Cross-resistance Relationships Research Articles

Sensitivity of Botrytis cinerea from Vineyards to Boscalid, Isofetamid, and Pydiflumetofen in Shandong Province, China.

Succinate dehydrogenase inhibitor (SDHI) fungicides are the most commonly and effectively used class of fungicides for controlling gray mold. Among them, only boscalid has been registered in China for controlling grape gray mold, whereas isofetamid and pydiflumetofen are two new SDHI fungicides that have demonstrated high efficacy against various fungal diseases. However, the sensitivity of Botrytis cinerea isolates from vineyards in China to these three fungicides is currently unknown. In this study, the sensitivity of 55 B. cinerea isolates from vineyards to boscalid, isofetamid, and pydiflumetofen was determined, with the effective concentrations for inhibiting 50% of spore germination (EC50) values ranging from 1.10 to 393, 0.0300 to 42.0, and 0.0990 to 25.5 μg ml-1, respectively. The resistance frequencies for boscalid, isofetamid, and pydiflumetofen were 60.0, 7.2, and 12.8%, respectively. Three mutations (H272R, H272Y, and P225F) were detected in the SdhB subunit, with H272R being the most prevalent (75.7%), followed by H272Y (16.2%) and P225F (8.1%). All three mutations are associated with resistance to boscalid, and of them, H272R mutants exhibited high resistance. Only P225F and H272Y mutants exhibited resistance to isofetamid and pydiflumetofen, respectively. A weakly positive cross-resistance relationship was observed between boscalid and pydiflumetofen (r = 0.38, P < 0.05). Additionally, the H272R mutants showed no significant fitness costs, whereas the remaining mutants exhibited reduced mycelial growth (P225F) and sporulation (H272Y and P225F). These results suggest that isofetamid and pydiflumetofen are effective fungicides against B. cinerea in vineyards, but appropriate rotation strategies must be implemented to reduce the selection of existing SDHI-resistant isolates.

Resistance risk assessment of Fusarium pseudograminearum from wheat to prothioconazole

Fusarium crown rot (FCR), primarily caused by Fusarium pseudograminearum, poses significant threats to cereal crops worldwide. Prothioconazole is a demethylation inhibitor (DMI) fungicide used to control FCR. However, the risk of resistance in F. pseudograminearum to prothioconazole has not yet been evaluated. In this study, the sensitivity of a total of 255 F. pseudograminearum strains obtained from Henan Province, China to prothioconazole were determined by the mycelial growth inhibition. The results showed that the effective concentration to 50% growth inhibition (EC50) of these strains ranged from 0.4228 μg/mL to 2.5284 μg/mL, with a mean EC50 value of 1.0692 ± 0.4527 μg/mL (mean ± SD). Thirty prothioconazole-resistant mutants were obtained out of six selected sensitive parental strains by means of fungicide taming. The resistant mutants exhibited defects in vegetative growth, conidia production, and pathogenicity on wheat seedlings compared to their parental strains. Under ion, cell wall, and temperature stress conditions but not osmotic stress, all the mutants exhibited decreased growth rates compared with their parental strains, which was consistent with the control treatment. Cross-resistance test showed that there was a cross-resistance relationship between prothioconazole and four DMI fungicides, including prochloraz, metconazole, tebuconazole and hexaconazole, but no cross-resistance was observed between prothioconazole and carbendazim, phenamacril, fludioxonil, or azoxystrobin. Although no site mutation occurred on Cyp51a and Cyp51b genes, the constitutive expression level of the Cyp51a gene was significantly increased in all mutants. After being treated with prothioconazole, the Cyp51a and Cyp51b genes were significantly increased in both the resistant mutants and their parents. These results suggested that the resistance to prothioconazole of the mutants may be attributed to the changes of the relative expression level of Cyp51a and Cyp51b genes. Taken together, these results could provide a theoretical basis for the scientific use of prothioconazole in the field and fungicide resistance management strategies.

