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.     

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.     

Sensitivity of Sclerotinia sclerotiorum to fludioxonil: In vitro determination of baseline sensitivity and resistance risk

This work determined the sensitivity of field populations of Sclerotinia sclerotiorum (Lib.) de Bary before exposure to the fungicide fludioxonil (= baseline sensitivity) and assessed the risk of fludioxonil resistance. The mean EC₅₀ (Effective Concentration) and Minimum inhibitory concentration (MIC) values for fludioxonil based on inhibition of mycelial growth of 120 wild-type isolates were 0.015 ± 0.005 μg/ml and <0.05 μg/ml, respectively. Positive cross-resistance was not detected between fludioxonil and benzimidazole fungicides but was detected between fludioxonil and dicarboximide fungicides which are considered as high resistance risk fungicides by FRAC, even though these fungicides have different molecular structures. By growing wild-type isolates on potato dextrose agar (PDA) containing sublethal concentrations of the fungicide, we obtained four fludioxonil-resistant mutants with resistance factors (EC₅₀ resistant/EC₅₀ sensitive phenotypes) >2000. The laboratory fludioxonil mutants were less fitter than their parental isolates in terms of mycelial radial growth, pathogenicity and sclerotial production. Moreover, on PDA amended with NaCl, the laboratory fludioxonil mutants grew more slowly than their fludioxonil-sensitive parents, especially at lower concentrations of NaCl. According to the fitness of mutants and the cross-resistance between fludioxonil and dicarboximide fungicides, phenylpyrroles can be considered to pose a moderate resistance risk. In a field trial, fludioxonil provided greater control (over 90% disease control) of S. sclerotiorum than iprodione.     

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。     

Potassium phosphite combined with reduced doses of fungicides provides efficient protection against potato late blight in large-scale field trials

Potato late blight caused by the oomycete Phytophthora infestans is a devastating disease of potato worldwide. Most of the potato cultivars grown in conventional agriculture are susceptible, or at best moderately resistant, and require frequent applications of fungicides to avoid heavy yield losses.In field trials spanning four years, we have investigated the effect of potassium phosphite, an inorganic salt on potato late blight. Potassium phosphite is known to induce defence responses in potato and to also have direct toxic effects on oomycetes, which in turn counteract late blight and tuber blight development. However, the use of this salt is not yet implemented and approved in European potato cultivation. We compared the effect of phosphite alone with fungicides currently used in Swedish potato cultivation. We also investigated the combined use of potassium phosphite and reduced doses of fungicides. Table potato cultivars and starch potato cultivars with different levels of resistance were used.We found that potassium phosphite in combination with reduced doses of fungicides results in the same level of protection as treatments with the recommended full dose of fungicides. These combined treatments reduce the need of traditional fungicides and may also decrease the selection pressure for fungicide resistance development in the pathogen. In relatively resistant starch potato cultivars using phosphite alone gave sufficient protection against late blight. Furthermore, in starch potato a combination of phosphite and fungicides at two-week intervals provided similar protection to weekly applications of fungicide at the recommended dose. Foliar treatment with phosphite also gave protection against tuber blight at similar levels to that of the best-performing fungicide. Our data suggests that potassium phosphite could be used in potato cultivation in temperate regions such as in Sweden, at least in combinations with reduced rates of fungicides. The implementation of the use of phosphite in practical potato crop protection as part of an IPM strategy is discussed. Doses, intervals and combinations could be adjusted to the level of cultivar resistance.     

