Carbendazim resistance in field isolates of Sclerotinia sclerotiorum in China and its management

The ascomycete fungus Sclerotinia sclerotiorum is a devastating necrotrophic plant pathogen with an unusually broad host range and worldwide distribution. The benzimidazole fungicide carbendazim has been widely used to control S. sclerotiorum in China for more than three decades and high levels of carbendazim resistance have been reported in eastern China. In this study, carbendazim sensitivity was assessed in a total of 5042 field isolates of S. sclerotiorum collected from different geographical regions of China from 2008 to 2013. Results showed that no carbendazim resistant isolates could be detected in northwestern and northeastern China. In 2013, 0.77% and 0.72% of the field isolates assayed were highly resistant to carbendazim in Hunan and Hubei provinces of central China, respectively. In Anhui province of eastern China, the frequencies of carbendazim resistance were 18.18%, 7.05% and 7.25% in 2008, 2011 and 2012, respectively. The decrease in frequency of carbendazim resistance in Anhui province from 2008 to 2012 was statistically significant (P = 0.001). Nevertheless, no significant differences (P ≥ 0.16) were found in fitness parameters such as mycelial growth on PDA media, virulence to oilseed rape plants and oxalic acid production between carbendazim resistant and sensitive isolates. A negative cross resistance pattern was detected between carbendazim and diethofencarb. Mixture of carbendazim and diethofencarb (4:1) applied at 200 μg/mL provided 100% and 91.5% preventive efficacy against carbendazim resistant and sensitive isolates, respectively, and 87.1% and 81.7% curative efficacy against resistant and sensitive isolates, respectively.     

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.     

Patterns of cross-resistance to fungicides in Botryotinia fuckeliana (Botrytis cinerea) isolates from French vineyards

Field strains of Botryotinia fuckeliana were isolated from diseased grape berries which were collected at the harvest, between 1993 and 1997, from French vineyards located in Alsace, Armagnac, Bordeaux, Champagne and Loire Valley. According to the inhibitory effects of fungicides towards spore germination, germ-tube elongation and mycelial growth, several phenotypes could be characterized. Thus, two types of benzimidazole (e.g. carbendazim, thiabendazole)-resistant strains were detected; negative cross-resistance towards diethofencarb, diphenylamine and dicloran was found only in one type. Most dicarboximide (e.g. iprodione, procymidone, vinclozolin)-resistant strains were also weakly resistant to aromatic hydrocarbon fungicides (e.g. chloroneb, dicloran, tolclofos-methyl) but remained sensitive to phenylpyrroles (e.g. fenpiclonil, fludioxonil). However, in some other dicarboximide-resistant strains, resistance was observed either as being restricted to dicarboximides or as extending weakly to phenylpyrroles. Towards anilinopyrimidines (e.g. cyprodinil, mepanipyrim, pyrimethanil), three resistant phenotypes have been identified. In the most resistant one, resistance was restricted to anilinopyrimidines, whereas in the others, resistance extended to various other groups of fungicides including dicarboximides, phenylpyrroles, sterol biosynthesis inhibitors (e.g. tolfanate, prochloraz, tebuconazole) and the hydroxyanilide derivative, fenhexamid. According to the pattern of cross-resistance, two phenotypes were characterized within these multifungicide-resistant strains. In vitro tests conducted with fenpropimorph, fenpropidin and spiroxamine, revealed the existence of strains highly susceptible to these sterol Δ¹⁴- reduction inhibitors. This property was correlated with a decreased sensitivity towards fenhexamid.     

