Anti-resistance Strategies Research Articles

Pest Management Challenges and Control Practices in Codling Moth: A Review.

The codling moth, Cydia pomonella L., is a serious insect pest in pome fruit production worldwide with a preference for apple. The pest is known for having developed resistance to several chemical groups of insecticides, making its control difficult. The control and management of the codling moth is often hindered by a lack of understanding about its biology and ecology, including aspects of its population genetics. This review summarizes the information about the origin and biology of the codling moth, describes the mechanisms of resistance in this pest, and provides an overview of current research of resistant pest populations and genetic research both in Europe and globally. The main focus of this review is on non-pesticide control measures and anti-resistance strategies which help to reduce the number of chemical pesticides used and their residues on food and the local environment. Regular monitoring for insecticide resistance is essential for proactive management to mitigate potential insecticide resistance. Here we describe techniques for the detection of resistant variants and possibilities for monitoring resistance populations. Also, we present our present work on developing new methods to maintain effective control using appropriate integrated resistance management (IRM) strategies for this economically important perennial pest.

Performance of chlorothalonil levels and spraying intervals on Asian rust control and soybean grain yield

ABSTRACT In an experiment conducted in the field, during the 2017/18 growing season, with the soybean cultivar Syn 1561 IPRO, the interaction of chlorothalonil levels with application intervals was evaluated for the control of Asian soybean rust, caused by Phakopsora pachyrhizi. The first fungicide application was performed in V8 stage, 44 days after emergence, with 1.85% rust leaflet incidence. The experiment consisted of a factorial arrangement with five fungicide levels (1.0, 1.5, 2.0, 2.5 and 3.0 L/ha) applied at 8, 12 and 16-day intervals, using randomized block treatments and four replicates. A self-propelled sprayer with 16 bars, XR11001VS nozzles and 150 L/ha volume was employed. Leaflet rust severity in R5.4 stage and grain yield were evaluated. Data were subjected to analysis of variance, and means were compared according to Tukey’s test. At eight-day intervals (six sprayings), control ranged from 75% to 93%; at 12-day intervals (four sprayings), it ranged from 35 to 63%, and at 16-day intervals (three sprayings), control ranged from 15 to 29% according to the sprayed levels. The longer the interval between applications, the lower the response of the used level for rust control and soybean grain yield. Chlorothalonil showed fungitoxicity to integrate a program of anti-resistance strategies to control soybean rust.

Contribution of Epithelial Plasticity to Therapy Resistance.

Therapy resistance is responsible for tumour recurrence and represents one of the major challenges in present oncology. Significant advances have been made in the understanding of the mechanisms underlying resistance to conventional and targeted therapies improving the clinical management of relapsed patients. Unfortunately, in too many cases, resistance reappears leading to a fatal outcome. The recent introduction of immunotherapy regimes has provided an unprecedented success in the treatment of specific cancer types; however, a good percentage of patients do not respond to immune-based treatments or ultimately become resistant. Cellular plasticity, cancer cell stemness and tumour heterogeneity have emerged as important determinants of treatment resistance. Epithelial-to-mesenchymal transition (EMT) is associated with resistance in many different cellular and preclinical models, although little evidence derives directly from clinical samples. The recognition of the presence in tumours of intermediate hybrid epithelial/mesenchymal states as the most likely manifestation of epithelial plasticity and their potential link to stemness and tumour heterogeneity, provide new clues to understanding resistance and could be exploited in the search for anti-resistance strategies. Here, recent evidence linking EMT/epithelial plasticity to resistance against conventional, targeted and immune therapy are summarized. In addition, future perspectives for related clinical approaches are also discussed.

QoI Resistance in Sugar Beet Powdery Mildew (Erysiphe betae) in Scandinavia

Powdery mildew caused by Erysiphe betae is one of the major fungal diseases in sugar beet in Denmark and Sweden. Frequent applications of fungicides mitigate the risk of powdery mildew epidemics and, consequently, reduce yield losses conferred by the disease. So far, mixtures of quinone outside inhibitors (QoIs) and triazoles have provided good efficacy against E. betae in field trials and common farming practice. However, development of fungicide resistance is a real risk, because only a limited number of active ingredients are available for the control of powdery mildew in sugar beet, and several other active ingredients are expected to be banned following reevaluation when the most recent European Union legislation is implemented. The G143A mutation associated with QoI resistance has been previously found in the United States. In this brief, its presence in Europe is reported for the first time. The current finding strongly encourages the adoption of anti-resistance strategies that minimize the spread of QoI resistance in sugar beet powdery mildew. Those strategies should be based on integrated pest management measures, including disease monitoring, the use of resistant cultivars, and the use of biological products. A sole reliance on QoI fungicides for sugar beet powdery mildew control should be avoided.

