Sensitivity Studies of Plasmopara Viticola to Carboxylic Acid Amide Fungicides: in Vivo Test and Molecular Studies of PvCesA3 Gene

Many oomycetes species are pathogens of plants, animals and humans; some of them are well studied because they cause significant economic losses in agriculture and acquaculture. The cell wall of oomycetes consists mainly of cellulose, β- (1,3) and β- (1,6)- glucan and in some species a small amount of chitin is also present. The biosynthesis of cellulose in oomycetes is still poorly studied and therefore misunderstood, although it’s the target of some fungicides that inhibit this process, such as carboxylic acid amides. Since 2010, single amino acid exchanges in CesA3 protein conferring CAA resistance in Plasmopara viticola have been identified. In this work, we initially provide the first evidence of the presence of mandipropamid resistant populations of Plasmopara viticola in commercial vineyards in Italy (paper I). We continue by studying the different activity of four CAAs fungicides (benthiavalicarb, dimethomorph, iprovalicarb, mandipropamid) toward P. viticola resistant strains (paper II). The results show that the G1105S mutation affects all four CAAs, but its impact is varied. These results confirm that they are cross resistant, although many gaps in the mode of action are still present. In order to confirm our previous findings, we performed a microscopical base method to assess the sensitivity of four CAAs, and preliminary microscopical data confirmed the different activity toward the CAA-resistant and CAA-sensitive populations (chapter ten). Furthermore, we present a study (chapter eleven) in which we randomly selected samples from paper I, in order to test them using a different approach, simulating a scenario more close to the field. With this test, we were able to confirm the data presented in the paper I

Fine tuning of Real Time PCR as a first tool for the detection of G146A substitution in Venturia inaequalis samples

none5noopenCeren Turan, Irene Maja Nanni, Lucia Landi, Alessandro Pirondi, Marina CollinaTuran, Ceren; Maja Nanni, Irene; Landi, Lucia; Pirondi, Alessandro; Collina, Marin

EFFECTIVENESS DIFFERENCES OF CARBOXYLIC ACID AMIDES FUNGICIDES TOWARDS PLASMOPARA VITICOLA POPULATIONS: IN VIVO TESTS AND MOLECULAR STUDIES ON PVCESA3 GENE

Plasmopara viticola is controlled by fungicides with different modes of action,including carboxylic acid amides (CAAs). Dimethomorph was the first CAA introduced in 1988, followed by iprovalicarb, flumorph, benthiavalicarb, mandipropamid, valifenalate and latest pyrimorph in 2010 . The mode of action of CAAs is linked to the inhibition of cellulose synthesis in the Oomycete plant pathogens. The mutations conferring CAA resistance in Plasmopara viticola located on the CesA3 gene are G1105S and G1105V.The aim of this work was to evaluate the activity of dimethomorph and mandipropamid on P. viticola populations. Bioassays on leaf discs, and on seedlings were carried out. In leaf discs assay, CAAs tested on Italian populations showed different level of activity and in particular dimethomorph showed lower levels of EC95 and this behavior was also confirmed from seedling tests. All CAA resistant populations carried the G1105S/V mutations, which were detected by RFLPPCR and qPCR . In order to gain a better understanding of the different behavior and to define the possible different resistant mechanism, molecular modelling and docking studies are ongoing

EFFICACY OF CARBOXYLIC ACID AMIDES (CAA) FUNGICIDES TOWARDS CAA SENSITIVE and CAA RESISTANT PLASMOPARA VITICOLA POPULATIONS: IN VIVO TESTS AND MOLECULAR STUDIES ON PVCESA3 GENE

Plasmopara viticola is controlled by fungicides with different modes of action, including carboxylic acid amides (CAAs). Dimethomorph was the first CAA introduced in 1988, followed by iprovalicarb, flumorph, benthiavalicarb, mandipropamid, valifenalate and latest pyrimorph in 2010 (Gisi et al. 2012). The mode of action of CAA compounds is linked to the inhibition of cellulose synthesis in the Oomycete plant pathogens. The mutations conferring CAA resistance in P. viticola located on the CesA3 gene are G1105S and G1105V.The aim of this work was to evaluate the activity of CAAs on CAA sensitive and CAA resistant isolates of Plasmopara viticola. Bioassays on leaf discs, on detached leaf and on grape plants trials were carried out. In leaf discs assay, CAAs tested on Italian strains showed different level of activity and in particular dimethomorph showed lower levels of EC95. The detached leaf tests showed under preventative conditions that the CAA-sensitive strains were fully controlled by all CAAs, while the CAA-resistant strains were best controlled by dimethomorph (Nanni et al. 2015). Under curative conditions all CAAs showed good activity on the sensitive strains, while the resistant strains were not controlled by any CAA. This finding confirms the cross resistance between CAAs. The good activity of dimethomorph on CAA resistant isolates under preventive conditions was also confirmed in greenhouse tests where whole plants were inoculated. All CAA resistant strains carried the G1105S/V mutations, which were detected by molecular techniques such as pyrosequencing, CAPS-PCR or qPCR. In order to gain a better understanding of the different behaviour among the CAA fungicides, molecular modelling and docking studies are still ongoing

