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Richard P Oliver - One of the best experts on this subject based on the ideXlab platform.
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Rapid in situ quantification of the strobilurin Resistance mutation G143A in the wheat pathogen Blumeria graminis f. sp. tritici
Scientific Reports, 2021Co-Authors: Kejal N. Dodhia, Richard P Oliver, Belinda A. Cox, Francisco J. Lopez-ruizAbstract:As the incidence of Fungicide Resistance in plant pathogens continues to increase, control of diseases and the management of Resistance would be greatly aided by rapid diagnostic methods. Quantitative allele-specific PCR (ASqPCR) is an ideal technique for the in-field analysis of Fungicide Resistance as it can quantify the frequency of mutations in Fungicide targets. We have applied this technique to the fungal pathogen Blumeria graminis f. sp. tritici ( Bgt ), the causal agent of wheat powdery mildew. In Australia, strobilurin-resistant Bgt was first discovered in 2016. Molecular analysis revealed a nucleotide transversion in the cytochrome b ( cytb ) gene in the cytochrome bc1 enzyme complex , resulting in a substitution of alanine for glycine at position 143 (G143A). We have developed an in-field ASqPCR assay that can quantify both the resistant (A143) and sensitive (G143) cytb alleles down to 1.67% in host and Bgt DNA mixtures, within 90 min of sample collection. The in situ analysis of samples collected during a survey in Tasmania revealed A143 frequencies ranging between 9–100%. Validation of the analysis with a newly developed laboratory based digital PCR assay found no significant differences between the two methods. We have successfully developed an in-field quantification method, for a strobilurin-resistant allele, by pairing the ASqPCR assay on a lightweight qPCR instrument with a quick DNA extraction method. The deployment of these type of methodologies in the field can contribute to the effective in-season management of Fungicide Resistance.
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when time really is money in situ quantification of the strobilurin Resistance mutation g143a in the wheat pathogen blumeria graminis f sp tritici
bioRxiv, 2020Co-Authors: Kejal N. Dodhia, Richard P Oliver, Belinda A. Cox, Francisco J LopezruizAbstract:Abstract Background There has been an inexorable increase in the incidence of Fungicide Resistance in plant pathogens in recent years. Control of diseases and the management of Resistance would be greatly aided by rapid diagnostic methods. Quantitative allele specific PCR (ASqPCR) is an ideal technique for the analysis of Fungicide Resistance in the field as it can both detect and quantify the frequency of mutations associated with Fungicide Resistance. We have applied this technique to the fungal pathogen Blumeria graminis f. sp. tritici (Bgt), an obligate biotrophic fungus that causes wheat powdery mildew and is responsible for up to 25% yield loss annually. In Australia, strobilurin resistant Bgt was first discovered in samples from Tasmania and Victoria in 2016. Molecular analysis revealed a nucleotide transversion in the cytochrome bc1 enzyme (cytb) complex, resulting in a substitution of alanine for glycine at position 143 (G143A) in Cytb. Results We have developed an in-field ASqPCR assay that can quantify both the resistant (A143) and sensitive (G143) cytb alleles down to 1.67% in host and Bgt DNA mixtures within 90 min of sample collection. The in situ analysis of field samples collected during a survey in Tasmania revealed A143 frequencies ranging between 9-100%. We validated the analysis with a newly developed laboratory based digital PCR assay and found no significant differences between the two methods. Conclusion We have successfully developed an in-field quantification method, for a QoI resistant allele, by pairing an ASqPCR assay on a lightweight qPCR instrument with a quick DNA extraction method. The deployment of this type of methodologies in the field can contribute to the effective in-season management of Fungicide Resistance.