Efficacy of fosetyl-Al,propamocarb, dimethomorph,cymoxanil, metalaxyl and metalaxyl-M in Czech Pseudoperonospora cubensis populations during the years 2005 through 2010

A total of 134 Czech Pseudoperonospora cubensis isolates originating from Cucumis sativus were used for fungicide resistance screening. Efficacy of six commonly used and registered fungicides was screened in Czech P. cubensis populations. The investigations covered the frequency and occurrence of sensitive, moderately resistant and resistant isolates to the individual fungicides during the period 2005–2010. Fosetyl-Al (Aliette 80 W) and propamocarb (Previcur 607 SL) were the most effective fungicides. All tested isolates were sensitive on all tested fosetyl-Al concentrations. However, some isolates expressed resistance (profuse sporulation) or moderate resistance (limited sporulation) to lower and/or even to recommended concentrations of propamocarb in the years 2006 and 2008–2010. Metalaxyl (Ridomil PLUS 48 WP) and metalaxyl-M (Ridomil Gold MZ 68 WP) were ineffective. Isolates collected in 2008 and 2009 showed large variation in moderate resistance or resistance even at high fungicide dosages. However, this result was not confirmed in 2010, when efficacy of these fungicides increased slightly, and 69% and 43% of isolates were controlled at the recommended concentration of metalaxyl-M and metalaxyl, respectively. Limited or profuse sporulation was observed only sporadically at high concentrations. Sensitivity of isolates to cymoxanil (Curzate K) differed among the studied years. While cymoxanil was ineffective in the years 2005–2008 and in 2010, 68% of isolates were controlled at the recommended concentration in 2009. During the years 2005 through 2010, a shift towards higher sensitivity to dimethomorph was observed at all screened concentrations.     

6种杀菌剂对马铃薯晚疫病菌的室内毒力测定

采用菌落直径法测定了金雷多米尔、杀毒矾、克露、克霜氰、大生M-45和进富6种杀菌剂对马铃薯晚疫病菌的毒力。结果表明供试的6种杀菌剂对马铃薯晚疫病菌丝生长都有较好的抑制作用,克露对晚疫病菌毒力最强,其EC50值为0.16μg·mL-1,毒力最差的是大生,其EC50值为2.89μg·mL-1,其余4种药剂按毒力大小依次为克霜氰、金雷多米尔、进富和杀毒矾,其EC50值分别为0.24μg.mL-1、0.28μg·mL-1、0.43μg·mL-1和1.17μg·mL-1。根据室内毒力测定结果及药剂特性,供试的6种杀菌剂在生产上可视病害的发生情况交替使用,以避免或延缓抗药性的产生。     

A rapid method to monitor fungicide sensitivity in the pecan scab pathogen, Fusicladium effusum

Commercial production of pecans in the southeastern U.S. relies on fungicide applications to control scab, caused by Fusicladium effusum. Under intense disease pressure, 10–15 applications may be made per season and the potential development of fungicide resistance is a major concern. A rapid method was developed to determine sensitivity of the pathogen to protectant chemicals based on conidia germination, and to fungicides active on post-appressorial pathogen stages based on micro-colony growth. This method uses conidia transferred directly from lesions to fungicide-amended media. Using this method, sensitivity profiles were determined for isolates from three non-treated orchards with little or no fungicide use histories and from 33 commercial orchards in 2008 with a history of fungicide use. Compared to the non-treated orchards, significant reductions in in vitro sensitivity were detected in 20 orchards to fentin hydroxide, in 4 orchards to dodine, in 6 orchards to thiophanate-methyl and in 20 orchards to propiconazole. The novel methodology will be useful for monitoring fungicide sensitivity of F. effusum populations and evaluating resistance management programs in commercial pecan orchards.     

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.     

Sensitivity to iprodione, difenoconazole and fludioxonil of Rhizoctonia cerealis isolates collected from wheat in China