A time-course investigation of resistance to the carboxylic acid amide mandipropamid in field populations of Plasmopara viticola treated with anti-resistance strategies.

Despite anti-resistance strategies being recommended to reduce selection pressure on insensitive strains, no information is available on fungal population dynamics following their application in real field conditions. In this study, the effects on Plasmopara viticola populations of two identical spray programs, differing only in including or not the carboxylic acid amide (CAA) mandipropamid in mixture and in alternation with an anti-resistance partner, were compared in terms of downy mildew control efficacy and mandipropamid sensitivity in two commercial vineyards for four seasons. Both programs effectively and similarly protected grapevine from downy mildew, despite different starting sensitivity levels of the P. viticola populations. In the vineyard where resistant strains were initially present, the frequency of mutations associated with resistance (G1105S/V) fluctuated within seasons in both programs and a shift towards sensitivity occurred after 3 years of the mandipropamid-free program. Where sensitivity was initially present, no changes occurred in the mandipropamid-free program and resistant strains were selected in the mandipropamid program in high disease pressure conditions. The anti-resistance strategy including mandipropamid in mixture showed a good field performance, but did not completely prevent an increase in the frequency of insensitive strains. This supports the need for appropriate planning to determine which mixtures should be used in the field. © 2018 Society of Chemical Industry.

Efficacy of acetamiprid and fipronil fly baits against the housefly (Musca domestica L.) under laboratory conditions.

Background:The housefly Musca domestica L. (Diptera: Muscidae) is permanent pests in livestock facilities. High fly density in livestock and poultry farms can increase the risks of economic loss and public health. Treatment with toxic baits is one of the methods for housefly control. However, development of resistance to insecticides makes it difficult to manage of flies. Anti-resistance strategies include the use of multiple pesticides with different modes of action.Aim:This study was conducted to estimate the efficacy of neonicotinoid acetamiprid and phenylpyrazole fipronil, applied alone or in the mixture, against adults of M. domestica and to evaluate the efficacy of fly bait formulations containing acetamiprid and fipronil under laboratory conditions.Materials and Methods:The adult flies, M. domestica of laboratory strain, were used in laboratory bioassays. The efficacy of acetamiprid and fipronil as technical substances, when applied alone and in the mixture, against adult flies was tested by no-choice feeding bioassays. The insecticidal efficacy of bait formulations (wet powder) with acetamiprid or fipronil or their mixture was tested against flies by choice feeding bioassays. The probit analysis was used to calculate lethal concentrations of insecticides, and the χ2 test was used to analyze the interaction between fipronil and acetamiprid in the mixture.Results:Fipronil was more toxic to adults of M. domestica than acetamiprid in laboratory tests. Lethal concentrations for 50% mortality (95% confidence interval) of flies were 0.0159% (0.0124-0.0205) of acetamiprid and 0.000119% (0.000039-0.0002640) of fipronil. The mixture containing fipronil at concentration 0.005% and acetamiprid at concentration 0.05% had the additive effect on fly mortality.Conclusion:The results of laboratory feeding bioassays indicate that the mixture of fipronil and acetamiprid might have a potential to use in toxic bait formulations against houseflies.

Biological and molecular characterization of field isolates of Alternaria alternata with single or double resistance to respiratory complex II and III inhibitors

