Menadione-induced oxidative stress re-shapes the oxylipin profile of Aspergillus flavus and its lifestyle

Aspergillus flavus is an efficient producer of mycotoxins, particularly aflatoxin B1, probably the most hepatocarcinogenic naturally-occurring compound. Although the inducing agents of toxin synthesis are not unanimously identified, there is evidence that oxidative stress is one of the main actors in play. In our study, we use menadione, a quinone extensively implemented in studies on ROS response in animal cells, for causing stress to A. flavus. For uncovering the molecular determinants that drive A. flavus in challenging oxidative stress conditions, we have evaluated a wide spectrum of several different parameters, ranging from metabolic (ROS and oxylipin profile) to transcriptional analysis (RNA-seq). There emerges a scenario in which A. flavus activates several metabolic processes under oxidative stress conditions for limiting the ROS-associated detrimental effects, as well as for triggering adaptive and escape strategies

Fungal pathogens associated with harvested table grapes in Lebanon, and characterization of the mycotoxigenic genera

Table grapes are exposed to fungal infections before and after harvest. In particular, Aspergillus, Penicillium, and Alternaria can cause decays and contamination by mycotoxins. The main fungi affecting Lebanese table grapes after harvest were assessed as epiphytic populations, latent infections, and rots. Effects of storage with and without SO2 generating pads were also evaluated. Representative isolates of toxigenic genera were characterised, and their genetic potential to produce ochratoxin A, fumonisins, and patulin was established. The epiphytic populations mainly included wound pathogens (Aspergillus spp. and Penicillium spp.), while latent infections and rots were mostly caused by Botrytis spp. The use of SO2 generating pads reduced the epiphytic populations and rots, but was less effective against latent infections. Characterization of Aspergillus, Penicillium, and Alternaria isolates showed that A. tubingensis, P. glabrum, and A. alternata were the most common species. Strains of A. welwitschiae and P. expansum were also found to be genetically able to produce, respectively, ochratoxin A plus fumonisins and patulin. These data demonstrate the need for effective measures to prevent postharvest losses caused by toxigenic fung

Postharvest Diseases of Pomegranate: Alternative Control Means and a Spiderweb Effect

The pomegranate is a fruit known since ancient times for its beneficial properties. It has recently aroused great interest in the industry and among consumers, leading to a significant increase in demand. Consequently, its cultivation has been boosted all over the world. The pomegranate crop suffers considerable yield losses, especially at the postharvest stage, because it is a “minor crop” with few permitted control means. To control latent (Alternaria spp., Botrytis spp., Coniella spp., Colletotrichum spp., and Cytospora spp.) and wound (Aspergillus spp., Penicillium spp., and Talaromyces spp.) fungal pathogens, different alternative compounds, previously evaluated in vitro, were tested in the field on pomegranate cv. Wonderful. A chitosan solution, a plant protein hydrolysate, and a red seaweed extract were compared with a chemical control treatment, all as preharvest (field application) and postharvest treatments and their combinations. At the end of the storage period, the incidence of stamen infections and external and internal rots, and the severity of internal decay were evaluated. Obtained data revealed that pre- and postharvest application of all substances reduced the epiphytic population on stamens. Preharvest applications of seaweed extract and plant hydrolysate were the most effective treatments to reduce the severity of internal pomegranate decays. Furthermore, the influence of spider (Cheiracanthium mildei) cocoons on the fruit calyx as a possible barrier against postharvest fungal pathogens was assessed in a ‘Mollar de Elche’ pomegranate organic orchard. Compared to no-cocoon fruit (control), the incidence of infected stamens and internal molds in those with spiderwebs was reduced by about 30%, and the mean severity of internal rots was halved. Spiderwebs analyzed via Scanning Electron Microscopy (SEM) disclosed a layered, unordered structure that did not allow for the passage of fungal spores due to its mean mesh size (1 to 20 µm ca). The aims of this research were (I) to evaluate alternative compounds useful to control postharvest pomegranate decays and (II) to evaluate the effectiveness of spiders in reducing postharvest fungal infections by analyzing related mechanisms of action. Alternative control means proposed in the present work and calyx spider colonization may be helpful to reduce postharvest pomegranate diseases, yield losses, and waste production in an integrated control strategy, satisfying organic agriculture and the planned goals of Zero Hunger Challenge launched by the United Nations