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improved detection and monitoring of Fungicide Resistance in blumeria graminis f sp hordei with high throughput genotype quantification by digital pcr
Frontiers in Microbiology, 2018Co-Authors: Katherine G Zulak, Richard P Oliver, Belinda A. Cox, Madeline Ann Tucker, Francisco J LopezruizAbstract:The increased occurrence of triazole Fungicide resistant strains of Blumeria graminis f. sp. hordei (Bgh) is an economic concern for the barley industry in Australia and elsewhere. High levels of Resistance to triazoles in the field are caused by two separate point mutations in the Cyp51 gene, Y136F and S509T. Early detection of these mutations arising in pathogen field populations is important as this allows time for changes in Fungicide practices to be adopted, thus mitigating potential yield losses due to Fungicide failure and preventing the Resistance from becoming dominant. A digital PCR assay has been developed for the detection and quantification of the Y136F and S509T mutations in the Bgh Cyp51 gene. Mutation levels were quantifiable as low as 0.2% in genomic DNA extractions and field samples. This assay was applied to the high throughput screening of Bgh field and bait trial samples from barley growing regions across Australia in the 2015 and 2016 growing seasons and identified the S509T mutation for the first time in the Eastern states of Australia. This is the first report on the use of digital PCR technology for Fungicide Resistance detection and monitoring in agriculture. Here we describe the potential application of dPCR for the screening of Fungicide Resistance mutations in a network of specifically designed bait trials. The combination of these two tools constitute an early warning system for the development of Fungicide Resistance that allows for the timely adjustment of management practices.
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Mixtures as a Fungicide Resistance management tactic
Phytopathology, 2014Co-Authors: F Bosch, Neil Paveley, P.h.f. Hobbelen, F. Berg, Richard P OliverAbstract:ABSTRACT We have reviewed the experimental and modeling evidence on the use of mixtures of Fungicides of differing modes of action as a Resistance management tactic. The evidence supports the following conclusions. 1. Adding a mixing partner to a Fungicide that is at-risk of Resistance (without lowering the dose of the at-risk Fungicide) reduces the rate of selection for Fungicide Resistance. This holds for the use of mixing partner Fungicides that have either multi-site or single-site modes of action. The resulting predicted increase in the effective life of the at-risk Fungicide can be large enough to be of practical relevance. The more effective the mixing partner (due to inherent activity and/or dose), the larger the reduction in selection and the larger the increase in effective life of the at-risk Fungicide. 2. Adding a mixing partner while lowering the dose of the at-risk Fungicide reduces the selection for Fungicide Resistance, without compromising effective disease control. The very few studies e...
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governing principles can guide Fungicide Resistance management tactics
Annual Review of Phytopathology, 2014Co-Authors: Frank Van Den Bosch, Richard P Oliver, Femke Van Den Berg, Neil PaveleyAbstract:Fungicide-Resistance management would be more effective if principles governing the selection of resistant strains could be determined and validated. Such principles could then be used to predict whether a proposed change to a Fungicide application program would decrease selection for resistant strains. In this review, we assess a governing principle that appears to have good predictive power. The principle states that reducing the product of the selection coefficient (defined as the difference between the per capita rate of increase of the sensitive and resistant strains) and the exposure time of the pathogen to the Fungicide reduces the selection for Resistance. We show that observations as well as modeling studies agree with the predicted effect (i.e., that a specific change to a Fungicide program increased or decreased selection or was broadly neutral in its effect on selection) in 84% of the cases and that only 5% of the experimental results contradict predictions. We argue that the selection coeffic...
Frank Van Den Bosch - One of the best experts on this subject based on the ideXlab platform.
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using epidemiological principles to explain Fungicide Resistance management tactics why do mixtures outperform alternations
Phytopathology, 2018Co-Authors: James Elderfield, Frank Van Den Bosch, Francisco J Lopezruiz, Nik J CunniffeAbstract:Whether Fungicide Resistance management is optimized by spraying chemicals with different modes of action as a mixture (i.e., simultaneously) or in alternation (i.e., sequentially) has been studied by experimenters and modelers for decades. However, results have been inconclusive. We use previously parameterized and validated mathematical models of wheat Septoria leaf blotch and grapevine powdery mildew to test which tactic provides better Resistance management, using the total yield before Resistance causes disease control to become economically ineffective ("lifetime yield") to measure effectiveness. We focus on tactics involving the combination of a low-risk and a high-risk Fungicide, and the case in which Resistance to the high-risk chemical is complete (i.e., in which there is no partial Resistance). Lifetime yield is then optimized by spraying as much low-risk Fungicide as is permitted, combined with slightly more high-risk Fungicide than needed for acceptable initial disease control, applying these Fungicides as a mixture. That mixture rather than alternation gives better performance is invariant to model parameterization and structure, as well as the pathosystem in question. However, if comparison focuses on other metrics, e.g., lifetime yield at full label dose, either mixture or alternation can be optimal. Our work shows how epidemiological principles can explain the evolution of Fungicide Resistance, and also highlights a theoretical framework to address the question of whether mixture or alternation provides better Resistance management. It also demonstrates that precisely how spray tactics are compared must be given careful consideration. [Formula: see text] Copyright © 2018 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .
