A Real-Time PCR Assay for the Quantification of Plasmopara viticola Oospores in Grapevine Leaves

Grapevine downy mildew caused by Plasmopara viticola is one of the most important diseases in vineyards. Oospores that overwinter in the leaf litter above the soil are the sole source of inoculum for primary infections of P. viticola; in addition to triggering the first infections in the season, the oospores in leaf litter also contribute to disease development during the season. In the current study, a quantitative polymerase chain reaction (qPCR) method that was previously developed to detect P. viticola DNA in fresh grapevine leaves was assessed for its ability to quantify P. viticola oospores in diseased, senescent grapevine leaves. The qPCR assay was specific to P. viticola and sensitive to decreasing amounts of both genomic DNA and numbers of P. viticola oospores used to generate qPCR standard curves. When the qPCR assay was compared to microscope counts of oospores in leaves with different levels of P. viticola infestation, a strong linear relationship (R2 = 0.70) was obtained between the numbers of P. viticola oospores per gram of leaves as determined by qPCR vs. microscopic observation. Unlike microscopic observation, the qPCR assay was able to detect significant differences between leaf samples with a low level of oospore infestation (25% infested leaves and 75% non-infested leaves) vs. samples without infestation, and this ability was not influenced by the weight of the leaf sample. The results indicate that the qPCR method is sensitive and provides reliable estimates of the number of P. viticola oospores in grapevine leaves. Additional research is needed to determine whether the qPCR method is useful for quantifying P. viticola oospores in grapevine leaf litter

Fungal contamination and aflatoxin content of maize, moringa and peanut foods from rural subsistence farms in South Haiti

Mycotoxins are toxic, low molecular weight compounds produced by fungi. Among them, aflatoxins are the most carcinogenic and they mainly impact on rural communities of developing countries. The present study supplies data on mycobiota and aflatoxin contamination in the most common food products consumed in Haiti. The study concerns analyses performed on 49 samples of meals and seeds collected in South Haiti and tested for fungal occurrence and aflatoxin content by HPLC-DAD technique. The results revealed that three main fungal genera affected Haitian food products: Aspergillus spp. (Section Flavi and Nigri), followed by Penicillium spp. and Fusarium spp. Aflatoxin was present in more than half of the samples of maize (Zea mays L.) kernels (55%), maize meal (57%) and moringa (Moringa oleifera Lam.) seeds (64%), and in 25% of peanut (Arachis hypogaea L.) samples. The tested food products were mostly contaminated by aflatoxin B1 (AFB1) followed by aflatoxin B2 (AFB2), while no aflatoxins type G were detected. The total concentration of aflatoxins in the positive samples was 228 mg/kg on average, i.e., fifty-seven and eleven times higher than the maximum levels allowed in Europe and USA, respectively. Both the presence of aflatoxigenic fungi and aflatoxin contamination in maize kernels seemed to be related to agricultural practices, such as weed control, irrigation and growing cycle length. These findings suggest that the Haitian population is strongly exposed to aflatoxin risk. This risk could be reduced by exploiting simple and accessible farming strategies for minimizing mycotoxin contamination, at least for maize

Pathogenicity variation in Fusarium verticillioides populations isolated from maize in northern Italy

One hundred and eighty one strains were selected among Fusarium verticillioides populations isolated from maize samples collected in three fields located in northern Italy. All the isolates were tested for their pathogenicity on maize seeds by assessing the seed germination percentages and the percentage infection indexes concerning seed colonization, radicle decay and coleoptile rot. Fusarium verticillioides strains did not affect seed germination even in presence of high seed colonization, but showed a variable pathogenic behavior according to the maize growth stages. Seedborne F. verticillioides population as well as strains isolated at maturity was effective in seed colonization and in inducing coleoptile rot, not causing however serious radicle decay. Only populations isolated at seedling and pre-silking stages showed high radicle decay ability. These results provide baseline information on F. verticillioides pathogenicity. They constitute an important input for further investigation of F. verticillioides biology in order to define its evolutionary potential

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, N e, 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. N e for mating type was 89 % of the count (total population) and the N e for male or hermaphrodite status was 55 %. The number of isolates that can function as the female parent limited N e 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

Characterization of Botrytis cinerea populations associated with treated and untreated cv. Moscato vineyards

Three Botrytis cinerea populations, isolated from three vineyards, one untreated and two treated twice a year, respectively, with fenhexamid or cyprodinil+fludioxonil, were investigated to evaluate the effect of repeated fungicide treatments on the presence and distribution of the transposons Boty and Flipper, and on the phenotypic traits of each pathogen community. The vacuma individuals lacking the two transposons represented the majority of the 390 B. cinerea isolates followed by transposa strains containing Boty and Flipper, while the remaining 67 isolates harboured respectively only Boty (60) or Flipper (7). This research has demonstrated that fungicide application did not influence the transposon distribution patterns, the sensitivity towards various botryticides, or the growth rate of the isolates belonging to the three different populations, but did induced overall reduction of the population size and selected isolates characterized by an enhanced pathogenicity, especially on Vitis vinifera leaves

Effetto del genotipo e del trattamento antipiralide sulla contaminazione da specie afferenti al clade Gibberella fujikuroi in mais coltivato in Lombardia

