Variation of Fungicide Resistance in Czech Populations of Pseudoperonospora cubensis

During the growing seasons between the years 2001 and 2004, 98 isolates of Pseudoperonospora cubensis from nine regions of Czech Republic were collected and screened for tolerance/resistance to the three frequently used fungicides (propamocarb, fosetyl‐Al, metalaxyl). Fungicides were tested in five different concentrations, using a floating disc bioassay. Fungicide effectiveness varied considerably. Propamocarb appeared most effective and all the isolates collected in the years 2001–2003 were found sensitive to all tested concentrations [607–9712 μg active ingredient (a.i.)/ml]. In 2004, some strains with increased resistance to propamocarb were detected. These strains were characterized by tolerance at the lowest concentrations (607 μg a.i./ml, eventually on 1214 μg a.i./ml); however, they were controlled by 2428 μg a.i./ml. Fosetyl‐Al was effective at the recommended concentration of 1600 μg a.i./ml against all isolates. However, the occurrence of isolates (collected in 2001) which sporulated at low concentrations (400 and 800 μg a.i./ml) indicated that the selection for tolerance occurs in the pathogen population. Nevertheless, this phenomenon was not confirmed with the P. cubensis isolates collected between the years 2002 and 2004. Metalaxyl was found ineffective, because 97% of the isolates showed the resistance to the recommended concentration (200 μg a.i./ml), and the other 3% of isolates expressed tolerant response. The majority of the isolates showed profuse and/or limited sporulation at higher concentrations (400 and 800 μg a.i./ml). A substantial shift to highly metalaxyl resistant strains was evident in the Czech P. cubensis populations during 2001–2004.     

Effect of combination between inducer resistance chemicals and systemic fungicides on management of sweet melon downy mildew

The efficiency of some inducer resistance chemicals (IRCs) like bion, chitosan, humic acid and salicylic acid as well as the fungicides like Folu-Gold, Galben Copper, Previcure-N and Redomil Gold Mancozeb on management of sweet melon downy mildew, caused by Pseudoperonospora cubensis was evaluated in vitro and in vivo. Also, the efficiency of the alternation between the sprayed two fungicides and IRCs on management of the disease and the produced fruit yield and its total soluble solids (TSS) under field conditions were assessed. The inhibitory effect of the IRCs and the tested fungicides on sporangial germination of P. cubensis resulted in a significant reduction in the germinated sporangia. In addition, IRCs were less effective than the fungicides. Disease management revealed the same trend of the in vitro experiment when they sprayed fungicides on sweet melon plants artificially inoculated with the sporangia of the causal fungus under greenhouse conditions. Furthermore, under field conditions, spraying sweet melon plants with the two tested fungicides was the most efficient in decreasing the disease and increasing fruit yield and its TSS, to somewhat, followed by the alternation between them and the tested IRCs. In addition, IRCs treatments showed the lowest efficiency in this regard.     

Double resistance by citrus green mould Penicillium digitatum to the fungicides guazatine and benomyl

In vitro dosage response data with different isolates of Penicillium digitatum and the fungicide guazatine indicated an approximate 10-fold shift in tolerance when compared with wild type strains. ED₅₀ values for resistant strains were approximately 0.5 μg/ml compared to approximately 0.05, μg/ml for the wild type strains. Colony growth of guazatine resistant isolates on selective media containing carbendazim showed that they were also resistant to the benzimidazole group of fungicides. In vivo tests in inoculated oranges with strains previously characterised by in vitro tests confirmed resistance to guazatine and benomyl. A combined treatment of these fungicides at 400 /μ/ml and 500 μg/ml respectively, which normally gives protection against decay, also failed to provide adequate mould control. Growth and pathogenicity of the resistant strains in these tests in oranges were indistinguishable from that of wild type strains.     

The rising threat of fungicide resistance in plant pathogenic fungi: Botrytis as a case study

The introduction of site-specific fungicides almost 50 years ago has revolutionized chemical plant protection, providing highly efficient, low toxicity compounds for control of fungal diseases. However, it was soon discovered that plant pathogenic fungi can adapt to fungicide treatments by mutations leading to resistance and loss of fungicide efficacy. The grey mould fungus Botrytis cinerea, a major cause of pre- and post-harvest losses in fruit and vegetable production, is notorious as a ‘high risk’ organism for rapid resistance development. In this review, the mechanisms and the history of fungicide resistance in Botrytis are outlined. The introduction of new fungicide classes for grey mould control was always followed by the appearance of resistance in field populations. In addition to target site resistance, B. cinerea has also developed a resistance mechanism based on drug efflux transport. Excessive spraying programmes have resulted in the selection of multiresistant strains in several countries, in particular in strawberry fields. The rapid erosion of fungicide activity against these strains represents a major challenge for the future of fungicides against Botrytis. To maintain adequate protection of intensive cultures against grey mould, strict implementation of resistance management measures are required as well as alternative strategies with non-chemical products.     

