Giovanni Venturini

The proteome of exudates from germinating Lupinus albus seeds is secreted through a selective dual-step process and contains proteins involved in plant defence

The general knowledge of defence activity during the first steps of seed germination is still largely incomplete. The present study focused on the proteins released in the exudates of germinating white lupin seeds. During the first 24 h, a release of proteins was observed. Initially (i.e. during the first 12 h), the proteins found in exudates reflected the composition of the seed, indicating a passive extrusion of pre‐formed proteins. Subsequently, when the rate of protein release was at its highest, the composition of the released proteome changed drastically. This transition occurred in a short time, indicating that more selective and regulated events, such as secretory processes, took place soon after the onset of germination. The present study considered: (a) the characterization of the proteome accumulated in the germinating medium collected after the appearance of the post‐extrusion events; (b) the biosynthetic origin and the modalities that are the basis of protein release outside the seeds; and (c) an assessment of antifungal activity of these exudates. The most represented protein in the exudate was chitinase, which was synthesized de novo. The other proteins are involved in the cellular mechanisms responding to stress events, including biotic ones. This exudate was effectively able to inhibit fungal growth. The results of the present study indicate that seed exudation is a dual‐step process that leads to the secretion of selected proteins and thus is not a result of passive leakage. The released proteome is involved in protecting the spermosphere environment and thus may act as first defence against pathogens.

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.

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.

Competition assays revealed Paenibacillus pasadenensis strain R16 as a novel antifungal agent

The development of new sustainable containment strategies of plant diseases is very important to guarantee food security while reducing the environmental impact of agriculture. Research of new biocontrol agents is a long and difficult process that involves several steps that start from the identification of possible candidates which, for example, show antibiotic activities, and ends with in field, large scale trials. In this study, the plant growth promoting potential and antifungal effect exerted by a novel, putative candidate biocontrol agent, strain R16, identified as Paenibacillus pasadenensis by sequence analysis of 16S rRNA and rpoB genes, against three important plant pathogenic fungi (Botrytis cinerea, Fusarium verticillioides, and Phomopsis viticola), were assessed. Biochemical assays to determine plant growth promoting potential gave negative results for siderophore production and phosphate solubilization, and positive results for ACC-deamination and IAA production. Further biochemical assays for endophytic lifestyle and antifungal activity gave positive results for catalase and chitinase activity, respectively. In vitro antagonism assays showed that strain R16 is effective against B. cinerea, reducing mycelial growth both in dual-culture and through volatile substances, characterized to be mostly composed by farnesol, and inhibiting conidia germination. Good antagonistic potential was also observed in vitro towards P. viticola, but not towards F. verticillioides. Moreover, in vivo assays confirmed the strain R16 activity reduced the infection rate on B. cinerea-inoculated berries. The obtained results firstly proved that P. pasadenesis strain R16 is a putative plant growth promoter and effective against phytopathogenic fungi. Further studies will be needed to investigate the possible application of this strain as a biocontrol agent.

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.