Ramularia mali associated with symptoms of dry lenticel rot, an emerging postharvest disease on apples in Italy.

Ramularia mali Videira & Crous is an emerging postharvest pathogen on apple (Malus × domestica Borkh.) in Italy and other apple producing countries (Prencipe et al. 2023). After 3 to 6 months of cold storage at 1 - 2 °C and low oxygen levels of 0.5 - 2 %, lenticels show black-brown speckled dry rot of 1 mm - 5 mm in diameter, without colonizing underlying tissue. The most affected cultivar (cv.) in South Tyrol (northern Italy) is Golden Delicious and postharvest losses due to characteristic lenticel spots range from 10 % to above 50 %. Four symptomatic fruits, originating from two orchards (Latsch/Laces and Bozen/Bolzano; South Tyrol, Italy), respectively, were sampled after cold storage (= ultra-low oxygen; 0.5 % O2 and 1 °C). After surface disinfection with 70 % EtOH for 1 min, sixteen explants from lenticel spots were cultivated on potato dextrose agar (PDA) at 25 °C. Two isolates, morphologically identified as Ramularia sp., were sequenced and showed high identities to R. mali type culture CBS 129581: 100 % and 99.31 % identity for ITS region (MH865432); 94.66 % and 91.41 % for TEF-1α (KJ504693); 97.22% and 97.40% for RpbII (KJ504649). Isolates were cultivated at 25 °C for 2 weeks and conidia were harvested with 3.0 mL 0.05 % Tween®20. Inoculation was performed in triplicate on 5-month cold stored fruits cv. Golden Delicious. After surface disinfection for 1 min with cotton swabs, which were immersed in 70 % EtOH, 10 µL spore suspension of each isolate (8.50 × 107 spores mL-1 in 0.05 % Tween®20) were injected horizontally beneath the epidermis with a syringe (Hamilton® model 710N). Also, a mixture of both isolates was used. Controls were carried out with 0.05 % Tween®20 only. Apples were stored either at 9 °C in the dark or at 1°C and 0.5 % oxygen for 4 months. First symptoms were observed for both spore concentrations after 2 weeks at 9 °C. The injection pathway changed to a brownish color, whereas the control did not show any change (Fig. 1). Final evaluation was carried out after 4 months, but the fruits did not show further symptom development. Fruits stored at 1°C for 5 months were simultaneously evaluated, confirming that the pathogen invaded the tissue surrounding the injection site, without penetrating deeper into the fruit flesh. (Fig. 2). Reisolation from artificially infected apples was successfully achieved, and sequence analysis was performed on the DNA extracts from the obtained isolates. Concatenated sequences of ITS (deposited to GenBank under the accession numbers: PP439643 -PP439647), TEF-1α (PP480231-PP480235), and RbpII (PP480226-PP480230) were subjected to multi-locus sequence analysis. References sequences of R. nyssicola CBS 127665, R. collo-cygni CBS 101181, R. vizellae CBS 115981, R. eucalypti CBS 120726, R. hydrangeae-macrophyllae CBS 122272, R. glennii CBS 129441 and R. mali CBS 129581 included and aligned by the CLUSTALW algorithm within the software Geneious® 11.1.5 (Biomatters Inc., New Zealand). Phylogeny was reconstructed with MEGAX (Version 10.2.6) (Kumar et al. 2018) based on the Maximum Likelihood (ML) algorithm (Fig. 3). Isolates from artificially infected fruit clustered with the R. mali type culture. Although Gianetti et al. (2012) and Lindner (2013), respectively, first described Ramularia sp. as a postharvest pathogen on apple, the present study demonstrated the reproduction of lenticel dry rot symptoms by R. mali.

Several secondary metabolite gene clusters in the genomes of ten Penicillium spp. raise the risk of multiple mycotoxin occurrence in chestnuts.

Penicillium spp. produce a great variety of secondary metabolites, including several mycotoxins, on food substrates. Chestnuts represent a favorable substrate for Penicillium spp. development. In this study, the genomes of ten Penicillium species, virulent on chestnuts, were sequenced and annotated: P. bialowiezense. P. pancosmium, P. manginii, P. discolor, P. crustosum, P. palitans, P. viridicatum, P. glandicola, P. taurinense and P. terrarumae. Assembly size ranges from 27.5 to 36.8 Mb and the number of encoded genes ranges from 9,867 to 12,520. The total number of predicted biosynthetic gene clusters (BGCs) in the ten species is 551. The most represented families of BGCs are non ribosomal peptide synthase (191) and polyketide synthase (175), followed by terpene synthases (87). Genome-wide collections of gene phylogenies (phylomes) were reconstructed for each of the newly sequenced Penicillium species allowing for the prediction of orthologous relationships among our species, as well as other 20 annotated Penicillium species available in the public domain. We investigated in silico the presence of BGCs for 10 secondary metabolites, including 5 mycotoxins, whose production was validated in vivo through chemical analyses. Among the clusters present in this set of species we found andrastin A and its related cluster atlantinone A, mycophenolic acid, patulin, penitrem A and the cluster responsible for the synthesis of roquefortine C/glandicoline A/glandicoline B/meleagrin. We confirmed the presence of these clusters in several of the Penicillium species conforming our dataset and verified their capacity to synthesize them in a chestnut-based medium with chemical analysis. Interestingly, we identified mycotoxin clusters in some species for the first time, such as the andrastin A cluster in P. flavigenum and P. taurinense, and the roquefortine C cluster in P. nalgiovense and P. taurinense. Chestnuts proved to be an optimal substrate for species of Penicillium with different mycotoxigenic potential, opening the door to risks related to the occurrence of multiple mycotoxins in the same food matrix.