Imazalil resistantPenicillium isolates from Spanish apple packinghouses

Imazalil tolerant isolates ofPenicillium spp. were recovered from sampling natural spore populations in storage rooms and apples collected from packinghouses in Lleida (Spain). ThePenicillium resistant strains belong to the speciesP. cyaneofulvum, P. variabile, P. rugulosum, P. minioluteum andP. pinophilum. 85% of the tested strains were resistant to imazalil in final concentrations of imazalil ranging from 4,500 µg/ml to 11,000 µg/ml. The resistance of these moulds to this fungicide was constant during successive subcultures. 89% ofPenicillium studied strains produced decay in the determination of parasitic fitness at 10 days.     

Azole-Resistant Invasive Aspergillosis: Relationship to Agriculture

Azole resistance in Aspergillus fumigatus has been increasingly reported particularly over the last decade. Two routes of acquisition are described: selection of resistance during long term azole therapy in the clinical setting, and primary acquisition of resistant isolates from the environment due to the considerable use of azole fungicides in agriculture and for material preservation. Three specific resistance genotypes have been found in azole naïve patients. Two of these have also been found in the environment and are characterized by a tandem repeat in the promoter region of the target gene coupled with point mutation(s) in CYP51A (TR₃₄/L98H and TR₄₆/Y121F/T289A). In the third a single target enzyme alteration (G432S) is found. These resistant “environmental” strains have been detected in many West-European countries as well as in the Asia-Pacifics. Noticeably, these two continents account for the highest fungicide use in the global perspective (37 % and 24 %, respectively). Among the 25 azole fungicides, five have been associated with the potential to select for the TR₃₄/L98H genotype; three of these are among those most frequently used. Although the number of antifungal fungicide compounds and classes available is impressive compared to the armamentarium in human medicine, azoles will remain the most important group in agriculture due to superior field performance and significant resistance in fungal pathogens to other compounds. Hence, further spread of environmental resistant Aspergillus genotypes may occur and will depend on the fitness of each resistant phenotype and the pattern of azole fungicide use.     

Activity of Ten Fungicides against Phytophthora capsici Isolates Resistant to Metalaxyl

Pepper Phytophthora blight (PPB), caused by Phytophthora capsici, is an important disease of pepper in China. The extensive application of metalaxyl has resulted in widespread resistance to this fungicide in field. This study has evaluated the activities of several fungicides against the mycelial growth and sporangium germination of metalaxyl‐sensitive and metalaxyl‐resistant P. capsici isolates by determination of EC₅₀ values. The results showed that the novel carboxylic acid amide (CAA) fungicide mandipropamid exhibited excellent inhibitory activity against PPB both in vitro and in vivo, with averagely EC₅₀ values of 0.075 and 0.004 μg/ml in mycelial growth and sporangium germination, respectively, and over 88% efficacy in controlling PPB. The other three CAA fungicides also provided over 70% efficacy in controlling PPB. The mycelial growth was less sensitive to quinone outside inhibitor (QoI) fungicides azoxystrobin and trifloxystrobin than that of sporangium germination in P. capsici isolates. However, azoxystrobin and trifloxystrobin provided over 80% efficacy in controlling PPB. It was noted that propamocarb and cymoxanil did not exhibit activity against the mycelial growth or sporangium germination of P. capsici isolates in the in vitro tests, with over 70% efficacy in controlling PPB. The new fungicide mixture 62.5 g/l fluopicolide + 625 g/l propamocarb (trade name infinito, 687.5 g/l suspension concentrate (SC)) produced over 88% efficacy in controlling PPB caused by both metalaxyl‐sensitive and metalaxyl‐resistant isolates. The data of this study also proved that there was obviously no cross‐resistance between metalaxyl and the other tested fungicides. Therefore, these fungicides should be good alternatives to metalaxyl for the control of PPB and management of metalaxyl resistance.     

