In the tripartite combination Botrytis cinerea-Arabidopsis-Eurydema oleracea, the fungal pathogen alters the plant-insect interaction via jasmonic acid signalling activation and inducible plant-emitted volatiles.

In ecosystems, plants are continuously challenged by combined stress conditions more than by a single biotic or abiotic factor. Consequently, in recent years studies on plant relationships with multiple stresses have aroused increasing interest. Here, the impact of inoculation with fungal pathogens with different lifestyles on Arabidopsis plants response to the following infestation with the invasive crop pest Eurydema oleracea was investigated. In particular, as fungal pathogens the necrotroph Botrytis cinerea and the biotroph Golovinomyces orontii were used. Plants exposed to B. cinerea, but not to G. orontii, showed reduced herbivore feeding damage. This difference was associated to different hormonal pathways triggered by the pathogens: G. orontii only induced the salicylate-mediated pathway, while B. cinerea stimulated also the jasmonate-dependent signalling, which persisted for a long time providing a long-term defence to further herbivore attack. In particular, the lower susceptibility of B. cinerea-infected Arabidopsis plants to E. oleracea was related to the stimulation of the JA-induced pathway on the production of plant volatile compounds, since treatment with VOCs emitted by B. cinerea inoculated plants inhibited both insect plant choice and feeding damage. These results indicate that necrotrophic plant pathogenic fungi modulate host volatile emission, thus affecting plant response to subsequent insect, thereby increasing the knowledge on tripartite plant-microbe-insect interactions in nature.

Metabolic and transcriptional changes associated with the use of Ascophyllum nodosum extracts as tools to improve the quality of wine grapes (Vitis vinifera cv. Sangiovese) and their tolerance to biotic stress.

BACKGROUND: Recent studies report that Ascophyllum nodosum extracts, once applied on the canopy of different crops, deliver positive effects, increasing yield, inducing tolerance to biotic stress, and improving the quality of products. However, the mechanisms of action are still unclear. In this research, vines subjected to multiple foliar applications of an A. nodosum extract (ANE) at label doses were compared with untreated vines (NTV) in accordance with a comparative approach. The investigation coupled a field experiment with a second trial conducted under semi-controlled conditions, to clarify the mechanisms of action involved. RESULTS: The biostimulant did not affect soluble solids or the acidity of grapes; instead, it improved their anthocyanin and phenolic concentrations and the respective profiles. At the time of harvest, anthocyanin, and phenolic concentration were increased by 10.4% and 14.5%, respectively, when compared to the NTV. These effects correlated with a specific modulation of genes involved in the flavonoid metabolic pathways. Moreover, grapes from ANE vines witnessed a significant reduction in the spreading of gray mold when they were either assessed in field conditions or in vitro, compared to the grapes of NTV vines. This was related to a significant upregulation of the defense-related genes of the plant. CONCLUSIONS: Overall, the results showed that A. nodosum extracts can be valuable tools in viticulture considering the emergence of challenging environmental conditions; hence, the regulation of specific metabolic pathways is the mechanism of action that leads to an increased tolerance of biotic stress and of changes in the content of grape metabolites. © 2019 Society of Chemical Industry.

Marketed Quinoa (Chenopodium quinoa Willd.) Seeds: A Mycotoxin-Free Matrix Contaminated by Mycotoxigenic Fungi.