Characterization of laboratory pyrimethanil-resistant mutants of Aspergillus flavus from groundnut in China

To assess the potential risk of resistance development in Aspergillus flavus to pyrimethanil, five highly pyrimethanil-resistant (Pyr^R) mutants (RF > 996.2) were obtained after UV-mutagenesis and tested for fitness parameters and aflatoxin B1 production. All five mutant strains had mycelial growth rate, sporulation and aflatoxin production similar to or even higher than the wild-type parent strain, which indicated that pyrimethanil possesses a high risk in the development of resistance in A. flavus. Comparing the sequences of four key enzymes cystathionine β-lyase (CBL), cystathionine γ-synthase (CGS), methionine sulfoxide reductase (MsrB), and sulfate permease (SP2) involved in the biosynthesis and metabolism of methionine and sulfate assimilation revealed that no amino acid difference was found between the mutant and wild-type parent strains, suggesting that the four enzymes might not be related to the anilinopyrimidines (APs) resistance in A. flavus.     

Characterization of fungicide resistant isolates of Phaeoacremonium aleophilum infecting grapevines in Spain

Fungicide resistance in Phaeoacremonium aleophilum, one of the most frequent fungal pathogens associated with grapevine trunk diseases, was investigated and found to exist in some isolates of the pathogen against a commercial formulation, Escudo^®. The effect of this compound and its two active substances, carbendazim and flusilazole, was first evaluated on the mycelial growth of P. aleophilum. Escudo^®-resistant isolates were estimated at a frequency of 24% in Spanish vineyards. Then, the two active substances were used individually to test their effect on mycelia growth of twelve single-spore cultures originating from six Escudo^®-resistant isolates. Flusilazole (DMI-triazole) did not inhibit mycelia growth of any single-spore cultures of P. aleophilum. Carbendazim (benzimidazole) used alone allowed the growth of the same single-spore cultures that were also resistant to Escudo^®. AFLP characterization of sensitive and resistant single-spore cultures showed genetic diversity within P. aleophilum isolates but no AFLP markers were associated with resistance. New primers set (L2/R1) were designed to partially amplify the exon 6 of the beta-tubulin gene of P. aleophilum. Two different point mutations resulted in glycine (GGC) or lysine (AAA) replacing the glutamic acid (GAG) at codon 198 of the beta-tubulin gene in some of the resistant single-spore cultures studied. Resistant single-spore cultures of P. aleophilum were shown to have different aggressiveness levels as sensitive single-spore cultures by inoculation of wood segments of Vitis vinifera in the presence and absence of fungicide.     

Assessing fungicide resistance in populations of Alternaria in Idaho potato fields

Early blight, caused by the fungus Alternaria solani and brown leaf spot, caused by Alternaria alternata, are important diseases of potato crops in Idaho. In recent years growers have reported a reduction in efficacy of fungicides traditionally used in the past decade to control early blight. In 2009, a collection of A. solani 39 isolates were screened for resistance to azoxystrobin, pyraclostrobin, boscalid and famoxadone. Fungicide sensitivity testing was done using spiral plate dilution gradients. Results showed that of 39 isolates screened, all were resistant to azoxystrobin and three were resistant to boscalid. None were resistant to pyraclostrobin or famoxadone. In summer 2010, more isolates were collected (9 A. alternata and 26 A. solani) and the survey was expanded to include more fungicides with four different modes of action that targeted succinate dehydrogenase (SDH), methionine biosynthesis, mitochondrial respiration and multi-site contact activity. New isolates of A. solani and A. alternata were also collected from two additional sites. The results showed that 57% of the isolates were resistant to boscalid as well as an average of 63% of the isolates being resistant to the strobilurin fungicides. Seven and 15% of isolates were resistant to penthiopyrad (an SDH inhibitor), and pyrimethanil (a methionine biosynthesis inhibitor), respectively. However, none of the isolates were resistant to fluopyram (an SDH inhibitor) or a mixture of fluopyram and pyrimethanil. Although there appears to be cross resistance developing in Alternaria spp. to some of the new SDH inhibitors like penthiopyrad, others such as fluopyram are still showing limited to no resistance development in Alternaria spp. in Idaho.     