First Report of Resistance to Cyflufenamid in Podosphaera xanthii, Causal Agent of Powdery Mildew, from Melon and Zucchini Fields in Italy

The fungicide cyflufenamid (phenyl-acetamide, Fungicide Resistance Action Committee [FRAC] code U6) was approved for use in Italy in 2011 as Takumi (Certis Europe, Utrecht, The Netherlands) to control Podosphaera xanthii (Castagne) U. Braun. & N. Shishkoff, the main causal agent of cucurbit powdery mildew. Considering that strains of this pathogen have developed resistance to strobilurin (5) and demethylation inhibitor (DMI) (4) fungicides, cyflufenamid represented a viable alternative to control this disease. However, this fungicide is also prone to resistance development as illustrated by resistance of P. xanthii in Japan (3). In the 2012 and 2013 growing seasons, significant declines in cyflufenamid efficacy were observed in two experimental fields in the Apulia (AP) and Emilia-Romagna (ER) regions of Italy on Cucumis melo and Cucurbita pepo, respectively. Takumi had been applied four times at the recommended field rate of 0.15 liter/ha (15 \u3bcg/ml of active ingredient [a.i.]) each growing season since 2010 in each field. Powdery mildew-infected leaf samples were collected in 2012 from both fields (25 isolates from AP and 19 from ER), and from five gardens (one isolate per garden); while in 2013, samples were collected only from the ER field (two polyconidial isolates). Isolates were maintained on detached zucchini cotyledons (1). Sensitivity of the isolates to cyflufenamid was determined by leaf disk bioassays (4) using Takumi at 0.01, 0.1, 1, 10, 20, and 50 \u3bcg a.i./ml. The 50% effective concentration (EC50) and the minimum inhibitory concentration (MIC) values were calculated (2). Isolates collected in ER and the gardens in 2012 all had an EC5050 \u3bcg/ml, and MIC values from 50 \u3bcg/ml; by 2013, the EC50 values of ER isolates ranged from 3.35 to >50 \u3bcg/ml. Based on the mean EC50 value of 0.0019 \u3bcg/ml for sensitive isolates of P. xanthii in Japan (2), isolates from both the ER field and gardens in 2012 were considered sensitive to cyflufenamid. Additionally, EC50 values of AP isolates from 2012 and ER isolates from 2013 were greater than those of sensitive isolates, indicating a shift in sensitivity toward resistance to cyflufenamid (resistance factor >100 [2]). Consequently, poor control of powdery mildew with cyflufenamid applications in the AP and ER trials was most likely a result of fungicide resistance. Isolates from these fields were exposed to selection pressure for fungicide resistance because cyflufenamid was applied more times than permitted in the label instructions. However, control of powdery mildew in 2013 was not as effective as in previous years in commercial fields in AP (C. Dongiovanni, personal communication). This observation, combined with proof of reduced sensitivity of some P. xanthii strains in Italy to cyflufenamid, highlights the need for implementing resistance management strategies to minimize the risk of fungicide resistant strains developing in cucurbit fields. References: (1) B. \uc1lvarez and J. A. Tor\ue9s. Bol. San. Veg. Plagas 23:283, 1997. (2) M. Haramoto et al. J. Pest. Sci. 31:397, 2006. (3) H. Hosokawa et al. Jpn. J. Phytopathol. 72:260, 2006. (4) M. T. McGrath et al. Plant Dis. 80:697, 1996. (5) M. T. McGrath and N. Shishkoff. Plant. Dis. 87:1007, 2003

Screening of sensitivity to mandipropamid of Plasmopara viticola populations from Italian vineyards by molecular and biological methods

We characterized Plasmopara viticola populations utilizing PCRRFLP technique to detect a point mutation known to cause resistance to carboxylic acid amides (CAA) fungicides. Sensitivity of these samples to the mandipropamid fungicide was assayed by a leaf-disc method. In this work, we provide the first evidence about the presence of mandipropamid-resistant populations of P. viticola from commercial vineyards in Italy. Improving the knowledge about development of resistant populations could enhance the current grapevine downy mildew management strategies and minimize the risk of the spread of mandipropamid and other CAA-resistant populations

DIMETHOMORPH ACITIVITY ON DIFFERENT OOMYCETE SPECIES OF ECONOMIC AND VETERINARY INTEREST

The class Oomycota comprises species that are pathogenic to plants and animals, including humans, and are able to cause severe economic losses in agriculture and aquaculture industry worldwide. Phytophthora infestans causes losses in potato and tomato crops for more than 6 billion per year, as well as damage to ecosystems; Plasmopara viticola is responsible for downy mildew, which is one of the most damaging grapevine diseases. The genus Phytium, includes species that are pathogenic to both plants and animals, particularly, Phytium insidiosum has been reported to cause disease in humans and in other mammals. Oomycetes of the order Saprolegniales are widely distributed in freshwater environments, causing infections in different taxa of aquatic animals. Among these, members of the genus Saprolegnia represent a severe problem in freshwater fish farms, where production losses from 10 to 50% are reported. Despite the wide distribution and the impact of oomycetes on economic activities and on animal health, there are no effective available molecules against these agents. Following the current classification of malachite green and formalin among carcinogens, there are limited possibilities to control oomycete infections in aquaculture. The aim of this work was to test in vitro the activity of dimethomorph, a fungicide used in agriculture to control oomycete infections, on different species of Saprolegnia and Pythium isolated from fish and aquatic environment. Although no complete inhibition was observed at tested concentrations (0.1; 1; 5; 10; 50; 100 mg/L) mycelial growth was slowed down at highest concentrations. Furthermore our results suggest a different susceptibility of dimethomorph on the tested oomycete species