Field isolates of Alternaria alternata collected from tomato processors were characterized for sensitivity to respiration inhibitors using in vitro mycelial growth assays. Pyraclostrobin (QoI), boscalid, fluopyram and isopyrazam (SDHIs) mean EC50 values were 0.32, 1.43, 2.21, and 3.53 μg/ml respectively. Of the 42 isolates, 36 were sensitive to all respiration inhibiting fungicides tested whereas three isolates were less sensitive to boscalid, one to pyraclostrobin and two were simultaneously resistant to both inhibitors and isopyrazam. Correlation analysis between fungicide sensitivities revealed a positive cross-resistance between pyraclostrobin and tebuconazole, and between cyprodinil and mancozeb. There was no cross-resistance between QoIs, SHDIs or any other mode of action. Sequencing of the QoI and SDHI targets revealed the G143A cytochrome b resistance mutation in all pyraclostrobin-resistant isolates while analysis of the succinate dehydrogenase coding gene revealed point mutations in two of three of the gene subunits analyzed in boscalid-resistant isolates. Specifically, two isolates carried the H277Y and three the H133Q resistance mutations located in the sdhB and sdhD subunits of the respiration complex II, respectively. Isolates bearing the H277Y mutation also carried the G143A cytochrome b resistance mutation. Boscalid and pyraclostrobin-resistant isolates exhibited greater pathogenicity and sporulation compared to sensitive isolates, respectively. Isolates with cross-resistance exhibited greater pathogenicity and sporulation but slower mycelial growth compared to sensitive isolates. This is the first report of field isolates of A. alternata with single or double resistance to QoIs and SDHIs in Greece and should be considered in planning and implementing effective anti-resistance strategies.

Frequency of fungicide-resistant Botrytis cinerea populations isolated from ‘Thompson Seedless’ table grapes in the Central Valley of Chile

During the 2013 and 2014 growth seasons, 526 single spore isolates of Botrytis cinerea were collected from naturally infected ‘Thompson Seedless’ table grape flowers from fifteen orchards in three regions of the Central Valley of Chile. The isolates were tested for resistance to azoxystrobin, boscalid, fenhexamid, fludioxonil and pyrimethanil. Among the 526 isolates, 106 (20.15%) were sensitive to all fungicides tested; from north to south, the frequency of sensitive isolates in the regions of Valparaiso, Metropolitana and O’Higgins ranged from 48.15% to 21.1% and 5.88%, respectively. Four hundred and twenty isolates (79%) showed resistance to single or multiple fungicides, 134 (25.4%) were simultaneously resistant to azoxystrobin and pyrimethanil. No fludioxonil-resistant isolates were found, indicating that fludioxonil has great potential for gray mold control in table grapes in Chile. From sixty randomly selected B. cinerea isolates, only the azoxystrobin-resistant isolates carried the G143A point mutation; according to the cytochrome b ( cyt b ) gene structure, the third intron Bcbi-143/144 was only detected in the azoxystrobin-sensitive isolates. The H272R and H272Y point mutations in the succinate dehydrogenase subunit B ( sdhB ) gene were associated only with the boscalid-resistant isolates. The F412S and F412V point mutations were found in the sequenced erg27 gene of randomly selected fenhexamid-resistant isolates. These results contribute to the knowledge of B. cinerea fungicide resistance for table grape vine crops in Central Chile, particularly for the development of multiple-resistance and the associated resistance mechanisms of azoxystrobin, boscalid and fenhexamid-resistant isolate populations. Antiresistance strategies are discussed in a general manner.

Does agricultural use of azole fungicides contribute to resistance in the human pathogen Aspergillus fumigatus?

Azole resistance in human fungal pathogens has increased over the past twenty years, especially in immunocompromised patients. Similarities between medical and agricultural azoles, and extensive azole (14α-demethylase inhibitor, DMI) use in crop protection, prompted speculation that resistance in patients with aspergillosis originated in the environment. Aspergillus species, and especially Aspergillus fumigatus, are the largest cause of patient deaths from fungi. Azole levels in soils following crop spraying, and differences in sensitivity between medical and agricultural azoles (DMIs), indicate weaker selection in cropping systems than in patients receiving azole therapy. Most fungi have just one CYP51 paralogue (isozyme CYP51B), but in Aspergillus sp. mutations conferring azole resistance are largely confined to a second paralogue, CYP51A. Binding within the active centre is similar for medical and agricultural azoles but differences elsewhere between the two paralogues may ensure selection depends on the DMI used on crops. Two imidazoles, imazalil and prochloraz, have been widely used since the early 1970s, yet unlike triazoles they have not been linked to resistance in patients. Evidence that DMIs are the origin, or increase the frequency, of azole resistance in human fungal pathogens is lacking. Limiting DMI use would have serious impacts on disease control in many crops, and remove key tools in anti-resistance strategies. © 2017 Society of Chemical Industry.