Resistance of Botrytis cinerea Pers. and Plasmopara viticola (Berk. & Curt.) Berl. and de Toni to fungicides in French vineyards

The control of Botrytis cinerea and Plasmopara viticola, two of the most important parasites on grapevines, is achieved by using fungicides. Recently several highly active compounds have been introduced but with some of them, failures of disease control have been observed in French vineyards. These phenomena are due to the build-up of resistance to benzimidazoles (benomyl, carbendazim, thiophanate-methyl) or to di-carboximides (iprodione, procymidone, vinclozolin) in Botrytis cinerea and to phenylamides (metalaxyl, ofurace) in Plasmopara viticola. The characteristics of the various resistant strains (levels of resistance, patterns of cross-resistance, fitness) are described. In 1982 and 1983 there has been monitoring for resistance in most French vineyards: the tests used and the results with regard to the frequencies of resistant strains are discussed.     

Sensitivity of Cercospora beticola isolates from Serbia to carbendazim and flutriafol

Cercospora beticola, causal agent of Cercospora leaf spot (CLS) of sugar beet, is primarily controlled by fungicides. Benzimidazole and demethylation inhibiting fungicides, including carbendazim and flutriafol, have been widely used in Serbia. Since these fungicide groups have a site-specific mode of action, there is a high risk for developing resistance in target organisms, which is the most important limiting factor in Cercospora leaf spot chemical control. A rapid identification of flutriafol and carbendazim resistance can help researchers in examining the potential of different fungicide resistance management practices, as well as in selection of fungicides for use in the areas where resistance has occurred. One hundred single-conidia isolates were collected from 70 representative locations of the sugar beet production region in Serbia. Evaluation of the isolates' sensitivity was based on the reduction of mycelial growth on medium amended with 1.25 μg mL⁻¹ flutriafol and 5 μg mL⁻¹ carbendazim. Resistance to flutriafol and carbendazim was detected in 16% and 96% of the tested isolates, respectively. All isolates resistant to flutriafol were resistant to carbendazim as well, which is the first report of a double resistance to fungicides in C. beticola. Detection of the isolates resistant to flutriafol and carbendazim using Cleaved Amplified Polymorphic Sequence (CAPS) markers confirmed the results of the in vitro tests. The efficacy of carbendazim, flutriafol, azoxystrobin, and tetraconazole at commercially recommended doses was evaluated in field trials where sugar beet plants in plots were inoculated with a mixture of isolates either sensitive and/or resistant to flutriafol and carbendazim. Carbendazim and flutriafol efficacy was very low in plots inoculated with isolates resistant to these fungicides. Presented results will contribute to development of a pathogen population sensitivity monitoring strategy that could be used for an effective CLS management in the region.     

油菜菌核病菌对腐霉利的抗性诱变及抗药菌株的生物学特性

从江苏省镇江市和扬州市采集菌核病植株的茎,从中分离获得油菜菌核病菌（Sclerotinia sclerotiorum）单菌核菌株。32个野生型菌株药剂敏感性（EC50）频率分布数据显示,病菌对腐霉利的敏感基线EC50值为0.1981 ± 0.0220 µg/mL,MIC（最小抑菌浓度）值为 1.1 ± 0.1595µg/mL。实验未发现田间抗腐霉利菌株。通过室内紫外线和药剂诱导,获得多株高抗腐霉利突变体（EC50>1000µg/mL）。这些抗性突变株经转管培养5~20代后抗药性稳定。它们对同类药剂异菌脲和有机磷类甲基立枯磷也表现高抗,但对苯并咪唑类多菌灵敏感。与野生型敏感菌株相比,抗腐霉利突变株菌丝生长较慢,对油菜茎杆和叶片的致病力均较弱。     

Virulence Complexity and High Levels of Fungicide Resistance Suggest Population Change of Phytophthora infestans in the Heilongjiang Province of China

