Chitosan-based films grafted with citrus waste-derived antifungal agents: An innovative and sustainable approach to enhance post-harvest preservation of citrus fruit.

This paper reports the synthesis, characterization, and properties of chitosan films (CHI) grafted with a natural antifungal agent with the aim of developing active films of natural origin to prevent post-harvest losses of citrus fruit. The antifungal agent was prepared by fermentation using lemon peel (AntiFun-LM), a citrus waste, and grafted on chitosan using different coupling agents (CHI/AntiFun-LM). Bioactive films were prepared by solvent casting. FTIR-ATR and ToF-SIMS analyses provided compelling evidence of the successful grafting process. TGA-DSC demonstrated that the films are stable after grafting. SEM studies showed the continuous and compact surface of the films. WCA measurements proved that CHI/AntiFun-LM films are more hydrophilic than CHI films. Moreover, the CHI/AntiFun-LM films showed stronger UV shielding effect when compared to CHI. The biological evaluation demonstrated that CHI/AntiFun-LM films gained considerable antifungal properties against most fungi responsible for post-harvest decay. Cytotoxicity tests showed that CHI/AntiFun-LM films did not cause any toxic effect against L929 fibroblasts. This study highlights the great potential of chemical grafting of antifungal agents produced from citrus waste to chitosan and preparation of natural-based films to act as a powerful alternative in post-harvest protection of citrus fruit in a perspective of circular economy.

Diversity of Mycotoxins and Other Secondary Metabolites Recovered from Blood Oranges Infected by Colletotrichum, Alternaria, and Penicillium Species.

This study identified secondary metabolites produced by Alternaria alternata, Colletotrichum gloeosporioides, and Penicillium digitatum in fruits of two blood orange cultivars before harvest. Analysis was performed by UHPLC-Q-TOF-MS. Three types of fruits were selected, asymptomatic, symptomatic showing necrotic lesions caused by hail, and mummified. Extracts from peel and juice were analyzed separately. Penicillium digitatum was the prevalent species recovered from mummified and hail-injured fruits. Among 47 secondary metabolites identified, 16, 18, and 13 were of A. alternata, C. gloeosporioides, and P. digitatum, respectively. Consistently with isolations, indicating the presence of these fungi also in asymptomatic fruits, the metabolic profiles of the peel of hail-injured and asymptomatic fruits did not differ substantially. Major differences were found in the profiles of juice from hail-injured and mummified fruits, such as a significant higher presence of 5,4-dihydroxy-3,7,8-trimethoxy-6C-methylflavone and Atrovenetin, particularly in the juice of mummified fruits of the Tarocco Lempso cultivar. Moreover, the mycotoxins patulin and Rubratoxin B were detected exclusively in mummified fruits. Patulin was detected in both the juice and peel, with a higher relative abundance in the juice, while Rubratoxin B was detected only in the juice. These findings provide basic information for evaluating and preventing the risk of contamination by mycotoxins in the citrus fresh fruit supply chain and juice industry.

DNA Metabarcoding and Isolation by Baiting Complement Each Other in Revealing Phytophthora Diversity in Anthropized and Natural Ecosystems.

Isolation techniques supplemented by sequencing of DNA from axenic cultures have provided a robust methodology for the study of Phytophthora communities in agricultural and natural ecosystems. Recently, metabarcoding approaches have emerged as new paradigms for the detection of Phytophthora species in environmental samples. In this study, Illumina DNA metabarcoding and a conventional leaf baiting isolation technique were compared to unravel the variability of Phytophthora communities in different environments. Overall, 39 rhizosphere soil samples from a natural, a semi-natural and a horticultural small-scale ecosystem, respectively, were processed by both baiting and metabarcoding. Using both detection techniques, 28 out of 39 samples tested positive for Phytophthora. Overall, 1,406,613 Phytophthora internal transcribed spacer 1 (ITS1) sequences and 155 Phytophthora isolates were obtained, which grouped into 21 taxa, five retrieved exclusively by baiting (P. bilorbang; P. cryptogea; P. gonapodyides; P. parvispora and P. pseudocryptogea), 12 exclusively by metabarcoding (P. asparagi; P. occultans; P. psycrophila; P. syringae; P. aleatoria/P. cactorum; P. castanetorum/P. quercina; P. iranica-like; P. unknown sp. 1; P. unknown sp. 2; P. unknown sp. 3; P. unknown sp. 4; P. unknown sp. 5) and four with both techniques (P. citrophthora, P. multivora, P. nicotianae and P. plurivora). Both techniques complemented each other in describing the variability of Phytophthora communities from natural and managed ecosystems and revealing the presence of rare or undescribed Phytophthora taxa.

