Crossover between the control of fungal pathogens in medicine and the wider environment,and the threat of antifungal resistance

Fungal propagules existing in the natural environment can easily be transmitted to the human body, mostly by inhalation of contaminated air or direct contact onto the skin, nails, and mucosa. Fungal infections in humans are, as compared to viral and bacterial infections, rarely serious (life-threatening) unless the immune system is weakened. Because azole fungicides (demethylation inhibitors, DMIs) are among the most important antifungal compounds used broadly in human and animal medicine as well as in agriculture and material protection, fungal propagules may come into contact with azoles almost everywhere, presenting a potential “crossover-use-pattern” and “cross-contamination-risk” for resistant propagules in all areas. A “hot-spot” in terms of the emergence of azole resistance in a fungal species is defined as a habitat in which the species is actively propagating and exposed to a fungicidally effective azole at available concentrations high enough to select for resistant individuals, potentially multiplying and spreading to other habitats. Intrinsic antifungal resistance may exist in less sensitive or insensitive species independent of previous exposure to antifungal compounds, whereas acquired antifungal resistance can evolve if triggered by the exposure of an originally sensitive species (or population) to agricultural or medical antifungal agents, resulting in the selection of resistant individuals. The origin and risks of these developments in medical settings and the wider environment are elucidated for the most relevant life-threatening fungal human pathogens, including several species of Cryptococcus, Candida, Pneumocystis, Aspergillus, Histoplasma, Coccidioides, Rhizopus, Mucor, Fusarium, and Scedosporium.     

Induced resistance: a tool for fungicide resistance management

Induced resistance to the apple scab fungus Venturia inaequalis was demonstrated in greenhouse tests with 12-day-old seedlings of the apple cultivar Golden Delicious treated with methyl 2,6-dichloro-isonicotinate or 3,5-dichlorosalicylic acid prior to inoculation with the causal fungus. Studies of the dose-response of flusilazole on induced resistant plants revealed synergistic effects between both crop protection principles. Therefore, the use of such resistance-inducing compounds in the field might allow a reduction in the number of fungicide applications, and possibly a reduction in dose, thus resulting in improved efficacy of fungicides. There was also evidence that induced resistance could prove to be a valid strategy for the treatment of pathogen populations with reduced sensitivity to a given fungicide. © 1998 SCI     

桃褐腐病菌抗药性分子机理研究进展

桃褐腐病是严重危害桃生产的重要病害之一。在实际生产中,该病害的控制主要以化学防治为主。由于杀菌剂的大量使用,桃褐腐病菌已对几类常用的杀菌剂产生了不同程度的抗药性。本文就桃褐腐病菌(Moniliniaspp.)对生产上使用的几大类杀菌剂的抗药性情况及抗药性分子机制研究进展进行简单概述和分析,以供国内同行借鉴与参考。     

植物病原菌对杀菌剂多药抗性的发生现状

近年来, 随着杀菌剂在农作物病害田间防治中的大量使用, 植物病原菌的多药抗性(multidrug resistance, MDR)现象发生越来越普遍。本文综述了在草坪币斑病、灰霉病、小麦叶枯病和苹果青霉病等病害防治中, 病原菌对杀菌剂多药抗性发生的情况。以及在紫外诱导和杀菌剂离体驯化下, 致病疫霉Phytophthora infestans、立枯丝核菌Rhizoctonia solani、指状青霉Penicillium digitatum的多药抗性发生情况。分析了导致植物病原菌多药抗性产生的外排转运蛋白过表达、解毒酶代谢和靶标位点变化等相关机制, 并在此基础上提出了多药抗性预防和治理的策略, 以期为田间杀菌剂的高效利用, 延缓药剂的抗性风险提供科学参考。     

5种链格孢属植物病原真菌对10种杀菌剂的敏感性比较

离体条件下测定并比较了10种杀菌剂对番茄早疫病菌、苹果斑点落叶病菌、梨黑斑病菌、烟草赤星病菌和甘蓝黑斑病菌等5种链格孢属植物病原真菌的抗菌活性。结果表明,不同种的链格孢属病菌对同一药荆的敏感性相近;咪鲜胺、啶菌恶唑、苯醚甲环唑、异菌脲、腐霉利等5种杀菌剂对供试病菌菌丝生长的抑制活性最好,其EC50在0．01～1．28μg／mL之间;百菌清对病菌分生孢子萌发的抑制活性最高,其次是醚菌酯和嘧菌酯。     