Inhibition of efflux transporter-mediated fungicide resistance in Pyrenophora tritici-repentis by a derivative of 4'-hydroxyflavone and enhancement of fungicide activity

Populations of the causal agent of wheat tan spot, Pyrenophora tritici-repentis, that are collected from fields frequently treated with reduced fungicide concentrations have reduced sensitivity to strobilurin fungicides and azole fungicides (C14-demethylase inhibitors). Energy-dependent efflux transporter activity can be induced under field conditions and after in vitro application of sublethal amounts of fungicides. Efflux transporters can mediate cross-resistance to a number of fungicides that belong to different chemical classes and have different modes of action. Resistant isolates can grow on substrata amended with fungicides and can infect plants treated with fungicides at levels above recommended field concentrations. We identified the hydroxyflavone derivative 2-(4-ethoxy-phenyl)-chromen-4-one as a potent inhibitor of energy-dependent fungicide efflux transporters in P. tritici-repentis. Application of this compound in combination with fungicides shifted fungicide-resistant P. tritici-repentis isolates back to normal sensitivity levels and prevented infection of wheat leaves. These results highlight the role of energy-dependent efflux transporters in fungicide resistance and could enable a novel disease management strategy based on the inhibition of fungicide efflux to be developed.     

Studies on the tolerance of Erysiphe graminis f. sp. hordei to systemic fungicides

Successive sowings of glasshouse-grown barley plants were treated with either tridemorph, ethirimol or both fungicides, and inoculated with an initially fungicide-sensitive isolate of Erysiphe graminis f. sp. hordei. The time taken for symptoms to appear, compared with that for untreated plants, decreased with successive sowings. This was interpreted as evidence for the increase in the frequency of fungicide-tolerant propagules in the pathogen population. Effective mildew control was obtained by the use of either or both fungicides in trial plot and field crops. Seedling assays for tolerance to tridemorph and ethirimol showed that tolerance was more evident in treated than in untreated crops in June. Some mildew populations partially tolerant to one fungicide also showed reduced sensitivity to the other. By July a response intermediate between tolerant and sensitive was recorded on all the plots, probably indicating the mixing of fungal populations from adjacent plots. Larger numbers of tolerant isolates were found in random samples from treated than from untreated crops.     

The potential for enhanced fungicide resistance in Beauveria bassiana through strain discovery and artificial selection

Our objectives were to determine the (1) natural variation in fungicide resistance among Beauveria bassiana strains, (2) potential to increase fungicide resistance in B. bassiana through artificial selection, and (3) stability of virulence in selected B. bassiana strains. Fungicides included dodine, fenbuconazole, and triphenyltin hydroxide, which are commonly used in pecan and other horticultural crops. Comparison of seven B. bassiana strains indicated some are substantially more resistant to fungicides than others; a commercial strain (GHA) was less resistant than all wild strains isolated from pecan orchards. Artificial selection resulted in enhanced fungicide resistance in the GHA strain but not in a mixed wild strain. Removal of selection pressure for three passages did not reduce the enhanced fungicide resistance. Sub-culturing with exposure to fungicides did not affect the GHA strain's virulence to pecan weevil, Curculio caryae, larvae, whereas fungicide exposure increased virulence in a mixed wild population of B. bassiana.     

Strategies for control of eyespot (Pseudocercosporella herpotrichoides) in UK winter wheat and winter barley

This review considers strategies for control of eyespot (Pseudocercosporella herpotrichoides) in winter wheat and winter barley in the UK. Varietal resistance to eyespot may take the form of direct resistance to the growth of the pathogen in the stem base or of indirect tolerance to eyespot through resistance to lodging. The French variety Cappelle-Desprez has been a source of resistance for most UK wheat varieties and a new source of resistance in wild goat grass has been discovered recently. Use of fungicides for control of eyespot increased rapidly after the introduction of the highly effective MBC fungicides in the 1970s, but has decreased recently because UK populations of P. herpotrichoides are now predominantly resistant to MBC fungicides and alternative fungicides are more expensive. Historically, cultural methods, especially crop rotation, have been important in the control of eyespot. The importance of cultural control of eyespot is now increasing because more non-cereal break crops are being grown and fungicide use is declining.     

Possible explanations of synergism in fungicide mixtures against Phytophthom infestans

Single foliar sprays of oxadixyl, mancozeb or cymoxanil alone or the mixtures oxadixyl/mancozeb and oxadixyl/cymoxanil were applied to tomato plants (cv. Baby) one day before inoculating with a sensitive or a phenylamide-resistant strain of Phytophthora infestans. Fungicide mixtures were far more effective in controlling both sensitive and resistant strains compared to the individual components alone, representing significant levels of synergistic interactions. When analysed by the Abbott method, synergy ratios continuously decreased with increasing amounts of fungicides in the mixture. All fungicide mixtures showed higher synergy ratios against resistant strains when using the Abbott method, whereas only mixtures containing cymoxanil showed this phenomenon when using the Wadley method for interpretation of interactions. Synergism significantly decreased when the components were used as foliar split applications with intervals of 48 and 72 h. A combination of a drench application of oxadixyl and a single spray application of mancozeb or cymoxanil resulted in a high level of synergism. The explanation of synergism may be that the exposure of sporangia to sublethal concentrations of one fungicide affects them to an extent that sublethal doses of the other fungicide becomes detrimental.     

