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.

First report of nut rot caused by Botryosphaeria dothidea on almond in Italy.

Almond (Prunus dulcis) is an important nut crop widely grown in the Mediterranean region, including Italy. In September 2021, almonds cv. Tuono showing dark lesions affecting the hull were collected in Villar San Costanzo (Piedmont, Northwestern Italy). The occurrence of symptoms in the orchard was estimated at 50% incidence. Two samples, each consisting of 50 fruits, were collected from the affected orchard. Small sections taken from the margins of the lesions were surface disinfected with 1% sodium hypochlorite for 1 min, rinsed in sterile water, dried on sterile filter paper, and placed on potato dextrose agar (PDA, VWR International, Leuven, Belgium), amended with streptomycin sulfate (25 mg/l) to inhibit bacterial growth. Plates were incubated at 25°C for 7 days under 12-h photoperiod. Botryosphaeria-like fungi were isolated with a frequency of 60%. Two representative isolates (21-06-F1A; 21-06-F4) were transferred onto new PDA plates to obtain pure cultures. Fungal colonies initially appeared white, then gradually turned dark grey and black in reverse as the colony aged. Abundant aerial mycelium was produced. Globose black pycnidia were produced on water agar supplemented with sterile pine needles (PNA; Smith et al. 1996) after 30 days of incubation at 25 ± 1°C under 12-h photoperiod. Conidia were one-celled, hyaline, elliptical, aseptate, 17.46 to 27.05 µm (average 23.51) long and 5.70 to 9.40 µm (average 7.48) wide (n = 50). Morphologically, the causal agent was identified as Botryosphaeria sp. Genomic DNA was extracted from the isolates using the E.Z.N.A. Fungal DNA mini kit (Omega Bio-Tek, Norcross, GA, USA) according to manufacturer instructions. The rDNA internal transcribed spacer (ITS), the partial translation elongation factor 1-alpha gene (tef-1α) and the partial beta-tubulin gene (tub2) were amplified and sequenced using primers ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), Bt2a/Bt2b (Glass and Donaldson 1995), respectively. BLAST analysis showed 100% identity of the obtained ITS sequences (GenBank accession nos. PP859471, PP859472), tub2 sequences (PP889527, PP889528) and tef-1α sequences (PP889529, PP889530) with those of the ex-type strain of B. dothidea (CBS 115476). The maximum likelihood method based on combined sequences of ITS, tef-1α and tub2 was performed, and both isolates clustered with high bootstrap support values (96) with the ex-type strain of B. dothidea. Pathogenicity was tested on fruits of almond cv Tuono inoculated on tree in a commercial orchard. Ten fruits were randomly chosen and inoculated for each isolate. Inoculation was performed by removing a piece of hull with a sterile cork borer (6 mm diameter) and applying mycelium plugs of the same diameter, taken from 7-day-old colonies grown on PDA, upside-down onto the wounds. Each fruit was inoculated with one mycelium plug and sealed with parafilm. Control fruits were inoculated with sterile PDA plugs. Dark lesions with softening pulp developed on inoculated almonds after 9 days. The mean diameter of the lesions was 3.89 ± 0.80 cm on almonds inoculated with the strain 21-06-F1A and 3.08 ± 0.56 cm on fruits inoculated with the strain 21-06-F4. The pathogen was successfully reisolated and morphologically identified as B. dothidea, fulfilling Koch's postulates. No symptoms were found on control fruits. Botryosphaeria dothidea was previously reported to affect other nut crops, causing nut rot on walnut (Li et al. 2023) and panicle and shoot blight on pistachio (Gusella et al. 2022). To the best of our knowledge, this is the first report of B. dothidea causing nut rot on almond in Italy.

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.

First report of almond decline syndrome caused by Neofusicoccum parvum in Italy.

