Silvia Laura Toffolatti

A new approach to modelling the dynamics of oospore germination in Plasmopara viticola

Oospores, the only overwintering structures of Plasmopara viticola, the causal agent of grapevine downy mildew, are the unique source of inoculum for primary infections in vineyards. We show that their germination dynamics depend on both climatic and endogenous factors. In particular, overwintering in controlled conditions suggests that low temperatures prolong the oospore germinability, while constant or gradually alternating water availability increases germination rates. However, wide fluctuations in germination percentage, observed both in naturally overwintered oospores, and under controlled conditions, indicate an important role for endogenous factors in the germination frequency. Ad hoc experimental assays and microscopic observations highlight an important role for calcium in the germination process. However, for a profound understanding of the biological mechanisms underlying oospore germination, mathematical models of the germination dynamics are needed. But, classical differential models of germination dynamics are, with current knowledge, prohibitive due both to the complexity of the underlying processes and knowledge incompleteness. Then, we propose a hybrid method derived from the integration of qualitative differential models and fuzzy systems.

Phenotypic and histochemical traits of the interaction between Plasmopara viticola and resistant or susceptible grapevine varieties

BackgroundGrapevine downy mildew, caused by Plasmopara viticola, is a very serious disease affecting mainly Vitis vinifera cultivated varieties around the world. Breeding for resistance through the crossing with less susceptible species is one of the possible means to reduce the disease incidence and the application of fungicides. The hybrid Bianca and some of its siblings are considered very promising but their resistance level can vary depending on the pathogen strain. Moreover, virulent strains characterized by high fitness can represent a potential threat to the hybrid cultivation.ResultsThe host response and the pathogen virulence were quantitatively assessed by artificially inoculating cv Chardonnay, cv Bianca and their siblings with P. viticola isolates derived from single germinating oospores collected in various Italian viticultural areas. The host phenotypes were classified as susceptible, intermediate and resistant, according to the Area Under the Disease Progress Curve caused by the inoculated strain. Host responses in cv Bianca and its siblings significantly varied depending on the P. viticola isolates, which in turn differed in their virulence levels. The fitness of the most virulent strain did not significantly vary on the different hybrids including Bianca in comparison with the susceptible cv Chardonnay, suggesting that no costs are associated with virulence. Among the individual fitness components, only sporangia production was significantly reduced in cv Bianca and in some hybrids. Comparative histological analysis revealed differences between susceptible and resistant plants in the pathogen diffusion and cytology from 48 h after inoculation onwards. Defence mechanisms included callose depositions in the infected stomata, increase in peroxidase activity, synthesis of phenolic compounds and flavonoids and the necrosis of stomata and cells immediately surrounding the point of invasion and determined alterations in the size of the infected areas and in the number of sporangia differentiated.ConclusionsSome hybrids were able to maintain an intermediate-resistant behaviour even when inoculated with the most virulent strain. Such hybrids should be considered for further field trials.

Evolution of Qol resistance in Plasmopara viticola oospores

QoI resistance in P. viticola was first detected in France and Italy in 1999. Molecular and biological assays have been carried out since 2000 in order to provide reliable methods of detecting and quantifying resistance. Oospores were collected in vineyards located in northern and southern Italy. QoI resistance was evaluated by the germination rate of oospores on azoxystrobin amended medium and the frequency of mutant alleles in the DNA extracted from oospores. Both methods correlated to each other and were used side by side to test QoI resistance. Due to the spontaneous occurrence of the G143A mutation in wild type populations and the immigration from surrounding vineyards, resistance frequencies up to 10% were found in samples collected from vineyards never treated with QoIs. Particularly high values, about 90%, were associated with the application of five to six QoI treatments within the same season, while lower percentages, about 30%, were detected in vineyards treated with QoI used in mixture with fungicides belonging to a different resistance group. A progressive decrease of resistance frequency was observed when QoI applications were reduced in number or completely suspended for at least one season. Therefore, a full recovery of sensitivity may be achieved even in vineyards characterized by high levels of resistance, if particular care is taken during disease control by using QoIs only in mixtures and reducing the number of QoI treatments.