Samples of Phytophthora infestans were isolated from 12 city and county fields in the main potato production areas of Heilongjiang province of China between 2004 and 2008. A total of 72 isolates were tested for virulence on a potato R gene differential set and 39 of these isolates were also analysed for their resistance to the fungicide metalaxyl. All 11 differential plants were infected by one or more of the isolates collected; the most commonly overcome differentials were R1, R3, R4, R7, R10, and R11. Most (89.7%) of the tested isolates were resistant to metalaxyl and the rest (10.3%) were sensitive. The proportion of metalaxyl-resistant isolates differed between sites (χ ²?=?25.42, df?=?10, p?<?0.005), but not between years (χ ²?=?4.63, df?=?4, p?>?0.05). Taken together and in comparison with earlier pathogen population studies, our findings suggest that the population of P. infestans in Heilongjiang is different from that existing in the province two decades ago and that at various locations within the province, the new population has a high frequency of resistance to metalaxyl.     

Five years' results from a long-term field experiment on carbendazim resistance of Pseudocercosporella herpotrichoides (Fron) Deighton

In a field monitoring experiment, winter wheat sown in large plots with or without applications of carbendazim was used to determine, during 5 years in succession, the development of carbendazim resistance in field populations of Pseudocercosporella herpotrichoides (Fron) Deighton, (Cercosporella herpotrichoides Fron). At the beginning of the experiment, one spore out of about 2·4 × 10⁹ was resistant. This figure increased at a linear rate that was highly statistically significant. Eventually, the frequency of carbendazim-resistant conidia was of the order of 10^?8. The differences in the frequency of resistant spores in the carbendazim-treated and in the untreated plots were highly significant, ranging from twofold to eightfold, depending on the year in question. In practice, there seems to be little danger from carbendazim resistance in the eyespot fungus if normal plant-protection recommendations are followed.     

Prevalence of isolates of Botrytis cinerea resistant to multiple fungicides in Chilean vineyards

Gray mold (Botrytis cinerea) is an important disease of grapevines (Vitis vinifera) and requires several fungicide treatments to achieve a satisfactory control in Chile. Furthermore, a high resistance risk has developed because of the extensive use of specific fungicides with a single-site mode of action. The aim of this study was to determine the presence of resistance to the multiple fungicides currently used against B. cinerea in Chile. During 2007–2009 and 2011, 214 isolates of B. cinerea from 36 commercial vineyards were examined for sensitivity to anilinopyrimidines (cyprodinil or pyrimethanil), demethylation inhibitors (DMIs) (tebuconazole), hydroxyanilides (fenhexamid) and phenylpyrroles (fludioxonil). Of all of the isolates, 62.7% were resistant to anilinopyrimidines, 7.2% to DMIs, 27.1% to hydoxyanilides and 44.8% to phenylpyrrole derivates. Overall, 16 (12.3%) of the isolates were sensitive; although none was resistant to all four of the fungicide classes, and 50 (38.5%), 51 (39.2%), and 13 (10.0%) isolates were resistant to one, two and three fungicides, respectively. In vitro, resistant isolates were capable of causing fruit rot on wounded apples pretreated with any one of the five fungicides at label concentrations. To the best of our knowledge, this is the first report of isolates having simultaneous resistance to anilinopyrimidines, DMIs, phenylpyrroles and hydroxyanilides in B. cinerea populations from grapevines in Chile. Therefore, fungicide resistance is a serious problem that questions the sustainability of the current gray mold control strategy, which relies almost exclusively on fungicides with single-site modes of action.     