Anti-resistance strategies for fungicides against wheat pathogen Zymoseptoria tritici with focus on DMI fungicides

Septoria tritici blotch (STB) caused by the fungal pathogen Zymoseptoria tritici is a global threat to sustainable wheat production. Applications of fungicide against STB are regarded as an essential means to minimise yield losses, however, fungicide resistance is developing and affecting fungicide efficacy greatly. Only a few fungicide classes are available for STB control. DMI fungicides are seen as the main group, but increasing problems with resistance is challenging their use. In 2015 and 2016, field trials were conducted testing fungicide spray strategies using one, two or three applications, alternations, mixtures of different DMIs and DMI mixed with other modes of action including a SDHI and a multi-site inhibitor. The strategies were tested for disease control, their impact on yield and their effect on resistance build-up, measured as CYP51 alterations responsible for decreased sensitivity and efficacy of DMIs. Strategies consisting of three treatments provided adequate control of STB and significant yield increases. The best results with regard to yield and control were attained by a diversified DMI strategy also including the SDHI boscalid and the multi-site inhibitor folpet. Spraying once or twice lowered selection yet compromised STB control and yield. CYP51 alteration I381V was the most predominated in all samples. Frequencies of alterations D134G, V136A and S524T increased significantly following applications with DMI fungicides. The more diversified a strategy, the less it selected for CYP51 alterations. EC50 values for epoxiconazole showed a tendency to be higher post-treatment in 2015. The results presented in this study encourage the adoption of mixing and alternating fungicides into spray strategies to minimise the risk of resistance build-up and to prolong the effective life of fungicides.

Cigarette smoke extract induces EGFR-TKI resistance via promoting EGFR signaling pathway and ROS generation in NSCLC cell lines

ObjectivesEpithelial growth factor receptor (EGFR) somatic-mutated non-small cell lung cancer (NSCLC) patients with smoking history always show a poor response to EGFR tyrosine kinase inhibitors (EGFR-TKIs). The aim of the study is to explore the molecular mechanism of EGFR-TKI resistance induced by cigarette smoke extract and investigate the novel anti-resistance strategies. MethodsThe effect of cigarette smoke extract (CSE) on gefitinib sensitivity, EGFR signaling, apoptosis and reactive oxygen species (ROS) levels were detected in vitro by MTT assays, western blot, flow cytometry and laser scanning confocal microscope, respectively. ResultsMTT assays presented that CSE claimed antagonistic effect on gefitinib sensitivity via the up-regulated half maximal inhibitory concentration (IC50) values, western blot showed that CSE instigated EGFR, AKT phosphorylation, while N-Acetyl-l-Cysteine (NAC) could alleviate gefitinib resistance and abort the aberrant phosphorylation in both PC-9 and A549 cells. Confocal microscope and flow cytometry displayed that ROS generation increased after CSE exposure in NSCLC cells and this change could be inhibited by NAC. ConclusionCigarette smoke extract induces EGFR-TKI resistance via promoting EGFR signaling and ROS generation in NSCLC cell lines which could be suppressed by NAC. Alternatively, combined NAC with EGFR-TKIs to treat EGFR mutated NSCLC patients with smoking history may be a potential choice in clinical setting.

Characterisation of Ramularia collo-cygni laboratory mutants resistant to succinate dehydrogenase inhibitors.

Ramularia collo-cygni (Rcc) is responsible for Ramularia leaf spot (RLS), a foliar disease of barley contributing to serious economic losses. Protection against the disease has been almost exclusively based on fungicide applications, including succinate dehydrogenase inhibitors (SDHIs). In 2015, the first field isolates of Rcc with reduced sensitivity to SDHIs were recorded in some European countries. In this study we established baseline sensitivity of Rcc to SDHIs in the United Kingdom and characterised mutations correlating with resistance to SDHIs in UV-generated mutants. Five SDHI-resistant isolates were generated by UV mutagenesis. In four of these mutants a single amino acid change in a target succinate dehydrogenase (Sdh) protein was associated with decrease in sensitivity to SDHIs. Three of these mutations were stably inherited in the absence of SDHI fungicide, and resistant isolates did not demonstrate a fitness penalty. There were no detectable declines in sensitivity in field populations in the years 2010-2012 in the United Kingdom. SDHIs remained effective in controlling Rcc in the United Kingdom in the years 2010-2012. However, given that the first isolates of Rcc with reduced sensitivity appeared in other European countries in 2015, robust antiresistance strategies need to be continuously implemented to maintain effective disease control. © 2016 Society of Chemical Industry.