Mating behavior of a Northern Italian population of Fusarium verticillioides associated with maize

Fusarium verticillioides, the most common causal organism of Fusarium stalk and ear rot of maize in Northern Italy, produces important mycotoxins such as fumonisins. Reproductive biology of F. verticillioides has been widely studied in numerous maize growing areas, but up to now no information is available on the mating behavior and genetic structure of this plant pathogen in Italy. Mating type and female fertility distribution and effective population number, Ne, were assessed for a population of 181 F. verticillioides strains isolated from three fields located in Lombardia region (Northern Italy) during 2007-2008 maize growing season. The ratio of MAT-1:MAT-2 was significantly different from the theoretical 1:1 ratio expected in an idealized population in which individuals mate at random. The frequency of hermaphroditic strains was 20 % of the total population. Ne for mating type was 89 % of the count (total population) and the Ne for male or hermaphrodite status was 55 %. The number of isolates that can function as the female parent limited Ne in the examined population. Under equilibrium cycle, assuming that female fertility has been lost due to selection and mutation rate during asexual reproduction, sexual reproduction needed to occur only once per 40 to 118 asexual generations to maintain this level of sexual fertility.

Assessing pigmented pericarp of maize kernels as possible source of resistance to fusarium ear rot, Fusarium spp. infection and fumonisin accumulation.

One of the purposes of maize genetic improvement is the research of genotypes resistant to fusarium ear rot (FER) and fumonisin accumulation. Flavonoids in the pericarp of the kernels are considered particularly able to reduce the fumonisin accumulation (FUM). The aim of this field study was to assess the effect of flavonoids, associated with anti-insect protection and Fusarium verticillioides inoculation, on FER symptoms and fumonisin contamination in maize kernels. Two isogenic hybrids, one having pigmentation in the pericarp (P1-rr) and the other without it (P1-wr), were compared. P1-rr showed lower values of FER symptoms and FUM contamination than P1-wr only if the anti-insect protection and the F. verticillioides inoculations were applied in combination. Fusarium spp. kernel infection was not influenced by the presence of flavonoids in the pericarp. Artificial F. verticillioides inoculation was more effective than anti-insect protection in enhancing the inhibition activity of flavonoids toward FUM contamination. The interactions between FUM contamination levels and FER ratings were better modeled in the pigmented hybrid than in the unpigmented one. The variable role that the pigment played in kernel defense against FER and FUM indicates that flavonoids alone may not be completely effective in the resistance of fumonisin contamination in maize.

Sensitivity to cymoxanil in Italian populations of Plasmopara viticola oospores.

BACKGROUND 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. RESULTS 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. CONCLUSION 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.

Genetic structure and fungicide sensitivity of Botrytis cinerea populations isolated from grapevine in northern Italy

Grey mould, caused by Botrytis cinerea, is a disease severely affecting grape production in northern Italy. However, little information is available on the variability of B. cinerea populations associated with grapevine. The mode of reproduction, sensitivity to fungicides and for the first time in Italy, the genetic structure of B. cinerea populations isolated from grapevine in a northern Italian region are reported. B. cinerea strains (317) were completely genotyped for six microsatellite loci and characterized for the presence of the transposable elements, Boty and Flipper, for the mating type and for resistance to cyprodinil, fludioxonil, boscalid and fenhexamid. All the strains were found to belong to B. cinerea Group II, indicating the absence of B. pseudocinerea in the investigated areas. The populations possess a high genotypic diversity, different frequencies of transposable elements and a mixed mode of reproduction. At a regional level, B. cinerea populations belong to a large and interconnected pathogen population, that includes the major grape-growing districts. The populations were generally sensitive to fungicides, with a low proportion (8 %) of strains resistant to cyprodinil, fludioxonil and boscalid. A small genetic distance was found between B. cinerea populations. However,the populations geographically isolated from the others by a mountain range showed a small but statistically significant genetic differentiation and a different pattern of fungicide resistance. The results show that northern Italian B. cinerea populations possess a high evolutionary potential and adaptive capacity. This article is protected by copyright. All rights reserved.