Resistance of Botrytis cinerea Pers. to dicarboximide fungicides — a literature review

The dicarboximides (iprodione, procymidone, vinclozolin) are a relatively new group of protectant fungicides which are used mainly for the control of Botrytis cinerea Pers. The literature documenting the emergence of dicarboximide-resistant Botrytis strains and the current situation, is reviewed. Although dicarboximide-resistant Botrytis can be produced readily in the laboratory, extensive monitoring did not reveal the emergence of such strains in the field until after 2–3 years of intensive use of dicarboximides. Today, resistant strains are foubd in many areas where the dicarboximides have been in constant use. Nevertheless, loss of control by these fungicides has not, so far, become a major field problem, possibly because of a loss of vigour and pathogenicity that is often linked to dicarboximide resistance in Botrytis strains. Because resistant strains also produce varying numbers of sensitive conidia, such strains are rapidly resensitized in the absence of dicarboximide fungicides. These findings suggest that the nature of dicarboximide resistance is quite different to that of carbendazim resistance, in that carbendazim-resistant Botrytis strains show no loss of vigour and are, moreover, very stable.     

Identification of mating types and metalaxyl resistance in North American populations ofPhytophthora infestans

The A² mating type ofPhytophthora infestans was first reported in the United States in 1990. Concurrently,P. infestans strains resistant to metalaxyl ere found in the Pacific Northwest. Collaborative surveys were undertaken during 1991–1993 to investigate the frequency of occurrence of A² mating types and metalaxyl resistant strains in populations ofP. infestans isolated from outbreaks of late blight in potato and tomato crops in North America.In vitro testing indicated that isolates from the northeastern U.S. and Atlantic Canada were primarily (52/55) metalaxyl sensitive and all were A¹ mating types. Among 85 isolates from late blight epidemics in Florida and Texas, greater than 61% were both metalaxyl resistant and A² mating type. Metalaxyl resistance and A² mating types were identified also in a few tomato isolates from North Carolina. Although the majority of 134 isolates from the Pacific Northwest (British Columbia and Washington) were metalaxyl resistant, only 2 isolates from Washington were A² mating types. Among 111 isolates from 2 sites in central Mexico, 63% and 77% were both metalaxyl resistant and A² mating types. The data indicate also a higher frequency of metalaxyl resistance in A² isolates, than in A¹ isolates, among isolates from Florida and Texas. Highest metalaxyl resistance levels were found, however, in A¹ isolates from California, where no A² isolates were recovered.     

Baseline sensitivity of Monilinia laxa from Greece to fenhexamid and analysis of fenhexamid-resistant mutants

Fenhexamid is a hydroxyanilide fungicide with excellent performance against Botrytis cinerea but also effective against Monilinia spp. which cause brown rot disease in apple and stone fruit. A total of 75 Monilinia laxa field isolates were utilized to determine baseline sensitivity while a number of fenhexamid-resistant laboratory mutants were used to evaluate the resistance risk associated with the longevity of the effectiveness of fenhexamid. Fenhexamid was found to be highly effective against all field isolates. EC₅₀ values ranged from 0.02 to 1 μg mL⁻¹ and were distributed unimodaly around an average of 0.1 μg mL⁻¹. M. laxa laboratory strains with moderate and high resistance levels to fenhexamid were isolated after UV mutagenesis. All fenhexamid-resistant strains showed parental sensitivity to carbendazim, iprodione, fludioxonil, pyraclostrobin, flusilazole and prochloraz. Interestingly, some of the mutant strains were also resistant to tridemorph, fenpropimorph and spiroxamine. Studies on fitness parameters of fenhexamid-resistant strains revealed a fitness cost on sporulation and pathogenicity but not on mycelial growth. These results suggest that fenhexamid should be a good alternative site-specific fungicide for the control of brown rot disease caused by M. laxa. However, appropriate anti-resistance strategies should be considered to ensure the successful commercial use of fenhexamid in the long run.     