灰霉病菌抗药位点及其分子检测方法研究进展

灰霉病由灰葡萄孢侵染所致,化学防治是目前最常用的治理方法,而随着杀菌剂的广泛使用,抗药菌株频繁出现。本文就近年来已研究报道的灰葡萄孢菌的抗药分子位点进行了系统总结,包括六大类杀菌剂,涉及5个基因;对灰霉病菌抗药位点的分子检测方法进行了综述,包括测序法、CAPS、ARMS、Tetra primer ARMS-PCR、AS real-time PCR、ASPPAA PCR和双杂交探针法,通过对不同检测方法进行比较分析,指出现有技术存在的问题并展望未来高通量的检测方法的发展方向。     

Fungicide efflux and the MgMFS1 transporter contribute to the multidrug resistance phenotype in Zymoseptoria tritici field isolates

Septoria leaf blotch is mainly controlled by fungicides. Zymoseptoria tritici, which is responsible for this disease, displays strong adaptive capacity to fungicide challenge. It developed resistance to most fungicides due to target site modifications. Recently, isolated strains showed cross‐resistance to fungicides with unrelated modes of action, suggesting a resistance mechanism known as multidrug resistance (MDR). We show enhanced prochloraz efflux, sensitive to the modulators amitryptiline and chlorpromazine, for two Z. tritici strains, displaying an MDR phenotype in addition to the genotypes CYP51^{I381V Y461H} or CYP51^{I381V ΔY459/G460}, respectively, hereafter named MDR6 and MDR7. Efflux was also inhibited by verapamil in the MDR7 strain. RNA sequencing lead to the identification of several transporter genes overexpressed in both MDR strains. The expression of the MgMFS1 gene was the strongest and constitutively high in MDR field strains. Its inactivation in the MDR6 strain abolished resistance to fungicides with different modes of action supporting its involvement in MDR in Z. tritici. A 519 bp insert in the MgMFS1 promoter was detected in half of the tested MDR field strains, but absent from sensitive field strains, suggesting that the insert is correlated with the observed MDR phenotype. Besides MgMfs1, other transporters and mutations may be involved in MDR in Z. tritici.     

Risk assessment studies on succinate dehydrogenase inhibitors, the new weapons in the battle to control Septoria leaf blotch in wheat

Chemical control of Septoria leaf blotch, caused by Mycosphaerella graminicola, is essential to ensure wheat yield and food security in most European countries. Mycosphaerella graminicola has developed resistance to several classes of fungicide and, with the efficacy of azoles gradually declining over time, new modes of action and/or improvements in host varietal resistance are urgently needed to ensure future sustainable disease control. Several new‐generation carboxamide fungicides with broad‐spectrum activity have recently been introduced into the cereal market. Carboxamides inhibit succinate dehydrogenase (Sdh) of the mitochondrial respiratory chain (complex II) but, because of their single‐site specificity, these fungicides may be prone to resistance development. The objective of this study was to assess the risk of resistance development to different Sdh inhibitor (SDHI) fungicides in M. graminicola. UV mutagenesis was conducted to obtain a library of carboxin‐resistant mutants. A range of SDHI resistance‐conferring mutations was found in Sdh subunits B, C and D. Pathogenicity studies with a range of Sdh variants did not detect any fitness costs associated with these mutations. Most of the amino acid residues identified (e.g. B‐S221P/T, B‐H267F/L/N/Y, B‐I269V and D‐D129E/G/T) are directly involved in forming the cavity in which SDHI fungicides bind. Docking studies of SDHI fungicides in structural models of wild‐type and mutated Sdh complexes also indicated which residues were important for the binding of different SDHI fungicides and showed a different binding for fluopyram. The predictive power of the model was also shown. Further diagnostic development, enabling the detection of resistant alleles at low frequencies, and cross‐resistance studies will aid the implementation of anti‐resistance strategies to prolong the cost‐effectiveness and lifetime of SDHI fungicides.     