Olive leachates affect germination of Colletotrichum godetiae conidia and the development of appressoria

The effects of nutrients and microorganisms from olive carpospheres on germination of conidia and the development of appressoria of Colletotrichum godetiae were investigated. The final germination ratio was the result of a dynamic equilibrium between the positive action of nutrients and the negative competition of indigenous microorganisms. In contrast, formation of appressoria was greatly increased by microorganisms and reduced by nutrients. Removal of the microbial fraction from olive leachates rich in natural nutrients amplified the vegetative phase after conidium germination, resulting in increased germtube length, and delayed and reduced production of appressoria. Three exogenous nutrients (sucrose, asparagine and glycine) increased germination of conidia and reduced the formation of appressoria. These results provide evidence that nutrients and microorganisms of the olive carposphere play important roles in the infection processes of C. godetiae. Since appressoria are necessary for successful host infection, microorganisms may favour the penetration of C. godetiae by stimulating the production of appressoria, reducing saprophyitic pre-infectional mycelial growth and reducing duration of the critical moist period required for host penetration

Mycotoxins as host tissue colonization factors

The biological role of mycotoxins remains unclear. It is believed that producing fungi might be better protected against organisms sharing the same trophic niche. However, some mycotoxins seemed to enhance fungal aggressiveness during host exploitation. For example, Alternaria alternata produces many toxic secondary metabolites, of which the most relevant is alternariol (AOH) and its derivative monomethyl ether (AME). Recently, the central role of the polyketide synthase gene pksI for the biosynthesis of AOH and AME has been established (Wenderoth et al., 2019, Mol. Microbiol. 112, 131-146). Moreover, the pksI-deleted Alternaria strain displayed reduced virulence on tomato, citrus and apple, suggesting AOH as virulence and colonization factor. A polyketide synthase is also the first step of the biosynthesis of the mycotoxin patulin, mainly associated to Penicillium expansum. The disruption of the 6-methyl-salicylic acid synthase allowed to obtain mutants that produced less patulin than their wild-type (WT) strain and showed a significantly reduced virulence on apples (Sanzani et al., 2012, Int. J. Food Microbiol. 153, 323-331). Moreover, when patulin was exogenously restored, mutants recovered their virulence as compared to that of the WT. Finally, mutants were susceptible to the antioxidant quercetin at 1/100 of the concentration needed for the WT. Finally, the quinone menadione was used as stressing agent for uncovering the molecular determinants driving Aspergillus flavus in challenging oxidative stress conditions by the host (Zaccaria et al., 2015, Toxins 7, 4315- 4329). Metabolic and transcriptional analyses were conducted. Under oxidative stress conditions, A. flavus proved to activate several metabolic processes for limiting the ROS associated detrimental effects, as well as for triggering adaptive and escape strategies, including aflatoxin B1 production. The results reported herein encourage investigation of mycotoxins from a plant pathologist perspectiv

First report of Stemphylium eturmiunum causing postharvest rot of sweet cherry in Italy