Cross-resistance of Colletotrichum acutatum s. lat. to Strobilurin Fungicides and Inhibitory Effect of Fungicides with Other Mechanisms on C. acutatum s. lat. Resistant to Pyraclostrobin

Colletotrichum acutatum s. lat. 20JDS8 sensitive and 20CDJ6 resistant to pylaclostrobin were used to investigate the cross-resistance with fungicides belonging to the strobilurins and the characteristics of fungicidal controlling activities with different mechanisms against the isolate resistant to the fungicide. The resistant isolate of 20CDJ6 also showed the resistance to azoxystrobin, trifloxystrobin, and kresoxim-methyl, suggesting that there is a cross-resistance relationship. All fungicides with different action mechanisms inhibited mycelial growth of both susceptible and resistant isolates of C. acutatum s. lat., but their disease control effects in fruits were different according to the fungicides. The disease control effect of isopyrazam against 20JDS8 and 20CDJ6 was very low, and fluazinam showed a control effect of 91.9% and 88.1% against 20JDS8 and 20CDJ6 only when it was treated before inoculation by spraying spore suspensions on pepper fruits without wounds. Tebuconazole and prochloraz effectively inhibited not only the mycelial growth of 20JDS8 and 20CDJ6 on potato dextrose agar medium, but also disease incidence in red pepper fruits. As a result of this study, C. acutatum s. lat. 20CDJ6 resistant to pyraclostrobin showed cross-resistance with other strobilurin fungicides. In addition, we think that fluazinam, tebuconazole, and prochloraz can be recommended as alternative fungicides for the control of red-pepper pyranthracnose pathogens resistant pyraclostrobin. However, fluazinam can be effective only if it is treated protectively before the occurrence of the disease.

CARACTERIZAÇÃO DA RESISTÊNCIA DE Chrysodeixis includens A DIAMIDAS

ABSTRACT In recent decades, soybean has been one of the most widely grown crops in Brazil. The soybean looper, Chrysodeixis includens (Walker, [1858]; Lepidoptera: Noctuidae), is one of the main defoliating pests of soybean that cause damage and affect the cost of production. The overall objective of the present study was to characterize the resistance of C. includens to diamide insecticides. To this end, the baseline susceptibility of C. includens to flubendiamide, chlorantraniliprole, and cyantraniliprole was characterized to estimate their concentrations for diagnostic monitoring of resistance in populations collected in commercial soybean crops in southern Brazil during the 2018/19 season. Under field conditions, evaluations were made of the residual activity of three diamide insecticides: chlorantraniliprole + lambda-cyhalothrin, teflubenzuron, methoxyfenozide, and bifenthrin for discrimination between flubendiamide-susceptible and resistant C. includens. Chrysodeixis includens was more tolerant to cyantraniliprole than to flubendiamide and chlorantraniliprole. The field populations had a higher survival rate than the susceptible population. The resistant population showed a resistance ratio to flubendiamide of 70.1-fold. Under field conditions, the residue of the study insecticides, except for bifenthrin, enabled discrimination between flubendiamide-susceptible and resistant C. includens. The data indicate the need to study the cross-resistance relationships between insecticides to improve the rotation recommendation. They also reinforce the importance of using other good management practices, e.g., the use of insecticides based on pest sampling and the choice of other control methods to prevent C. includens from becoming resistant to diamide insecticides.

Laboratory evolution of Mycobacterium on agar plates for analysis of resistance acquisition and drug sensitivity profiles

Drug-resistant tuberculosis (TB) is a growing public health problem. There is an urgent need for information regarding cross-resistance and collateral sensitivity relationships among drugs and the genetic determinants of anti-TB drug resistance for developing strategies to suppress the emergence of drug-resistant pathogens. To identify mutations that confer resistance to anti-TB drugs in Mycobacterium species, we performed the laboratory evolution of nonpathogenic Mycobacterium smegmatis, which is closely related to Mycobacterium tuberculosis, against ten anti-TB drugs. Next, we performed whole-genome sequencing and quantified the resistance profiles of each drug-resistant strain against 24 drugs. We identified the genes with novel meropenem (MP) and linezolid (LZD) resistance-conferring mutation, which also have orthologs, in M. tuberculosis H37Rv. Among the 240 possible drug combinations, we identified 24 pairs that confer cross-resistance and 18 pairs that confer collateral sensitivity. The acquisition of bedaquiline or linezolid resistance resulted in collateral sensitivity to several drugs, while the acquisition of MP resistance led to multidrug resistance. The MP-evolved strains showed cross-resistance to rifampicin and clarithromycin owing to the acquisition of a mutation in the intergenic region of the Rv2864c ortholog, which encodes a penicillin-binding protein, at an early stage. These results provide a new insight to tackle drug-resistant TB.

