Genetic Control of Resistance to the Piperidine Fungicide Piperalin in Ustilago maydis

Mutants of Ustilago maydis resistant to the piperidine fungicide piperalin were isolated in a mutation frequency of 2.4 × 10^–5 after UV-irradiation and selection on media containing 50gml^–1 piperalin. Genetic analysis with 15 such mutant isolates resulted in the identifications of two unlinked chromosomal loci, the U/ppl-1 locus with two allelic genes (U/ppl-1A and U/ppl-1B) and the U/ppl-2 locus. The U/ppl-2 and U/ppl-1A mutations are responsible for two levels of moderate and high resistance to piperalin (resistance factor, Rf: 54 and 135, respectively, based on effective concentration causing a 50% reduction in the growth rate, EC₅₀), while the U/ppl-1B mutation gives only a small reduction (approximately 8-fold) in piperalin sensitivity. Cross-resistance studies with other SBIs shows that the major gene (U/ppl-2 and U/ppl-1A) mutants are resistant to fenpropidin (Rf: 43 and 68), fenpropimorph (Rf: 261 and 283) and tridemorph (Rf: 9 and 10), but not to the inhibitors of C-14 demethylase (DMIs) and squalene epoxidase. The minor gene mutation U/ppl-1B codes a low-level of resistance (approximately 5—12-fold) to the above morpholine-type fungicides, but in contrast with the major gene mutations it increases 2–10 times the sensitivity to triazoles: triadimefon, triadimenol, propiconazole and flusilazole. Crosses between mutants carrying the U/ppl-genes with compatible isolates carrying the U/fpd, U/fpm or U/tdm mutations, which have been identified in previous genetic works for resistance to morpholine-type fungicides, yielded, with the exception of U/ppl-2 ×U/fpm-2 cross, a large number of recombinants with wild-type sensitivity, indicating that the mutant genes involved in these crosses, were not allelic. An additive gene effect was observed only between nonallelic minor genes U/ppl-1B and U/fpm-1B or U/tdm-1,2. Studies of the fitness of piperalin-resistant isolates showed that the reduced sensitivity of major gene mutants was not associated with changes on the phytopathogenic fitness determining characteristics, such as growth in liquid culture and pathogenicity on young corn plants. Conversely, the minor gene mutation U/ppl-1B appeared to be pleiotropic, having significantly adverse effects on the phytopathogenic fitness.     

Genetic control of resistance to fenpropimorph in Ustilago maydis

Fenpropimorph-resistant mutants of Ustilago maydis were obtained at high frequency (30 × 10⁻⁶) after UV-irradiation followed by selection on media containing fenpropimorph (50 μ g mL⁻¹). Genetic analysis of 30 such mutants resulted in the identification of two unlinked chromosomal loci, the U/fpm -1 locus with two allelic genes ( U/fpm- 1A and U/fpm -1B) and the U/fpm -2 locus. The mutant genes U/fpm- 1A and U/fpm -2 are responsible for high resistance levels (Rf: 75–100 or 257–286 based on MICs or ED_50s, respectively), while the U/fpm -1B mutation gives only a small reduction (approximately 7–10-fold) in fenpropimorph sensitivity. Cross-resistance studies with other SBIs showed that the major gene ( U/fpm- 1A and U/fpm -2) mutants were cross-resistant to the related compound fenpropidin (Rf: 15–20 or 53–66 based on MICs or ED_50s values, respectively) and to tridemorph (Rf: 5 or 7.1–9.5 based on MICs or ED_50s values, respectively), but not to the inhibitors of steps of ergosterol biosynthesis preceding the Δ¹⁴-reductase. The minor gene ( U/fpm -1B) mutants also had low-level resistance (approximately 5-fold) to tridemorph and to fenpropidin, but in contrast with the major gene mutants they were 2–10 times more sensitive to the triazoles studied (triadimefon, triadimenol, propiconazole and flusilazole) and to the pyridine, pyrifenox.
Studies of the fitness of U. maydis mutants showed that in major gene mutants, resistance was not associated with changes in growth rate in liquid culture or pathogenicity on young maize plants. The minor gene mutation reduced significantly the growth rate in liquid culture and the pathogenicity, either in homozygous or heterozygous condition in dikaryotic mycelium.     