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using epidemiological principles to explain Fungicide Resistance management strategies why do mixtures outperform alternations
bioRxiv, 2017Co-Authors: James Elderfield, Frank Van Den Bosch, Francisco J Lopezruiz, Nik J CunniffeAbstract:Whether Fungicide Resistance management is optimised by spraying chemicals with different modes of action as a mixture (i.e. simultaneously) or in alternation (i.e. sequentially) has been studied by experimenters and modellers for decades, largely inconclusively. We use previously-parameterised and validated mathematical models of wheat septoria leaf blotch and grapevine powdery mildew to test which strategy provides better Resistance management, using the total yield before Fungicide-Resistance causes disease control to become economically-ineffective ('lifetime yield') to measure effectiveness. Lifetime yield is optimised by spraying as much low-risk Fungicide as is permitted, combined with slightly more high-risk Fungicide than needed for acceptable initial disease control, applying these Fungicides as a mixture. This is invariant to model parameterisation and structure, as well as the pathosystem in question. However if comparison focuses on other metrics, for example lifetime yield at full label dose, either mixtures or alternation can be optimal. Our work shows how epidemiological principles can explain the evolution of Fungicide Resistance, and highlights a theoretical framework to address the question of whether mixtures or alternation provide better Resistance management. Our work also demonstrates that precisely how spray strategies are compared must be given extremely careful consideration.
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Fungicide Resistance risk assessment based on traits associated with the rate of pathogen evolution
Pest Management Science, 2015Co-Authors: M K Grimmer, Frank Van Den Bosch, Stephen J Powers, N D PaveleyAbstract:BACKGROUND A new Fungicide Resistance risk assessment method is described, based on traits (of pathogens, Fungicides and agronomic systems) that are associated with rapid or slow occurrence of Resistance. Candidate traits tested for their predictive value were those for which there was a mechanistic rationale that they could be determinants of the rate of Resistance evolution. RESULTS A dataset of 61 European cases of Resistance against single-site-acting Fungicides was assembled. For each case, the number of years from product introduction to first detection of Resistance (the FDR time) was quantified – varying from 2 to 24 years. Short and long predicted FDR times represent high and low Resistance risk respectively. Regression analysis identified traits that were statistically associated with FDR time. A model combining these traits explained 61% of the variation in FDR time. Validation showed that this predictive power was highly unlikely to have occurred by chance. CONCLUSION Unlike previous methods, trait-based risk assessment can be used to assess Resistance risk for Fungicides with new modes of action, when there is no prior knowledge of Resistance behaviour. Risk predictions using the new method provide a more reliable basis for Resistance management decisions. © 2014 Society of Chemical Industry
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governing principles can guide Fungicide Resistance management tactics
Annual Review of Phytopathology, 2014Co-Authors: Frank Van Den Bosch, Richard P Oliver, Femke Van Den Berg, N D PaveleyAbstract:Fungicide-Resistance management would be more effective if principles governing the selection of resistant strains could be determined and validated. Such principles could then be used to predict whether a proposed change to a Fungicide application program would decrease selection for resistant strains. In this review, we assess a governing principle that appears to have good predictive power. The principle states that reducing the product of the selection coefficient (defined as the difference between the per capita rate of increase of the sensitive and resistant strains) and the exposure time of the pathogen to the Fungicide reduces the selection for Resistance. We show that observations as well as modeling studies agree with the predicted effect (i.e., that a specific change to a Fungicide program increased or decreased selection or was broadly neutral in its effect on selection) in 84% of the cases and that only 5% of the experimental results contradict predictions. We argue that the selection coefficient and exposure time principle can guide the development of Resistance management tactics.