FIRST REPORT OF FUSARIUM TEMPERATUM CAUSING EAR ROT ON MAIZE IN ITALY

A two-year survey (2011-2012) was conducted to determine the population composition of Fusarium species on maize (Zea mays L.) in Lombardy (northern Italy). Maize ears exhibiting typical symptoms of Fusarium ear rot (FER) (about 30%), such as white to pink colored mycelia on the tip or scattered all over the ear, were collected at harvest. Rotted kernels were surface sterilized, rinsed in sterile water, dried and placed on potato dextrose agar (PDA) amended with dichloran and antibiotics. Developed Fusarium colonies were single-spore purified and grown on Spezieller Nahrstoffarmer agar (Leslie and Summerell, 2006). Based on morphological characteristics, 15 Fusarium spp. strains were identified as Fusarium subglutinans. Colonies on PDA showed a cottony aerial mycelium, initially white, becoming pinkish white. No microconidia chains were detected, conidiophores were erect and terminated in one to three phialides. Macroconidia were falcate, three to five septate, with a slightly beaked apical cell and a barely or distinct footlike basal cell. The translation elongation factor-1α (EF-1α) gene was partially sequenced for all the F. subglutinans strains using primers EF1 and EF2 (Scauflaire et al., 2011). BLASTn analysis showed that the nucleotide sequence (673 bp) of one strain (isolate GV2188, GenBank Accession No. KX156836) shared 100% sequence identity with F. temperatum (HM067689), a recently described species closely related to F. subglutinans (Scauflaire et al., 2011). Using silk channel inoculation method, F. temperatum pathogenicity was assessed on 10 maize ears (LG 32.85 hybrid) under greenhouse conditions. After 30 days, FER symptoms, i.e. whitish pink to lavender fungal growth on kernels, were observed only on inoculated ears and not on water controls. Kochs postulates were fulfilled by re-isolating the fungus from infected kernels. To our knowledge, this is the first report of F. temperatum in Italy associated with FER.

Unique resistance traits against downy mildew from the center of origin of grapevine ( Vitis vinifera )

The Eurasian grapevine (Vitis vinifera), an Old World species now cultivated worldwide for high-quality wine production, is extremely susceptible to the agent of downy mildew, Plasmopara viticola. The cultivation of resistant V. vinifera varieties would be a sustainable way to reduce the damage caused by the pathogen and the impact of disease management, which involves the economic, health and environmental costs of frequent fungicide application. We report the finding of unique downy mildew resistance traits in a winemaking cultivar from the domestication center of V. vinifera, and characterize the expression of a range of genes associated with the resistance mechanism. Based on comparative experimental inoculations, confocal microscopy and transcriptomics analyses, our study shows that V. vinifera cv. Mgaloblishvili, native to Georgia (South Caucasus), exhibits unique resistance traits against P. viticola. Its defense response, leading to a limitation of P. viticola growth and sporulation, is determined by the overexpression of genes related to pathogen recognition, the ethylene signaling pathway, synthesis of antimicrobial compounds and enzymes, and the development of structural barriers. The unique resistant traits found in Mgaloblishvili highlight the presence of a rare defense system in V. vinifera against P. viticola which promises fresh opportunities for grapevine genetic improvement.

A time-course investigation of resistance to the carboxylic acid amide mandipropamid in field populations of Plasmopara viticola treated with anti-resistance strategies: Fungicide resistance and disease epidemics in the field

BACKGROUND 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. RESULTS 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. CONCLUSION 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.