Efficacy of Essential Oil Vapours in Reducing Postharvest Rots and Effect on the Fruit Mycobiome of Nectarines.

Nectarines can be affected by many diseases, resulting in significant production losses. Natural products, such as essential oils (EOs), are promising alternatives to pesticides to control storage rots. This work aimed to test the efficacy of biofumigation with EOs in the control of nectarine postharvest diseases while also evaluating the effect on the quality parameters (firmness, total soluble solids, and titratable acidity) and on the fruit fungal microbiome. Basil, fennel, lemon, oregano, and thyme EOs were first tested in vitro at 0.1, 0.5, and 1.0% concentrations to evaluate their inhibition activity against Monilinia fructicola. Subsequently, an in vivo screening trial was performed by treating nectarines inoculated with M. fructicola, with the five EOs at 2.0% concentration by biofumigation, performed using slow-release diffusers placed inside the storage cabinets. Fennel, lemon, and basil EOs were the most effective after storage and were selected to be tested in efficacy trials using naturally infected nectarines. After 28 days of storage, all treatments showed a significant rot reduction compared to the untreated control. Additionally, no evident phytotoxic effects were observed on the treated fruits. EO vapors did not affect the overall quality of the fruits but showed a positive effect in reducing firmness loss. Metabarcoding analysis showed a significant impact of tissue, treatment, and sampling time on the fruit microbiome composition. Treatments were able to reduce the abundance of Monilinia spp., but basil EO favored a significant increase in Penicillium spp. Moreover, the abundance of other fungal genera was found to be modified.

Soil, rhizosphere, and root microbiome in kiwifruit vine decline, an emerging multifactorial disease.

Kiwifruit vine decline syndrome (KVDS) is characterized by severe root system impairment, which leads to irreversible wilting of the canopy. Plants usually collapse rapidly from the appearance of the first aboveground symptoms, without recovery even in the following seasons. The syndrome has been negatively impacting kiwifruit yield in different areas of Italy, the main producing European country, since its first outbreak in 2012. To date, a unique, common causal factor has yet to be found, and the syndrome is referred to as multifactorial. In this article, we investigated the whole biotic community (fungi, bacteria, and oomycetes) associated with the development of KVDS in three different belowground matrices/compartments (soil, rhizosphere, and root). Sampling was performed at both healthy and affected sites located in the main kiwifruit-producing area of Northwestern Italy. To address the multifactorial nature of the syndrome and to investigate the potential roles of abiotic factors in shaping these communities, a physicochemical analysis of soils was also performed. This study investigates the associations among taxonomic groups composing the microbiome and also between biotic and abiotic factors. Dysbiosis was considered as a driving event in shaping KVDS microbial communities. The results obtained from this study highlight the role of the oomycete genus Phytopythium, which resulted predominantly in the oomycete community composition of diseased matrices, though it was also present in healthy ones. Both bacterial and fungal communities resulted in a high richness of genera and were highly correlated to the sampling site and matrix, underlining the importance of multiple location sampling both geographically and spatially. The rhizosphere community associated with KVDS was driven by a dysbiotic process. In addition, analysis of the association network in the diseased rhizosphere revealed the presence of potential cross-kingdom competition for plant-derived carbon between saprobes, oomycetes, and bacteria.

Molecular Characterization and Pathogenicity of Alternaria spp. Associated with Black Rot of Sweet Cherries in Italy.

Black rot is limiting the production of sweet cherries in Italy. Dark brown to black patches and sunken lesions on fruits are the most common symptoms of Alternaria black rot on sweet cherry fruits. We isolated 180 Alternaria spp. from symptomatic cherry fruits 'Kordia', 'Ferrovia', and 'Regina' harvested in Northern Italy, over three years, from 2020 to 2022. The aim was to identify and characterize a selection of forty isolates of Alternaria spp. based on morphology, pathogenicity, and combined analysis of rpb2, Alt-a1, endoPG and OPA10-2. The colonies were dark greyish in the center with white margins. Ellipsoidal or ovoid shaped conidia ranging from 19.8 to 21.7 µm in length were observed under a microscope. Based on the concatenated session of four gene regions, thirty-three out of forty isolates were identified as A. arborescens species complex (AASC), and seven as A. alternata. Pathogenicity was evaluated on healthy 'Regina' sweet cherry fruits. All the tested strains were pathogenic on their host. This study represents the first characterization of Alternaria spp. associated with black rot of cherries in Italy and, to the best of our knowledge, it is also the first report of AASC as an agent of black rot of sweet cherries in Italy.