Resistance to inhibitors of sterol biosynthesis in field isolates or laboratory strains of the eyespot pathogen Pseudocercosporella herpotrichoides

Strains of Pseudocercosporella herpotrichoides collected in France on winter wheat give either fast-growing mycelial colonies with regular margins or slow-growing mycelial colonies with irregular margins. Most of the fastgrowing isolates were sensitive to triadimenol (EC₅₀ below 2mg litre^?1), but some of them were resistant to this inhibitor of sterol C-14 demethylation. In contrast, all the slow-growing strains were highly resistant to triadimenol (EC₅₀ greater than 100 mg litre^?1). This resistance was also expressed in inhibition of germ-tube elongation. Positive cross-resistance was observed between most of the inhibitors of sterol C-14 demethylation, with the exception of some imidazole derivatives (clotrimazole, prochloraz). All the fast-growing strains were tolerant to fenpropimorph and fenpropidin whereas the slow-growing ones were susceptible; the reverse was true with piperalin and tridemorph. All the field isolates were inhibited to the same extent by the inhibitors of squalene-epoxidase, nafifine and terbinafine. Two types of mutant resistant to triadimenol have been induced under laboratory conditions from sensitive fast-growing strains. The most common mutants were resistant to all the inhibitors of sterol C–14 demethylation and also in some conditions to fenpropimorph, tridemorph and the inhibitors of squalene-epoxidase. The other mutants were characterised by a reduced spectrum of cross-resistance between triadimenol and the other inhibitors of sterol biosynthesis. The field isolates and laboratory mutants resistant to triadimenol and propiconazole were also resistant to each of the four enantiomers of these two fungicides.     

Resistance to fungicides which inhibit ergosterol biosynthesis in laboratory strains of Botrytis cinerea and Ustilago maydis

The strains of Botrytis cinerea or Ustilago maydis selected on fenarimol, triarimol, or triadimefon were also resistant to the other inhibitors of sterol C-14 demethylation; the sterol composition of the strains was normal. Among the isolates of U. maydis resistant to dodemorph, fenpropidin, fenpropimorph and tridemorph, some were resistant to the 15-azasteroid A 25822B and did not contain ergosterol. The other strains remained sensitive to A 25822B and had a normal sterol composition. All the resistant isolates and the wild-type were inhibited to the same extent by nystatin and pimaricin.     

Studies on the Inherent Resistance Risk to Fenhexamid in Botrytis cinerea

After chemical mutagenesis with N-methyl-N-nitrosoguanidine (MNNG) two phenotypes that were highly or moderately resistant to fenhexamid, were isolated from a wild-type strain of Botrytis cinerea, at a mutation frequency of 0.9 × 10^–5. Resistance factors, based on EC⁵⁰ values, were 460–570 and 10–15, respectively. The mutation(s) for resistance to fenhexamid did not affect the sensitivity of mutant strains to the benzimidazole benomyl, the phenylpyridinamine fluazinam, the anilinopyrimidine cyprodinil, the guanidine iminoctadine or to the sterol-biosynthesis-inhibiting fungicides fenarimol, fenpropimorph and tridemorph. On the contrary, an increased sensitivity (EC⁵⁰ ratios of 0.2–0.6) of fenhexamid-resistant strains to the phenylpyrrole fludioxonil and the dicarboximide iprodione was observed. Study of fitness parameters of fenhexamid-resistant isolates of both phenotypic classes showed that these mutation(s) had no effect on mycelial growth and sensitivity to high osmolarity, but they did affect one or more of some other characteristics, such as sporulation, conidial germination and sclerotia production. In tests on cucumber seedlings under greenhouse conditions, all highly fenhexamid-resistant isolates tested presented decreased infection ability compared with the wild-type. Preventive applications of a commercial formulation of fenhexamid, Teldor 50 WP, were effective against lesion development on cotyledons by the wild-type, but ineffective, even in high concentrations, against disease caused by the fenhexamid-resistant isolates. The risk of resistance problems arising during commercial use of fenhexamid is discussed.     