Fusarium ear rot (FER) of maize is caused by Fusarium species belonging to Gibberella fujikuroi species complex (GFC) well known as fumonisin (FUM) producers. European corn borer (ECB), the most common maize pest in northern Italy, favors the infection of maize by GFC strains. Since ECB control is not completely effective in lowering fumonisin contamination, additional preventive means, such as increased maize resistance, are required in order to assure a more effective protection. Flavonoids in maize pericarp seem to be able to reduce the fumonisin accumulation. The goal of this study was to assess the effect of flavonoids associated with insecticides on ECB and FER rating under field conditions in Lombardy. Two maize hybrids were sown in 2011 and 2012, one hybrid characterized by the presence of flavonoids in pericarp and the other without pigmentation. The ECB damage incidence, frequency and severity of FER, together with the incidence of GFC strains and FUM in kernels, were assessed in each genotype. In 2012, the ECB control associated with flavonoids presence reduced all the measured parameters except FUM contamination. FUM contamination seemed to be more influenced by the local GFC population and the environment rather than pesticides and pericarp pigmentation

Identification of the First Oomycete Mating-type Locus Sequence in the Grapevine Downy Mildew Pathogen, Plasmopara viticola

Mating types are self-incompatibility systems that promote outcrossing in plants, fungi, and oomycetes. Mating-type genes have been widely studied in plants and fungi but have yet to be identified in oomycetes, eukaryotic organisms closely related to brown algae that cause many destructive animal and plant diseases. We identified the mating-type locus of Plasmopara viticola, the oomycete responsible for grapevine downy mildew, one of the most damaging grapevine diseases worldwide. Using a genome-wide association approach, we identified a 570-kb repeat-rich non-recombining region controlling mating types, with two highly divergent alleles. We showed that one mating type was homozygous, whereas the other was heterozygous at this locus. The mating-type locus encompassed 40 genes, including one encoding a putative hormone receptor. Functional studies will, however, be required to validate the function of these genes and find the actual determinants of mating type. Our findings have fundamental implications for our understanding of the evolution of mating types, as they reveal a unique determinism involving an asymmetry of heterozygosity, as in sex chromosomes and unlike other mating-type systems. This identification of the mating-type locus in such an economically important crop pathogen also has applied implications, as outcrossing facilitates rapid evolution and resistance to harsh environmental conditions

NoPv1: a synthetic antimicrobial peptide aptamer targeting the causal agents of grapevine downy mildew and potato late blight

Grapevine (Vitis vinifera L.) is a crop of major economic importance. However, grapevine yield is guaranteed by the massive use of pesticides to counteract pathogen infections. Under temperate- humid climate conditions, downy mildew is a primary threat for viticulture. Downy mildew is caused by the biotrophic oomycete Plasmopara viticola Berl. & de Toni, which can attack grapevine green tissues. In lack of treatments and with favourable weather conditions, downy mildew can devastate up to 75% of grape cultivation in one season and weaken newly born shoots, causing serious economic losses. Nevertheless, the repeated and massive use of some fungicides can lead to environmental pollution, negative impact on non-targeted organisms, development of resistance, residual toxicity and can foster human health concerns. In this manuscript, we provide an innovative approach to obtain specific pathogen protection for plants. By using the yeast two-hybrid approach and the P. viticola cellulose synthase 2 (PvCesA2), as target enzyme, we screened a combinatorial 8 amino acid peptide library with the aim to identify interacting peptides, potentially able to inhibit PvCesa2. Here, we demonstrate that the NoPv1 peptide aptamer prevents P. viticola germ tube formation and grapevine leaf infection without affecting the growth of non-target organisms and without being toxic for human cells. Furthermore, NoPv1 is also able to counteract Phytophthora infestans growth, the causal agent of late blight in potato and tomato, possibly as a consequence of the high amino acid sequence similarity between P. viticola and P. infestans cellulose synthase enzymes

Not Just a Pathogen? : Description of a Plant-Beneficial Pseudomonas syringae Strain

Plants develop in a microbe-rich environment and must interact with a plethora of microorganisms, both pathogenic and beneficial. Indeed, such is the case of Pseudomonas, and its model organisms P. fluorescens and P. syringae, a bacterial genus that has received particular attention because of its beneficial effect on plants and its pathogenic strains. The present study aims to compare plant-beneficial and pathogenic strains belonging to the P. syringae species to get new insights into the distinction between the two types of plant-microbe interactions. In assays carried out under greenhouse conditions, P. syringae pv. syringae strain 260-02 was shown to promote plant-growth and to exert biocontrol of P. syringae pv. tomato strain DC3000, against the Botrytis cinerea fungus and the Cymbidium Ringspot Virus. This P. syringae strain also had a distinct volatile emission profile, as well as a different plant-colonization pattern, visualized by confocal microscopy and gfp labeled strains, compared to strain DC3000. Despite the different behavior, the P. syringae strain 260-02 showed great similarity to pathogenic strains at a genomic level. However, genome analyses highlighted a few differences that form the basis for the following hypotheses regarding strain 260-02. P. syringae strain 260-02: (i) possesses nonfunctional virulence genes, like the mangotoxin-producing operon Mbo; (ii) has different regulation pathways, suggested by the difference in the autoinducer system and the lack of a virulence activator gene; (iii) has genes encoding DNA methylases different from those found in other P. syringae strains, suggested by the presence of horizontal-gene-transfer-obtained methylases that could affect gene expression