Molecular Characterization and Pathogenicity of Diaporthe Species Causing Nut Rot of Hazelnut in Italy.

Hazelnut (Corylus avellana), a nut crop that is rapidly expanding worldwide, is endangered by a rot. Nut rot results in hazelnut defects. A survey was conducted in north-western Italy during 2020 and 2021 to identify the causal agents of hazelnut rots. Typical symptoms of black rot, mold, and necrotic spots were observed on hazelnut nuts. The prevalent fungi isolated from symptomatic hazelnut kernels were Diaporthe spp. (38%), Botryosphaeria dothidea (26%), Diplodia seriata (14%), and other fungal genera with less frequent occurrences. Among 161 isolated Diaporthe spp., 40 were selected for further analysis. Based on morphological characterization and multi-locus phylogenetic analysis of the ITS, tef1- α, and tub2, seven Diaporthe species were identified as D. eres, D. foeniculina, D. novem, D. oncostoma, D. ravennica, D. rudis, and D. sojae. D. eres was the main species isolated from hazelnut rots, in particular from moldy nuts. Pathogenicity test performed on hazelnut nuts 'Tonda Gentile del Piemonte' using a mycelium plug showed that all the Diaporthe isolates were pathogenic on their original host. To our knowledge, this work is the first report of D. novem, D. oncostoma and D. ravennica on hazelnut nuts worldwide. Diaporthe foeniculina, D. rudis, and D. sojae were reported for the first time as agents of hazelnut nut rot in Italy. Future studies should focus on the comprehension of epidemiology and climatic conditions favoring the development of Diaporthe spp. on hazelnut. Prevention and control measures should target D. eres, representing the main causal agents responsible for defects and nut rot of hazelnuts in Italy.

Efficacy and Mechanisms of Action of Essential Oils' Vapours against Blue Mould on Apples Caused by Penicillium expansum.

Biofumigation with slow-release diffusers of essential oils (EOs) of basil, oregano, savoury, thyme, lemon, and fennel was assessed for the control of blue mould of apples, caused by Penicillium expansum. In vitro, the ability of the six EOs to inhibit the mycelial growth was evaluated at concentrations of 1.0, 0.5, and 0.1%. EOs of thyme, savoury, and oregano, at all three concentrations, and basil, at 1.0 and 0.5%, were effective in inhibiting the mycelial growth of P. expansum. In vivo, disease incidence and severity were evaluated on 'Opal' apples artificially inoculated with the pathogen and treated at concentrations of 1.0% and 0.5% of EOs. The highest efficacy in reducing blue mould was observed with EOs of lemon and oregano at 1.0% after 60 days of storage at 1 ± 1 °C (incidence of rot, 3 and 1%, respectively) and after a further 14 days of shelf-life at 15 ± 1 °C (15 and 17%). Firmness, titratable acidity, and total soluble solids were evaluated at harvest, after cold storage, and after shelf-life. Throughout the storage period, no evident phytotoxic effects were observed. The EOs used were characterised through GC-MS to analyse their compositions. Moreover, the volatile organic compounds (VOCs) present in the cabinets were characterised during storage using the SPME-GC-MS technique. The antifungal effects of EOs were confirmed both in vitro and in vivo and the possible mechanisms of action were hypothesised. High concentrations of antimicrobial and antioxidant compounds in the EOs explain the efficacy of biofumigation in postharvest disease control. These findings provide new insights for the development of sustainable strategies for the management of postharvest diseases and the reduction of fruit losses during storage.

Effects of a prothioconazole- and tebuconazole-based fungicide on the yield, silage characteristics, and fungal mycobiota of corn harvested and conserved as whole-crop and high-moisture ear silages.

AIMS: To analyze the effect of a prothioconazole- and tebuconazole-based fungicide on the yield and silage characteristics of whole-crop corn (WCC) and high-moisture ear corn (HMC) silages and on the fungal community dynamics from the harvest to aerobic exposure. METHODS AND RESULTS: Corn were untreated (NT) or treated (T) with a prothioconazole- and tebuconazole-based fungicide and harvested as WCC and HMC. Silages were conserved for 60 and 160 d and subjected to an aerobic stability test. The fungicide increased the yield per hectare however, it did not affect the main nutritional characteristics of WCC or HMC. The main chemical, fermentative and microbial characteristics, dry matter (DM) losses and aerobic stability were mainly affected by the conservation time, regardless of the treatment. Fusarium, Alternaria, Aspergillus, and Penicillium genera were identified as dominant before ensiling, but Aspergillus and Penicillium became dominant after silo opening and aerobic exposure. Yeast population during ensiling and aerobic deterioration resulted in a simplification, with Pichia and Kazachstania genera being dominant. CONCLUSIONS: The application of fungicide improved the DM, starch, and net energy for lactation (NEL) yield per hectare but had no consistent effect on the microbial and fermentative silage quality and aerobic stability.