Genetics of responses to morpholine-type fungicides and of avirulences inErysiphe graminis f. sp.hordei

The genetics of the responses of the barley powdery mildew pathogen,Erysiphe graminis f.sp.hordei, to three morpholine-type fungicides were studied. Resistances to a phenylpropylamine fungicide, fenpropidin, and to a morpholine, fenpropimorph, co-segregated in crosses of a sensitive isolate, DH14, with each of two resistant ones, CC151 and CC152. In the cross CC151×DH14, the results were consistent with resistance to both fungicides being controlled by a single gene, at a locus namedFenl. In the other cross, CC152×DH14, the genetics of resistance were more complicated; the data were consistent with the segregation of two complementary, unlinked genes which each conferred resistance to both fungicides. Fenpropidin-resistant progeny of CC151×DH14 were significantly more resistant to fenpropimorph than were fenpropidin-resistant progeny of CCI 52×DH14, although the resistant progeny of the two crosses did not differ significantly in their level of fenpropidin resistance. Fenpropidin-resistant progeny of CC151×DH14 were significantly more resistant to another morpholine, tridemorph, than were fenpropidin-sensitive progeny, but this was not the case for CC152×DH14. Resistance to triadimenol, a C14 demethylation-inhibitor (DMI) fungicide, segregated in both crosses. Triadimenol resistance appeared to be controlled by one gene in each cross and was not associated with morpholine resistance. CC151×DH14 also segregated for eight avirulence genes. Two of these matched theMla6 resistance, while one gene matched a previously unknown resistance in a Pallas near-isogenic line, P17, which also carries a known resistance gene,Mlk. Fenl was not significantly linked to the triadimenol resistance gene,Tdl(a), or to any of the eight avirulence genes.Avr _a6 1, Avr _a12 ,Avr _La ,Avr _p17 andTdl(a) were linked, as wereAvr _a 10 andAvr _k .Abbreviations ED₅₀ median effective dose - Fpd fenpropidin - Fpm fenpropimorph - PCA principal components analysis - Tdm tridemorph     

Mechanism of inhibition of sterol biosynthesis enzymes by N-substituted morpholines

Tridemorph, fenpropimorph and derivatives have been tested in vitro on Δ⁸→Δ⁷-sterol isomerase (SI) from maize seedlings. The results show that these N-substituted morpholines strongly inhibit this enzyme (I₅₀: 0.4 μM for fenpropimorph, 0.6 μM for tridemorph). In fenpropimorph, where a chiral centre is present at C-2 of the alkyl chain, good enantioselectivity was observed for enzyme inhibition. Inhibition of SI and of the cycloeucalenol-obtusifoliol isomerase (COI), another enzyme of plant sterol biosynthesis, is probably due to protonation of the N-substituted morpholines, giving a positively charged nitrogen atom, at the pH (7.4) of the enzyme assays. In order to test this hypothesis, the pH dependence of the inhibition constants has been measured. Fenpropimorph, (pK_a 7.5) which can be protonated to give a morpholinium cation and N-methylfenpropimorph, the corresponding quaternary ammonium derivative, have been tested as inhibitors in a pH range from 6 to 8.5. The I₅₀ value of fenpropimorph toward the SI increased 20 times as the pH increased from 6 to 8.5. In contrast the I₅₀ value of fenpropimorph for COI did not change significantly in this pH range. While the I₅₀ value of N-methyl fenpropimorph towards COI decreased more than 50 times as the pH increased from 6 to 8.5, its I₅₀ value for SI only varied slightly in this pH range. Taken together, these results suggest the existence of an interaction of the morpholinium cations with one or several enzyme amino acid residues of a much higher pK_a in the case of COI than SI. Moreover for maize COI, the pK_a of the amino acid residue(s) interacting with these morpholines derivatives is probably close to that of these molecules.     

Impact of fludioxonil resistance on fitness and cross-resistance profiles of Altrenaria solani laboratory mutants