Almond (Prunus dulcis Mill. D. A. Webb) is considered a major crop in the Mediterranean countries. In Italy, almond orchards cover an area of 54,939 ha, with a yield of 82,146 t, with the cultivation widely distributed across several areas, such as southern regions. During the summer of 2023, 3-year-old almond plants cultivated in a commercial orchard in southern Piedmont (44°32'41.92'' N; 7°57'25.7'' E) showed wilting and sudden decline of the whole plant with an incidence of 15%. Main symptoms were cankers on the trunk characterized by a chocolate-brown discoloration of wood, cambium and even bark and occasional presence of gummy exudate. Symptomatic bark samples were rinsed with water, then blotted dry, and briefly soaked in ethanol for 15 s. Small pieces (5 × 5 mm) from the margin of necrotic areas were cut and placed on potato dextrose agar (PDA) plates amended with streptomycin sulphate (150 µg/ml). After incubation in the dark at 25 °C for 4 days fungal colonies emerging from the plated fragments were subcultured as monohyphal fungal cultures. They were fast-growing, turning from withish to smoky grey or grey-olivaceous after a week. Stromatic conidiomata were produced in pine needle agar, with pycnidia forming hyaline, globose to ellipsoidal, unicellular conidia, ranging in size from 16.9-17.3 µm × 5.4-5.6 µm. The causal agent was preliminary identified as Neofusicoccum sp. based on morphology. Two representative isolates (MTB3, MTC4) were identified by sequencing the ITS region and translation elongation factor 1-alpha (TEF1-α) gene with the primer pairs ITS1/ITS4 (White et al. 1990) and EF1-728F/EF1-986R (Carbone and Kohn 1999). The sequences of the two isolates were deposited in NCBI GenBank with accession numbers PP930646 / PP930647 (ITS), and PP933185 / PP933186 (TEF-1α). BLAST analysis revealed 99% similarity with reference sequences of Neofusicoccum parvum isolate CMW9081. A maximum-likelihood phylogenetic tree was constructed using MEGA7 software based on the combined ITS and TEF1-α dataset. The phylogenetic tree demonstrated that the two isolates grouped with N. parvum in a clade with the ex-type CMW9081. The pathogenicity of both isolates was evaluated on 1-year-old 'Vairo' almond potted plants grafted on GF677 rootstock, to assess Koch's postulate. For the inoculation, 5 mm in diameter mycelium plugs taken from a 7-days-old PDA culture were placed on the surface of fresh wounds made with a sterile cork-borer (5 mm-diameter) on the stem. Control plants were inoculated with sterile PDA plugs. The test was conducted twice. Three blocks of three plants per isolate and control were arranged in a randomized block design. All inoculated plants showed symptoms of shoot blight and cankers 2-weeks after inoculation. Additionally, gumming, pycnidia production and internal wood discoloration developed on the inoculation sites, with lesion length spreading for 16.8 ± 1.6 and 15.4 ± 3.0 cm for MTB3 and MTC4, respectively. Most of the plants died one month after inoculation. Control plants showed no symptoms. The pathogen was consistently reisolated from inoculated plants and the identity was confirmed through TEF-1α sequencing. Thus, results of Koch postulate clearly identify N. parvum as the etiological agent of the observed disease of almond in Piedmont. N. parvum was recently reported in the same area on hazelnut (Waqas et al. 2021) and blueberry (Guarnaccia et al. 2021), and as causal agent of band canker and branch dieback in almond trees in US and Spain (Moral et al. 2019). To our knowledge, this is the first report of N. parvum as the causal agent of almond decline syndrome in Italy. Considering the increasing of almond cultivation in Italy, this disease represents a threat for the future production.

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.

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.

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.

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.

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.

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.

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 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 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.

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.

Different Phenotypes, Similar Genomes: Three Newly Sequenced Fusarium fujikuroi Strains Induce Different Symptoms in Rice Depending on Temperature.

Bakanae, caused by the hemibiotrophic fungus Fusarium fujikuroi, is one of the most important diseases of rice and is attributed to up to 75% of losses, depending on the strain and environmental conditions. Some strains cause elongation and thin leaves, whereas others induce stunting and chlorotic seedlings. Differences in symptoms are attributed to genetic differences in the strains. F. fujikuroi strains Augusto2, CSV1, and I1.3 were sequenced with Illumina MiSeq, and pathogenicity trials were conducted on rice cultivar Galileo, which is susceptible to bakanae. By performing gene prediction, single nucleotide polymorphism (SNP) calling, and structural variant analysis with a reference genome, we show how an extremely limited number of polymorphisms in genes not commonly associated with bakanae disease can cause strong differences in phenotype. CSV1 and Augusto2 were particularly close, with only 21,887 SNPs between them, but they differed in virulence, reaction to temperature, induced symptoms, colony morphology and color, growth speed, fumonisin, and gibberellin production. Genes potentially involved in the shift in phenotype were identified. Furthermore, we show how temperature variation may result in different symptoms even in rice plants inoculated with the same F. fujikuroi strain. Moreover, all of the F. fujikuroi strains became more virulent at higher temperatures. Significant differences were likewise observed in gibberellic acid production and in the expression of both fungal and plant gibberellin biosynthetic genes.

