Agostino Brunelli

Resistance to Dicarboximide Fungicides in Stemphylium vesicarium of Italian Pear Orchards

Brown spot, caused by Stemphylium vesicarium, is the main fungal disease of pear in northern Italy where it may cause severe crop losses and it requires numerous fungicide applications. Monitoring work was performed by collecting fungal populations in Po valley between 1995 and 2003 in order to study the dicarboximides resistance already detected in the 1990s for procymidone as a result of control failures in field. Sensitivity tests showed that the resistant strains occur all over the monitored areas. Where present the efficacy of procymidone in field is completely lost in spite of what is observed often in other fungi. In most of the isolates (phenotype R1), S. vesicarium resistance level to procymidone (Sialex) was shown to be very high (RF≅3000) whereas it was lower towards the other dicarboximides iprodione (Rovral), vinclozolin (Ronilan) and chlozolinate (Serinal) (RF≅10). Therefore the resistance is partially crossed even if a high level of resistance was rarely observed for all dicarboximides (phenotype R2). At least two different mechanisms of resistance seem to be involved: one that may provide a moderate resistance and the other that may give a high resistance level. Monospore isolate sensitivity tests confirmed the qualitative response suggested by such high resistance factors.

Sensitivity to strobilurin fungicides of Italian Venturia inaequalis populations with different origin and scab control

BACKGROUND Venturia inaequalis (Cooke) Winter with reduced sensitivity to strobilurins has been reported in several countries, including Italy. This study aimed to characterise the sensitivity to strobilurins of three different types of V. inaequalis population: (a) wild types; (b) from commercial orchards satisfactorily managed with strobilurins; (c) from an experimental orchard with control failures by trifloxystrobin and kresoxim-methyl. In vitro sensitivity tests included antigerminative activity on population conidia and mycelial growth inhibition on monoconidial isolates. Cleaved amplified polymorphic sequence (CAPS) analysis was used for the detection of G143A substitution. RESULTS Wild-type populations showed EC(50) values lower than 0.031 mg L(-1), while those of orchards with good performance by strobilurins presented EC(50) values never higher than 0.063 mg L(-1). Samples with scab control failures showed a strongly reduced population sensitivity. Similar differences were confirmed in monoconidial isolates. The G143A substitution was always detected in low-sensitivity populations, only sometimes in well-controlled populations and generally not in wild types. CONCLUSIONS In vitro sensitivity assays were able to discriminate the three population types with different scab management, while the qualitative PCR analysis (CAPS) was only partially reliable. High sensitivity differences among V. inaequalis populations with good and poor field control by strobilurins were observed.

Occurrence in Italy and characteristics of lettuce downy mildew (Bremia lactucae) resistant to phenylamide fungicides

Control of lettuce downy mildew (Bremia lactucae) with phenylamide fungicides has failed in some intensive lettuce-producing areas in Northern Italy since Spring 1993. Before then, these chemicals and particularly metalaxyl, provided the best disease control. The sensitivity of Bremia lactucae isolates collected in such areas to metalaxyl was evaluated in the laboratory. These strains grew and sporulated profusely on lettuce seedlings treated with 100 and 200 ppm of metalaxyl, whereas sensitive control strains were completely inhibited when treated with fungicide concentrations ranging from 0.5–1 ppm. Thus in Italy occurrence of resistance to phenylamide fungicides in Bremia lactucae has also been demonstrated, as in almost all the countries where these chemicals were previously authorised. Subsequently, a demonstration of occurrence of resistance was made and the virulence pattern of several strains (resistant and sensitive to metalaxyl) was characterised using differential NL series containing the 13 DM resistant genes or R factors. The results suggest the occurrence of a new pathotype in Italy different from all the 16 NL Bremia lactucae races studied.

Differences in the efficacy of carboxylic acid amide fungicides against less sensitive strains of Plasmopara viticola.

BACKGROUND Plasmopara viticola is controlled by fungicides with different modes of action, including carboxylic acid amides (CAAs). The aim of this study was to evaluate differences in the response of CAA-resistant P. viticola strains towards CAAs. RESULTS The G1105S mutation affects all four CAAs, but with different impacts. While this confirms that they have the same mode of action, it shows that differences between CAAs can occur. CONCLUSION Further molecular modelling and docking studies are needed to gain a better understanding of the different behaviours reported here.

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 μg/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 μg 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 EC50< 0.01 μg/ml, and the MIC ranged from <0.01 to <1 μg/ml. Isolates from AP in 2012 had elevated EC50 values, from 0.230 to >50 μg/ml, and MIC values from <10 to >50 μg/ml; by 2013, the EC50 values of ER isolates ranged from 3.35 to >50 μg/ml. Based on the mean EC50 value of 0.0019 μg/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. Álvarez and J. A. Torés. 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.