Impact of benzimidazole resistance on fitness parameters and fumonisin B1 production in Fusarium verticillioides (Sacc) Niremberg

Fusarium verticillioides laboratory isolates resistant to carbendazim were readily obtained after mutagenesis with UV-irradiation. Three resistant phenotypes that included highly (HR, Resistant factors>30), moderately (MR, Rf: 14–25) and low (LR, Rf: 2–5) resistant isolates to carbendazim were identified by fungitoxicity tests in vitro. Mutant isolates were simultaneously resistant to thiabendazole and benomyl but not to the also tubulin-blocking fungicides diethofencarb and zoxamide. No cross resistance relationship was found between carbendazim and fungicides with different modes of action such as the dicarboximide iprodione, the QoI pyraclostrobin, the imidazole prochloraz, the triazoles flusilazole and epoxiconazole. Mutation(s) for resistance to benzimidazoles did not significantly affect mycelial growth rate whereas sporulation and pathogenicity were adversely affected in most of the resistant mutants. Moreover, low temperatures suppressed the expression of carbendazim-resistance in all resistant phenotypes. Most mutant isolates produced Fumonisin B1 in similar or less quantities than the wild-type isolate both in vitro and in vivo in the absence of carbendazim with the exception of one HR isolate which had up to 3-fold increased FB1 production. Sub lethal doses of carbendazim in the growth medium in vitro resulted in a significant reduction in FB1 levels in all cases even though most mutant isolates produced higher FB1 quantities than the wild type. A significant correlation was found between F. verticillioides sporulation and FB1 production on maize seeds. The potential risk of food and feed contamination by FB1 producing F. verticillioides isolates resistant to benzimidazoles and implications on resistance management strategies are discussed.

Resistance of Spodoptera frugiperda (Lepidoptera: Noctuidae) to spinetoram: inheritance and cross-resistance to spinosad.

The use of spinosyn insecticides is one of the major control strategies of the fall armyworm, Spodoptera frugiperda (J. E. Smith) in Brazil. In this study, we selected a spinetoram-resistant strain from a field-population of S. frugiperda to characterize the inheritance of the resistance and cross-resistance relationship between spinosyn insecticides. The values of LC50 (95% CI) obtained from concentration-response bioassays were 0.63 (0.55-0.73) μg spinetoram mL-1 for the susceptible strain (SUS), and 1170.96 (1041.61-1323.89) μg spinetoram mL-1 for the strain resistant to spinetoram (SPT-R). These values resulted in a resistance ratio of 1844-fold. The SPT-R strain showed cross-resistance with spinosad (resistance ratio = 1196-fold). The reciprocal crosses showed LC50 values of 3.91 (2.97-5.84) and 5.37 (4.52-6.52) μg spinetoram mL-1 , suggesting that the resistance of S. frugiperda to spinetoram is autosomal and incompletely recessive. The backcrosses of the F1 progeny with the SPT-R strain suggest a resistance with polygenic effect. Estimates of the effective number of loci with equal contributions to the resistance effect were from 1.18 to 1.76, suggesting that resistance to spinetoram is associated with a few genes. The inheritance pattern of resistance of S. frugiperda to spinetoram was characterized as autosomal, incompletely recessive and polygenic. Cross-resistance between spinosyns was confirmed in S. frugiperda. The importance of this information for implementing insect resistance management strategies is discussed in this paper. © 2020 Society of Chemical Industry.

Baseline sensitivity of Botrytis cinerea to fluazinam and cross‐resistance

AbstractGrey mould, caused by the fungus Botrytis cinerea Pers ex Fr., is a very destructive and important disease worldwide. Fluazinam is a phenylpyridinamine fungicide with broad‐spectrum activities. The baseline sensitivity of B. cinerea to fluazinam is yet to be established in Henan Province, China. In this study, a total of 117 field isolates of B. cinerea were collected from 49 commercial greenhouses in different locations of Henan Province, in 2016, and the sensitivities of these isolates to fluazinam were determined based on mycelial growth. The effective concentration for 50% (EC50) values ranged from 0.0038 to 0.0441 μg/ml, and the mean EC50 value was 0.0201 ± 0.0081 μg/ml for mycelial growth. The frequency distribution range presented a unimodal curve. To define the cross‐resistance relationships, the linear correlation coefficients of the EC50 values between fluazinam and carbendazim, procymidone, pyrimethanil or boscalid were analysed. The results showed that no correlation was observed between fluazinam and the other tested fungicides. These results provide important information to growers for the prevention and control of grey mould.