A super absorbent polymer containing copper to control Plenodomus tracheiphilus the causative agent of mal secco disease of lemon.

The aim of this study was to determine the effectiveness of a Super absorbent polymer (SAP) containing copper (SAP-Cu) in controlling mal secco disease (MSD) of lemon caused by the fungus Plenodomus tracheiphilus. Super absorbent polymer containing copper was characterized by atomic absorption spectrometry (AAS) and UV-VIS spectroscopy. In vitro tests were performed to determine the inhibitory effects of SAP-Cu against the pathogen on both potato-dextrose-agar medium and naturally infected lemon cuttings. Super absorbent polymer was able to absorb up to about 200 and 30 times its weight of ionized water and copper (II) sulfate solution (Cu2+ ions at the concentration 236 mM), respectively. The distribution of copper released on twigs after 24 h of contact with SAP-Cu was determined by secondary ion mass spectrometry with time-of-flight analyzer (ToF-SIMS). Super absorbent polymer containing copper significantly inhibited the viability of P. tracheiphilus in lemon twigs. Overall, the results of this study showed that the SAP could be a suitable carrier of antifungal compounds.

Natural Biostimulants Elicit Plant Immune System in an Integrated Management Strategy of the Postharvest Green Mold of Orange Fruits Incited by Penicillium digitatum.

This study was aimed at testing the integrated use of a natural biostimulant based on seaweed (Ascophyllum nodosum) and plant (alfalfa and sugarcane) extracts and reduced dosages of the conventional synthetic fungicide Imazalil (IMZ) to manage postharvest rots of orange fruits. The following aspects were investigated: (i) the effectiveness of postharvest treatment with natural biostimulant alone or in mixture with IMZ at a reduced dose against green mold caused by Penicillium digitatum; (ii) the differential expression of defense genes in orange fruits treated with the natural biostimulant both alone and in combination with a reduced dose of IMZ; (iii) the persistence of the inhibitory activity of both biostimulant and the mixture biostimulant/IMZ against green mold; and (iv) the residue level of fungicide in citrus peel when applied alone or in combination with the biostimulant. Treatments with the chemical plant resistance-inducer potassium phosphite, alone or with a reduced dose of IMZ, were included for comparison. The mixture of natural biostimulant and IMZ at a low dose consistently reduced the incidence and severity of fruit green mold and induced a significant increase of the expression level of ß-1,3-glucanase-, peroxidase (PEROX)-, and phenylalanine ammonia-lyase (PAL)-encoding genes in fruit peel, suggesting that the natural biostimulant elicits a long-lasting resistance of citrus fruits to infections by P. digitatum. Interestingly, the residual concentration of IMZ in fruits treated with the biostimulant/fungicide mixture was significantly lower than that of IMZ in fruits treated only with the fungicide at the same dose and by far below the threshold values set by the European Union. This study laid the foundations for (i) conceiving a practical and more eco-friendly alternative to the conventional postharvest management of green mold of citrus fruits, based almost exclusively on the use of synthetic fungicide IMZ, alone or mixed with potassium phosphite and (ii) providing a better insight into the mechanisms of disease resistance induction by biostimulants.

Inhibition of Mycotoxigenic Fungi in Different Vegetable Matrices by Extracts of Trichoderma Species.

Post-harvest fungal diseases of plant products are a serious concern leading to economic losses and health risks. Moreover, the use of synthetic chemical fungicides to prevent these diseases is limited due to toxic residues. This study aimed at determining the effective dose of extracts of Trichoderma asperellum IMI393899 (TE1) and Trichoderma atroviride TS (TE2) in inhibiting the contamination by mycotoxigenic fungi on different plant matrices. Extracts were tested on tomatoes contaminated by Fusarium verticillioides and Fusarium graminearum, wheat contaminated by Penicillium verrucosum and maize contaminated by Aspergillus flavus. The efficacy of extracts was evaluated at two time intervals after treatment, 4 and 11 days for tomato, and 10 and 20 days for both wheat and maize. Both extracts showed a significant inhibitory activity on mycotoxigenic pathogens and significantly reduced Log CFU/g compared to the control. Moreover, the extracts reduced mycotoxin production in a dose dependent manner and with a long-lasting effect. The ochratoxin A was reduced by both extracts but only the extract TE2 was effective in reducing aflatoxins, whereas TE1 treatment increased their synthesis.