Update on mechanisms of azole resistance in Mycosphaerella graminicola and implications for future control

This review summarises recent investigations into the molecular mechanisms responsible for the decline in sensitivity to azole (imidazole and triazole) fungicides in European populations of the Septoria leaf blotch pathogen, Mycosphaerella graminicola. The complex recent evolution of the azole target sterol 14α‐demethylase (MgCYP51) enzyme in response to selection by the sequential introduction of progressively more effective azoles is described, and the contribution of individual MgCYP51 amino acid alterations and their combinations to azole resistance phenotypes and intrinsic enzyme activity is discussed. In addition, the recent identification of mechanisms independent of changes in MgCYP51 structure correlated with novel azole cross‐resistant phenotypes suggests that the further evolution of M. graminicola under continued selection by azole fungicides could involve multiple mechanisms. The prospects for azole fungicides in controlling European M. graminicola populations in the future are discussed in the context of these new findings. Copyright © 2012 Society of Chemical Industry     

The drug transporter MgMfs1 can modulate sensitivity of field strains of the fungal wheat pathogen Mycosphaerella graminicola to the strobilurin fungicide trifloxystrobin

BACKGROUND: The major facilitator superfamily (MFS) drug transporter MgMfs1 of the wheat pathogen Mycosphaerella graminicola (Fuckel) J Schroeter is a potent multidrug transporter with high capacity to transport strobilurin fungicides in vitro. The data presented in this paper indicate that, in addition to the predominant cause of strobilurin resistance, cytochrome b G143A subsititution, MgMfs1 can play a role in sensitivity of field strains of this pathogen to trifloxystrobin. RESULTS: In a major part of field strains of M. graminicola (collected in the Netherlands in 2004) containing the cytochrome b G143A substitution, the basal level of expression of MgMfs1 was elevated as compared with sensitive strains lacking the G143A substitution. Induction of MgMfs1 expression in wild-type isolates upon treatment with trifloxystrobin at sublethal concentrations proceeded rapidly. Furthermore, in disease control experiments on wheat seedlings, disruption mutants of MgMfs1 displayed an increased sensitivity to trifloxystrobin. CONCLUSION: It is concluded that the drug transporter MgMfs1 is a determinant of strobilurin sensitivity of field strains of M. graminicola.     

Resistance profiles of Botrytis cinerea populations to several fungicide classes on greenhouse tomato and strawberry in Lebanon

Tomato and strawberry are the most important protected crops in Lebanon and are seriously affected by grey mould disease, caused by Botrytis cinerea. In the present study, the fungicide sensitivity assays revealed medium to high frequencies of B. cinerea isolates resistant to benzimidazoles, dicarboximides, and anilinopyrimidines on tomato and strawberry. Fludioxonil- and boscalid-resistant mutants were uncommonly found at generally low frequency on both crops. Resistance to fenhexamid was detected in only one site on tomato but in most sites on strawberry with high frequencies, and the occurrence of resistance to QoI fungicides was ascertained on both crops. The majority of the tested isolates (>90%) exhibited multiple fungicide resistance, and isolates resistant to the seven antibotrydial fungicide classes were detected on strawberry in three locations. A high level of resistance was shown by B. cinerea mutants resistant to boscalid, fenhexamid, and QoI fungicides, while two levels of moderate and high resistance to anilinopyrimidines were identified. Genetic analysis revealed point mutations in the target genes commonly associated with resistance in B. cinerea isolates, with all mutants resistant to dicarboximides, fenhexamid, boscalid, and QoI fungicides carrying single-nucleotide polymorphims in BcOS1 (I365S/N, Q369P, and N373S), Erg27 (F412V/I), SdhB (H272R/Y), and cytb (G143A) genes, respectively. The general incorrect use of fungicides has caused the development and spread of fungicide resistance as a widespread phenomenon on protected tomato and strawberry in Lebanon. The implementation of appropriate antiresistance strategies is highly recommended.     