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evaluation of a matrix to calculate Fungicide Resistance risk
Pest Management Science, 2014Co-Authors: M K Grimmer, Frank Van Den Bosch, Stephen J Powers, N D PaveleyAbstract:BACKGROUND In the European Union, assessments of Resistance risk are required by the regulatory authorities for each Fungicide product and are used to guide the extent of anti-Resistance strategies. This paper reports an evaluation of a widely used ‘risk matrix’, to determine its predictive value. Sixty-seven unique cases of Fungicide Resistance in Europe were identified for testing the risk assessment scheme, where each case was the first occurrence of Resistance in a pathogen species against a Fungicide group. RESULTS In most cases, high-, moderate- and low-risk categories for Fungicide, pathogen and agronomic systems were each associated with significant differences in the number of years from Fungicide introduction to the first detection of Resistance (FDR time). The combined risk, calculated by multiplying the individual risk factors using the risk matrix, had useful predictive power (72.8% of FDR time variance accounted for; VAF) for all Fungicides, but only limited predictive power (25.8% VAF) for single-site acting Fungicides (the predominant type). CONCLUSION The Resistance risk matrix has significant, but limited, predictive value. New Fungicide modes of action, or pathogens that have become newly prevalent, cannot be assigned to risk categories until new methods of Resistance risk assessment are developed. © 2013 Society of Chemical Industry
Matthias Hahn - One of the best experts on this subject based on the ideXlab platform.
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botrytis fragariae a new species causing gray mold on strawberries shows high frequencies of specific and efflux based Fungicide Resistance
Applied and Environmental Microbiology, 2017Co-Authors: Sabrina Rupp, Guido Schnabel, Roland W S Weber, Cecilia Plesken, Sibylle Rumsey, Madeline E Dowling, Matthias HahnAbstract:Botrytis cinerea causes pre- and postharvest decay of many fruit and vegetable crops. A survey of German strawberry fields revealed Botrytis strains that differed from B. cinerea in diagnostic PCR markers and growth appearance. Phylogenetic analyses showed that these strains belong to an undescribed species in Botrytis clade 2, named Botrytisfragariae sp. nov. Isolates of Bfragariae were detected in strawberry fields throughout Germany, sometimes at frequencies similar to those of B. cinerea, and in the southeastern United States. Bfragariae was isolated from overwintering strawberry tissue but not from freshly infected fruit. Bfragariae invaded strawberry tissues with an efficiency similar to or lower than that of B. cinerea but showed poor colonization of inoculated nonhost plant tissues. These data and the exclusive occurrence of this fungus on strawberry plants indicate that Bfragariae is host specific and has a tissue preference different from that of B. cinerea Various Fungicide Resistance patterns were observed in Bfragariae populations. Many Bfragariae strains showed Resistance to one or several chemical classes of Fungicides and an efflux-based multidrug Resistance (MDR1) phenotype previously described in B. cinerea Resistance-related mutations in Bfragariae were identical or similar to those of B. cinerea for carbendazim (E198A mutation in tubA), azoxystrobin (G143A in cytB), iprodione (G367A+V368F in bos1), and MDR1 (gain-of-function mutations in the transcription factor mrr1 gene and overexpression of the drug efflux transporter gene atrB). The widespread occurrence of Bfragariae indicates that this species is adapted to Fungicide-treated strawberry fields and may be of local importance as a gray mold pathogen alongside B. cinereaIMPORTANCE Gray mold is the most important fruit rot on strawberries worldwide and requires Fungicide treatments for control. For a long time, it was believed to be caused only by Botrytis cinerea, a ubiquitous pathogen with a broad host range that quickly develops Fungicide Resistance. We report the discovery and description of a new species, named Botrytisfragariae, that is widely distributed in commercial strawberry fields in Germany and the southeastern United States. It was observed on overwintering tissue but not on freshly infected fruit and seems host specific on the basis of its occurrence and artificial infection tests. Bfragariae has also developed Resistance to several Fungicides that is caused by mutations similar to those known in B. cinerea, including an efflux-based multidrug Resistance. Our data indicate that Bfragariae could be of practical importance as a strawberry pathogen in some regions where its abundance is similar to that of B. cinerea.