OCCURRENCE OF CHASMOTHECIA AND MATING TYPE DISTRIBUTION OF PODOSPHAERA XANTHII, A CAUSAL AGENT OF CUCURBIT POWDERY MILDEW IN NORTHERN ITALY

Powdery mildew is a widespread disease that causes important losses to cucurbit production. The main agents of the disease are Podosphaera xanthii and Golovinomyces orontii. To determine the occurrence of chasmothecia as overwintering forms of both fungal species in northern Italy, powdery mildew-infected samples from cultivated cucurbits were collected in different locations of the provinces of Bologna and Mantova during 2010, 2011 and 2012. Only the sexual stage of P. xanthii was found, indicating that in northern Italy, contrary to what reported from other areas of the Mediterranean basin, the pathogen overwinters as chasmothecia. In parallel, to determine the frequency and distribution of both MAT 1-1-1 and MAT 1-2-1 P. xanthii idiomorphs, a multiplex- PCR with MAT idiomorph-specific primers was carried out on 147 monoconidial isolates obtained from infected leaf samples. The obtained frequencies were tested for random mating. Results showed a MAT ratio that tended to 1:1, supporting the finding of the sexual stage thus suggesting the occurrence of actively mating populations and that sexual reproduction plays a significant role in the life cycle of P. xanthii in this area. The lack of G. orontii chasmothecia suggests that this species might have alternative overwintering strategies.

New rapid DNA extraction method with Chelex from Venturia inaequalis spores.

The objective of this study was to develop a rapid method to isolate DNA from Venturia inaequalis spores for use in diagnostic DNA mutation analysis. Chelex-100 resin was evaluated and compared with a well established DNA exctraction method, utilizing CTAB in order to have a robust comparison. In this research we demonstrated that Chelex-100 efficiently makes extraction of the DNA from V. inaequalis spores available for direct use in molecular analyses. Also, the quantity and quality of extracted DNA were shown to be adequate for PCR analysis. Comparatively, the quality of DNA samples isolated using Chelex method was better than those extracted using CTAB. In conclusion, the Chelex method is recommended for PCR experiments considering its simplicity and cost-effectiveness.

Genetic diversity analysis of the cucurbit powdery mildew fungus Podosphaera xanthii suggests a clonal population structure.

The sexual stage of Podosphaera xanthii is rarely found worldwide. However, chasmothecia are frequently recorded in northern Italy, suggesting the presence of an actively mating population. With the aim of investigating the genetic structure of the Italian population with respect to populations from other countries, genetic diversity analysis was performed both on 92 isolates from European and American countries by multilocus sequence typing (MLST) and on 59 isolates by amplified fragment length polymorphism (AFLP) methods. Mating type frequencies were tested for random mating and two-locus linkage disequilibrium (LD) analysis was performed. Results showed very low levels of genetic diversity: MLST showed no variations in eight housekeeping gene fragments and, accordingly, UPGMA dendrogram from AFLP data showed a high similarity (0.91-1.00 simple matching similarity coefficient) between isolates. Moreover, the random mating test showed no deviations from mating-type 1:1 ratio in the Italian population but deviations were observed in populations from Europe and American countries while two-locus LD analysis showed the presence of significant LD. The results suggest that the populations of P. xanthii are likely to be predominantly clonal, and asexual reproduction, producing a huge amount of conidia, appears to be the predominant type of reproduction of the species.

Field strains of Stemphylium vesicarium with a resistance to dicarboximide fungicides correlated with changes in a two-component histidine kinase

In Stemphylium vesicarium, four phenotypes were recognized according to their in vitro responses to dicarboximide fungicides: S (sensitive), S+ (low resistant to iprodione and procymidone but moderately resistant to vinclozolin), R1 (moderately resistant to iprodione and vinclozolin but highly resistant to procymidone), R2 (highly resistant to all dicarboximides). Cross-resistance was observed between dicarboximides and aromatic hydrocarbon fungicides in all cases while cross-resistance to phenylpyrroles was only detected in R2 phenotype. Moreover, no changes were noted in sensitivity to oxidative and osmotic stress inducers. An osmosensing histidine kinase gene, homologous to OS1 from Neurospora crassa, was sequenced from several field isolates of Stemphylium vesicarium. This gene is predicted to encode a 1,329 amino acid protein, comprising a conserved histidine-kinase domain in the C-terminal region and six tandem repeats of about 90 amino acids at the N-terminal end. In S+ and R1 phenotype isolates, a single amino acid substitution was observed in the first amino acid repeat; F267L and L290S respectively. For the R2 isolates, the exchanges T765R or Q777R were located within the histidine-kinase domain.

QTLS FOR BROWN SPOT RESISTANCE IN EUROPEAN PEAR

Brown spot (Stemphylium vesicarium) is one of the most serious fungal disease that affects the pear tree orchards in the Po valley. In this region, which produces 90% of the pears in Italy, several antifungal treatments must be repeated along the period between flowering and harvest. In addition, many of the most widely grown varieties of pear (e.g., ‘Abbe Fetel’) are very susceptible to this fungus, while others, such as William and his mutated sports are substantially resistant. With the aim to develop molecular markers linked with brown spot resistance, plants derived from the cross ‘Abbe Fetel’ × ‘Max Red Bartlett’ were challenged for two consecutive years with a suspension of conidia both on leaves and fruits of each seedling. As a control, both the parents of the progeny and the varieties ‘Conference’ and ‘William’ were inoculated. On the genetic maps of the two parents QTL analysis revealed the presence of two QTLs, one for resistance to brown spot in ‘Max Red Bartlett’ and another for the susceptibility in ‘Abbe Fetel’. The availability of molecular markers in the regions of QTLs is an important step forward towards the selection of new pear genotypes resistant to brown spot by Marker-Assisted Selection (MAS).