Sensitivity and inherent resistance risk of Alternaria solani to fludioxonil, cross-resistance profiles and the potential implications of resistance mutations on fitness parameters were investigated. Fludioxonil was highly effective against a wild type A. solani field strain both in vitro (EC₅₀?=?0.05 μg/mL) and in preventive applications on artificially inoculated tomato fruit. Mutants with low [Resistance factor (Rf): 15 based on EC₅₀], medium (Rf: 150–300) and high (Rf: > 1000) levels of phenylpyrrole resistance were isolated from the wild type strain at high frequencies following mutagenesis with UV irradiation and selection on fludioxonil containing medium. Resistant isolates retained their resistance levels even after 9 subcultures on fungicide-free growth medium while they could express their resistant phenotypes in planta. Investigation of cross-resistance relationships showed that fludioxonil resistance mutations also reduce the sensitivity of mutant strains to the aromatic hydrocarbon fungicide quintozene as well as the dicarboximides iprodione and vinclozolin. No cross-resistance was observed between fludioxonil and fungicides with different modes of action such as the sterol biosynthesis inhibitors (DMIs) imazalil and flusilazole and the carboxamide boscalid. All fludioxonil resistant isolates were more sensitive to the anilinopyrimidine pyrimethanil, while only two isolates were less sensitive to the QoI pyraclostrobin compared to the wild-type strain. Study of fitness determining parameters showed that resistance mutation(s) had no adverse effects on mycelial growth, conidial germination and sensitivity to osmotic stress while they had a pleiotropic effect on virulence and conidia production in resistant mutants. Results of the present study indicate that fludioxonil is a highly effective fungicide against A. solani, while the risk of resistance development to this fungicide is considered to be medium making fludioxonil an ideal alternative to high risk fungicides such as boscalid and pyraclostrobin whose performance against early blight has already been compromised by resistance development.

     

Effect of Phenylpyrrole-resistance Mutations on Ecological Fitness of <Emphasis Type="Italic">Botrytis cinerea</Emphasis> and their Genetical Basis in <Emphasis Type="Italic">Ustilago maydis</Emphasis>

Mutants of Botrytis cinerea and Ustilago maydis highly resistant to fludioxonil were isolated at a high frequency, after nitrosoguanidine or UV mutagenesis, respectively, and selection on media containing fludioxonil. Tests on the response of mutant strains to high osmotic pressure resulted in the identification of two fludioxonil-resistant phenotypes (FLD_osm/s and FLD_osm/r), regarding the sensitivity to high osmolarity. Approximately 95% of fludioxonil-resistant mutants were found to be more sensitive to high osmotic pressure than the wild-type parent strains. Genetic analysis of phenylpyrrole-resistance in the phytopathogenic basidiomycete U. maydis, showed that fludioxonil-resistance was coded by three unlinked chromosomal loci (U/fld-1, U/fld-2 and U/fld-3), from which only the U/fld-1 mutation coded an osmotic sensitivity similar to that of the wild-types. Cross-resistance studies with fungicides from other chemical groups showed that the mutations for resistance to phenylpyrroles affect the sensitivity of mutant strains to the aromatic hydrocarbon and dicarboximide fungicides, but not to the benzimidazoles, anilinopyrimidines, phenylpyridinamines, hydroxyanilides or the sterol biosynthesis inhibiting fungicides. A study of fitness parameters in the wild-type and fludioxonil-resistant mutants of B. cinerea, showed that all osmotic sensitive (B/FLD_osm/s) isolates had significant reductions in the characteristics determining saprophytic fitness such as mycelial growth, sporulation, conidial germination and sclerotial production. Contrary to that, with the exception of mycelial growth, the fitness parameters were unaffected or only slightly affected in most of the osmotic resistant (B/FLD_osm/r) isolates. Tests on cucumber seedlings showed that the osmotic-sensitive strains were significantly less pathogenic compared with the wild-type and B/FLD_osm/r strains of B. cinerea. Preventative applications of the commercial products Saphire 50 WP (fludioxonil) and Rovral 50 WP (iprodione) were effective against lesion development on cotyledons by the wild-type and the mutant strains of B. cinerea that were resistant to the anilinopyrimidine cyprodinil (B/CPL-27) and to the hydroxyanilide fenhexamid (B/FNH-21), but ineffective, even at high concentrations, against disease caused by the fludioxonil-resistant isolates (B/FLD) and a mutant strain resistant to the dicarboximide iprodione (B/IPR-1). Experiments on the stability of the fludioxonil-resistant phenotype showed a reduction of resistance, mainly in osmotic-sensitive isolates, when the mutants were grown on inhibitor-free medium. A rapid recovery of the high resistance was observed after mutants were returned to the selection medium. Studies on the competitive ability of mutant isolates against the wild-type parent strain of B. cinerea, by applications of a mixed conidial population, showed that, in vitro, all mutants were less competitive than the wild-type strain. However, the competitive ability of osmotic-resistant mutants was higher than the osmotic-sensitive ones. Furthermore, competition tests, in planta, showed a significant reduction of the frequency of both phenylpyrrole-resistant phenotypes, with a respective increase in the population of the wild-type strain of the pathogen.     