Baseline sensitivity and efficacy of fluopyram against Botrytis cinerea from table grape in Italy

ABSTRACTSuccinate dehydrogenase inhibitor (SDHI) fungicides constitute a relatively recent fungicide class registered for the treatment of grey mould on grapevine in Italy. The sensitivity profile to a novel compound fluopyram was established for a set of 203 Botrytis cinerea isolates collected from Sicilian vineyards within 2009–2012 prior its introduction into market. In addition, its performances were compared in in vitro and in vivo assays with other registered SDHI fungicide boscalid, to evaluate their frequency distributions EC50 values and cross‐resistance patterns. Results of the article showed that EC50 values for fluopyram ranged from 0.05 to 1.98 µg mL−1. Although EC50 values of boscalid ranged from 0.01 to 89.52 µg mL−1, no cross‐resistance relationship was observed between the two fungicides (r = 0.003; P = 0.964) within our B. cinerea population. On further confirming these data, boscalid failed in controlling grey mould infections when boscalid‐resistant isolates were inoculated on grape berries whereas fluopyram exhibited a good efficacy against the same isolates. This study represents the first report on the baseline sensitivity to fluopyram within B. cinerea population from Sicilian table grape vineyards in Italy, and it clearly shows the lack of cross‐resistance in vitro and in vivo between fluopyram and boscalid for the field pathogen isolates. These results provided useful information for managing of fungicide resistance suggesting that fluopyram could be a valid alternative to boscalid for the control of grey mould of table grape.

Pyrethroid resistance in Phytoseiulus macropilis (Acari: Phytoseiidae): cross-resistance, stability and effect of synergists

Phytoseiulus macropilis Banks (Acari: Phytoseiidae) is an effective predator of Tetranychus urticae Koch (Acari: Tetranychidae). The objectives of this research were to study the stability of fenpropathrin resistance and the cross-resistance relationships with different pyrethroids, and also to evaluate the effect of synergists [piperonyl butoxide (PBO), diethyl maleate (DEM) and S,S,S-tributyl phosphorotrithioate (DEF)] on fenpropathrin resistant and susceptible strains of this predaceous mite. The stability of fenpropathrin resistance was studied under laboratory conditions, using P. macropilis populations with initial frequencies of 75 and 50% of resistant mites. The percentages of fenpropathrin resistant mites were evaluated monthly for a period of up to 12 months. A trend toward decreased resistance frequencies was observed only during the first 3-4 months. After this initial decrease, the fenpropathrin resistance was shown to be stable, maintaining constant resistance frequencies (around 30%) until the end of the evaluation period. Toxicity tests carried out using fenpropathrin resistant and susceptible strains of P. macropilis indicated strong positive cross-resistance between fenpropathrin and the pyrethroids bifenthrin and deltamethrin. Bioassays with the synergists DEM, DEF and PBO were also performed. The maximum synergism ratio (SR = LC50 without synergist/LC50 with synergist) detected for the three evaluated synergists (PBO, DEM, DEF) was 5.86 (for DEF), indicating low influence of enzyme detoxification processes in fenpropathrin resistance.

Detection of isolates of Alternaria alternata with multiple-resistance to fludioxonil, cyprodinil, boscalid and pyraclostrobin in California pistachio orchards

In this study, 229 isolates of Alternaria alternata collected from commercial pistachio orchards with (n = 198) and without (n = 31) a prior history of Switch (cyprodinil + fludioxonil) applications were examined for their sensitivity to fludioxonil and cyprodinil using in vitro mycelial growth assays. The 50% effective dose (EC50) values for the baseline isolates for cyprodinil and fludioxonil ranged from 0.001 to 1.184 μg/mL (average = 0.214) and from 0.010 to 4.875 μg/mL (average = 0.770), respectively. Out of 72 isolates assayed from the exposed population, 93% had EC50 values for fludioxonil ranging from 0.001 to 5.370 μg/mL (average = 0.472), but five isolates were highly resistant to this fungicide (EC50 > 100 μg/mL). About 98.55% of these isolates were sensitive to cyprodinil (0009 < EC50 < 0.415 μg/mL; average = 0.098). Only one isolate was resistant to cyprodinil (EC50 > 10 μg/mL). No cross-resistance relationship between cyprodinil and fludioxonil was observed (P = 0.2925, r = −0.1074). To determine the occurrence of multiple resistances, the radial growths of 126 isolates, collected from orchards with a history of Switch, Pristine, and strobilurin exposures, were measured on agar media amended with a single discriminatory dose of 10 μg fungicide/mL. High proportions of these isolates were resistant to boscalid (86.3%) and pyraclostrobin (92.9%). Most isolates were still sensitive to fludioxonil (77.8%) and cyprodinil (89.7%), but some were found to be independently or simultaneously resistant to all fungicides tested. Under conditions of excessive exposure to Switch resistant phenotypes may develop in A. alternata populations and ultimately affect fungicide performance. Therefore, monitoring studies must be continued and strict adherence to anti-resistance strategies followed and implemented in at risk orchards.