Detection of sdhB Gene Mutations in SDHI-Resistant Isolates of Botrytis cinerea Using High Resolution Melting (HRM) Analysis.

Botrytis cinerea, is a high risk pathogen for fungicide resistance development. Pathogen’ resistance to SDHIs is associated with several mutations in sdh gene. The diversity of mutations and their differential effect on cross-resistance patterns among SDHIs and the fitness of resistant strains necessitate the availability of a tool for their rapid identification. This study was initiated to develop and validate a high-resolution melting (HRM) analysis for the identification of P225H/F/L//T, N230I, and H272L/R/Y mutations. Based on the sequence of sdhB subunit of resistant and sensitive isolates, a universal primer pair was designed. The specificity of the HRM analysis primers was verified to ensure against the cross-reaction with other fungal species and its sensitivity was evaluated using concentrations of known amounts of mutant’s DNA. The melting curve analysis generated nine distinct curve profiles, enabling the discrimination of all the four mutations located at codon 225, the N230I mutation, the three mutations located in codon 272, and the non-mutated isolates (isolates of wild-type sensitivity). Similar results were obtained when DNA was extracted directly from artificially inoculated strawberry fruit. The method was validated by monitoring the presence of sdhB mutations in samples of naturally infected strawberry fruits and stone fruit rootstock seedling plants showing damping-off symptoms. HRM analysis data were compared with a standard PIRA–PCR technique and an absolute agreement was observed suggesting that in both populations the H272R mutation was the predominant one, while H272Y, N230I, and P225H were detected in lower frequencies. The results of the study suggest that HRM analysis can be a useful tool for sensate, accurate, and rapid identification of several sdhB mutations in B. cinerea and it is expected to contribute in routine fungicide resistance monitoring or assessments of the effectiveness of anti-resistance strategies implemented in crops heavily treated with botryticides.

Comparison of the sensitivity of Monilinia fructicola isolates to tebuconazole in Brazil using three methods

The frequency of strains of Monilinia fructicola that are resistant to the demethylation inhibiting fungicide tebuconazole in Brazil was determined by three methods. The first involved testing 295 isolates for relative growth under a discriminatory dose of 0.3 µg mL−1. The second and the third methods involved testing 120 isolates for the effective concentration for 50% population inhibition (EC50); one method employed the discriminatory dose of 0.3 µg mL−1 and the other method utilized the baseline dose of 0.046 µg mL−1 to separate the strains based on sensitivity. The frequencies of resistance among the isolates based on the three methods were determined by measuring mycelial growth in fungicide-amended media. The shifts in M. fructicola sensitivities were determined by comparing the EC50 density distributions of the baseline (2000–2004, n=31), intermediate (2005–2008, n=88), and current (2009–2011, n=120) isolate populations. The frequency of resistance to tebuconazole varied according to the assessment method; the resistance frequencies were 18.3% for the relative growth method, 40% for the baseline EC50 method, and 8.33% for the EC50 method segregated by the dose of 0.3 µg mL−1. The application of the EC50 method involving the discriminatory dose of 0.3 µg mL−1 revealed a decrease in the frequency of resistant genotypes. However, the baseline EC50 method is recommended for future resistance surveys because this method was originally used to determine the mean sensitivity prior to the use of tebuconazole in Brazil. Additionally, a shift of the current population toward sensitivity was observed compared with the intermediate population. Finally, São Paulo was found to be the state with the highest EC50 values (0.186 µg mL−1) compared with Paraná (0.053 µg mL−1) and Rio Grande do Sul (0.026 µg mL−1). These results suggest that the build-up of fungicide resistance in Monilinia fructicola has been prevented by the use of anti-resistance strategies in the main peach production areas of Brazil; however, additional studies are required to demonstrate how resistance instability and fitness are affecting isolate sensitivity.

