scholarly journals QoI Resistance in Fusicladium carpophilum Populations from Almond in California and Evaluation of Molecular Resistance Mechanisms

Plant Disease ◽  
2013 ◽  
Vol 97 (10) ◽  
pp. 1322-1330 ◽  
Author(s):  
Y. Luo ◽  
L. Hou ◽  
H. Förster ◽  
J. E. Adaskaveg
Keyword(s):  
Cytochrome B ◽  
High Resistance ◽  
Quantitative Polymerase Chain Reaction ◽  
Resistance Mechanisms ◽  
Cytochrome B Gene ◽  
Analysis Of Covariance ◽  
Mitochondrial Cytochrome B ◽  
Moderately Resistant ◽  
Qoi Resistance

Disease management failures have been reported in California for almond scab caused by Fusicladium carpophilum following quinone outside inhibitor (QoI) applications. Resistance in the pathogen populations was found to be common and at high incidence in the major almond-growing regions beginning in 2003, 4 years after registration of azoxystrobin on this crop. Two levels of azoxystrobin resistance, moderate and high, were identified with 50% effective concentration (EC50) values between 0.15 and 10 μg/ml or >40 μg/ml, respectively. Reference isolates collected before resistance was detected had EC50 values <0.05 μg/ml. High-resistance was associated with a G143A mutation in the mitochondrial cytochrome b gene. For the less commonly found moderately resistant isolates, no mutations in the gene were detected between codons 122 and 212. Using primers targeting the G143A mutation or the cytochrome b gene of all F. carpophilum isolates in quantitative polymerase chain reaction (qPCR) analyses, the frequency of highly resistant isolates was accurately determined in mixtures of conidia with selected ratios of sensitive and resistant isolates. The frequency of high resistance in bulked samples of scab lesions, however, was generally underestimated compared with in vitro testing of fungicide sensitivity of fungal isolates from the same lesions. Competition experiments using conidial suspensions demonstrated stability of the highly resistant genotype in the presence of different amounts of sensitive and moderately resistant genotypes. Analysis of covariance of linear regressions of cycle threshold values on DNA concentrations derived from qPCR amplifications using two primer pairs for cytochrome b alleles with and without the G143 mutation showed that several isolates differed in their slopes and midpoints. Thus, heteroplasmy of mitochondrial-inherited QoI resistance is suggested as a likely cause for incongruence in estimating resistance frequencies using the two methods.

scholarly journals In vitro splicing of the terminal intervening sequence of Saccharomyces cerevisiae cytochrome b pre-mRNA.

1987 ◽  
Vol 7 (7) ◽  
pp. 2545-2551 ◽  
Author(s):  
A Gampel ◽  
A Tzagoloff
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cytochrome B ◽  
Cytochrome B Gene ◽  
Group I Introns ◽  
Vitro Assay ◽  
Group I ◽  
Mitochondrial Cytochrome B ◽  
Nuclease Mapping

A region of the Saccharomyces cerevisiae mitochondrial cytochrome b gene encompassing the entire terminal intron plus flanking exonic sequences has been cloned in an SP6 vector. A runoff transcript prepared from this construct as well as the native cytochrome b pre-mRNA containing the terminal intervening sequence were found to act as substrates for the autocatalytic excision of the intervening sequence in vitro. This reaction proceeds under conditions previously shown by Cech and co-workers to promote protein-independent excision of the Tetrahymena rRNA intervening sequence. The 5' and 3' termini of the excised intervening sequence, determined by S1 nuclease mapping and sequence analysis, are consistent with the known sequence of the cytochrome b mRNA. The same region of the cytochrome b gene from a yeast mutant, defective in splicing due to a mutation in a critical sequence inside the terminal intron, has also been cloned in an SP6 vector. The mutant transcript fails to self-splice in the in vitro assay. These observations provide strong presumptive evidence that in vivo processing of the terminal intervening sequence of the cytochrome b pre-mRNA occurs by an autocatalytic mechanism analogous to that shown for other group I introns. In vivo processing of the terminal intervening sequence of the cytochrome b pre-mRNA, however, exhibits complete dependence on a protein factor previously shown to be encoded by the nuclear gene CBP2.