Fitness and cross-resistance of Alternaria alternata field isolates with specific or multiple resistance to single site inhibitors and mancozeb

42 field isolates of Alternaria alternata collected from infected tomato fruit of processing plants located in the region of Pelloponisos, South Greece, were characterized in terms of sensitivity to mancozeb, tebuconazole, fludioxonil, iprodione and cyprodinil. Fungitoxicity tests in vitro revealed that 63 % of the isolates tested were sensitive to all the above fungicides whereas the remainder of the isolates exhibited reduced sensitivity to one or more of these fungicides. The observed resistance phenotypes were confirmed by in vivo experiments on artificially inoculated tomato fruit. EC50 in vitro values ranged from 2.34 to >100 μg/ml for mancozeb, 0.16 to >10 μg/ml for iprodione, 0.43 to 20 μg/ml for tebuconazole, 0.007 to >5 μg/ml for cyprodinil and from 0.002 to >10 μg/ml for fludioxonil. Sensitivity distributions of A. alternata isolates to mancozeb, iprodione, cyprodinil and fludioxonil was bimodal indicating a probable initiation of a discrete shift of the population towards reduced sensitivity to these fungicides. Correlation analysis of respective EC50 values showed positive cross-resistance relationships between fludioxonil and iprodione while positive correlation was observed between mancozeb and cyprodinil, mancozeb and tebuconazole, and cyprodinil and tebuconazole. Interestingly, a number of A. alternata isolates were specifically resistant to the multi-site inhibitor mancozeb, a fact reported for the first time. Isolates resistant to mancozeb were more aggressive while those with reduced sensitivity to both fludioxonil and iprodione produced significantly less conidia than sensitive ones. A considerable portion of the sample exhibited reduced sensitivity and no apparent fitness penalties, a fact that should be considered in future planning and implementation of anti-resistance strategies.

Insecticide resistance monitoring and correlation analysis to select appropriate insecticides against Nilaparvata lugens (Stål), a migratory pest in Korea

Nilaparvata lugens Stål is one of the important migratory pests of rice paddy fields in Korea. Resistance levels to nine insecticides were monitored in 12 local strains and correlation analysis was conducted to determine cross-resistance relationships among the tested insecticides. The local strains revealed 1.3- to 28.0-, 1.6- to 6.0-, 2.8- to 237.0-, 0.6- to 0.9-, and 0.7- to 1.3-fold resistance to carbamates, organophosphates, neonicotinoids, fipronil and etofenprox, respectively. Organophosphate insecticides revealed moderate correlations with benzofuranyl methylcarbamate (r=0.566–0.614, p>0.01). Three neonicotinoids were not correlated with each other, but imidacloprid and clothianidin were moderately correlated with several benzofuranyl methylcarbamate and organophosphate insecticides (r=0.590–0.705, p>0.05), indicating that unknown common factors (such as detoxification enzymes) might contribute to resistance to both insecticides. Fipronil and etofenprox exhibited low levels of resistance and cross-resistance with other insecticides, suggesting their potential as an effective insecticide for field application. Resistance level monitoring and correlation analysis would be valuable for the selection of appropriate insecticides to control insecticide-resistant N. lugens, a typical migratory pest in Korea.

Effect of phenylpyrrole-resistance on fitness parameters and ochratoxin production in Aspergillus carbonarius

The risk of resistance development to fludioxonil and the potential implications of resistance mutations to ochratoxin production in Aspergillus carbonarius were investigated. Mutants of A. carbonarius highly resistant to phenylpyrroles were isolated at a high mutation frequency after N-MNTG-mutagenesis and selection on media containing fludioxonil. A highly reduced sensitivity to fungicides belonging to the same cross-resistance group (AHDs and phenylpyrroles) such as the aromatic hydrocarbon tolclofos-methyl and the dicarboximide fungicides iprodione and vinclozolin was also observed. No cross-resistance relationships were found between fludioxonil and the triazole epoxiconazole, the anilinopyrimidine cyprodinil and the chloronitrile chlorothalonil. Interestingly, fludioxonil-resistant isolates were highly sensitive to the QoI fungicide pyraclostrobin compared to the wild-type parental strain. Fitness studies revealed that resistance mutation(s) had a negative effect on mycelial growth, resistance to osmotic stress and pathogenicity of the fludioxonil-resistant strains. Mycotoxin analysis showed that most fludioxonil-resistant strains produce less quantities of ochratoxin A (OTA) than the wild-type strain both when grown on artificial medium and on grapes. Increased osmotic sensitivity and reduced pathogenicity of the mutant strains were significantly correlated with reduced ochratoxin production in vivo but not in vitro. The above-mentioned data indicate that fludioxonil is an excellent fungicide for the control of A. carbonarius in grapes and a valuable asset for farmers in terms of resistance management and ochratoxin contamination of grapes, vine products and wines.