Italy is the first producing country of sweet cherries in Europe. In June 2019, sweet cherry fruit showing rot symptoms were collected from retailers located in Apulia (Southern Italy). Marginal pieces of rotted tissue of surface-sterilized fruit were plated onto semi-selective PDA medium. Colonies were further purified and grown on Potato Carrot Agar (PCA), looking initially whitish and then turning pale olive green to light brown. Conidia were septate and broadly ovoid or ellipsoid. Based on its micro- and macro-morphological features, the pathogen was identified as Stemphylium eturmiunum E.G. Simmons. A multi-locus approach was applied to confirm the identification. ITS-rDNA region and portion of genes coding for the glyceraldehyde-3-phosphate dehydrogenase and the calmodulin were sequenced. Both nBLAST homology and phylogenetic analysis confirmed the pathogen as S. eturmiunum. To fulfil Koch's postulates, surface-sterilized sweet cherry fruit, cv. Ferrovia, were inoculated with the strain. Typical disease symptoms were recorded after 7 days and the pathogen's identity was confirmed by re-isolation and characterization. Because of the resemblance with the symptoms caused by Alternaria spp., reasonably the incidence of S. eturmiunum infections might have been underestimated. This is the first report of S. eturmiunum as causal agent of postharvest rot of sweet cherries in Italy. © 2020 Elsevier Lt

Evaluation of alternative means to control postharvest Rhizopus rot of peaches

The objective of the present research was to test the activity of calcium chloride and lemongrass oil, alone or in combination, against Rhizopus stolonifer on peaches. The inhibitory effect was evaluated both in vitro and in vivo. Results showed that in vitro pathogen growth decreased as treatment's concentration increased, reaching a complete inhibition at 1.5 ml/l and 20 g/l for lemongrass and calcium chloride, respectively. However, taking into account phytotoxicity phenomena, lower concentrations were tested in vivo. A 70% reduction of both rot incidence and severity was achieved using lemongrass oil at 1.5 ml/l; whereas, in presence of CaCl2, the disease reduction was much lower, reaching a maximum at 1.5 g/l of 30 and 59% for incidence and severity, respectively. The combination of the two treatments gave the best performance against rot, and the control effect proved to be synergic as far as disease severity concerns. The observation using scanning electron microscopy (SEM) confirmed the ultra-structure modification in R. stolonifer after treatment. New strategies are needed to reach the critical goal of controlling Rhizopus rot of peaches with no fungicide residues on fruit. In this context, the integration of calcium chloride with lemongrass essential oil might be promising, although further trials are needed

First report of Pilidiella granati causing postharvest fruit rot on pomegranate in southern Italy

In 2015, rot symptoms were observed during storage on pomegranate fruit (Punica granatum L.) cvs Wonderful and Mollar de Elche from a packinghouse in Apulia (Southern Italy). Symptoms, observed on 26% of fruit, consisted of circular brownish-yellow lesions, beginning in the crown area and quickly expanding to entire fruit, with softening of the tissues including arils. Tissue portions were cut from surface-sterilized fruit and incubated on semi-selective PDA at 28±1°C in the dark. Colonies were white to creamy, leathery, and covered by abundant dark-greenish- brown to black spherical pycnidia (80-140 μm in diameter) with thin membranous walls. Hyphae were septate, and conidia hyaline, one-celled, 10-17.5×2-5 μm, ellipsoid to fusiform, straight or slightly curved. These characteristics corresponded to Pilidiella granati (Saccardo) (syn. Coniella granati Sacc.) Petr. & Syd.). For molecular confirmation, fungal DNA was amplified using universal primers ITS5/ITS4. BLAST analysis of the 506 bp amplicon (GenBank accession No. KU821701) showed 100% identity with other P. granati ITS sequences. For pathogenicity tests, surface-sterilized fruit of both cvs were wounded (5×5 mm), inoculated by a mycelial plug and incubated as reported above. Sterile plugs were used as controls. Lesions were visible after five days only on inoculated fruit. The re-isolated fungus corresponded to P. granati, which was reported as pomegranate postharvest rot agent in Spain (Palou et al., 2010) and recently associated with a crown rot in Italy (Pollastro et al., 2016). To our knowledge, this is the first report of P. granati rot on harvested pomegranate fruit in Southern Italy that might represent a serious threat for marketing of this promising crop