Evolution of fungicide resistance in the cereal eyespot fungi Tapesia yallundae and Tapesia acuformis in France

Field isolates of the cereal eyespot pathogen can be divided into two groups which are now considered as two species: Tapesia yallundae and Tapesia acuformis. In both species the first case of acquired resistance was observed with benzimidazole fungicides in the early 1980s. At the same time, a number of sterol C-14 demethylation inhibitors (DMIs), such as the imidazole prochloraz and several triazoles, including flusilazole, were introduced. Surprisingly T. acuformis appeared intrinsically resistant to the triazole derivatives in comparison to T. yallundae, but both species were sensitive to prochloraz. The intensive use of these DMIs led to the development of acquired resistance towards triazoles in T. yallundae and towards prochloraz in T. acuformis. Today all the strains in both species appear equally sensitive to the anilinopyrimidine cyprodinil. ©1997 SCI     

RNA interference technology in crop protection against arthropod pests,pathogens and nematodes

Scientists have made significant progress in understanding and unraveling several aspects of double‐stranded RNA (dsRNA)‐mediated gene silencing during the last two decades. Now that the RNA interference (RNAi) mechanism is well understood, it is time to consider how to apply the acquired knowledge to agriculture and crop protection. Some RNAi‐based products are already available for farmers and more are expected to reach the market soon. Tailor‐made dsRNA as an active ingredient for biopesticide formulations is considered a raw material that can be used for diverse purposes, from pest control and bee protection against viruses to pesticide resistance management. The RNAi mechanism works at the messenger RNA (mRNA) level, exploiting a sequence‐dependent mode of action, which makes it unique in potency and selectivity compared with conventional agrochemicals. Furthermore, the use of RNAi in crop protection can be achieved by employing plant‐incorporated protectants through plant transformation, but also by non‐transformative strategies such as the use of formulations of sprayable RNAs as direct control agents, resistance factor repressors or developmental disruptors. In this review, RNAi is presented in an agricultural context (discussing products that have been launched on the market or will soon be available), and we go beyond the classical presentation of successful examples of RNAi in pest‐insect control and comprehensively explore its potential for the control of plant pathogens, nematodes and mites, and to fight against diseases and parasites in beneficial insects. Moreover, we also discuss its use as a repressor for the management of pesticide‐resistant weeds and insects. Finally, this review reports on the advances in non‐transformative dsRNA delivery and the production costs of dsRNA, and discusses environmental considerations. © 2017 Society of Chemical Industry     

A review on the potential effects of temperature on fungicide effectiveness

Fungicides are one possible way to manage fungal and oomycete plant pathogens in order to safeguard yield and quality of crops and to improve shelf-life of produce in agriculture and horticulture. However, global warming and the resulting temperature increase may affect the effectiveness of some important fungicides, including efficacy and duration of plant disease control. Nevertheless, according to our literature survey, there is little specific information available on whether and how temperature influences the effectiveness of fungicides. The very few publications that show specific data are summarized herein. Specific data are mainly gained under controlled conditions, both based on in vitro and in planta experiments. Field data are more or less missing. Most researchers assume that indirect effects of temperature on fungicide efficacy are particularly important. For example, temperature effects on pathogen spore germination and hyphal growth (optimal versus sub- and supra-optimal), whereby optimal temperature conditions can improve pathogen fitness, thereby increasing the tolerance of pathogens to fungicides. Presumably, these indirect effects are often more important than the direct effects of temperature on fungicide performance. However, the data needed to prove this assumption are lacking. Therefore, it would be beneficial to conduct more in-depth laboratory, greenhouse and field experiments in order to investigate the potential direct and indirect influence of temperature on the effectiveness of important fungicides. This would enable the establishment of appropriate recommendations for fungicide use in an increasingly warmer world and would assist the development of future fungicide solutions, based on improved knowledge.     