Resistance to pyraclostrobin, boscalid and multiple resistance to Pristine® (pyraclostrobin + boscalid) fungicide in Alternaria alternata causing alternaria late blight of pistachios in California

Pristine® (pyraclostrobin + boscalid) is a fungicide registered for the control of alternaria late blight in pistachio. A total of 95 isolates of Alternaria alternata collected from orchards with and without a prior history of Pristine® sprays were tested for their sensitivity towards pyraclostrobin, boscalid and Pristine® in conidial germination assays. The EC₅₀ values for 35 isolates from orchards without Pristine® sprays ranged from 0·09 to 3·14 µg mL^?1 and < 0·01 to 2·04 µg mL^?1 for boscalid and Pristine®, respectively. For pyraclostrobin, 27 isolates had EC₅₀ < 0·01 µg mL^?1 and six had low resistance (mean EC₅₀ value = 4·71 µg mL^?1). Only one isolate was resistant to all three fungicides tested, with EC₅₀ > 100 µg mL^?1. Among 59 isolates from the orchard with a history of Pristine® sprays, 56 were resistant to pyraclostrobin; only two were sensitive (EC₅₀ < 0·01 µg mL^?1) and one was weakly resistant (EC₅₀ = 10 µg mL^?1). For the majority of these isolates EC₅₀ values ranged from 0·06 to 4·22 µg mL^?1 for boscalid and from 0·22 to 7·74 µg mL^?1 for Pristine®. However, seven isolates resistant to pyraclostrobin were also highly resistant to boscalid and Pristine® and remained pathogenic on pistachio treated with Pristine®. Whereas strobilurin resistance is a common occurrence in Alternaria of pistachio, this is the first report of resistance to boscalid in field isolates of phytopathogenic fungi. No cross resistance between pyraclostrobin and boscalid was detected, suggesting that Pristine® resistance appears as a case of multiple resistance.     

Sensitivity of<Emphasis Type="Italic">Botrytis cinerea</Emphasis> from greenhouse vegetables to DMIs and fenhexamid

Two hundred isolates ofBotrytis cinerea were collected from greenhouse vegetables between 2003 and 2006 to determine their baseline sensitivity to triadimefone, penconazole, tebuconazole and fenhexamid. Mean values of 50% effective concentrations (EC₅₀) of inhibiting growth were 4.853±5.102, 0.41±0.215, 0.19±0.099 and 0.36±0.891 mgl ⁻¹, respectively (mean±SD). Individuals ofB. cinerea in the population differed by a factor (EC₅₀ of the least sensitive isolate/EC₅₀ of the most sensitive isolate) of 6625, 20, 603 and 1800, respectively. Naturally fenhexamid-resistant isolates were detected with an unexpected high frequency of 10% although the pathogen population had never been exposed to this fungicide. The resistance level (mean EC₅₀ of resistant isolates / mean EC₅₀ of sensitive isolates) was 19.5. These naturally resistant isolates also were resistantin vivo, and there was no significant difference in growth rate, conidial production or pathogenicity ability between naturally resistant and wild sensitive isolates. These results indicated that there was a potential risk of practical resistance if fenhexamid was applied alone. Negative cross-resistance was observed between fenhexamid and tebuconazole in 90% of the naturally resistant isolates. Moreover, an obvious synergism of the antifungal activity of fenhexamid by tebuconazole was demonstrated in some of the naturally fenhexamid-resistant isolates. http://www.phytoparasitica.org posting May 9, 2007.     