Inhibitory Activity of Shrimp Waste Extracts on Fungal and Oomycete Plant Pathogens.

(1) Background: This study was aimed at determining the in vitro inhibitory effect of new natural substances obtained by minimal processing from shrimp wastes on fungi and oomycetes in the genera Alternaria, Colletotrichum, Fusarium, Penicillium, Plenodomus and Phytophthora; the effectiveness of the substance with the highest in vitro activity in preventing citrus and apple fruit rot incited by P. digitatum and P. expansum, respectively, was also evaluated. (2) Methods: The four tested substances, water-extract, EtOAc-extract, MetOH-extract and nitric-extract, were analyzed by HPLC-ESI-MS-TOF; in vitro preliminary tests were carried out to determine the minimal inhibitory/fungicidal concentrations (MIC and MFC, respectively) of the raw dry powder, EtOAc-extract, MetOH-extract and nitric-extract for each pathogen. (3) Results: in the agar-diffusion-assay, nitric-extract showed an inhibitory effect on all pathogens, at all concentrations tested (100, 75, 50 and 25%); the maximum activity was on Plenodomus tracheiphilus, C. gloeosporioides and Ph. nicotianae; the diameters of inhibition halos were directly proportional to the extract concentration; values of MIC and MFC of this extract for all pathogens ranged from 2 to 3.5%; the highest concentrations (50 to 100%) tested in vivo were effective in preventing citrus and apple fruit molds. (4) Conclusions: This study contributes to the search for natural and ecofriendly substances for the control of pre- and post-harvest plant pathogens.

Trichoderma Counteracts the Challenge of Phytophthora nicotianae Infections on Tomato by Modulating Plant Defense Mechanisms and the Expression of Crinkler, Necrosis-Inducing Phytophthora Protein 1, and Cellulose-Binding Elicitor Lectin Pathogenic Effectors.

Decoding the mechanisms of plant defense against plant pathogens in a scenario where antagonistic activity and the plant growth-promoting effects of useful organisms intervene simultaneously is a new frontier of plant pathology. Here, we demonstrated that (i) two selected strains of Trichoderma asperellum and Trichoderma atroviride promoted tomato (Solanum lycopersicum) growth and reduced the severity of disease caused by the oomycete Phytophthora nicotianae and (ii) the genetic patterns of the components of the experimental model system tomato-Trichoderma spp.-P. nicotianae were differentially expressed. The beneficial effects in both the promotion of the growth of host plant and the biological control of the pathogen by two selected strains of different Trichoderma species were tested both in planta and in vitro. In both respects, T. atroviride demonstrated to be more effective than T. asperellum. Additionally, the simultaneous transcriptional reprogramming of several plant defense-related genes, pathogen effectors, and mycoparasitism-related genes in tomato, P. nicotianae, and Trichoderma spp., respectively, was evaluated during the three-component interaction. Results support the hypothesis that Trichoderma spp. elicit the expression of plant defense-related genes. As expected, a mycoparasitism-related gene was significantly up-regulated in Trichoderma-colonizing tomato plants infected by P. nicotianae. Finally, a marked up-regulation of the genes encoding two necrosis-inducing effectors was observed in P. nicotianae infecting tomato plants colonized by Trichoderma. In conclusion, this study is a contribution toward understanding the genetic pathways related with the ability of Trichoderma spp. to counteract the challenge of P. nicotianae infections on tomato. Additionally, the experiments revealed the beneficial effects in the tomato growth promotion of a new T. atroviride strain and its good antagonistic effectiveness in the biological control of root and crown rot incited by P. nicotianae, confirming that Trichoderma spp. can be a powerful tool in integrated pest management strategies of Phytophthora diseases of horticultural crops.

First Report of Root Rot Caused by Phytophthora bilorbang on Olea europaea in Italy.

Leaf chlorosis, severe defoliation and wilt associated with root rot were observed on mature olive trees cv. Nera di Gonnos in an experimental orchard at Mirto Crosia (Calabria, southern Italy). An oomycete was consistently isolated from rotten roots of symptomatic olive trees. It was identified as Phytophthora bilorbang by morphological characters and sequencing of Internal Transcribed Spacer (ITS) regions of ribosomal DNA (rDNA). Pathogenicity was verified by inoculating potted two-month-old rooted cuttings of Olea europaea var. Nera di Gonnos in a soil infestation trial. P. bilorbang was re-isolated from roots of symptomatic, artificially inoculated olive cuttings to fulfill Koch's postulates. This is the first report of P. bilorbang on O. europaea L. and on a species of the Oleaceae family worldwide.