Simona Marianna Sanzani

Study on the role of patulin on pathogenicity and virulence of Penicillium expansum

Although the antibacterial activity and toxicity to humans and animals of the mycotoxin patulin are well known, its role in the postharvest decay of apples by Penicillium expansum has never been investigated. In the present study the gene disruption technique was used to alter the sequence of 6-methyl-salicylic acid synthase, an enzyme involved in the first committed step of patulin biosynthesis. Thirty-nine mutants were obtained, however only two of them (M5 and M21) passed the sub-cultural and molecular confirmation tests. They proved to produce 33-41% less patulin than their wild-type (WT) strain, although no difference in the growth and morphology of the colony was observed. Moreover, the mutants showed a significantly reduced pathogenicity and virulence on artificially inoculated apples. In particular, a 33-34% and 47-54% reduction of disease incidence and severity were recorded for M5 and M21, respectively. As confirmation, when the biomass of the mutants was quantified in vivo by Real-time PCR, a significant difference was recorded as compared to the WT and even between mutants. Moreover, when patulin production potential of mutants was restored by exogenous application of the mycotoxin, their ability to cause the disease was not significantly different from that of WT. Finally, mutants showed an increased susceptibility to the application of the antioxidant quercetin, their pathogenicity and virulence being significantly reduced at only 1/100 of the concentration needed for the WT. Based on these findings, patulin seems to have a role in the development of blue mold decay on apples.

DEVELOPMENT OF QUANTITATIVE PCR DETECTION METHODS FOR PHYTOPATHOGENIC FUNGI AND OOMYCETES

SUMMARY In recent years quantitative PCR (qPCR) detection methods have been widely utilised to detect phytopathogenic fungi and oomycetes and have greatly contributed to the advancement of knowledge in plant pathology. However, major drawbacks and common errors, most typical of earlier reports, still affect many methods currently available in the literature. Errors can be made throughout the entire process for the development of qPCR methods, at the level of selection of appropriate DNA extraction and purification protocols, identification of suitable target regions, choice of the chemistry, design and validation of specific primers and probes, analysis of sensitivity, choice of an absolute and/or relative quantification approach and analysis of the risk of detecting target DNA from dead sources. In the present review the above mentioned steps are analysed, highlighting their critical aspects and providing a practical guide for the users.

Effect of quercetin and umbelliferone on the transcript level of Penicillium expansum genes involved in patulin biosynthesis

Penicillium expansum is commonly associated with patulin accumulation in pome fruits. In in vitro studies, two phenolic compounds (quercetin and umbelliferone) proved to be effective in reducing patulin accumulation, particularly when applied in combination, without consistently affecting mycelial growth. To investigate the mode of action of quercetin and umbelliferone, the expression of five genes likely involved in patulin biosynthesis was evaluated using real-time PCR in the presence and absence of the tested phenolic compounds. The relative expression of genes coding isoepoxydon dehydrogenase (IDH), 6-methylsalicylic acid synthase (msas) and an ATP-binding cassette transporter (peab1) proved to be down-regulated when quercetin and umbelliferone were added in combination. Furthermore, the relative expression of two putative cytochrome P450 monooxygenases (p450-1 and p450-2) was reduced by all treatments, although the combination of the two substances was the most effective. These results provide evidence that quercetin and umbelliferone reduce patulin accumulation by acting on the transcription level of the tested genes.

Genetic structure and natural variation associated with host of origin in Penicillium expansum strains causing blue mould.

Blue mould, caused by Penicillium expansum, is one of the most economically damaging postharvest diseases of pome fruits, although it may affect a wider host range, including sweet cherries and table grapes. Several reports on the role of mycotoxins in plant pathogenesis have been published, but few focussed on the influence of mycotoxins on the variation in host preference amongst producing fungi. In the present study the influence of the host on P. expansum pathogenicity/virulence was investigated, focussing mainly on the relationship with patulin production. Three P. expansum strain groups, originating from apples, sweet cherries, and table grapes (7 strains per host) were grown on their hosts of isolation and on artificial media derived from them. Strains within each P. expansum group proved to be more aggressive and produced more patulin than the other two groups under evaluation when grown on the host from which they originated. Table grape strains were the most aggressive (81% disease incidence) and strongest patulin producers (up to 554μg/g). The difference in aggressiveness amongst strains was appreciable only in the presence of a living host, suggesting that the complex pathogen-host interaction significantly influenced the ability of P. expansum to cause the disease. Incidence/severity of the disease and patulin production proved to be positively correlated, supporting the role of patulin as virulence/pathogenicity factor. The existence of genetic variation amongst isolates was confirmed by the High Resolution Melting method that was set up herein, which permitted discrimination of P. expansum from other species (P. chrysogenum and P. crustosum) and, within the same species, amongst the host of origin. Host effect on toxin production appeared to be exerted at a transcriptional level.