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spread of botrytis cinerea strains with multiple Fungicide Resistance in german horticulture
Frontiers in Microbiology, 2017Co-Authors: Sabrina Rupp, Roland W S Weber, Daniel Rieger, Peter Detzel, Matthias HahnAbstract:Botrytis cinerea is a major plant pathogen, causing gray mold rot in a variety of cultures. Repeated Fungicide applications are common but have resulted in the development of fungal populations with Resistance to one or more Fungicides. In this study, we have monitored Fungicide Resistance frequencies and the occurrence of multiple Resistance in Botrytis isolates from raspberries, strawberries, grapes, stone fruits and ornamental flowers in Germany in 2010 to 2015. High frequencies of Resistance to all classes of botryticides was common in all cultures, and isolates with multiple Fungicide Resistance represented a major part of the populations. A monitoring in a raspberry field over six seasons revealed a continuous increase in Resistance frequencies and the emergence of multiresistant Botrytis strains. In a cherry orchard and a vineyard, evidence for immigration of multiresistant strains from neighboring fields was obtained. Inoculation experiments with Fungicide-treated leaves in the laboratory and with strawberry plants cultivated in the greenhouse or outdoors revealed a nearly complete loss of Fungicide efficacy caused by multiple Resistance. B. cinerea field strains carrying multiple Resistance mutations against all classes of site-specific Fungicides were found to show similar fitness as sensitive field strains under laboratory conditions, based on their vegetative growth, reproduction, stress Resistance, virulence and competitiveness in mixed infection experiments. Our data confirm an alarming increase in the occurrence of multiResistance in gray mold populations from different cultures, which presents a major threat to the chemical control of gray mold.
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population structure Fungicide Resistance profile and sdhb mutation frequency of botrytis cinerea from strawberry and greenhouse grown tomato in greece
Plant Disease, 2015Co-Authors: Sotirios Konstantinou, Matthias Hahn, Michaela Leroch, Thomas Veloukas, George Menexes, G S KaraoglanidisAbstract:Botrytis cinerea is a pathogen with high genetic variability that has also shown high risk for Fungicide Resistance development. In total, 1,169 isolates obtained from strawberry (n = 297) and tomato (n = 872) in five geographic regions of Greece were tested for their sensitivity to several botryticides. A high frequency of isolates with multiple Resistance to carbendazim, cyprodinil, pyraclostrobin, and boscalid was found in isolates from strawberry. In the isolates from tomato, the predominant phenotype was that of dual Resistance to carbendazim and cyprodinil in the Crete island, of single Resistance to carbendazim in the region of Preveza, and of sensitive isolates in the region of Kyparissia. None of the tested isolates was found to be fludioxonil resistant. High frequencies of boscalid-resistant phenotypes were observed in the strawberry isolates, while boscalid-Resistance frequency in the tomato isolates was lower. H272R was the predominant sdhB mutation, associated with Resistance to boscalid, in all the sampled isolates, while other sdhB mutations were found at low frequencies. B. cinerea group S, identified by the presence of a 21-bp insertion in the transcription factor mrr1 gene, was predominant within the tomato isolates obtained from all three sampled regions, with frequencies ranging from 62 to 75% of the isolates; whereas, within strawberry isolates, B. cinerea was predominant, with frequencies of 75 to 95%. Correlations of isolate genotype and Fungicide Resistance profile showed that B. cinerea sensu stricto isolates were more prone to the development of Resistance to boscalid compared with the Botrytis group S isolates, which may explain the observed predominance of B. cinerea sensu stricto in strawberry fields.
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The rising threat of Fungicide Resistance in plant pathogenic fungi: Botrytis as a case study
Journal of Chemical Biology, 2014Co-Authors: Matthias HahnAbstract:The introduction of site-specific Fungicides almost 50 years ago has revolutionized chemical plant protection, providing highly efficient, low toxicity compounds for control of fungal diseases. However, it was soon discovered that plant pathogenic fungi can adapt to Fungicide treatments by mutations leading to Resistance and loss of Fungicide efficacy. The grey mould fungus Botrytis cinerea, a major cause of pre- and post-harvest losses in fruit and vegetable production, is notorious as a ‘high risk’ organism for rapid Resistance development. In this review, the mechanisms and the history of Fungicide Resistance in Botrytis are outlined. The introduction of new Fungicide classes for grey mould control was always followed by the appearance of Resistance in field populations. In addition to target site Resistance, B. cinerea has also developed a Resistance mechanism based on drug efflux transport. Excessive spraying programmes have resulted in the selection of multiresistant strains in several countries, in particular in strawberry fields. The rapid erosion of Fungicide activity against these strains represents a major challenge for the future of Fungicides against Botrytis. To maintain adequate protection of intensive cultures against grey mould, strict implementation of Resistance management measures are required as well as alternative strategies with non-chemical products.