Sensitivity of Sclerotinia sclerotiorum from oilseed crops to boscalid in Jiangsu Province of China

Between 2006 and 2008, 120 isolates of Sclerotinia sclerotiorum from the Jiangsu Province of China were characterized for the baseline sensitivity to boscalid, a new active ingredient that interferes with succinate ubiquinone reductase in the electron transport chain of fungi. The isolates collected between 2006 and 2008 all showed similar sensitivity to boscalid. Baseline sensitivities were distributed as unimodal curves with mean EC₅₀ values of 0.17 ± 0.09 μg ml⁻¹ for inhibition of mycelial growth. Inhibition of mycelial growth of isolates whether they had resistance to carbendazim or, dimethachlon or dual resistance to both carbendazim and dimethachlon was similar, suggesting that there was no cross-resistance between boscalid and MBC/dimethachlon already in commercial use. In field trials, the treatments boscalid (50% WG), at 125, 187.5 and 250 g ai ha⁻¹ provided higher control efficacies of 65.2, 69.0 and 75.1%, respectively, than the treatments carbendazim (50% WP), at 750 g ai ha⁻¹ and dimethachlon (46% WP), at 690 g ai ha⁻¹ with control efficacies of 18.9 and 44.6%, respectively.     

Sensitivity to fluopicolide of wild type isolates and biological characteristics of fluopicolide-resistant mutants in Pseudoperonospora cubensis

Cucurbit downy mildew caused by the oomycete pathogen Pseudoperonospora cubensis is a devastating disease that is distributed worldwide and affects cucumber in open fields and greenhouses. Fluopicolide, which was a novel systemic fungicide and was released in 2008, it is very effective in controlling downy mildew on cucumber and grape, potato late blight and pepper Phythophthora blight and reduces the loss caused by the diseases, but so far the potential for P. cubensis to develop resistance to fluopicolide has not been investigated. Hence, a laboratory study was undertaken to assess the risk of P. cubensis developing resistance to fluopicolide. Baseline sensitivity to fluopicolide was determined by using 75 P. cubensis isolates collected from cucumber-growing greenhouses in Hebei province, where no fluopicolide had been used for control of cucumber downy mildew before. Values of effective concentrations for 50% inhibition (EC₅₀) of sporulation ranged from 0.02 to 0.40 μg ml⁻¹ and were distributed as a unimodal curve, indicating that all 75 isolates were sensitive to fluopicolide. Sporangia of nine sensitive isolates were ultraviolet (UV)-irradiated, and four fluopicolide-resistant mutants were acquired at a mutation frequency of 7.4 × 10⁻⁷. Seven mutants resistant to fluopicolide were obtained from seven isolates by sporangia adaptation on fluopicolide-treated leaves of cucumber. The EC₅₀ values for all eleven fluopicolide-resistant mutants ranged from 3.37 to 13.06 μg ml⁻¹ with mean resistance factors of 7.9–118.0. After 10 sporangia transfers on fungicide-free leaves of cucumber, all eleven resistant mutants remained resistant to fluopicolide with mean resistance factors of 8.2–81.3. Seven resistant mutants from the selection for resistance and one resistant mutant from UV mutagenesis exhibited stable resistance; however, the other three resistant mutants from UV irradiation became significantly less resistant. Compared to their respective sensitive parents, the eleven resistant mutants exhibited diversity in latent period, infection frequency, lesion extension and sporulation ability. Five out of the eleven resistant mutants exhibited prolonged latent period and three out of the eleven resistant mutants provided decreased infection frequency (IF) compared to their respective parents, indicating that in some cases, resistance mutation might affect the latent period and IF of P. cubensis. There were significant differences in pathogenicity and ability to produce sporangia, but this seemed not to be caused by resistance mutation. No cross-resistance was detected between fluopicolide and azoxystrobin, metalaxyl, dimethomorph, or cymoxanil. In all, there could be a moderate to high risk of field populations of P. cubensis developing resistance to fluopicolide, and populations of P. cubensis should be monitored regularly for their shift of sensitivity over years of application.     

Association of tcdA+/tcdB+ Clostridium difficile Genotype with Emergence of Multidrug-Resistant Strains Conferring Metronidazole Resistant Phenotype

Background:

Reduced susceptibility of Clostridium difficile to antibiotics is problematic in clinical settings. There is new evidence indicating the cotransfer of toxin-encoding genes and conjugative transposons encoding resistance to antibiotics among different C. difficile strains. To analyze this association, in the current study, we evaluated the frequency of toxigenic C. difficile among the strains with different multidrug-resistant (MDR) profiles in Iran.