Presence of Powdery Mildew Caused by Erysiphe corylacearum on Hazelnut (Corylus avellana) in Italy.

Hazelnut (Corylus avellana) is widely grown in Italy, which is the second largest producer worldwide with 132,700 tonnes harvested from 78,593 hectares (FAOSTAT, 2018 ). Powdery mildew caused by Phyllactinia guttata has been reported in Italy and in other European countries, but recently in Austria, Switzerland and in central Europe a new species was discovered (Voglmayr et al., 2020; Beenken, 2020). During summer 2020, in Villar Fioccardo (Torino province, Piedmont, Italy) on hazelnut (cv. 'Tonda Gentile') growing on the edges of private gardens and parks, an extensive colonization of the adaxial side of the leaves with white powdery mycelium covering more than 80% of the surface was observed. Also, the abaxial side of the leaves showed the scattered presence of powdery, white, and thin mycelium. The powdery fungal pathogen collected from leaves had amphigenous, hyaline, branched, septate 1.5 to 3.7 µm wide mycelium; lobed, solitary hyphal appressoria; vertically elevated above the mycelium 53 to 82 µm long and 5 to 12 µm wide conidiophores (n = 30); hyaline, ellipsoid, ovoid to doliform conidia, solitary on conidiophores, 21 to 36 µm long, 15 to 21 µm wide (average 28 to 18 µm) (n = 50). Chasmothecia appeared in late September 2020 and they were spherical, single or in groups, 83 to 138 (average 100) µm in diameter (n = 50); 7 to 15 aseptate appendages were straight, sometimes flexuous, 55 to 111 (average 73) µm long (n = 50), with four to five times dichotomous branched apexes and recurved tips. In each chasmothecium, there were three to five ellipsoid, ovoid to subglobose asci with a length of 41 to 60 µm and a width of 28 to 56 µm (average 52 to 44 µm) (n = 30). Asci contained four to eight ascospores, 15 to 26 µm long and 10 to 17 µm wide (average 19 to 12 µm) (n =50). Mycelia were carefully scraped from the leaves with a scalpel and DNA was extracted by using the E.Z.N.A. Fungal DNA Mini Kit (Omega Bio-Tek, Darmstadt, Germany). Partial rDNA internal transcribed spacer region (ITS) of two isolates (DB20SET01, DB20SET01) was amplified using specific primers PMITS1/PMITS2 (Cunnington et al. 2003) and sequenced. Obtained sequences were deposited in GenBank (Accession Nos. MW045425, MW045426). BLAST analysis of the obtained 749-bp fragments showed 100% identity to ITS rDNA sequences of Erysiphe corylacearum from Switzerland (MN822721) and Azerbaijan (LC270863). One-year-old plants of C. avellane cv. Tonda Gentile were artificially inocuated by dusting conidia from infected leaves. Inoculated plants were incubated under controlled conditions at 23°C ± 1 and 70 to 80% relative humidity. Typical symptoms (white bloom) appeared on the upper surface of the leaves at 8 to 10 days after inoculation. No symptoms were found on control plants treated with sterile water. The fungus isolated from inoculated leaves was morphologically identical to the original isolates from diseased plants collected from Villar Fioccardo. Erysiphe corylacearum causes a new and aggressive form of powdery mildew. Since the first observation in north-eastern Turkey in 2013, it has spread rapidly throughout the Black Sea region, causing significant economic losses (Sezer et al., 2017). It has also been reported in Iran, Azerbaijan, and Ukraine (Arzanlou et al. 2018; Heluta et al., 2018). The disease has been observed sporadically in Piedmont, Italy, during summer 2020 (Regione Piemonte & Agrion, 2020) in some hazelnut growing areas, but presently, doesn't appear to impact yield. This is the first report of E. corylacearum, causing an aggressive powdery mildew on hazelnut in Italy, and as such, may more severely affect hazelnut groves in Italy and cause considerable yield losses. Literature cited Arzanlou M et al. 2018. Forest Pathology, 48:e12450. https://doi.org/10.1111/efp.12450. Beenken L et al. 2020. New Disease Reports 41, 11. http://dx.doi.org/10.5197/j.2044-0588.2020.041.011. Cunnington JH et al. 2003. Australasian Plant Pathology, 32, 421-428. Food and Agriculture Organization (FAO). 2018. http://www.fao.org/faostat/en/#home Heluta V.P. et al.2019. Ukrainian Botanical Journal, 2019, 76(3), 252-259. Regione Piemonte SFR & Agrion. 2020. https://www.regione.piemonte.it/web/sites/default/files/media/documenti/2020-10/mal_bianco_nocciolo_da_erysiphe_corylacearum.pdf Sezer AD et al. 2017. Phytoparasitica, 45, 577-581. Voglmayr H et al. 2020. New Disease Reports, 42, 14 http://dx.doi.org/10.5197/j.2044-0588.2020.042.014.