Since the 1980s wheat sharp eyespot (WSE) caused mainly by Rhizoctonia cerealis has become one of serious diseases of wheat in China. In this study, the sensitivity of 89 R. cerealis isolates to different fungicides was evaluated using mycelial growth inhibition assays. The results showed that all R. cerealis isolates tested were sensitive to iprodione, difenoconazole and fludioxonil with mean EC₅₀ (effective concentration that results in 50% of mycelial growth inhibition) values of 0.419, 0.062 and 0.033 μg/ml, respectively. To evaluate the risk of fungicide resistance in R. cerealis, an attempt was made to induce resistant mutants in the laboratory. Although difenoconazole- and fludioxonil-resistant mutants were not obtained, we obtained seven independently iprodione-resistant (IR) mutants from 89 parental isolates. The EC₅₀ values for these IR mutants were greater than 100 μg/ml, whereas those for the original wild-type counterparts were less than 1 μg/ml. After having been subcultured on PDA for 10 generations, the IR mutants did not show any decrease in resistance to iprodione. Additionally, these IR mutants also showed resistance to fludioxonil but remained sensitive to difenoconazole. Osmotic sensitivity tests showed that the IR mutants were hypersensitive to osmotic stress generated by NaCl. Inoculation tests showed that all the IR mutants lost their ability to infect the host plant. Taken together, these results indicate that the current population of R. cerealis is sensitive to these fungicides and a fitness cost is associated with iprodione-resistant mutants of R. cerealis in both osmotic stress and pathogenicity. The information obtained in this study is useful in monitoring and managing fungicide resistance in R. cerealis populations in China.     

Sensitivity,resistance stability,and cross-resistance of Plasmopara viticola to four different fungicides

Baseline sensitivity to fungicides was determined in 105 Plasmopara viticola isolates using the floating leaf disk test. The mean EC₅₀ values were 0.372 ± 0.104, 0.604 ± 0.215, 0.306 ± 0.101, and 0.922 ± 0.209 μg mL⁻¹ for fluxapyroxad, benthiavalicarb-isopropyl, ametoctradin, and famoxadone, respectively, which we regarded as the baseline sensitivity to the four fungicides. Resistant mutants can be generated using a median effective concentration or a minimum inhibitory concentration to fluxapyroxad, benthiavalicarb-isopropyl, ametoctradin, and famoxadone respectively, but the resistance stability of resistant mutants from the minimum inhibitory concentration (MIC) was significantly lower than that from the median effective concentration (EC₅₀). In brief, high resistant mutants obtained was at the higher concentration, the resistance stability of the mutants recovered was more quickly. But lower resistant mutants obtained was at the lower concentration, the resistance stability of the mutants recovered was slower. Even if the resistance level of the mutants is low, it is more capable of stably heritable. These data indicate that the highest dosage is not used, resistance will still develop. In a correlation analysis, no cross-resistance to each other of these four fungicides was observed.     

不同杀菌剂对马铃薯晚疫病的防治效果

通过田间试验调查,研究了7种化学药剂对马铃薯植株和薯块晚疫病的防治效果。结果表明:7种药剂对马铃薯晚疫病均可达到显著防治效果。防治效果最好的4种药剂为25%瑞凡、50%福帅得、0.5%苦参碱和30%甲霜.嘧菌酯,防治效果均达到80%以上;其次为45%三苯乙酸锡和68.75%银法利,防治效果达到70%以上;较差为52.5%抑快净,防治效果达到65%。建议在生产上交替使用,避免产生抗药性。     

Genetic variability in the fungi and the problem of fungicide resistance

The way in which fungal populations respond to fungicides used for their control, and hence the risk of disease control failures due to resistance, depends primarily on the type of genetic variability available. Existing evidence indicates that the type of variability differs with the chemical rather than with the organism. With most systemic fungicides and antifungal antibiotics, high resistance can be obtained by mutation of a major gene. The population response is then qualitative and a high or moderate resistance risk is involved, depending on the effect of the major gene on fitness. Mathematical models are available, on the basis of which the establishment of resistance and the loss of effectiveness of a fungicide of this group can be predicted. With a few systemics and some protectants, a practically significant decrease of sensitivity requires the interaction of several genes, each of a rather minor effect: the response is quantitative and the resistance risk is low th moderate. In this situation, the combination of degree of resistance and fitness loss varies greatly and this has not permitted the construction of suitable mathematical models so far. Finally, there is no evidence that pathogenic fungi possess genes for resistance to most protectant fungicides, which involve a low resistance risk, if any. Selection of forms less sensitive to fungicides of this group does not seem to have taken place, in spite of many years of repeated use.     