Genetic correlations in resistance to morpholine and piperidine fungicides inPyrenophora teres populations

Highly significant genetic variation (P<0.001) in resistance to the morpholine fungicides fenpropimorph, tridemorph and dodemorph and the piperidine fungicide, fenpropidin was found in different populations ofPyrenophore teres in North America and Europe which had not been previously exposed to these fungicides. Resistance phenotypes were continuously distributed for each fungicide in each population. Cross resistance relationships were determined by estimating genetic correlation coefficients in resistance to all pairwise combinations of fungicides. The majority of the correlation coefficients were highly positive for all fungicide combinations in all populations; eight of 36 (22%) coefficients were not significantly different from 1 (P>0.05). This result is consistent with the hypothesis that many of the same genes, or genes in gametic disequilibrium, control resistance to more than one fungicide in most populations ofP. teres and that these fungicides comprise a single cross resistance group. Three of 36 (8%) correlation coefficients were not significantly different from 0 (P>0.05) indicating that, in these populations, independent genes controlled resistance to these fungicides. The results of this study indicate that although most of the same genes control resistance to morpholine and piperidine fungicides inP. teres, differences in frequencies of these genes among populations can result in different cross resistance relationships from one population to another.     

白术黑斑病菌对甲基硫菌灵和腐霉利的抗性检测及对咪鲜胺敏感性基线的建立

黑斑病是影响白术种植的主要病害之一,2015—2017年从浙江省磐安、天台、新昌和嵊州4地采集、分离得到137株白术黑斑病菌Alternaria alternata,采用菌丝生长速率法检测其对甲基硫菌灵和腐霉利的抗性。结果表明:供试的黑斑病菌群体 (n = 137) 对甲基硫菌灵的高水平抗性频率为77.4%,中等水平抗性频率为22.6%,未检测到敏感菌株;对腐霉利的抗性频率为20.4%,含21株低水平抗性菌株和7株中等水平抗性菌株。7种杀菌剂的室内毒力测定结果表明:咪鲜胺对白术黑斑病菌的活性最高,EC₅₀值为0.15 mg/L。供试的137株白术黑斑病菌对咪鲜胺的EC₅₀值分布在0.05~0.87 mg/L之间,平均EC₅₀值为 (0.29 ± 0.11) mg/L,可作为敏感性基线。     

Occurrence and distribution of resistance to QoI fungicides in populations of Podosphaera fusca in south central Spain

Cucurbit powdery mildew caused by Podosphaera fusca limits crop production in Spain. Since its management is strongly dependent on chemicals, the rational design of control programmes requires a good understanding of the fungicide resistance phenomenon in field populations. Fifty single-spore isolates of P. fusca were tested for sensitivity to three quinone-outside inhibiting (QoI) fungicides: azoxystrobin, kresoxim-methyl and trifloxystrobin. Minimum inhibitory concentration (MIC) values for QoI-sensitive isolates were found to range from 0.25 to 10 μg ml⁻¹ for azoxystrobin to 5–25 μg ml⁻¹ for kresoxim-methyl, using a leaf disc-based bioassay. High levels of cross-resistance to QoI fungicides were found. Eleven isolates showed resistance to the three QoI fungicides tested with MIC and EC₅₀ values >500 μg ml⁻¹ resulting in RF values as high as >715 and >1000 for trifloxystrobin and azoxystrobin, respectively. A survey of P. fusca QoI resistance was carried out in different provinces located in the south central area of Spain during the cucurbit growing seasons in 2002, 2003 and 2004. Examination of a collection of 250 isolates for QoI resistance revealed that 32% were resistant to the three fungicides tested; the provinces of Ciudad Real, Córdoba and Murcia being the locations with the highest frequencies of resistance (44–74%). By contrast, no resistance was found in Badajoz, and relatively low frequencies were observed in Almería and Valencia (10–13%). Nearly 50% of resistant isolates were collected from melon plants. Based on these data, recommendations about the use of QoI fungicides for cucurbit powdery mildew management in the sampled areas are made.     

Sensitivity to iprodione and boscalid of Sclerotinia sclerotiorum isolates collected from rapeseed in China