In vitro splicing of the terminal intervening sequence of Saccharomyces cerevisiae cytochrome b pre-mRNA

1987 ◽  
Vol 7 (7) ◽  
pp. 2545-2551
Author(s):  
A Gampel ◽  
A Tzagoloff
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cytochrome B ◽  
Cytochrome B Gene ◽  
Group I Introns ◽  
Vitro Assay ◽  
Group I ◽  
Mitochondrial Cytochrome B ◽  
Nuclease Mapping

A region of the Saccharomyces cerevisiae mitochondrial cytochrome b gene encompassing the entire terminal intron plus flanking exonic sequences has been cloned in an SP6 vector. A runoff transcript prepared from this construct as well as the native cytochrome b pre-mRNA containing the terminal intervening sequence were found to act as substrates for the autocatalytic excision of the intervening sequence in vitro. This reaction proceeds under conditions previously shown by Cech and co-workers to promote protein-independent excision of the Tetrahymena rRNA intervening sequence. The 5' and 3' termini of the excised intervening sequence, determined by S1 nuclease mapping and sequence analysis, are consistent with the known sequence of the cytochrome b mRNA. The same region of the cytochrome b gene from a yeast mutant, defective in splicing due to a mutation in a critical sequence inside the terminal intron, has also been cloned in an SP6 vector. The mutant transcript fails to self-splice in the in vitro assay. These observations provide strong presumptive evidence that in vivo processing of the terminal intervening sequence of the cytochrome b pre-mRNA occurs by an autocatalytic mechanism analogous to that shown for other group I introns. In vivo processing of the terminal intervening sequence of the cytochrome b pre-mRNA, however, exhibits complete dependence on a protein factor previously shown to be encoded by the nuclear gene CBP2.

scholarly journals Characterization of Colletotrichum graminicola Isolates Resistant to Strobilurin-Related QoI Fungicides

Plant Disease ◽  
2003 ◽  
Vol 87 (12) ◽  
pp. 1426-1432 ◽  
Author(s):  
Cruz Avila-Adame ◽  
Gilberto Olaya ◽  
Wolfram Köller
Keyword(s):  
Cytochrome B ◽  
Cytochrome B Gene ◽  
In Vitro Test ◽  
Colletotrichum Graminicola ◽  
Wild Type ◽  
Qoi Fungicides ◽  
Test Conditions ◽  
Vitro Test ◽  
Qoi Resistance

Isolates of Colletotrichum graminicola were collected from annual bluegrass or bent grass turf in Japan and the United States, and their sensitivities to QoI fungicides (QoIs) as well as their cytochrome b sequences were characterized. Five isolates sampled from turf treated repeatedly with azoxystrobin were highly QoI resistant under both in vivo and in vitro test conditions. The nucleotide sequences of a large cytochrome b gene segment involving the binding site of QoIs were fully homologous for all resistant isolates and contained the G143A target site mutation known to confer QoI resistance in other pathogens. QoI-sensitive isolates collected prior to treatments with QoIs were more diverse with regard to their cytochrome b gene sequences and their phenotype responses to QoIs. All wild-type isolates retained a glycine in position 143 of cytochrome b. Three of the four QoI-sensitive isolates were, in addition, distinguished by leucines in positions 95, 130, and 141, which were exchanged to threonine in all resistant but also in one of the sensitive isolates. In addition to a more pronounced divergence of cytochrome b sequences, the sensitive wild-type isolates also were diverse with regard to the induction of alternative respiration in response to QoI action, as indicated by comparisons of QoI sensitivities displayed in the absence or presence of the alternative oxidase inhibitor salicylhydroxamic acid. These different phenotype responses expressed under in vitro test conditions had no or only a slight impact on anthracnose control in protective applications of azoxystrobin. Isolate responses in vitro were very similar for trifloxystrobin, indicating cross-resistance among the class of QoIs. Our results imply that C. graminicola falls into the class of pathogens with a potential for rapid selection of highly QoI-resistant phenotypes. Frequent monitoring of population sensitivities will be required to determine the status of population responses toward practical QoI resistance.

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