Methods:

Antimicrobial susceptibility patterns and minimal inhibitory concentrations (MIC) of the isolates were determined against metronidazole, imipenem, ceftazidime, amikacin, and ciprofloxacin by agar dilution method. The association of the resistance profiles and toxigenicity of the strains were studied by PCR targeting tcdA and tcdB genes.

Results:

Among 86 characterized strains, the highest and lowest resistance rates were related to ciprofloxacin (97%) and metronidazole (5%), respectively. The frequency of resistance to other antibiotics was as follow: imipenem (48%), ceftazidime (76%), and amikacin (76.5%). Among the resistant strains, double drug resistance and MDR phenotypes were detected in the frequencies of 10.4% and 66.2%, respectively. All of the metronidazole-resistant strains belonged to tcdA ⁺/tcdB ⁺ genotype with triple or quintuple drug resistance phenotypes. MIC₅₀ and MIC₉₀ for this antibiotic was equally ≤ 8 μg/ml.

Conclusion:

These results proposed the association of tcdA ⁺/tcdB ⁺ genotype of C. difficile and the emergence of resistance strains to broad-spectrum antibiotics and metronidazole. Key Words: Multidrug resistance, Clostridium difficile, Metronidazole     

Sensitivity of Botryosphaeria dothidea from apple to tebuconazole in China

The white rot disease caused by Botryosphaeria dothidea threatens apple production in Bohai bay area and along the Yellow River of China, where disease control is largely dependent on fungicides such as tebuconazole. A total of 146 isolates of B. dothidea obtained from different apple orchards in six provinces were tested for their sensitivity to tebuconazole, carbendazim and iprodione. The EC₅₀ values of all tested isolates for tebuconazole were from 0.035 to 1.415 μg/mL. The broad range of EC₅₀ values of tebuconazole suggests an obvious variation among the 146 isolates. Isolate HB13 (EC₅₀ = 1.415 μg/mL) showed reduced sensitivity to tebuconazole, with an EC₅₀ value significantly higher than those of the other 145 isolates tested. The low sensitivity of HB13 was stable after 15 generations, and this isolate showed similar pathogenicity as susceptible strains. EC₅₀ correlation analysis indicates no cross resistance between tebuconazole and carbendazim and iprodione. Field efficacy trials showed that tebuconazole remains very effective for apple white rot control in China.     

Occurrence of resistant strains of Botrytis cinerea to anilinopyrimidine fungicides in table grapes in Chile

Effort to control gray mold caused by Botrytis cinerea failed in a table grapevine (Vitis vinifera) vineyard near Santiago, Chile where cyprodinil (Vangard 50 WP), a new fungicide of the anilinopyrimidine group, had been applied alone up to four times per year during two growing seasons. A relatively high frequency (38.5%) of resistant isolates of B. cinerea (EC₅₀ for mycelial growth inhibition varied from 2.9 to 4.84 μg ml⁻¹) may explain the partial loss of field control efficacy obtained. Resistance was correlated with a complete loss of in vivo sensitivity to cyprodinil. Resistant isolates of B. cinerea showed cross resistance to the anilinopyrimidines fungicides mepanipyrim and pyrimethanil. Cyprodinil partially impaired conidia germination and differentially affected conidial germination of resistant and sensitive isolates. Significant differences (p<0.05) in growth rate, sclerotia production and osmotic sensitivity were found among isolates of B. cinerea, but no correlation could be drawn between these biological differences and resistance or sensitivity. This indicates a disruptive selection characteristic of monogenic resistance. Thus, strategies were implemented to avoid the further development and spread of resistance in B. cinerea to the anilinopyrimidine fungicides. To our knowledge this constitutes the first mention of resistance in B. cinerea populations to anilinopyrimidine fungicides in South America.