Characterization of Phytopythium Species Involved in the Establishment and Development of Kiwifruit Vine Decline Syndrome.

Since 2012, the kiwifruit vine decline syndrome (KVDS) has progressively compromised Italian kiwifruit orchards. Different abiotic and biotic factors have been associated with the establishment and development of KVDS. During monitoring of orchards affected by KVDS in north-western Italy during 2016-2019, 71 Phytopythium spp. were isolated. Based on maximum likelihood concatenated phylogeny on the ITS1-5.8S-ITS2 region of the rDNA, large subunit rDNA, and cytochrome oxidase I, isolates were identified as P. vexans (52), P. litorale (10), P. chamaehyphon (7) and P. helicoides (2). Phytopythium litorale and P. helicoides are reported for the first time as agents of KVDS in Italy. To demonstrate pathogenicity and fulfil Koch's postulates, representative isolates of P. vexans, P. litorale, P. chamaehyphon and P. helicoides were inoculated in potted plants. In these trials, waterlogging was applied to stress plant with a temporary anoxia and to favour the production of infective zoospores by the oomycetes. In experiments in vitro, the four species showed the highest growth at 25-30 °C, depending on the media used. P. helicoides was able to grow also at 40 °C. The four species were able to grow in vitro at a pH ranging from 5.0 to 8.0, showing that pH had less effect on growth than temperature. The present study suggests a strong role of different species of Phytopythium in the establishment and development of KVDS. Phytopythium spp. could be favoured by the average increase in soil temperatures during summer, associated with global warming.

A Quantitative Real-Time PCR Assay for Early Detection and Quantification of Ramularia mali, an Emerging Pathogen of Apple Causing Dry Lenticel Rot.

Ramularia mali is an emerging pathogen of apple (Malus domestica) causing dry lenticel rot. No preventive measures have been adopted due to the lack of knowledge about the life cycle and epidemiology of this pathogen. In a preliminary survey to identify the agents of dry lenticel rot of apple, R. mali was constantly associated with the disease. Using isolates from this survey, a SYBR Green quantitative PCR (qPCR) assay was developed, using calmodulin as target gene, for the detection and quantification of R. mali in apple fruit. The qPCR assay was validated in terms of specificity, sensitivity, repeatability, and reproducibility following the international European and Mediterranean Plant Protection Organization standard PM 7/98. The primers amplified a region of 237 bp specific to R. mali. The specificity was validated with 20 fungal species commonly found on apple, and 36 strains of R. mali and closely related species of the R. eucalypti species complex. Positive amplifications were obtained only with DNA of R. mali and no cross-reaction was detected with the other fungal species. Sensitivity was assessed with serial dilutions of target DNA and the limit of detection was 100 fg. No influence of host DNA was observed when target DNA was diluted on the DNA of Ambrosia and Golden Delicious apple. The assay permitted to detect and quantify R. mali in symptomatic and asymptomatic fruit. The presence of R. mali on asymptomatic Ambrosia and Golden Delicious apple fruit was demonstrated, and the presence of the pathogen was reported for the first time on Jeromine, Gala, Opal, and Story Inored fruit. This assay could be useful in clarifying the life cycle of this pathogen in order to build up an effective disease management strategy. Furthermore, the early detection of the pathogen on asymptomatic apple fruit could be used to forecast the development of dry lenticel rot, supporting the packinghouse operators in deciding the storage length of apple lots.

Pathogenicity, Molecular Characterization, and Mycotoxigenic Potential of Alternaria spp. Agents of Black Spots on Fruit and Leaves of Pyrus communis in Italy.