Independent action of fungicides and its consequences for strategies to retard the evolution of fungicide resistance

Several authors have discussed the evolution of fungicide resistance in a pathogenic fungus which contains a distinct resistant genotype to one fungicide (A), but not to another (B). Each author predicts when alternating use of the two fungicides will retard the evolution of resistance to A more than use of a mixture, but their conclusions differ. This is partly because they use in different ways the concept that the fungicides act independently in the mixture. A parameter ψ is introduced that quantifies the extent to which component B modifies the evolution of resistance to A. If admixture of B has no effect, ψ = 1 and A and B are independent with respect to the development of resistance. ψ is defined as the ratio of the change in frequency of the resistant genotype when a mixture of A and B is applied for a given time, to the change when A is applied alone. It is useful for interpreting the results of experiments and the predictions of mathematical models. Small values of ψ indicate mixtures that greatly retard the evolution of resistance. These are more likely to occur if fungicides, with the other properties assumed, act at the same stage of the life cycle of the fungus. Values <0·5 indicate that mixtures will be more favourable than alternation in the absence of cross resistance.     

Influence of fungicide spray schedules on the sensitivity of Cercospora beticola to the sterol demethylation-inhibiting fungicide flutriafol

A field experiment was conducted over a three-year period (1997–1999) to study the efficacy of several fungicide spray-schedules and their effects on the sensitivity of Cercospora beticola populations to the DMI fungicide flutriafol. Spray applications of flutriafol, either alone at the recommended dose, or at a reduced dose in mixtures with maneb, or in alternation with tank mixed fentin-acetate and maneb, were included in the spray programs. Applications of flutriafol at the recommended dose showed a significantly greater control efficacy in comparison with the other treatments, while applications of flutriafol alternated with tank mixed fentin-acetate and maneb showed lower efficacy in comparison with the remaining flutriafol treatments. Fungal populations from plots continuously treated with flutriafol, either alone at the full dose or at reduced dose with maneb, had lower sensitivity to flutriafol in comparison with populations from plots treated alternatively with flutriafol and tank mixed fentin-acetate and maneb. Repeated applications of flutriafol, at full or reduced doses, favoured the selection of highly resistant strains. Since applications of flutriafol in alternation with tank mixed fentin-acetate and maneb do not maintain a high level of disease control, the only available anti-resistance strategy would be the restriction of the number of flutriafol treatments, by applying them only when environmental conditions are favourable for disease development and by using alternative fungicides during the rest of the growing season.     

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.     

Synergy of cymoxanil and mancozeb when used to control potato late blight

Summary Synergism between fungicides could be used to reduce the application dosage. Synergism between cymoxanil and mancozeb was studied using potted potato plants under field conditions. In three experiments the preventive efficacy of the fungicides and fungicide mixtures in the treatments were studied using a bio-assay. Potato plants were sprayed with the fungicides or fungicide mixtures. Leaves were picked from the potato plants with regular intervals after fungicide application. The efficacy of the fungicides was studied in a bioassay by inoculating the leaves withPhytophthora infestans zoosporangia. The Abbot method was used to show synergistic effects of the cymoxanil/mancozeb mixtures tested. In two of three experiments synergy of the fungicide mixtures was found.     

Penthiopyrad applied in close proximity to Rhizoctonia solani provided effective disease control in sugar beet