Fungicide resistance: facing the challenge - a review

Fungicide resistance continues to generate disease control problems in many crops. Experience amassed over the past fifty years has emphasised the importance of diversity in modes of action in anti-resistance strategies. Because of losses, not only from resistance, but increasingly from environmental and health concerns, the number of modes of action has become dangerously small. This paper considers three challenges facing crop protection in the search for durable disease control systems. Greater understanding of the biochemistry surrounding fungal development and pathogenicity provides opportunities for the discovery and development of novel modes of action, and some recent advances in this area are discussed. To ensure sufficient resources available to take a novel discovery forward to a commercial product, a second challenge facing manufacturers involves early assessment of resistance risk. A third challenge facing researchers, manufacturers and growers requires translation of resistance risk into effective and durable disease control strategies in actual crops. At the core of this challenge is using resistance risk evidence obtained in laboratory and glasshouse studies using individual isolates of target pathogens, to evaluate the fitness cost of resistance in pathogen populations in field crops. Increasingly, management of resistance is seen as the integration of fungicides with non-chemical disease control methods. But success of any Integrated Disease Management (IDM) strategy ultimately depends on the ability of growers to maintain production and profitability.

Detection of isolates of Alternaria alternata with multiple-resistance to fludioxonil, cyprodinil, boscalid and pyraclostrobin in California pistachio orchards

In this study, 229 isolates of Alternaria alternata collected from commercial pistachio orchards with (n = 198) and without (n = 31) a prior history of Switch (cyprodinil + fludioxonil) applications were examined for their sensitivity to fludioxonil and cyprodinil using in vitro mycelial growth assays. The 50% effective dose (EC50) values for the baseline isolates for cyprodinil and fludioxonil ranged from 0.001 to 1.184 μg/mL (average = 0.214) and from 0.010 to 4.875 μg/mL (average = 0.770), respectively. Out of 72 isolates assayed from the exposed population, 93% had EC50 values for fludioxonil ranging from 0.001 to 5.370 μg/mL (average = 0.472), but five isolates were highly resistant to this fungicide (EC50 > 100 μg/mL). About 98.55% of these isolates were sensitive to cyprodinil (0009 < EC50 < 0.415 μg/mL; average = 0.098). Only one isolate was resistant to cyprodinil (EC50 > 10 μg/mL). No cross-resistance relationship between cyprodinil and fludioxonil was observed (P = 0.2925, r = −0.1074). To determine the occurrence of multiple resistances, the radial growths of 126 isolates, collected from orchards with a history of Switch, Pristine, and strobilurin exposures, were measured on agar media amended with a single discriminatory dose of 10 μg fungicide/mL. High proportions of these isolates were resistant to boscalid (86.3%) and pyraclostrobin (92.9%). Most isolates were still sensitive to fludioxonil (77.8%) and cyprodinil (89.7%), but some were found to be independently or simultaneously resistant to all fungicides tested. Under conditions of excessive exposure to Switch resistant phenotypes may develop in A. alternata populations and ultimately affect fungicide performance. Therefore, monitoring studies must be continued and strict adherence to anti-resistance strategies followed and implemented in at risk orchards.

A pyrosequencing-based method to quantify genetic substitutions associated with resistance to succinate dehydrogenase inhibitor fungicides in Botrytis spp. populations

The genetic underlying resistance mechanisms in the population of the phytopathogenic fungus Botrytis cinerea are well documented. Specifically, several genetic substitutions associated with succinate dehydrogenase inhibitor (SDHI)-based fungicide resistance have been identified in the succinate dehydrogenase gene. The objective of the present work was to develop a molecular tool for accurate quantification of these genetic substitutions within Botrytis populations. A test using the PyroMark Q24 instrument was designed to detect and quantify five genetic substitutions associated with SDHI resistance. The technique is based on sequencing by synthesis, and it generated quantitative and accurate data with a limit of quantification of a minimum of 500 spores. There was a linear relationship between the known and estimated percentages of spores with the targeted genetic substitutions and wild-type strains at ratios of 0-100%, with a 20% increment. With the pyrosequencing assay developed in this study, a large number of Botrytis spp. individuals can be characterised in a timely fashion with greater accuracy than by commonly used methods. Hence, pyrosequencing-based methods will be useful for improving our understanding of fungicide resistance, detecting the arrival of new genetic substitutions, monitoring shifts in fungal populations and assessing the effectiveness of antiresistance strategies, and for routine monitoring of fungicide resistance.