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Fungicide Resistance phenotypes of botrytis cinerea isolates from commercial vineyards in south west germany
Journal of Phytopathology, 2011Co-Authors: Michaela Leroch, Matthias Kretschmer, Matthias HahnAbstract:Fungicide Resistance frequencies of Botrytis cinerea populations in the German Wine Road region were determined for 4 years. Strains showing specific Resistance against carbendazim, iprodione or fenhexamid were found to occur wide-spread, but at low frequencies. In contrast, cyprodinil Resistance increased from 5.4% in 2006 to 21.9% in 2008 and 16% in 2009, and strains resistant to boscalid increased from 2% in 2006 to 26.7% in 2009. Strains with multidrug Resistance (MDR) phenotypes were found at high frequencies. One of the three MDR phenotypes, MDR1, with reduced sensitivity to cyprodinil and fludioxonil, was dominating, representing 19% to 35% of the total population. Strains with a combination of cyprodinil Resistance and MDR1 were found to be strongly increasing in 2008 and 2009.
D A Rosenberger - One of the best experts on this subject based on the ideXlab platform.
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do some ipm concepts contribute to the development of Fungicide Resistance lessons learned from the apple scab pathosystem in the united states
Pest Management Science, 2015Co-Authors: Janna L Beckerman, George W Sundin, D A RosenbergerAbstract:One goal of integrated pest management (IPM) as it is currently practiced is an overall reduction in Fungicide use in the management of plant disease. Repeated and long-term success of the early broad-spectrum Fungicides led to optimism about the capabilities of Fungicides, but to an underestimation of the risk of Fungicide Resistance within agriculture. In 1913, Paul Ehrlich recognized that it was best to ‘hit hard and hit early’ to prevent microbes from evolving Resistance to treatment. This tenet conflicts with the Fungicide reduction strategies that have been widely promoted over the past 40 years as integral to IPM. The authors hypothesize that the approaches used to implement IPM have contributed to Fungicide Resistance problems and may still be driving that process in apple scab management and in IPM requests for proposals. This paper also proposes that IPM as it is currently practiced for plant diseases of perennial systems has been based on the wrong model, and that conceptual shifts in thinking are needed to address the problem of Fungicide Resistance. © 2013 Society of Chemical Industry
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do some ipm concepts contribute to the development of Fungicide Resistance lessons learned from the apple scab pathosystem in the united states
Pest Management Science, 2015Co-Authors: Janna L Beckerman, George W Sundin, D A RosenbergerAbstract:One goal of integrated pest management (IPM) as it is currently practiced is an overall reduction in Fungicide use in the management of plant disease. Repeated and long-term success of the early broad-spectrum Fungicides led to optimism about the capabilities of Fungicides, but to an underestimation of the risk of Fungicide Resistance within agriculture. In 1913, Paul Ehrlich recognized that it was best to 'hit hard and hit early' to prevent microbes from evolving Resistance to treatment. This tenet conflicts with the Fungicide reduction strategies that have been widely promoted over the past 40 years as integral to IPM. The authors hypothesize that the approaches used to implement IPM have contributed to Fungicide Resistance problems and may still be driving that process in apple scab management and in IPM requests for proposals. This paper also proposes that IPM as it is currently practiced for plant diseases of perennial systems has been based on the wrong model, and that conceptual shifts in thinking are needed to address the problem of Fungicide Resistance.
Francisco J Lopezruiz - One of the best experts on this subject based on the ideXlab platform.