Assessing the risk of resistance in Pseudoperonospora cubensis to the fungicide flumorph in vitro

BACKGROUND: The oomycete fungicide flumorph is a recently introduced carboxylic acid amide (CAA) fungicide. In order to evaluate the risk of developing field resistance to flumorph, the authors compared it with dimethomorph and azoxystrobin with respect to the ease of obtaining resistant isolates to these fungicides, the level of resistance and their fitness in the laboratory. RESULTS: Mutants with a high level of resistance to azoxystrobin were isolated readily by adaptation and UV irradiation, and their fitness was as good as that of the parent isolates. Attempts to generate mutants of Pseudoperonospora cubensis (Burk. & MA Curtis) Rostovsev resistant to flumorph and dimethomorph by sporangia adaptation on fungicide-treated leaves were unsuccessful. However, moderately resistant mutants were isolated using UV mutagenesis, but their resistance level [maximum resistance factor (MRF) < 100] was much lower than that of the azoxystrobin-resistant mutant (MRF = 733). With the exception of stability of resistance, all mutants showed low pathogenicity and sporulation compared with wild-type isolates and azoxystrobin-resistant mutants. There is cross-resistance between flumorph and dimethomorph, suggesting that they have the same resistance mechanism. CONCLUSION: The above results suggest that the resistance risk of flumorph may be similar to that of dimethomorph but lower than that of azoxystrobin and can be classified as moderate. Thus, it can be managed by appropriate product use strategies.     

A critical evaluation of the role of alternative oxidase in the performance of strobilurin and related fungicides acting at the Qo site of complex III

Mitochondrial respiration conserves energy by linking NADH oxidation and electron-coupled proton translocation with ATP synthesis, through a core pathway involving three large protein complexes. Strobilurin fungicides block electron flow through one of these complexes (III), and disrupt energy supply. Despite an essential need for ATP throughout fungal disease development, strobilurins are largely preventative; indeed some diseases are not controlled at all, and several pathogens have quickly developed resistance. Target-site variation is not the only cause of these performance difficulties. Alternative oxidase (AOX) is a strobilurin-insensitive terminal oxidase that allows electrons from ubiquinol to bypass Complex III. Its synthesis is constitutive in some fungi but in many others is induced by inhibition of the main pathway. AOX provides a strobilurin-insensitive pathway for oxidation of NADH. Protons are pumped as electrons flow through Complex I, but energy conservation is less efficient than for the full respiratory chain. Salicylhydroxamic acid (SHAM) is a characteristic inhibitor of AOX, and several studies have explored the potentiation of strobilurin activity by SHAM. We present a kinetic-based model which relates changes in the extent of potentiation during different phases of disease development to a changing importance of energy efficiency. The model provides a framework for understanding the varying efficacy of strobilurin fungicides. In many cases, AOX can limit strobilurin effectiveness once an infection is established, but is unable to interfere significantly with strobilurin action during germination. A less stringent demand for energy efficiency during early disease development could lead to insensitivity towards this class of fungicides. This is discussed in relation to Botrytis cinerea, which is often poorly controlled by strobilurins. Mutations with a similar effect may explain evidence implicating AOX in resistance development in normally well-controlled plant pathogens, such as Venturia inaequalis.     

Significance of ABC transporters in fungicide sensitivity and resistance

ATP-binding cassette (ABC) transporters are members of a protein superfamily which can be responsible for efflux of drugs from cells of target organisms. In this way, the transporters may provide a mechanism of protection against cytotoxic drugs. In laboratory-generated mutants of fungi, overproduction of ABC transporters can cause multi-drug resistance to azoles and other non-related toxicants. The impact of this mechanism of resistance in field populations with decreased sensitivity to azoles remains to be established. Inhibitors of ABC transporter activity may synergize activity of azoles to populations of both sensitive and azole-resistant pathogens. The natural function of ABC transporters in plant pathogenic fungi may relate to transport of plant-defence compounds or fungal pathogenicity factors. Therefore, inhibitors of ABC transporter activity may act as disease control agents with an indirect mode of action. ©1997 SCI     

Mating type and fungicide resistance in populations of Podosphaera xanthii in south Italy