Rhizoctonia solani Kühn is an important pathogen of sugar beet (Beta vulgaris L.) that can cause damping-off and crown and root rot. Commercial cultivars which are highly resistant to the pathogen are not as high yielding as susceptible cultivars under low or absent disease pressure. These resistant cultivars often do not have resistance to other common pathogens such as Aphanomyces cochlioides, Cercospora beticola, and Fusarium oxysporum. Fungicides, such as azoxystrobin which belongs to the quinone outside inhibitors (QoI) class, are necessary for controlling Rhizoctonia solani, but there are concerns about the buildup of fungicide-resistant strains in the targeted pathogen population. There is a need to find effective fungicides from different chemical groups so they can be rotated with the current widely-used azoxystrobin to manage R. solani. The objective of this greenhouse study was to evaluate the efficacy of penthiopyrad, a succinate dehydrogenase inhibitor (SDHI), in managing R. solani on sugar beet using three different application methodologies. Penthiopyrad effectively controlled R. solani on sugar beet when applied at 210, 280, 420, or 550 g a.i./ha in-furrow at planting and as a soil drench at the 4-leaf stage. However, foliar application of penthiopyrad failed to provide disease control. These trials indicated that penthiopyrad needs to be in close proximity or direct contact with R. solani in the soil to provide effective control. Penthiopyrad has the potential to be used as an effective alternate partner with azoxystrobin for controlling R. solani and to help in mitigating the development of fungicide resistant isolates of R. solani.     

Multiple-fungicide resistance to carbendazim,diethofencarb, procymidone,and pyrimethanil in field isolates of Botrytis cinerea from tomato in Henan Province,China

Botrytis cinerea, the fungal pathogen responsible for causing gray mold in tomatoes, frequently exhibits multiple fungicide resistance. In this study, the sensitivity to carbendazim (Car), diethofencarb (Die), procymidone (Prc), and pyrimethanil (Pyr) was determined for 263 isolates of B. cinerea from diseased fruit and leaves of tomato plants from 87 commercial greenhouses in Henan Province of China during 2013 and 2014. Of the isolates tested, 89%, 93%, 85%, and 86% were resistant to Car, Die, Prc, and Pyr, respectively. Twelve phenotypes were found having different sensitivity patterns, and multi-fungicide resistance was found to one, two, three or four fungicides. The percentage of multiple fungicide resistant isolates (Car^RDie^RPrc^RPyr^R) was 68%. Mycelial growth, spore production, and mycelial dry weight were not significantly different between resistant and sensitive phenotypes of the B. cinerea isolates. However, the virulence of Car^SDie^RPrc^SPyr^S isolates was higher than that of the other fungicide-resistance phenotypes. All of the carbendazim and diethofencarb resistant phenotypes carried a point mutation at codon 198 (E198A or E198K) or 200 (F200Y) in the β-tubulin gene, and all of the procymidone resistant phenotypes carried point mutations at codons 369 (Q369P) and 373 (N373S) in the Bos1 gene. These results provided important reference data for assessment of resistance risk of B. cinerea isolates from commercial tomato transplants in Henan Province, indicating the existence of a high frequency of resistance to carbendazim, diethofencarb, procymidone, and pyrimethanil revealing the necessity of a more integrated control of gray mold.     

A comparison of the biochemical and physiological effects of the systemic fungicide cyprofuram with those of the related compounds metalaxyl and metolachlor

Strains of Phytophthora megasperma insensitive to the fungicide metalaxyl showed limited cross-resistance to the structurally related fungicide cyprofuram and to metolachlor, a herbicide with antifungal properties. The three compounds each inhibited nucleic acid synthesis, but the wide variation in resistance factors and in the degree of nucleic acid inhibition at the concentrations inhibiting mycelial growth by 50% indicated that cyprofuram and metolachlor possess alternative mechanisms of toxicity. Studies with protoplasts suggested that cyprofuram has a direct effect on the plasma membrane which may be important against those resistant strains where nucleic acid synthesis is least affected. Metolachlor also was extremely effective in causing protoplast lysis. Sporangia production by P. palmivora was stimulated by low concentrations of cyprofuram, and germination was delayed. As with other ‘acylalanine-type’ fungicides, inhibition of nucleic acid synthesis is considered to be the main mode of action. Some degree of cross-resistance between cyprofuram and other acylalanines is likely to occur in the field but the dual mode of action of cyprofuram might delay its onset. The effect of increased sporogenesis on the development of resistance is more difficult to assess.