Baseline sensitivity of Sclerotinia sclerotiorum, causal agent of stem rot of rapeseed, to a dicarboximide fungicide iprodione was determined using 50 isolates (historic population) collected in 2001 from the rapeseed fields without a previous history of dicarboximide usage. The 50% effective concentration (EC₅₀) values to iprodione of these wild-type isolates ranged from 0.163 to 0.734 μg/ml with a mean of 0.428 μg/ml. In 2007 and 2008, 111 isolates (current population) were collected from rapeseed fields with 4–5 years of iprodione application. The EC₅₀ values of these 111 isolates ranged from 0.117 to 0.634 μg/ml. The historic and current populations were not significantly (P > 0.05) different in sensitivity to iprodione. The EC₅₀ values of these 161 isolates to a carboxamide fungicide boscalid ranged from 0.002 to 0.391 μg/ml with a mean of 0.042 μg/ml. In the laboratory, three iprodione-resistant (IR) isolates HA17-IR, SZ31-IR, and SZ45-IR were induced from wild-type isolates HA17, SZ31, and SZ45, respectively. The EC₅₀ values of the IR isolates were 200-fold higher than those of the original wild-type parents. The IR isolates showed an increase in osmotic sensitivity. The IR isolate HA17-IR lost its ability to produce sclerotia, and showed a significantly lower virulence on rapeseed leaves than its parent isolate HA17. In contrast, the IR isolate SZ31-IR had a significantly higher virulence than its wild-type parent SZ31. PCR assays showed that the partial two-component histidine kinase (os-1) gene, which is the putative target gene of iprodione, was deleted in the low virulent IR isolate HA17-IR. DNA sequence analysis showed that each of the other two IR isolates SZ31-IR and SZ45-IR had two point mutations in their partial os-1 genes. These results indicate that the mutations in os-1 gene may be associated with dicarboximide sensitivity, sclerotial development, and virulence in S. sclerotiorum.     

番茄灰霉病菌对咯菌腈的抗性诱变及抗药突变体的生物学特性

为评估番茄灰霉病菌Botrytis cinerea对咯菌腈的抗性风险,就室内经紫外照射获得抗药突变体的方法及抗性突变体的生物学性状进行了研究。结果表明:番茄灰霉病菌分生孢子的紫外照射亚致死时间为90~120 s;经亚致死时间紫外照射后,4个亲本菌株中有2个菌株共产生了6个抗咯菌腈的突变体,其EC50值是亲本菌株的310倍以上,抗性突变频率为3.13×10-7;经紫外照射诱变获得的所有抗性突变体在菌丝生长速率、产孢量、产菌核能力及其在番茄果实上的致病性方面均比其亲本菌株明显降低。相关分析显示,所得抗咯菌腈突变体对氟啶胺、啶菌唑、啶酰菌胺和嘧霉胺无交互抗性。表明番茄灰霉病菌对咯菌腈的抗药性风险较低。     

Cross-Resistance Patterns Among Sterol Biosynthesis Inhibiting Fungicides (SBIs) in Cercospora beticola

Thirty single-spore isolates of Cercospora beticola, collected from several fields in northern Greece, representing a broad spectrum sensitivity to the sterol demethylation-inhibiting (DMIs) fungicide flutriafol, were tested for sensitivity to eleven other sterol biosynthesis-inhibiting (SBI) fungicides and to the guanidine fungicide dodine. Sensitivity was measured as EC₅₀ values for each fungicide and log-transformed EC₅₀ values to each fungicide were pairwise correlated and the correlation coefficient estimated. These pairwise comparisons showed high correlation coefficients between the DMIs suggesting a cross-resistance relationship between these fungicides. However, the degree of cross-resistance between DMIs varied greatly. Conversely, low correlation coefficients were obtained for the pair-wise comparisons with the morpholine fungicide fenpropimorph suggesting a lack of cross-resistance between morpholines and DMIs in C. beticola. Similarly, there was no correlation between the sensitivity (EC₅₀ values) to dodine and all the other fungicides tested, indicating that there was no negative cross-resistance relationship between dodine and SBIs in C. beticola. Based on these results, combinations or alternations of fungicides which show no cross-resistance relationship should be used to control the disease in areas where reduced sensitivity to DMIs has been already observed.     

Resistance to metalaxyl-M and cymoxanil in a dominant clonal lineage of <Emphasis Type="Italic">Phytophthora infestans</Emphasis> in Huánuco,Peru, an area of continuous potato production

Fifty-eight isolates of Phytophthora infestans were evaluated in vitro and on detached leaves of potato for their sensitivities to metalaxyl-M and cymoxanil. The isolates belonged to the clonal lineage, EC-1, which is dominant on potato in Peru and Ecuador. All isolates were collected in Huánuco, Peru, an area of year-round potato production, where the potential for development of fungicide resistance is high. All isolates were resistant to metalaxyl-M, with in vitro EC₅₀ values ranging from 468.30—813.57 mg l⁻¹. In contrast, we found no evidence for resistance to cymoxanil for which in vitro EC₅₀ values ranged from 0.03—1.11 mg l⁻¹. Resistance to each fungicide was also evaluated for five isolates in a detached leaf assay in which the fungicide was sprayed on the leaf surface prior to inoculation. With metalaxyl-M, the range of EC₅₀ values was 158.85—828.29 mg l⁻¹, similar to that for the in vitro assay. For cymoxanil, EC₅₀ values ranged from 1.41 to 2.31 mg l⁻¹, which was higher than in the in vitro assay but still two orders of magnitude lower than the concentration applied by farmers in the field.     