Chlorfenapyr resistance in the spider mite Tetranychus urticae: stability, cross-resistance and monitoring of resistance

The two-spotted spider mite, Tetranychus urticae Koch, is a key pest of many agricultural crops. Studies of stability of resistance, cross-resistance relationships and monitoring of chlorfenapyr resistance were carried out with T. urticae to provide basic information necessary to define effective acaricide resistance management strategies for this pest. Chlorfenapyr resistance was shown to be stable in the absence of selection pressure under laboratory conditions. The activities of seven different acaricides against chlorfenapyr-resistant and -susceptible strains of T. urticae were evaluated. The results indicated possible positive cross-resistance between chlorfenapyr and the acaricides abamectin, propargite and etoxazole. No cross-resistance was detected for the acaricides milbemectin, fenpyroximate and diafenthiuron. A possible negatively correlated cross-resistance was observed between chlorfenapyr and spiromesifen. The evaluation of 21 T. urticae populations from several crops in the States of Sao Paulo, Mato Grosso, Goias, and Bahia, in Brazil, indicated that the susceptibility of mites to chlorfenapyr was variable, with percentages of resistant mites ranging from 0.0 to 86.5%. The highest resistance frequencies were observed in ornamental plants in the State of Sao Paulo. Some populations from cotton and papaya also presented high frequencies of chlorfenapyr resistance. This is the first report on chlorfenapyr resistance in T. urticae on cotton and papaya in Brazil. Strategies for the management of acaricide resistance are discussed.

Molecular characterization, fitness and mycotoxin production of benzimidazole-resistant isolates of Penicillium expansum

Penicillium expansum field-strains resistant to benzimidazole fungicides were isolated in high frequency from decayed apple fruit collected from packinghouses and processing industries located in the region of Imathia, N. Greece. In vitro fungitoxicity tests resulted in the identification of two different resistant phenotypes: highly (BEN-HR) and moderately (BEN-MR) carbendazim-resistant. Thirty seven percent of the isolated P. expansum strains belonged to the BEN-HR phenotype, carried no apparent fitness penalties and exhibited resistance levels higher than 60 based on EC50 values. Cross resistance studies with other benzimidazole fungicides showed that all BEN-HR and BEN-MR isolates were also less sensitive to benomyl and thiabendazole. Fungitoxicity tests on the response of BEN-HR isolates to fungicides belonging to other chemical classes revealed no cross-resistance relationships between benzimidazoles and the phenylpyrrole fludioxonil, the dicarboximide iprodione, the anilinopyrimidine cyprodinil, the QoI pyraclostrobin, the imidazole imazalil and the triazole tebuconazole, indicating that a target-site modification is probably responsible for the BEN-HR phenotype observed. Contrary to the above, some BEN-MR isolates exhibited an increased sensitivity to cyprodinil compared to benzimidazole-sensitive ones. BEN-MR isolates had fitness parameters similar to the benzimidazole-sensitive isolates except for conidia production which appeared significantly decreased. Analysis of mycotoxin production (patulin and citrinin) showed that all benzimidazole-resistant isolates produced mycotoxins at concentrations significantly higher than sensitive isolates both on culture medium and on artificially inoculated apple fruit. Comparison of the β-tubulin gene DNA sequence between resistant and sensitive isolates revealed a point mutation resulting from the E198A substitution of the corresponding protein in most but not all HR isolates tested. Molecular analysis of the β-tubulin gene in moderately resistant isolates did not reveal any amino acid substitution. This is the first report on the existence and distribution of highly mycotoxigenic field isolates of P. expansum resistant to the benzimidazoles indicating a high potential risk of increased mycotoxin contamination of pome fruit and by-products.