Powdery mildew is one of the most common and severe diseases of cucurbits, causing heavy yield losses in all growing areas when not successfully controlled. Two different fungal species, Podosphaera xanthii and Golovinomyces orontii, are generally recognized as causal agents. The results of monitoring carried out in 2016 and 2018 confirmed that P. xanthii is the exclusive pathogen causing cucurbit powdery mildew (CPM) in southern Italy. P. xanthii is a bipolar heterothallic fungus; a PCR-based method for distinguishing MAT1-1 and MAT1-2 idiomorphs was applied for assessing mating type distribution in fungal populations present on cucurbits in different sites. The idiomorph MAT1-2 was prevalent over the MAT1-1 in 2016, whereas they were approximately in a 1:1 ratio in 2018; this finding corroborated the hypothesis that the MAT1-1 idiomorph was more recently introduced in the area. Cyflufenamid-resistant isolates were widespread in commercial greenhouses and field plantings even though use of this fungicide had been drastically reduced by the farmers 1 year before the monitoring due to the effectiveness losses observed in CPM control. Occurrence of cyflufenamid resistance and its impact on efficacy were evaluated in a field trial comparing different fungicide spray schedules. Cyflufenamid-resistant isolates were detected even at the first appearance of symptoms on leaves, increasing over time. Isolates resistant to cyflufenamid showed a resistance factor as high as 900. Generally, P. xanthii was better controlled when cyflufenamid was used in integrated strategies rather than in spray schedules based on the exclusive use of the fungicide.     

安徽省番茄灰霉菌抗药性测定和治理

测定了安徽省番茄灰霉病菌对多菌灵、腐霉利、乙霉威的抗性。结果表明,田间有B_SD_SN_S、B_SD_SN_LR、B_SD_SN_HR、B_HRD_SN_S、B_MRD_LRN_S、B_HRD_LRN_S、B_HRD_LRN_LR7种抗性类型,对多菌灵、腐霉利、乙霉威具有抗性的菌株分别占试验总菌株数的28.3%、20%和73.3%。     

Modulators of membrane drug transporters potentiate the activity of the DMI fungicide oxpoconazole against Botrytis cinerea

Modulators known to reduce multidrug resistance in tumour cells were tested for their potency to synergize the fungitoxic activity of the fungicide oxpoconazole, a sterol demethylation inhibitor (DMI), against Botrytis cinerea Pers. Chlorpromazine, a phenothiazine compound known as a calmodulin antagonist, appeared the most potent compound. Tacrolimus, a macrolide compound with immunosuppressive activity, was also active. The synergism of chlorpromazine negatively correlated with the sensitivity of the parent strain and mutants of B. cinerea. The synergism was highest in a mutant that overexpressed the ATP-binding cassette transporter BcatrD, known to transport DMI fungicides such as oxpoconazole. The synergism of chlorpromazine positively correlated with its potency to enhance the accumulation of oxpoconazole in BcatrD mutants. These results indicate that chlorpromazine is a modulator of BcatrD activity in B. cinerea and suggest that mixtures of DMI fungicides with modulators may represent a perspective for the development of new resistance management strategies.     

Characterization of the cytochrome b gene fragment of Puccinia species responsible for the binding site of QoI fungicides

The fragment of the cytochrome b (cyt b) gene responsible for the binding site of QoI fungicides was sequenced for different Puccinia species by using DNA and RNA as template for PCR and RT-PCR, respectively. Degenerated primers for the cyt b gene amplified in P. recondita f.sp. tritici a 450 bp fragment, which was cloned and sequenced. At cDNA level, several Thermal Asymmetric InterLaced (TAIL)-PCR cycles were needed to produce a 996 bp long fragment, which corresponded to almost the whole cyt b gene (about 1160-1180 bp, without introns). This fragment was sequenced and specific primers were designed. Amplification with cyt b specific primers using genomic DNA as template revealed the presence of an intron of about 1500 bp length after the codon for glycine at amino acid position 143. By using the same primer pair, the cyt b gene fragment was amplified and sequenced both at cDNA and genomic DNA level also for other rust species, including P. graminis f.sp. tritici (length: 506 bp), P. striiformis f.sp. tritici (755 bp), P. coronata f.sp. avenae (644 bp), P. hordei (660 bp), P. recondita f.sp. secalis (687 bp), P. sorghi (709 bp), and P. horiana (478 bp). At the same position as for P. recondita f.sp. tritici, an intron of about 1500-1600 bp length was detected also in all other Puccinia species. High homologies were observed among all Puccinia species for both the exonic and intronic fragments of the cyt b gene. Specific primers for the cyt b gene of all eight Puccinia species were developed, which easily amplified the fragment of the gene including all possible mutations known to confer resistance to QoIs in several plant pathogens. However, in all tested isolates of the Puccinia species included in this study, the sequence of cyt b gene fragment did not contain any point mutations.