Brown and black spots, caused by Stemphylium and Alternaria species, are important fungal diseases affecting European pear (Pyrus communis) in orchards. Both fungal genera cause similar symptoms, which could favor misidentification, but Alternaria spp. are increasingly reported due to the changing climatic conditions. In this study, Alternaria spp. were isolated from symptomatic leaves and fruits of European pear, and their pathogenicity was evaluated on pear fruits from cultivar Abate Fétel, and molecular and chemical characterization were performed. Based on maximum likelihood phylogenetic analysis, 15 of 46 isolates were identified as A. arborescens species complex (AASC), 27 as A. alternata, and four as Alternaria sp. Both species were isolated from mature fruits and leaves. In pathogenicity assays on pear fruits, all isolates reproduced the symptoms observed in the field, by both wound inoculation and direct penetration. All but one isolate produced Alternaria toxins on European pears, including tenuazonic acid and alternariol (89.1% of the isolates), alternariol monomethyl ether (89.1%), altertoxin I (80.4%), altenuene (50.0%), and tentoxin (2.2%). These isolates also produced at least two mycotoxins, and 43.5% produced four mycotoxins, with an average total concentration of the Alternaria toxins exceeding 7.58 × 106 ng/kg. Our data underline the potential risks for human health related to the high mycotoxin content found on fruits affected by black spot. This study also represents the first report of AASC as an agent of black spot on European pear in Italy.

Essential Oils Reduce Grey Mould Rot of Apples and Modify the Fruit Microbiome during Postharvest Storage.

Botrytis cinerea is the causal agent of grey mould rot of apples. The efficacy of biofumigation with thyme (Thymus vulgaris), savoury (Satureja montana), and basil (Ocimum basilicum) essential oils (EOs) at 1%, 0.5%, and 0.1% concentrations were tested against B. cinerea. In vitro, the results showed 100% growth inhibition at 1% concentration for all oils. Subsequent biofumigation experiments on apples of cultivar 'Opal' with 1% EOs showed that, after 60 d storage, thyme and savoury EOs significantly reduced grey mould rot incidence (average incidence 2% for both treatments) compared to the control (7%). Analyses of quality indicated slightly higher fruit firmness for 1% thyme at 30 d and slightly higher titratable acidity for 1% thyme and savoury at 60 d. Sampling of the atmosphere inside the cabinets was performed to characterize and quantify the volatile components of EOs released through biofumigation. Though thymol and p-cymene were the main components of thyme EO, the antimicrobial activity was mainly due to the presence of thymol and, to a lower extent, of carvacrol. In savoury EO, carvacrol and p-cymene were the main components, whereas in basil EO, linalool and estragole were mainly present. Metabarcoding analyses showed that the epiphytic microbiome had higher richness and evenness compared to their endophytic counterpart. By the end of shelf-life, treatments with thyme EO reduced B. cinerea abundance compared to the inoculated control for both endophytes (from 36.5% to 1.5%) and epiphytes (from 7.0% to 0.7%), while favouring a significant increase in Penicillium species both in endophytes (from 0.2% to 21.5%) and epiphytes (from 0.5% to 18.6%). Results indicate that thyme EO (1%) and savoury EO (1%) are equally effective in hampering grey mould rot development in vivo.

Sequencing of non-virulent strains of Fusarium fujikuroi reveals genes putatively involved in bakanae disease of rice.

Bakanae, one of the most important diseases of rice, is caused by the fungal pathogen Fusarium fujikuroi. The elongation of internodes is the most common symptom induced by the pathogen, and it is related to the production of gibberellins. Despite this, the pathogenicity mechanism of F. fujikuroi is still not completely clear, and there are some strains inducing stunting instead of elongation. Even if there are relatively many genomes of F. fujikuroi strains available in online databases, none of them belongs to an isolate of proven non-virulence, and therefore there has been no comparative genomics study conducted between virulent and non-virulent strains. In the present work, the genomes of non-virulent strain SG4 and scarcely virulent strain C2S were compared to the ones of 12 available virulent isolates. Genes present in the majority of available virulent strains, but not in the non-virulent one, underwent functional annotation with multiple tools, and their expression level during rice infection was checked using pre-existing data. Nine genes putatively related to pathogenicity in F. fujikuroi were identified throughout comparative and functional analyses. Among these, many are involved in the degradation of plant cell wall, which is poorly studied in F. fujikuroi-rice interactions. Three of them were validated through qPCR, showing higher expression in the virulent strain and low to no expression in the low virulent and non virulent strains during rice infection. This work helps to clarify the mechanisms of pathogenicity of F. fujikuroi on rice.

First Report of Brown Rot Caused by Monilinia polystroma on Apple in Italy.