Metabarcoding Analysis of Phytophthora Diversity Using Genus-Specific Primers and 454 Pyrosequencing

A metabarcoding method based on genus-specific primers and 454 pyrosequencing was utilized to investigate the genetic diversity of Phytophthora spp. in soil and root samples of potted plants, from eight nurseries. Pyrosequencing enabled the detection of 25 Phytophthora phylotypes distributed in seven different clades and provided a much higher resolution than a corresponding cloning/Sanger sequencing approach. Eleven of these phylotypes, including P. cactorum, P. citricola s.str., P. palmivora, P. palmivora-like, P. megasperma or P. gonapodyides, P. ramorum, and five putative new Phytophthora species phylogenetically related to clades 1, 2, 4, 6, and 7 were detected only with the 454 pyrosequencing approach. We also found an additional 18 novel records of a phylotype in a particular nursery that were not detected with cloning/Sanger sequencing. Several aspects confirmed the reliability of the method: (i) many identical sequence types were identified independently in different nurseries, (ii) most sequence types identified with 454 pyrosequencing were identical to those from the cloning/Sanger sequencing approach and/or perfectly matched GenBank deposited sequences, and (iii) the divergence noted between sequence types of putative new Phytophthora species and all other detected sequences was sufficient to rule out sequencing errors. The proposed method represents a powerful tool to study Phytophthora diversity providing that particular attention is paid to the analysis of 454 pyrosequencing raw read sequences and to the identification of sequence types.

Effectiveness of Phenolic Compounds against Citrus Green Mould

Stored citrus fruit suffer huge losses because of the development of green mould caused by Penicillium digitatum. Usually synthetic fungicides are employed to control this disease, but their use is facing some obstacles, such public concern about possible adverse effects on human and environmental health and the development of resistant pathogen populations. In the present study quercetin, scopoletin and scoparone—phenolic compounds present in several agricultural commodities and associated with response to stresses—were firstly tested in vitro against P. digitatum and then applied in vivo on oranges cv. Navelina. Fruits were wound-treated (100 µg), pathogen-inoculated, stored and surveyed for disease incidence and severity. Although only a minor (≤13%) control effect on P. digitatum growth was recorded in vitro, the in vivo trial results were encouraging. In fact, on phenolic-treated oranges, symptoms appeared at 6 days post-inoculation (DPI), i.e., with a 2 day-delay as compared to the untreated control. Moreover, at 8 DPI, quercetin, scopoletin, and scoparone significantly reduced disease incidence and severity by 69%–40% and 85%–70%, respectively, as compared to the control. At 14 DPI, scoparone was the most active molecule. Based on the results, these compounds might represent an interesting alternative to synthetic fungicides.

Detection of Ochratoxin a Using Molecular Beacons and Real-Time PCR Thermal Cycler

We developed a simple and cheap assay for quantitatively detecting ochratoxin A (OTA) in wine. A DNA aptamer available in literature was used as recognition probe in its molecular beacon form, i.e., with a fluorescence-quenching pair at the stem ends. Our aptabeacon could adopt a conformation allowing OTA binding, causing a fluorescence rise due to the increased distance between fluorophore and quencher. We used real-time PCR equipment for capturing the signal. With this assay, under optimized conditions, the entire process can be completed within 1 h. In addition, the proposed system exhibited a good selectivity for OTA against other mycotoxins (ochratoxin B and aflatoxin M1) and limited interference from aflatoxin B1 and patulin. A wide linear detection range (0.2–2000 µM) was achieved, with LOD = 13 nM, r = 0.9952, and R2 = 0.9904. The aptabeacon was also applied to detect OTA in red wine spiked with the same dilution series. A linear correlation with a LOD = 19 nM, r = 0.9843, and R2 = 0.9708 was observed, with recoveries in the range 63%–105%. Intra- and inter-day assays confirmed its reproducibility. The proposed biosensor, although still being finalized, might significantly facilitate the quantitative detection of OTA in wine samples, thus improving their quality control from a food safety perspective.