Sensitivity ofFusarium graminearum to fungicide JS399-19:In vitro determination of baseline sensitivity and the risk of developing fungicide resistance

The baseline sensitivity ofFusarium graminearum Schwade [teleomorph =Gibberella zeae (Schweinitz) Petch] to the fungicide JS399-19 (development code no.) [2-cyano-3-amino-3-phenylacrylic acetate] and the assessment of risk to JS399-19 resistancein vitro are presented. The mean EC₅₀ values for JS399-19 inhibiting mycelial growth of three populations of wild-typeF. graminearum isolates were 0.102±0.048, 0.113±0.035 and 0.110±0.036 μg ml⁻¹, respectively. Through UV irradiation and selection for resistance to the fungicide, we obtained a total of 76 resistant mutants derived from five wild-type isolates ofF. graminearum with an average frequency of 1.71 × 10⁻⁷% and 3.5%, respectively. These mutants could be divided into three categories of resistant phenotypes with low (LR), moderate (MR) and high (HR) level of resistance, determined by the EC₅₀ values of 1.5–15.0 μg ml⁻¹, 15.1–75.0 μg ml⁻¹ and more than 75.0 μg ml⁻¹, respectively. There was no positive cross-resistance between JS399-19 and fungicides belonging to other chemical classes, such as benzimidazoles, ergosterol biosynthesis inhibitors and strobilurins, suggesting that JS399-19 presumably has a new biochemical mode of action. Although the resistant mutants appeared to have comparable pathogenicity to their wild-type parental isolates, they showed decreased mycelial growth on potato-sucrose-agar plates and decreased sporulation capacity in mung bean broth. Nevertheless, most of the resistant mutants possessed fitness levels comparable to their parents and had MR or HR levels of resistance. As these studies yielded a high frequency of laboratory resistance inF. graminearum, appropriate precautions against resistance development in natural populations should be taken into account. http://www.phytoparasitica.org posting August 7, 2008.     

Occurrence and molecular characterization of azoxystrobin resistance in cucumber downy mildew in Shandong province of China

Cucumber downy mildew caused byPseudoperonospora cubensis (Berk. and Curt.) Rostov. limits crop production in Shandong Province of China. Since management of downy mildew is strongly dependent on fungicides, a rational design of control programs requires a good understanding of the fungicide resistance phenomenon in field populations of the pathogen. A total of 106 and 97 isolates ofP. cubensis were obtained in 2006 and 2007, respectively. The EC₅₀ values for the growth of all the 106 isolates collected in 2006 were 0.0063–0.0688μg ml⁻¹ (average: 0.0196±0.0048μg ml⁻¹) azoxystrobin and these were therefore considered sensitive isolates. However, 57 field isolates ofP. cubensis of the 97 collected in 2007 with EC₅₀ values that ranged from 0.609 to >51.2μg ml⁻¹ were considered resistant to azoxystrobin. Fragments of the fungicide-targeted mitochondrial cytochromeb gene from total pathogen DNA were amplified using polymerase chain reaction and their sequences analyzed to elucidate the molecular mechanism of resistance. A single point mutation (GGT to GCT) in the cytochromeb gene, resulting in substitution of glycine by alanine at position 143, was found in the three selected azoxystrobin-resistant isolates of downy mildew. This substitution in cytochromeb exhibited different resistance levels, with the resistance factor from 21.15 to greater than 2618.9. In addition, the different resistance levels seemed to appear within 1 year (between 2006 and 2007). Therefore, growers of Shandong Province in China now are faced with a challenge in managing the azoxystrobin resistance in cucumber downy mildew. http://www.phytoparasitica.org posting March 10, 2008.