Brown rot is a common apple disease in Italy, caused by Monilinia fructicola, M. laxa and M. fructigena (Martini et al. 2013). In September 2020, in a 'Jeromine' apple orchard under integrated pest management located in Scarnafigi (44°39'N, 7°33'E, north-western of Italy), fruits (8.6%) showing brown to blackish firm lesions (6.0 to 8.0 cm diameter) were observed. In some fruits, rots were covered by yellowish stromata. Two isolates (MPI1; MPI2) were obtained from two symptomatic apples and cultured on potato dextrose agar (PDA) for 7 days at 25°C in 12-h light/12-h dark regime. A white-to-greyish mycelium with slightly undulate margins and irregular, black stromata developed on PDA after 12 days incubation. Conidia, observed in branched monilioid chains, (Suppl. Fig. 1) were one-celled, globose, limoniform, hyaline, 38 to 58 µm (mean: 48) × 20 to 44 µm (mean: 33). Based on morphology, the isolates were tentatively identified as Monilinia polystroma (G.C.M. Leeuwen) Kohn. A polymerase chain reaction with primers ITS1 and ITS4 was performed on internal transcribed spacer (ITS) region 1 and 2 and 5.8S gene. The sequenced amplicons (435 bp - 445 bp; GenBank Accession No. MW600854; MW600855) showed 100% identity to the reference isolate of M. polystroma (HQ846944) and to other isolates from apples (AM937114; JX315717) and plum (GU067539). The ITS region of M. polystroma had five nucleotides to distinguish it from the closest species M. fructigena (Zhu et al. 2016; MH862738) (Suppl. Fig. 2). The pathogenicity of both isolates was tested on mature 'Jeromine' apples (10.1% total soluble solids). Three replicates of six apples per isolate were surface disinfected with 1% NaClO. A mycelial plug (5 mm) from colony grown on PDA was inserted using a cork borer into a hole (6 mm) in each fruit (Vasic et al. 2016). Apples inoculated with sterile PDA plugs were used as control. Fruits were placed at 22 ± 1 °C, 85% relative humidity and 12 h light/12 h dark regime. Lesion size was measured after 3, 6 and 9 days of incubation. All inoculated fruits developed typical brown rot symptoms 6 days after inoculation and yellowish stromata appeared on the surface; control fruit remained healthy (Suppl. Fig. 3). The virulence of both isolates was statistically similar (Suppl. Table 1). M. polystroma was reisolated from all inoculated fruits and confirmed by molecular methods. This is the first report of M. polystroma on apple in Italy. M. polystroma was previously reported on apple in Hungary (Petróczy et al. 2009), on apricot in Switzerland (Hilber-Bodmer et al. 2012), on peach and pear in Italy (Martini et al. 2014; 2015), on plum in China (Zhu et al. 2016), and on apple in Serbia (Vasic et al. 2018). The emergence of this pathogen for pome and stone fruit production in Europe stimulates to study its biology and epidemiology, and its fitness and management, as compared to the other endemic Monilinia species.

CRISPR-Cas9-Based Discovery of the Verrucosidin Biosynthesis Gene Cluster in Penicillium polonicum.

Penicillium polonicum, commonly found on food matrices, is a mycotoxigenic species able to produce a neurotoxin called verrucosidin. This methylated α-pyrone polyketide inhibits oxidative phosphorylation in mitochondria and thereby causes neurological diseases. Despite the importance of verrucosidin as a toxin, its biosynthetic genes have not been characterized yet. By similarity analysis with the polyketide synthase (PKS) genes for the α-pyrones aurovertin (AurA) and citreoviridin (CtvA), 16 PKS genes for putative α-pyrones were identified in the P. polonicum genome. A single PKS gene, verA, was found to be transcribed under verrucosidin-producing growth conditions. The annotated functions of the genes neighboring verA correspond to those required for verrucosidin biosynthesis. To prove the involvement of verA in verrucosidin biosynthesis, the clustered regularly interspaced short palindrome repeats (CRISPR) technology was applied to P. polonicum. In vitro reconstituted CRISPR-Cas9 was used to induce targeted gene deletions in P. polonicum. This approach allowed identifying and characterizing the verrucosidin biosynthetic gene cluster. VerA deletion mutants were no longer able to produce verrucosidin, whereas they were displaying morphological characteristics comparable with the wild-type strain. The available CRISPR-Cas9 technology allows characterizing the biosynthetic potential of P. polonicum as a valuable source of novel compounds.

First Report of Stemphylium eturmiunum causing postharvest rot on tomato (Solanum lycopersicum) in Italy.