설부소립균핵병균 Typhula incarnata의 살균제 감수성

설부소립균핵병 방제를 위한 살균제 반응을 조사하기 위하여 Typhula incarnata 균주를 2012년 용평에 3곳 골프장에서 채집하여 propiconazole, tebuconazole 및 azoxystrobin 살균제에 대하여 감수성을 평가하였다. 50개의 균주에 대한 실내에서 감수성을 평가를 위하여 4개의 다른 농도로 수행하였다. 균사생장을 효과적으로 50%까지 억제하는 농도인 <TEX>$EC_{50}$</TEX>은 tebuconazole이 가장 낮았다. Triazole 계인 tebuconazole의 평균 <TEX>$EC_{50}$</TEX>은 0.0048 <TEX>${\mu}g\;ml^{-1}$</TEX>이며, 최소 <TEX>$EC_{50}$</TEX>은 0.0005 <TEX>${\mu}g\;ml^{-1}$</TEX> 농도이며, 최대 <TEX>$EC_{50}$</TEX>의 농도는 0.014 <TEX>${\mu}g\;ml^{-1}$</TEX>이었다. 반면, Propiconazole의 평균 <TEX>$EC_{50}$</TEX>은 0.5825 <TEX>${\mu}g\;ml^{-1}$</TEX>이고, <TEX>$EC_{50}$</TEX>의 범위는 0.078 <TEX>${\mu}g\;ml^{-1}$</TEX>에서 1.651 <TEX>${\mu}g\;ml^{-1}$</TEX>이었다. Strobilurin 계인 azoxystrobin은 평균 <TEX>$EC_{50}$</TEX>은 0.0278 <TEX>${\mu}g\;ml^{-1}$</TEX>이고, 최소와 최대 <TEX>$EC_{50}$</TEX>은 범위는 0.0017 <TEX>${\mu}g\;ml^{-1}$</TEX>에서 0.131 <TEX>${\mu}g\;ml^{-1}$</TEX>이었다. Propiconazole, azoxystrobin 및 tebuconazole 살균제간의 <TEX>$EC_{50}$</TEX> 사이에 상관 관계가 나타나지 않았으며, 이들 균주와 이들 살균제 간에 교차저항성도 없었다. 이 연구 결과 용평에서 채집한 T. incarnata 균주는 3종의 살균제에 대하여 실내감수성 시험에서 저항성 균주가 없다는 것을 확인할 수 있었다. In response of fungicides for control of gray snow mold, isolates of Typhula incarnata were collected from three golf courses in 2012 Yongpyeong, Korea and tested for sensitivity to propiconazole, tebuconazole and azoxystrobin fungicides. Four discriminatory concentrations were used to detect in vitro sensitivity of 50 isolates. Mean 50% effective concentration inhibiting mycelial growth (<TEX>$EC_{50}$</TEX>) values for tebuconazole was the lowest among the three fungicides. The <TEX>$EC_{50}$</TEX> value of tebuconazole ranged from 0.0005 <TEX>${\mu}g\;ml^{-1}$</TEX> to 0.014 <TEX>${\mu}g\;ml^{-1}$</TEX> with a mean of 0.0048 <TEX>${\mu}g\;ml^{-1}$</TEX>. The mean <TEX>$EC_{50}$</TEX> values of propiconazole in triazole family was 0.5825 (0.78-1.651) <TEX>${\mu}g\;ml^{-1}$</TEX>. <TEX>$EC_{50}$</TEX> value of azoxystrobin ranged from 0.0017 <TEX>${\mu}g\;ml^{-1}$</TEX> to 0.131 <TEX>${\mu}g\;ml^{-1}$</TEX> with a mean of 0.0278 <TEX>${\mu}g\;ml^{-1}$</TEX>. There was no correlation among <TEX>$EC_{50}$</TEX> values for propiconazole, azoxystrobin and tebuconazole indicating no cross-resistance relationships with each other. Results of this study were confirmed no resistance isolates in vitro sensitivity of T. incarnata of three fungicides in Yongpyeong.

Cross‐resistance relationships of the sulfoximine insecticide sulfoxaflor with neonicotinoids and other insecticides in the whiteflies Bemisia tabaci and Trialeurodes vaporariorum