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when time really is money in situ quantification of the strobilurin Resistance mutation g143a in the wheat pathogen blumeria graminis f sp tritici
bioRxiv, 2020Co-Authors: Kejal N. Dodhia, Richard P Oliver, Belinda A. Cox, Francisco J LopezruizAbstract:Abstract Background There has been an inexorable increase in the incidence of Fungicide Resistance in plant pathogens in recent years. Control of diseases and the management of Resistance would be greatly aided by rapid diagnostic methods. Quantitative allele specific PCR (ASqPCR) is an ideal technique for the analysis of Fungicide Resistance in the field as it can both detect and quantify the frequency of mutations associated with Fungicide Resistance. We have applied this technique to the fungal pathogen Blumeria graminis f. sp. tritici (Bgt), an obligate biotrophic fungus that causes wheat powdery mildew and is responsible for up to 25% yield loss annually. In Australia, strobilurin resistant Bgt was first discovered in samples from Tasmania and Victoria in 2016. Molecular analysis revealed a nucleotide transversion in the cytochrome bc1 enzyme (cytb) complex, resulting in a substitution of alanine for glycine at position 143 (G143A) in Cytb. Results We have developed an in-field ASqPCR assay that can quantify both the resistant (A143) and sensitive (G143) cytb alleles down to 1.67% in host and Bgt DNA mixtures within 90 min of sample collection. The in situ analysis of field samples collected during a survey in Tasmania revealed A143 frequencies ranging between 9-100%. We validated the analysis with a newly developed laboratory based digital PCR assay and found no significant differences between the two methods. Conclusion We have successfully developed an in-field quantification method, for a QoI resistant allele, by pairing an ASqPCR assay on a lightweight qPCR instrument with a quick DNA extraction method. The deployment of this type of methodologies in the field can contribute to the effective in-season management of Fungicide Resistance.
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parallel evolution of multiple mechanisms for demethylase inhibitor Fungicide Resistance in the barley pathogen pyrenophora teres f sp maculata
bioRxiv, 2019Co-Authors: Wesley J Mair, Geoffrey J Thomas, Kejal Dodhia, Andrea L Hills, Kithsiri W Jayasena, Simon R Ellwood, Richard Oliver, Francisco J LopezruizAbstract:The demethylase inhibitor (DMI) or group 3 Fungicides are the most important class of compounds for the control both of plant and human fungal pathogens. The necrotrophic fungal pathogen Pyrenophora teres f. sp. maculata (Ptm), responsible for spot form of net blotch (SFNB), is currently the most significant disease of barley in Australia, and a disease of increasing concern worldwide. The main basis for management of SFNB is by Fungicide application, and in Australia the DMIs predominate. Although reduced sensitivity to DMI Fungicides has recently been described in the closely related pathogen P. teres f. sp. teres (Ptt), the mechanisms of DMI Resistance have not thus far been described for Ptm. In this study, several different levels of sensitivity to DMI Fungicides were identified in Western Australian strains of Ptm from 2016 onwards, and reduced sensitivity phenotypes were correlated with a number of distinct mutations in both the promoter region and coding sequence of the DMI target gene encoding cytochrome P450 sterol 14α-demethylase (Cyp51A). Five insertions elements of 134-base pairs in length were found at different positions within the upstream regulatory region of Cyp51A in both highly DMI-resistant (HR) and select moderately DMI-resistant (MR1) Ptm isolates. The five insertion elements had at least 95% sequence identity and were determined to be Solo-LTR (Long Terminal Repeat) elements, all deriving from Ty1/Copia-family LTR Retrotransposons. The 134-bp elements contained a predicted promoter sequence and several predicted transcription factor binding sites, and the presence of an insertion element was correlated with constitutive overexpression of Cyp51A. The substitution of phenylalanine by leucine at position 489 of the predicted amino acid sequence of CYP51A was found in both HR and select moderately DMI-resistant (MR2) Ptm isolates. The same F489L amino acid substitution has been previously reported in Western Australian strains of Ptt, where it has also been associated with reduced sensitivity to DMI Fungicides. In Ptm, the F489L amino acid change was associated with either of three different single nucleotide polymorphisms in codon 489. This suggests that, in contrast to Ptt, in Ptm the F489L mutation has emerged as a result of three distinct mutational events. Moderately DMI-resistant isolates had one or the other of the F489L substitution or a promoter insertion mutation, whereas highly DMI-resistant isolates were found to have combinations of both mechanisms together. Therefore, multiple mechanisms acting both alone and in concert were found to contribute to the observed phenomena of DMI Fungicide Resistance in Ptm. Moreover, these mutations have apparently emerged repeatedly and independently in Western Australian Ptm populations, by a process of convergent or parallel evolution.