Italy is the largest tomato (Solanum lycopersicum)-producing country in Europe with a cultivated area of 97,092 ha and a production of 5,798,103 tons/year in 2018 (FAOSTAT, 2020). During July 2020, a postharvest rot occurred in fresh tomatoes 'Piccadilly' cultivated in Sicily (Pachino, RG) and commercialized in Northern Italy (Torino, TO). Affected fruit showed circular black rot on the blossom end. The rot had an average incidence of 7% of the fruits, in three batches of 100 tomatoes each. Isolation was carried out by cutting pieces of symptomatic rotten fruits. The fragments were surface-disinfected with 1% sodium hypochlorite for 30 s, rinsed in sterile water and air-dried. Five fragments were cut and plated onto Potato Dextrose Agar (PDA) supplemented with streptomycin, and incubated at 24±1°C in the dark for 5 days. Representative colonies were transferred onto PCA and morphological observations were performed as described by Woudenberg et al. (2017) after 7 and 14 days. Colonies were olive-green, flat with regular margins, while conidia were mid to deep brown, solitary, ovoid or ellipsoid (17.39 µm ± 2.04 × 10.59 ± 3.30 µm) with transverse and longitudinal septa. Based on morphological observations the isolates were identified as Stemphylium eturmiunum (Simmons, 2001). Species identification was confirmed by sequencing rDNA internal transcribed spacer (ITS) using primers ITS1/ITS4 (White et al. 1990), cmdA gene region using primers CALDF1/CALDR2 (Lawrence et al. 2013) and gapdh gene region with primers gpd1/gpd2 (Berbee et al. 1999). Six amplified sequences per region (ANos. from MW158387 to MW158398 and from MW159746 to MW159751) were BLAST-searched in GenBank, obtaining >99 % identity with ex-type strain of S. eturmiunum strain CBS 109845 (AN° KU850541) for ITS, and 100% identity (ANos. KU850831 and KU850689) for cmdA and gapdh, respectively. To confirm the species, DNA sequences were aligned with CLUSTAL W with closely related species of Stemphylium reported in the last revision of the genus (Woudenberg et al., 2017), and a phylogenetic analysis with the Neighbor Joining method based on Tamura Nei model + Gamma distribution (bootstrap 1,000) was performed. The phylogenetic tree confirmed the identity of the isolates as S. eturmiunum (Suppl. Fig. 1). To fulfil Koch's postulates, pathogenicity tests were conducted on S. lycopersicum cv. Piccadilly fruits. Tomatoes were surface sterilized with 1% sodium hypochlorite and air-dried. Fruits (5 fruits per isolates) were wounded (two injuries of 3 mm each) and inoculated with a spore suspension of 1x105 cell/mL obtained from 15 days-old PCA cultures, as in Spadoni et al. (2020. Negative controls were wounded and inoculated with sterile deionized water. Symptoms occurred on all fruits inoculated after 12 days at 24±1°C and S. eturmiunum was re-isolated from inoculated fruits on PCA (Suppl. Fig. 2), control remained symptomless. Re-isolated colonies were molecularly identified as S. eturmiunum. In Italy a different species, S. vesicarium, was reported on tomato (Porta-Puglia, 1981), while S. eturmiunum was described as a postharvest pathogen of tomato in China, Greece, New Zealand and the United States (Woudenberg et al., 2017; Vaghefi et al., 2020), and from fruits commercialized in Danish and Spanish markets (Andersen and Frisvad, 2004). To the best of our knowledge, this is the first report of S. eturmiunum causing postharvest rot on tomato in Italy. The occurrence of this pathogen further stresses the importance of careful handling to prevent fruit crackings and of preharvest control strategies.

First Multi-Target Application of Exclusion Net in Nectarine Orchards: Effectiveness against Pests and Impact on Beneficial Arthropods, Postharvest Rots and Fruit Quality.

Over the past few years, there has been an increasing interest in the development of alternative pest control strategies to reduce environmental impact. In this contest, exclusion nets have been evaluated as a sustainable alternative to pesticides. In this study, the use of a photoselective exclusion net was investigated in semi-field conditions as a potential strategy to protect nectarine orchards from different pests (i.e., fruit moths, Halyomorpha halys and Drosophila suzukii) in NW Italy. The presence and abundance of pest populations inside and outside the net, as well as the damage they caused on fruits, were evaluated. Moreover, any possible effects of the net on beneficial arthropods, postharvest rots and fruit quality and nutraceutical parameters were considered. The exclusion net significantly reduced pest populations. At harvest, fruit damage caused by Grapholita molesta and H. halys in netted plots was reduced up to 90% and to 78%, respectively, compared with insecticide-treated plots. The exclusion net allowed the production of healthier fruits with a strong reduction of insecticide treatments (up to seven less) and of their related costs without any negative impact on postharvest rots, neither fruit quality nor nutraceutical properties.

Global analysis of the apple fruit microbiome: are all apples the same?

We present the first worldwide study on the apple (Malus × domestica) fruit microbiome that examines questions regarding the composition and the assembly of microbial communities on and in apple fruit. Results revealed that the composition and structure of the fungal and bacterial communities associated with apple fruit vary and are highly dependent on geographical location. The study also confirmed that the spatial variation in the fungal and bacterial composition of different fruit tissues exists at a global level. Fungal diversity varied significantly in fruit harvested in different geographical locations and suggests a potential link between location and the type and rate of postharvest diseases that develop in each country. The global core microbiome of apple fruit was represented by several beneficial microbial taxa and accounted for a large fraction of the fruit microbial community. The study provides foundational information about the apple fruit microbiome that can be utilized for the development of novel approaches for the management of fruit quality and safety, as well as for reducing losses due to the establishment and proliferation of postharvest pathogens. It also lays the groundwork for studying the complex microbial interactions that occur on apple fruit surfaces.