A total of 25 marketed quinoa seed samples different for origin, farming system and packaging were analyzed for the presence of mycotoxigenic fungi (by isolation both on Potato Dextrose Agar and with the deep-freezing blotter method) and relative contamination by mycotoxins (by LC-MS/MS analysis). Fungal microorganisms, but not mycotoxins, were detected in all the samples, and 25 isolates representative of the mycobiota were obtained. Morphological and molecular characterization and, for some isolates, the in vitro mycotoxigenic profile, allowed the identification of 19 fungal species within five different genera: Alternaria, Aspergillus, Penicillium, Cladosporium and Fusarium. Among the identified species, Alternaria abundans, A. chartarum, A. arborescens, Cladosporium allicinum, C. parasubtilissimum, C. pseudocladosporioides, C. uwebraunianum, Aspergillus jensenii, A. tubingensis, Penicillium dipodomyis, P. verrucosum and P. citreosulfuratum were first reported on quinoa, and Alternaria infectoria and Fusarium oxysporum were first reported on quinoa seeds. The geographical origin, farming system and packaging were showed to affect the amount and type of the isolated fungal species, highlighting that the level of fungal presence and their related secondary metabolites is conditioned by different steps of the quinoa supply chain. However, despite the presence of mycotoxigenic fungi, the marketed quinoa seeds analyzed resulted in being free from mycotoxins.

Phytopathological Threats Associated with Quinoa (Chenopodium quinoa Willd.) Cultivation and Seed Production in an Area of Central Italy.

In 2017, in a new Chenopodium quinoa cultivation area (Central Italy), emergence failures of the Titicaca, Rio Bamba, and Real varieties, whose seeds were obtained the previous year (2016) in the same location, were observed. Moreover, leaf disease symptoms on the Regalona variety, whose seeds came from Chile, were detected. Visual and microscopic analyses showed the presence of browning/necrotic symptoms on the seeds of the three varieties whose emergence in the field had failed. In addition, their in vitro germination rates were strongly compromised. Fusarium spp. was isolated with high incidence from Titicaca, Rio Bamba, and Real seeds. Among the detected Fusarium species, in the phylogenetic analysis, the dominant one clustered in the sub-clade Equiseti of the Fusarium incarnatum-equiseti (FIESC) species complex. Instead, the pathogen associated with Regalona leaf symptoms was identified, by morphological and molecular features, as Peronospora variabilis, the causal agents of downy mildew. This is the first report of both P. variabilis and F. equiseti on C. quinoa in Italy. Species-specific primers also detected P. variabilis in Regalona seeds. These results underline the importance of pathogen monitoring in new quinoa distribution areas, as well as of healthy seed production and import for successful cultivation.

Aspergillus, Penicillium and Cladosporium species associated with dried date fruits collected in the Perugia (Umbria, Central Italy) market.

A total of 20 dried date samples, chosen as representative among those available on the Perugia (Umbria, Central Italy) market, were analyzed for the possible occurrence of fungal species and related contamination by fungal secondary metabolites. Twenty-six isolates, representative of the total mycobiota, were obtained and morphologically identified as belonging to the genera Aspergillus, Penicillium and Cladosporium. Inside each genus, molecular characterization (by partial sequencing of ITS region and/or ß-tubulin and calmodulin regions for Aspergillus and Penicillium isolates or actin region for Cladosporium isolates) and in vitro mycotoxigenic profile characterization (by LC-MS/MS analysis) showed the presence of the following species: A. flavus, A. tubingensis, P. brevicompactum, P. chrysogenum, P. crustosum, P. glabrum, P. solitum, P. venetum, C. cladosporioides, C. limoniforme and C. halotolerans, with A. tubingensis as the prevalent species and P. crustosum, P. solitum, P. venetum and C. limoniforme first reported here on dates. Date packaging and format showed an effect on the incidence of isolated fungi, with the lowest incidence recovered from whole dates and in hermetic bag packaging. These findings can be useful both for dried dates producers and consumers, guiding them towards choices of packaging and format with a lower risk of mycotoxigenic species presence. However, no fungal metabolites were detected in the dried date samples analyzed, which were therefore regarded as safe for human consumption, underlining the absence of correspondence between fungal isolation and mycotoxin contaminations.

An Arabidopsis thaliana leucine-rich repeat protein harbors an adenylyl cyclase catalytic center and affects responses to pathogens.