SENSITIVITY OF PLASMOPARA VITICOLA POPULATIONS AND PRESENCE OF SPECIFIC AND NON-SPECIFIC RESISTANCE MECHANISMS

The Oomycete Plasmopara viticola is controlled by fungicides with different modes of action, which include cyazofamid and ametoctradin. Both fungicides are inhibitors of the mitochondrial complex III, with different binding behaviour. Cyazofamid1 is classified in the QiI group and ametoctradin2 in the QoSI group. Compounds with a site-specific mode of action, are more prone to provoke resistance due to the occurrence of target site mutations. However, the first identified resistance mechanism for these two modes of action was a non-specific adaptation phenomenon, which affected all the inhibitors of the complex III. This mechanism is the overexpression of the alternative oxidase (AOX), which forms a bypass of the complex III. The aim of this work was to test the sensitivities of P. viticola populations from different North\u2013Italian regions towards cyazofamid and ametoctradin and the underlying resistance mechanisms. Bioassays on leaf discs were carried out on sixty-four populations, and performed with both fungicides and with and without adding propylgallate (PPG), an inhibitor of AOX. From this trial approach it could be concluded if a reduced sensitivity was caused by an AOX overxpression and/or by a specific resistance. Samples with a specific resistance phenotype were analysed for mutations in the cytochrome b gene. In our samples the mutation L201S or an insertion of two amino acids (E203 VE V204) were detected as mechanisms for specific target site resistance to cyazofamid. Overexpression of AOX has also been been found in our samples, which affected the sensitivity to all complex III inhibitors. If the described resistance types lead to fitness penalties needs further investigation

Genome sequence of Stemphylium vesicarium, the causal agent of Brown Spot disease of Pear

Stemphylium vesicarium (Wallr.) E. Simmons is the causal agent of several plant diseases as well Brown Spot of Pear (BSP), which is one of the most economically important fungal diseases in European pear-production areas. Moreover, conidia widespread from plant material infected by the pathogen can trigger respiratory allergy. We were aimed to provide the first available genome sequence of S. vesicarium.The resource is presented accompanied by genome properties.Genomic DNA was extracted from the monoconidial strain 1731a13FI1M3 isolated from pear, then de novo sequenced by shotgun on Illumina Miseq platform v3. 1,127 contigs were assembled and a total assembly length of 38.66 Mb were obtained. The size was similar to those of Stemphylium lycopersici,the only other genome currently available for the Stemphilium genus. Gene prediction resulted in 12,309 putative genes.A primary functional annotation provided information about Orthologous Groups, Gene Ontology terms, KEGG pathways, and SMART/Pfam domains for each group. Furthermore, combined prediction of transmembrane topology and signal peptide were carried out, and genome sequence was also analyzed for the automatic genomic identification and analysis of biosynthetic gene clusters.Among predicted genes it was also possible to identify several orthologues required in other Ascomycota as related to: pathogenesis or full virulence on plants, signal transduction, cell respiration.Furthermore, the annotation was able to detect the orthologues of alt a 1 major and alt a 7 minor A. alternata allergens. The availability of this genome opens a new scenario in the investigation of S.vesicarium lifestyle and molecular plant/pathogen interaction, and could be fundamental to design more effective and sustainable fungicides management strategies to control BSP and other plant diseases. The genome sequence has been deposited in the GenBank under the accession number:QXCR00000000 (BioProject: PRJNA470620, BioSample: SAMN09098503)

Genome sequence resource for Stemphylium vesicarium, causing Brown Spot Disease of Pear

none5noStemphylium vesicarium is the causal agent of several plant diseases as well Brown Spot of Pear (BSP), which is one of the most economically important fungal diseases in European pear-production areas. In addition to the relevance of the economic impact, conidia widespread from plant material infected by the pathogen can trigger respiratory allergy. Here, we report the first genome of a S. vesicarium strain, the 173-1a13FI1M3, isolated from pear and sensitive to the mostly used fungicide classes currently authorized in Europe against BSP. The availability of this draft genome could represent a first important step in understanding the physiology and the infection mechanism of the pathogen. Furthermore, this contribution could be fundamental in order to design more effective and sustainable strategies to control the disease.openKatia Gazzetti, Elena L. Diaconu, Irene M. Nanni, Alessandro Ciriani, Marina CollinaKatia Gazzetti, Elena L. Diaconu, Irene M. Nanni, Alessandro Ciriani, Marina Collin