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improved detection and monitoring of Fungicide Resistance in blumeria graminis f sp hordei with high throughput genotype quantification by digital pcr
Frontiers in Microbiology, 2018Co-Authors: Katherine G Zulak, Richard P Oliver, Belinda A. Cox, Madeline Ann Tucker, Francisco J LopezruizAbstract:The increased occurrence of triazole Fungicide resistant strains of Blumeria graminis f. sp. hordei (Bgh) is an economic concern for the barley industry in Australia and elsewhere. High levels of Resistance to triazoles in the field are caused by two separate point mutations in the Cyp51 gene, Y136F and S509T. Early detection of these mutations arising in pathogen field populations is important as this allows time for changes in Fungicide practices to be adopted, thus mitigating potential yield losses due to Fungicide failure and preventing the Resistance from becoming dominant. A digital PCR assay has been developed for the detection and quantification of the Y136F and S509T mutations in the Bgh Cyp51 gene. Mutation levels were quantifiable as low as 0.2% in genomic DNA extractions and field samples. This assay was applied to the high throughput screening of Bgh field and bait trial samples from barley growing regions across Australia in the 2015 and 2016 growing seasons and identified the S509T mutation for the first time in the Eastern states of Australia. This is the first report on the use of digital PCR technology for Fungicide Resistance detection and monitoring in agriculture. Here we describe the potential application of dPCR for the screening of Fungicide Resistance mutations in a network of specifically designed bait trials. The combination of these two tools constitute an early warning system for the development of Fungicide Resistance that allows for the timely adjustment of management practices.
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using epidemiological principles to explain Fungicide Resistance management tactics why do mixtures outperform alternations
Phytopathology, 2018Co-Authors: James Elderfield, Frank Van Den Bosch, Francisco J Lopezruiz, Nik J CunniffeAbstract:Whether Fungicide Resistance management is optimized by spraying chemicals with different modes of action as a mixture (i.e., simultaneously) or in alternation (i.e., sequentially) has been studied by experimenters and modelers for decades. However, results have been inconclusive. We use previously parameterized and validated mathematical models of wheat Septoria leaf blotch and grapevine powdery mildew to test which tactic provides better Resistance management, using the total yield before Resistance causes disease control to become economically ineffective ("lifetime yield") to measure effectiveness. We focus on tactics involving the combination of a low-risk and a high-risk Fungicide, and the case in which Resistance to the high-risk chemical is complete (i.e., in which there is no partial Resistance). Lifetime yield is then optimized by spraying as much low-risk Fungicide as is permitted, combined with slightly more high-risk Fungicide than needed for acceptable initial disease control, applying these Fungicides as a mixture. That mixture rather than alternation gives better performance is invariant to model parameterization and structure, as well as the pathosystem in question. However, if comparison focuses on other metrics, e.g., lifetime yield at full label dose, either mixture or alternation can be optimal. Our work shows how epidemiological principles can explain the evolution of Fungicide Resistance, and also highlights a theoretical framework to address the question of whether mixture or alternation provides better Resistance management. It also demonstrates that precisely how spray tactics are compared must be given careful consideration. [Formula: see text] Copyright © 2018 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .
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using epidemiological principles to explain Fungicide Resistance management strategies why do mixtures outperform alternations
bioRxiv, 2017Co-Authors: James Elderfield, Frank Van Den Bosch, Francisco J Lopezruiz, Nik J CunniffeAbstract:Whether Fungicide Resistance management is optimised by spraying chemicals with different modes of action as a mixture (i.e. simultaneously) or in alternation (i.e. sequentially) has been studied by experimenters and modellers for decades, largely inconclusively. We use previously-parameterised and validated mathematical models of wheat septoria leaf blotch and grapevine powdery mildew to test which strategy provides better Resistance management, using the total yield before Fungicide-Resistance causes disease control to become economically-ineffective ('lifetime yield') to measure effectiveness. Lifetime yield is optimised by spraying as much low-risk Fungicide as is permitted, combined with slightly more high-risk Fungicide than needed for acceptable initial disease control, applying these Fungicides as a mixture. This is invariant to model parameterisation and structure, as well as the pathosystem in question. However if comparison focuses on other metrics, for example lifetime yield at full label dose, either mixtures or alternation can be optimal. Our work shows how epidemiological principles can explain the evolution of Fungicide Resistance, and highlights a theoretical framework to address the question of whether mixtures or alternation provide better Resistance management. Our work also demonstrates that precisely how spray strategies are compared must be given extremely careful consideration.