Adenylyl cyclases (ACs) catalyze the formation of the second messenger cAMP from ATP. Here we report the characterization of an Arabidopsis thaliana leucine-rich repeat (LRR) protein (At3g14460; AtLRRAC1) as an adenylyl cyclase. Using an AC-specific search motif supported by computational assessments of protein models we identify an AC catalytic center within the N-terminus and demonstrate that AtLRRAC1 can generate cAMP in vitro. Knock-out mutants of AtLRRAC1 have compromised immune responses to the biotrophic fungus Golovinomyces orontii and the hemibiotrophic bacteria Pseudomonas syringae, but not against the necrotrophic fungus Botrytis cinerea. These findings are consistent with a role of cAMP-dependent pathways in the defense against biotrophic and hemibiotrophic plant pathogens.

Arabidopsis flower specific defense gene expression patterns affect resistance to pathogens.

We investigated whether the Arabidopsis flower evolved protective measures to increase reproductive success. Firstly, analyses of available transcriptome data show that the most highly expressed transcripts in the closed sepal (stage 12) are enriched in genes with roles in responses to chemical stimuli and cellular metabolic processes. At stage 15, there is enrichment in transcripts with a role in responses to biotic stimuli. Comparative analyses between the sepal and petal in the open flower mark an over-representation of transcripts with a role in responses to stress and catalytic activity. Secondly, the content of the biotic defense-associated phytohormone salicylic acid (SA) in sepals and petals is significantly higher than in leaves. To understand whether the high levels of stress responsive transcripts and the higher SA content affect defense, wild-type plants (Col-0) and transgenic plants defective in SA accumulation (nahG) were challenged with the biotrophic fungus Golovinomyces cichoracearum, the causal agent of powdery mildew, and the necrotrophic fungus Botrytis cinerea. NahG leaves were more sensitive than those of Col-0, suggesting that in leaves SA has a role in the defense against biotrophs. In contrast, sepals and petals of both genotypes were resistant to G. cichoracearum, indicating that in the flower, resistance to the biotrophic pathogen is not critically dependent on SA, but likely dependent on the up-regulation of stress-responsive genes. Since sepals and petals of both genotypes are equally susceptible to B. cinerea, we conclude that neither stress-response genes nor increased SA accumulation offers protection against the necrotrophic pathogen. These results are interpreted in the light of the distinctive role of the flower and we propose that in the early stages, the sepal may act as a chemical defense barrier of the developing reproductive structures against biotrophic pathogens.

Role of pathogen-induced volatiles in the Nicotiana tabacum-Golovinomyces cichoracearum interaction.

Plant injuries activate signal transduction cascades mediated by the plant hormones, which lead to enhanced expression of defence related genes and/or to changes in the emission of volatile organic compounds that can act as semiochemicals. In this research we demostrated that infection with the biotrophic pathogen Golovinomyces cichoracearum (DC.) V.P. Heluta (ex Erysiphe cichoracearum DC.), the causal agent of powdery mildew, led in the susceptible host Nicotiana tabacum L. cv Havana 425 to an increased emission of volatile compounds including Methyl-jasmonate (MeJA), (E)-2-hexenal and (E)-ß-ocimene. Furthermore we investigated the role of these volatiles in the plant-pathogen interaction. Exogenous application of MeJA induced in tobacco an increase in the transcripts level of the defence related genes lipoxygenase, allene oxide cyclase and defensin and a decrease in the severity of the infection. Qualitative and quantitative differences in volatile compounds emission were showed also in MeJA-treated plants, where the emission of (E)-ß-ocimene was significantly increased instead (E)-2-hexenal was not detected. Application of (E)-2-hexenal reduced the severity of powdery mildew while application of (E)-ß-ocimene did not. Since (E)-2-hexenal did not activate in tobacco the accumulation of the above reported genes transcripts and the plant cell death, the reduction of the infection severity could be attributable to its inhibitory activity on the fungal germ tube growth. Our data highlight the contributions of natural substances that can act, directly or indirectly, against phytopathogens. In the global context of sustainability, food safety and environmental protection, such semiochemicals represent an alternative and promising approach to integrated pest management.