AsSdhB elucidated point mutations cytosine (C) to thymine (T) at nucleotide 990 and adenine (A) to guanine (G) at nucleotide 991, leading to an exchange from histidine to tyrosine (H278Y) or arginine (H278R), respectively, at codon 278. The H278R exchange was detected in 4 of 10 A. solani isolates moderately resistant to boscalid, exhibiting EC50 values of 6 to 8 μg ml–1. Further genetic analysis also confirmed this mutation in isolates with high and very high EC50 values for boscalid of 28 to 500 μg ml–1. Subsequent sequencing of AsSdhC and AsSdhD genes confirmed the presence of additional mutations from A to G at nucleotide position 490 in AsSdhC and at nucleotide position 398 in the AsSdhD, conferring H134R and H133R exchanges in AsSdhC and AsSdhD, respectively. The

H134R exchange in AsSdhC was observed in A. solani isolates with sensitive, moderate, highly resistant, and very highly resistant boscalid phenotypes, and the AsSdhD H133R exchange was observed in isolates with both moderate and very high EC50 value boscalid phenotypes. Detection and differentiation of point mutations in AsSdhB resulting in H278R and H278Y exchanges in the AsSdhB subunit were facilitated by the development of a mismatch amplification mutation assay. Detection of these two mutations in boscalid-resistant isolates, in addition to mutations in AsSdhC and AsSdhD resulting in an H134R and H133R exchange, respectively, was achieved by the development of a multiplex polymerase chain reaction to detect and differentiate the sensitive and resistant isolates based on the single-nucleotide polymorphisms present in all three genes. A single A. solani isolate with resistance to boscalid did not contain any of the above-mentioned exchanges but did contain a substitution of aspartate to glutamic acid at amino acid position 123 (D123E) in the AsSdhD subunit. Among A. solani isolates possessing resistance to boscalid, point mutations in AsSdhB were more frequently detected than mutations in genes coding for any other subunit.

Early blight control in potatoes using disease orientated threshold values. / Leiminger, J.H. and Hausladen H.

Year published: 2012

Journal: Plant Disease 96 (1), 124-130

Abstract: Epidemics of early blight caused by Alternaria species can cause significant economic damage to potato production if not adequately controlled. In order to improve control of Alternaria spp. in potatoes, studies were conducted to identify the optimal fungicide strategy and, if possible, to reduce the number of fungicide applications per growing season. Therefore, a disease threshold-based framework was tested to define the optimal timing of fungicide application. The initiation and subsequent applications of fungicides were based on increases in disease incidence or severity. Adequate disease control was achieved by a three time application with azoxystrobin, given that the applications were carried out at pivotal times in the epidemic. Targeted applications of fungicides reduced the number of sprayings required to protect starch yield. Results indicate early blight can be effectively managed by using fungicide application thresholds based on disease progress.

Occurrence of the F129L mutation in Alternaria solani populations in Germany in response to QoI application, and its effect on sensitivity. / Leiminger, J.H., Adolf, B. and Hausladen H.

Year published: 2013

Journal: Plant Pathology 63 (3), 640-650

Abstract: 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. We assume that the application of QoIs has selected for the occurrence of F129L mutations, which may contribute to loss of fungicide efficacy.

Genetic variability among Alternaria solani isolates from potatoes in Southern Germany based on RAPD-profiles. / Leiminger, J.H., Auinger, H.-J., Wenig, M., Bahnweg, G and Hausladen H.

Year published: 2013

Journal: Journal of Plant Diseases and Protection 4/2013 120:164-172

Abstract: One of the problems encountered when studying the population genetics of fungal plant pathogens is defining what constitutes a “population”. Potato early blight is a major disease of potatoes and other species of the Solanaceae. The causal agent is the ascomycete Alternaria solani (Sorauer), which infects leaves and stems of potato plants leading to premature defoliation. In this study, spatial genetic diversity within A. solani populationsfrom potato leaves were investigated using RAPD markers in order to reveal the degree of homo- or heterogeneity. Analysis of RAPD profiles revealed distinct genetic diversity among isolates originating from the same field. In addition, pronounced genetic variability was found for isolates from different years. These results indicate a surprising genetic heterogeneity within the population of A. solani, which must be kept in mind when designing protective measures for agriculture.

Phenotyping early blight resistance in potato cultivars and breeding clones. / F. Odilbekov, U. Carlson-Nilsson, E. Liljeroth

Year published: 2014

Journal: Euphytica (2014) DOI 10.1007/s10681-013-1054-4

Abstract: Thirty-two potato cultivars/clones were evaluated for resistance to early blight using different methods. The evaluations were performed under field and greenhouse conditions. In the field experiments, plants were evaluated for disease symptoms, and the relative area under the disease progress curve and percent defoliation were determined. In the greenhouse experiments, leaf lesion sizes were determined on either intact plants or detached leaves after drop inoculation with Alternaria solani. The effect of leaf position (lower, middle or upper part of the plant) on lesion size was investigated. There was no correlation between lesion sizes on lower leaves and upper leaves after inoculation. However, significant correlations between lesion sizes on lower and middle leaves and also between middle and upper leaves were found. Furthermore, we found significant correlation between the results of resistance studies in the field and in intact plant inoculation experiments in the greenhouse. In contrast, results from the detached-leaf experiment were not correlated with either greenhouse intact plant tests or field results. The results indicate that using detached-leaf assays for screening potato for early blight resistance is not accurate. We found significant differences in resistance to A. solani among cultivars/ clones in both the field and in greenhouse experiments.