Insecticides are important tools for managing damaging insect pests. Compounds that are effective against pests such as the whiteflies Bemisia tabaci and Trialeurodes vaporariorum, which show resistance to a range of insecticidal modes of action (MOA), have particular value as components of resistance management programmes. The sulfoximine insecticides are chemically unique as the first to incorporate a sulfoximine functional group. Sulfoxaflor is the first sulfoximine compound under commercial development for the control of sap-feeding insects. Its cross-resistance relationships were investigated by comparing the responses of field-collected strains with those of insecticide-susceptible laboratory strains of B. tabaci and T. vaporariorum. Sulfoxaflor exhibited very low (less than threefold) resistance ratios (RR) when tested against strains of B. tabaci that produced RR of up to 1000-fold to imidacloprid and cross-resistance to other neonicotinoid insecticides. Similarly, sulfoxaflor was not cross-resistant in a strain of B. tabaci exhibiting resistance to a pyrethroid (deltamethrin) and an organophosphate (profenophos). No cross-resistance was observed between sulfoxaflor and imidacloprid in T. vaporariorum. One population of the three field strains tested showed slightly reduced susceptibility to sufloxaflor with an RR of 4.17. By comparison, this same population exhibited an RR of more than 23.8-fold for imidacloprid relative to the susceptible population. In spite of sharing a target site with neonicotinoids (the nicotinic acetylcholine receptor), sulfoxaflor was largely unaffected by existing cases of neonicotinoid resistance in B. tabaci and T. vaporariorum. Neonicotinoid resistance mechanisms in these whitefly species are known to be primarily based on enhanced detoxification of insecticide. This lack of cross-resistance indicates that sulfoxaflor is a valuable new tool for the management of sap-feeding pests already resistant to established insecticide groups.

PCR-RFLP detection of the E198A mutation conferring resistance to benzimidazoles in field isolates of Monilinia laxa from Greece

Sensitivity to benzimidazoles in isolates of the brown rot pathogen (Monilinia laxa) collected from stone fruit in central and northern Greece was evaluated and the molecular basis for resistance was investigated. M. laxa isolates were classified as benzimidazole – sensitive (S) or highly resistant (HR) based on their sensitivity profiles to carbendazim. Thirty seven percent of the isolates belonged to the HR phenotype, carried no apparent fitness penalties and exhibited resistance factor values (based on EC50 values) greater than 500. Highly resistant isolates were also less sensitive to the benzimidazoles benomyl and thiophanate-methyl but more sensitive to the N-phenylcarbamate diethofencarb and the benzamide zoxamide compared to isolates belonging to the S phenotype. Fungitoxicity tests with fungicides belonging to other chemical classes revealed no cross resistance relationships between benzimidazoles and the dicarboximide iprodione, the phenylpyrrole fludioxonil, the hydroxyanilide fenhexamid, the carboxamide boscalid, the triazole tebuconazole and the strobilurin-type fungicide pyraclostrobin, indicating that a target site modification is probably responsible for the resistant phenotypes observed. Comparison of the β-tubulin gene DNA sequences between resistant and sensitive isolates revealed a point mutation resulting from the E198A substitution of the corresponding protein in all HR isolates tested. An Eco31I restriction site in the β-tubulin gene, which was destroyed in HR M. laxa isolates, allowed the development of a PCR-RFLP diagnostic for the detection of the E198A resistance mutation.

Biological activity of the succinate dehydrogenase inhibitor fluopyram against Botrytis cinerea and fungal baseline sensitivity

Succinate dehydrogenase inhibitors (SDHIs) constitute a fungicide class with increasing relevance in crop protection. These fungicides could play a crucial role in successful management of grey mould disease. In the present study the effect of fluopyram, a novel SDHI fungicide, on several developmental stages of Botrytis cinerea was determined in vitro, and the protective and curative activity against the pathogen was determined on strawberry fruit. Furthermore, fungal baseline sensitivity was determined in a set of 192 pathogen isolates. Inhibition of germ tube elongation was found to be the most sensitive growth stage affected by fluopyram, while mycelial growth was found to be the least sensitive growth stage. Fluopyram provided excellent protective activity against B. cinerea when applied at 100 µg mL(-1) 96, 48 or 24 h before the artificial inoculation of the strawberry fruit. Similarly, fluopyram showed a high curative activity when it was applied at 100 µg mL(-1) 24 h post-inoculation, but, when applications were conducted 48 or 96 h post-inoculation, disease control efficacy was modest or low. The measurement of baseline sensitivity showed that it was unimodal in all the populations tested. The individual EC(50) values for fluopyram ranged from 0.03 to 0.29 µg mL(-1). In addition, no correlation was found between sensitivity to fluopyram and sensitivity to other fungicides, including cyprodinil, fenhexamid, fludioxonil, iprodione, boscalid and pyraclostrobin. The obtained biological activity, baseline sensitivity and cross-resistance relationship data suggest that fluopyram could play a key role in grey mould management in the near future and encourage its introduction into spray programmes.