First report of Erysiphe corylacearum, agent of powdery mildew, on hazelnut (Corylus avellana) in Romania.

Romania has an area dedicated to hazelnut (Corylus avellana L.), covering 890 hectares as of 2019. During October 2020, powdery mildew symptoms were observed on the upper side of leaves of hazelnut 'Tonda di Giffoni' in two commercial orchards in Dudeștii Vechi, Romania (Fig. 1). The disease was present on 70% of the trees in planting, with at least 5 leaves per tree having powdery mildew. Micromorphological examination revealed amphigenous, hyaline, branched, septate mycelial patches of 2.3 to 3.6 µm in diameter. Conidiophores measured 24-60 × 5-6 (average: 45 × 6) µm and consisted of erect, cylindrical to flexuous foot cells, followed by 1-2 shorter cells. Ellipsoid, ovoid to doliform conidia were produced singly and they measured 19-35 × 16-24 (average: 28 × 19) µm. Chasmothecia were spherical, 75 to 107 (average: 88) µm in diameter. Nine to thirteen straight, sometimes flexuous, appendages measured 54 to 92 (average: 66) µm in length and they had five times dichotomous branched apices with curved tips (Fig. 2). Each chasmothecium contained three to five ellipsoid, ovoid to subglobose asci measuring 41-58 × 29-55 µm (average 52 × 43) µm. The asci contained four to eight ascospores measuring 13-24 × 11-15 (average 18 × 14) µm. Morphological identification was confirmed by sequencing the ITS-region of rDNA using two isolates from leaves, stored as frozen mycelium at -20°C. PCR was performed with Erysiphales-specific primer pair PMITS1/PMITS2 (Cunnington et al. 2003). The obtained sequences were deposited in GenBank (Accession n° MW423075, MW423076). Blast analysis of both sequences had 100% identity to ITS rDNA sequences of Erysiphe corylacearum from Azerbaijan (Abasova et al. 2018; Accession n° LC270863), Turkey (Sezer et al. 2017; KY082910), Switzerland (Beenken et al. 2020; MN82272), Iran (Arzanlou et al. 2018; MH047243), Italy (Mezzalama et al. 2020; MW045425) and 99% identity from Georgia (Meparishvili et al. 2019; MK157199). The sequences had a lower percent identity (83%) to Phyllactinia guttata (Accession n° AB080558) (Fig. 3). Pathogenicity was verified on one-year-old plants of C. avellana 'Tonda di Giffoni', which were artificially inoculated with a conidial suspension from infected leaves (n = 25). Inoculated plants were incubated at 20 to 28°C with 70 to 80% relative humidity. White mycelium appeared on the upper surface of the leaves at 8 to 10 days after inoculation. No symptoms were found on control plants sprayed with sterile water. The fungus present on inoculated leaves was morphologically identical to the original isolates from diseased trees from the field. E. corylacearum is native to East Asia and was previously reported in Japan on wild species of Corylus (Takamatsu et al. 2015; Accession n° LC009928). The pathogen most likely spread into Europe from east to west of Europe (Heluta et al. 2019), through the Caucasus, starting from Turkey, Azerbaijan, Georgia, and Iran. P. guttata was considered the only causal agent of powdery mildew on hazelnut in most countries, including Romania (Brown 1995). Compared to P. guttata, which generally develops a mycelium on the underside of leaves, E. corylacearum grows with a white mycelium on the upper side of the leaves. Recently, E. corylacearum on C. avellana was reported also in Ukraine (Heluta et al. 2019), from which it could have moved to Romania. Crop protection strategies for hazelnut should be revised according to the new pathogen occurrence.

Metagenomics Approaches for the Detection and Surveillance of Emerging and Recurrent Plant Pathogens.

Globalization has a dramatic effect on the trade and movement of seeds, fruits and vegetables, with a corresponding increase in economic losses caused by the introduction of transboundary plant pathogens. Current diagnostic techniques provide a useful and precise tool to enact surveillance protocols regarding specific organisms, but this approach is strictly targeted, while metabarcoding and shotgun metagenomics could be used to simultaneously detect all known pathogens and potentially new ones. This review aims to present the current status of high-throughput sequencing (HTS) diagnostics of fungal and bacterial plant pathogens, discuss the challenges that need to be addressed, and provide direction for the development of methods for the detection of a restricted number of related taxa (specific surveillance) or all of the microorganisms present in a sample (general surveillance). HTS techniques, particularly metabarcoding, could be useful for the surveillance of soilborne, seedborne and airborne pathogens, as well as for identifying new pathogens and determining the origin of outbreaks. Metabarcoding and shotgun metagenomics still suffer from low precision, but this issue can be limited by carefully choosing primers and bioinformatic algorithms. Advances in bioinformatics will greatly accelerate the use of metagenomics to address critical aspects related to the detection and surveillance of plant pathogens in plant material and foodstuffs.