Soybean and casein hydrolysates induce grapevine immune responses and resistance against Plasmopara viticola

Plasmopara viticola, the causal agent of grapevine downy mildew, is one of the most devastating grape pathogen in Europe and North America. Although phytochemicals are used to control pathogen infections, the appearance of resistant strains and the concern for possible adverse effects on environment and human health are increasing the search for alternative strategies. In the present investigation, we successfully tested two protein hydrolysates from soybean (soy) and casein (cas) to trigger grapevine resistance against P. viticola. On Vitis vinifera cv. Marselan plants, the application of soy and cas reduced the infected leaf surface by 76 and 63%, as compared to the control, respectively. Since both hydrolysates might trigger the plant immunity, we investigated their ability to elicit grapevine defense responses. On grapevine cell suspensions, a different free cytosolic calcium signature was recorded for each hydrolysate, whereas a similar transient phosphorylation of two MAP kinases of 45 and 49 kDa was observed. These signaling events were followed by transcriptome reprogramming, including the up-regulation of defense genes encoding pathogenesis-related (PR) proteins and the stilbene synthase enzyme responsible for the biosynthesis of resveratrol, the main grapevine phytoalexin. Liquid chromatography analyses confirmed the production of resveratrol and its dimer metabolites, δ- and ε-viniferins. Overall, soy effects were more pronounced as compared to the cas ones. Both hydrolysates proved to act as elicitors to enhance grapevine immunity against pathogen attack.

Abiotic diseases of olive

SUMMARY Several non-parasitic diseases are of primary significance for olive trees growing in the Mediterranean area and other warm-temperate regions of the world. As for other crops, they are generally poorly understood and often completely unrecognized, especially in olive which is considered a crop of marginal lands. The origin may rest with a lack/excess of essential nutrients or an excess of non-essential elements; it may be an unsatisfactory environment: too cold or hot, too wet or dry, or too windy; there may be unsuitable soil characteristics such as poor physical condition, water-logging, salinity, improper pH, and so on. Also pollution in the environment, spray and fire damage, and climatic extremes like lightning, hail, and snow can cause heavy losses to olive. This review focuses on the most important disorders of olive caused by environmental, physical, and chemical stresses that can affect the normal physiological processes in trees. An integrated view is utilized to highlight interactions with plant healthiness, production, and quality.

Characterization of Citrus-Associated Alternaria Species in Mediterranean Areas

Alternaria brown spot is one of the most important diseases of tangerines and their hybrids worldwide. Recently, outbreaks in Mediterranean areas related to susceptible cultivars, refocused attention on the disease. Twenty representatives were selected from a collection of 180 isolates of Alternaria spp. from citrus leaves and fruit. They were characterized along with reference strains of Alternaria spp. Micro- and macroscopic characteristics separated most Alternaria isolates into six morphotypes referable to A. alternata (5) and A. arborescens (1). Phylogenetic analyses, based on endopolygalacturonase (endopg) and internal transcribed spacer (ITS), confirmed this finding. Moreover, a five-gene phylogeny including two anonymous genomics regions (OPA 1–3 and OPA 2–1), and the beta-tubulin gene (ß-tub), produced a further clustering of A. alternata into three clades. This analysis suggested the existence of intra-species molecular variability. Investigated isolates showed different levels of virulence on leaves and fruit. In particular, the pathogenicity on fruit seemed to be correlated with the tissue of isolation and the clade. The toxigenic behavior of Alternaria isolates was also investigated, with tenuazonic acid (TeA) being the most abundant mycotoxin (0.2–20 mg/L). Isolates also synthesized the mycotoxins alternariol (AOH), its derivate alternariol monomethyl ether (AME), and altenuene (ALT), although to a lesser extent. AME production significantly varied among the six morphotypes. The expression of pksJ/pksH, biosynthetic genes of AOH/AME, was not correlated with actual toxin production, but it was significantly different between the two genotypes and among the four clades. Finally, ten isolates proved to express the biosynthetic genes of ACTT1 phytotoxin, and thus to be included in the Alternaria pathotype tangerine. A significant correlation between pathogenicity on leaves and ACTT1 gene expression was recorded. The latter was significantly dependent on geographical origin. The widespread occurrence of Alternaria spp. on citrus fruit and their ability to produce mycotoxins might represent a serious concern for producers and consumers.