Fitness and cross-resistance of Alternaria alternata field isolates with specific or multiple resistance to single site inhibitors and mancozeb

42 field isolates of Alternaria alternata collected from infected tomato fruit of processing plants located in the region of Pelloponisos, South Greece, were characterized in terms of sensitivity to mancozeb, tebuconazole, fludioxonil, iprodione and cyprodinil. Fungitoxicity tests in vitro revealed that 63 % of the isolates tested were sensitive to all the above fungicides whereas the remainder of the isolates exhibited reduced sensitivity to one or more of these fungicides. The observed resistance phenotypes were confirmed by in vivo experiments on artificially inoculated tomato fruit. EC50 in vitro values ranged from 2.34 to >100 μg/ml for mancozeb, 0.16 to >10 μg/ml for iprodione, 0.43 to 20 μg/ml for tebuconazole, 0.007 to >5 μg/ml for cyprodinil and from 0.002 to >10 μg/ml for fludioxonil. Sensitivity distributions of A. alternata isolates to mancozeb, iprodione, cyprodinil and fludioxonil was bimodal indicating a probable initiation of a discrete shift of the population towards reduced sensitivity to these fungicides. Correlation analysis of respective EC50 values showed positive cross-resistance relationships between fludioxonil and iprodione while positive correlation was observed between mancozeb and cyprodinil, mancozeb and tebuconazole, and cyprodinil and tebuconazole. Interestingly, a number of A. alternata isolates were specifically resistant to the multi-site inhibitor mancozeb, a fact reported for the first time. Isolates resistant to mancozeb were more aggressive while those with reduced sensitivity to both fludioxonil and iprodione produced significantly less conidia than sensitive ones. A considerable portion of the sample exhibited reduced sensitivity and no apparent fitness penalties, a fact that should be considered in future planning and implementation of anti-resistance strategies.

Developments in Integrated Pest Management in Italy.

In order to implement Directive 2009/128/EC and to reduce the risks arising from the use of plant protection products, a National Action Plan has been developed in Italy. The plan calls for several actions, including low pesticide-input pest management. Each producer will have to consider the many aspects that contribute to the production process, such as appropriate cultivation techniques, use of certified propagating material and balanced fertilization. Producers will also need tools to protect beneficial organisms, prevent the spread of harmful organisms, implement monitoring and alert systems, define thresholds and establish anti-resistance strategies. Advanced knowledge about plant protection products will also be required of professional users, with the introduction of specific certified training programmes; only authorized users will be allowed to purchase plant protection products classified for professional use. The spraying equipment will have to pass functional tests. Italy has articulated Integrated Pest Management (IPM) on two levels: one mandatory and one voluntary. The latter will be supported by specific funding, in line with what has already been implemented as part of the Common Agricultural Policy. With regard to the voluntary level, the enforcement of national IPM guidelines (concerning defence and weeding technical standards) that have so far characterized the application of IPM in Italy will serve as the medium-term reference tool. This paper demonstrates how IPM has evolved in Italy over the last 20 years, and why Italian producers have already been applying the requirements of Directive 2009/128/EC for some time.

Sensitivity to cymoxanil in Italian populations of Plasmopara viticola oospores.

The level of sensibility towards cymoxanil was quantified by oospore germination assays in 28 populations of Plasmopara viticola (Berk. et Curt.) Berlese and De Toni sampled from different Italian regions from 2009 to 2012. The populations showed good sensitivity levels, with EC50 values often lower than 10 mg AI L(-1) and percentages of resistant individuals lower than 16%. Only three populations, sampled at the end of the 2012 grapevine growing season, were characterised by high resistance levels. Field trials carried out in two of these vineyards showed that, at the beginning of the 2013 grapevine growing season, the EC50 values of P. viticola populations as measured in the sporangial assay were higher than those observed with oospores. At the end of the season, in plots where cymoxanil was not applied, the populations fully reverted to sensitivity, while the EC50 values remained high where 3-6 applications were performed. Oospore germination assays provide valuable information on the sensitivity of populations in vineyards also at the quantitative level. The results obtained during the grapevine growing season confirm those obtained on the oospores, and that cymoxanil resistance is unstable, indirectly suggesting that the application of the fungicide according to antiresistance strategies can lead to good disease control.