Sensitivity to Fludioxonil of Botrytis cinerea Isolates from Tomato in Henan Province of China and Characterizations of Fludioxonil‐resistant Mutants

Grey mould, caused by Botrytis cinerea Pers ex Fr., is one of the most common diseases of tomato worldwide. Fludioxonil belongs to the phenylpyrrole fungicides, which have high activity against B. cinerea. The sensitivity of fludioxonil was evaluated on the basis of the level of inhibition of mycelium growth in 274 B. cinerea isolates collected from different locations (untreated with this fungicide) in Henan Province, China. The EC₅₀ values for fludioxonil ranged from 0.0033 to 0.0415 mg/l, and the average EC₅₀ values were 0.0156 ± 0.0078 mg/l. Three fludioxonil‐resistant mutants were obtained by subculturing fludioxonil‐sensitive wild‐type isolates on continuously increasing fludioxonil concentrations. For the cross‐resistance assay, fludioxonil revealed positive cross‐resistance with procymidone but did not reveal cross‐resistance with pyrimethanil, boscalid and trifloxystrobin. Mycelial growth, conidial production, hyphal dry weight and pathogenicity were diminished significantly between the fludioxonil‐resistant mutants and their sensitive wild‐type parental isolates. This study shows for the first time that fludioxonil‐resistant isolates of B. cinerea are still not present in Henan Province because this fungicide is an attractive and effective fungicide for chemical control. Recommendations can be made to growers to use fludioxonil to control grey mould and to consider the potential moderate resistance risk of using this fungicide.     

Susceptibility of eight potato cultivars to tuber infection by Phytophthora erythroseptica and Pythium ultimum and its relationship to mefenoxam-mediated control of pink rot and leak

In addition to cultural practices, the application of the fungicide mefenoxam is an important disease management tactic used to control both pink rot and leak on potato tubers grown in the USA. Mefenoxam resistance has been identified in many of the potato growing regions, and therefore resistance management strategies are very important for retaining this fungicide as a tool to manage these storage rot diseases. The relationship between mefenoxam efficacy and cultivar susceptibility to pink rot and leak was assessed in post‐harvest inoculation studies. Mefenoxam was applied to potato (Solanum tuberosum) cultivars known to express varying levels of susceptibility to pink rot and leak caused by Phytophthora erythroseptica and Pythium ultimum, respectfully. Tubers harvested from plants treated with in‐furrow and foliar applications of mefenoxam were inoculated with isolates sensitive to the fungicide. Incidence and severity of both diseases ranged widely among cultivars. Russet Norkotah was the most susceptible to infection by P. erythroseptica, while cvs Pike and Atlantic were the most resistant. Cultivars Dark Red Norland, Russet Norkotah, Goldrush and Russet Burbank were most susceptible to infection by P. ultimum whereas Snowden was most resistant. Control of pink rot differed significantly among cultivars following mefenoxam treatment, ranging from 28% (cv. Goldrush) to 67% (cv. Snowden) and generally provided the greatest level of disease control on susceptible and moderately susceptible cultivars such as Russet Norkotah and Snowden, respectively. In contrast, the impact of mefenoxam on leak development was minimal and disease control did not differ significantly among the cultivars. The fungicide failed to control leak in the susceptible cvs Atlantic and Pike and control ranged from 1.7% to 5.2% in cvs Goldrush, Russet Norkotah, Dark Red Norland, Russet Burbank and Kennebec. The greatest level of leak control was achieved with the moderately resistant cv., Snowden, at 12.7%. Cultivars most likely to benefit from mefenoxam treatments should be targeted as part of a pink rot management programme. Judicious use of the fungicide, when matched with the level of cultivar susceptibility, may prove to be an efficient and effective approach to reduce infection rates and possibly manage mefenoxam resistance thereby maintaining longevity of the compound.     

Integrated Control of Clubroot

Clubroot caused by Plasmodiophora brassicae affects the Brassicaceae family of plants, including many important vegetable and broadacre crops. In the last 20 years increasing intensity of vegetable production and the rapid growth in popularity of oilseed rape as a broadacre or arable break crop have increased the severity of clubroot and the area of land affected in both the vegetable and broadacre industries. Resting spores of P. brassicae are long-lived in soil, but the number of spores can be reduced through crop rotation, fallowing, chemical application, and management of brassica weeds. The host-pathogen system is responsive to a range of control measures, including calcium and boron amendments, manipulation of soil pH, and fungicide application. Molecular tests have been developed to predict disease and resistant cultivars are available for some crops. Increasingly, a multifaceted or integrated approach is being used to manage clubroot. This approach has been particularly successful in vegetable production systems.