Development of a Sensitive TaqMan qPCR Assay for Detection and Quantification of Venturia inaequalis in Apple Leaves and Fruit and in Air Samples.

A TaqMan quantitative PCR (qPCR) assay based on the translation elongation factor 1-α gene was developed for the quantification of Venturia inaequalis in leaves and fruits of Malus × domestica and in spore trap samples. The designed primers and hydrolysis probe amplified a specific 86-bp fragment for V. inaequalis. The specificity of the assay was tested using 35 strains of V. inaequalis and 20 different fungal species, including common pathogens of apple and other species of Venturia. The limit of detection was 20 fg, which is lower than a single genome of V. inaequalis. The selectivity of the assay was tested using DNA from three cultivars of Malus × domestica, and no influence on pathogen amplification was found. The assay was also validated for repeatability and reproducibility. With this assay, it was possible to detect and quantify V. inaequalis in four cultivars (Ambrosia, Florina, Golden Delicious, and Mondial Gala) in both symptomatic and asymptomatic leaves and in symptomatic Golden Delicious apple fruit stored for 2 months. Furthermore, the assay was successfully tested on spore trap samples originating from apple orchards. The quantification of the molecular assay when compared with the estimated number of V. inaequalis cells, using an optical microscope, showed a correlation coefficient of 0.8186. The developed technique could be used to detect V. inaequalis in asymptomatic samples without any cross-reaction with other fungal species. Furthermore, to improve the efficacy of disease management with a timely application of fungicides, this assay could be used for the analysis of spore trap samples by using an implemented extraction method.

Increase in aflatoxins due to Aspergillus section Flavi multiplication during the aerobic deterioration of corn silage treated with different bacteria inocula.

The growth of Aspergillus flavus and the production of aflatoxins (AF) during the aerobic deterioration of corn silage represent a problem for animal and human health. This experiment was conducted to evaluate whether the presence of A. flavus and AF production originate from the field or additional AF are produced during the fermentation phase or during aerobic deterioration of corn silage. The trial was carried out in northern Italy on corn at a dry matter (DM) level of 34%. The fresh herbage was either not treated (C) or treated with a Lactobacillus buchneri (LB) NCIMB 40788 [(at 3 × 105 cfu/g of fresh matter (FM)], Lactobacillus hilgardii (LH) CNCM I-4785 (at 3 × 105 cfu/g of FM), or their combination (LB+LH; at 1.5 × 105 cfu/g of FM of each strain) ensiled in 20-L silos and opened after 250 d of ensiling. After silo opening, the aerobic stability was evaluated and samples were taken after 7 and 14 d of air exposure. The pre-ensiled material, the silages at silo opening, and the aerobically exposed silages were analyzed for DM content, fermentative profiles, microbial count, nutritive characteristics, DM losses, and AFB1, AFB2, AFG1, and AFG2 contents. Furthermore, a subsample of colonies with macromorphological features of Aspergillus section Flavi was selected for AF gene pattern characterization and in vitro AF production. The presence of A. flavus was below the detection limit (<1.00 log10 cfu/g) in the fresh forage before ensiling, whereas it was found in 1 out of 16 silage samples at silo opening at a level of 1.24 log10 cfu/g. The AF were found in both the fresh forage and at opening in all the samples, with a predominance of AFB2 (mean value of 1.71 µg/kg of DM). The inoculation of lactic acid bacteria determined a reduction in the lactic-to-acetic ratio compared with the control. A larger amount of acetic acid resulted in a lower yeast count and higher aerobic stability in the treated silages than in the control ones. At the beginning of aerobic deterioration, the yeasts increased to over 5 log10 cfu/g, whereas the molds were close to the value observed at silo opening. When the inhibiting conditions were depleted (pH and temperature higher than 5 and 35°C, respectively), both the total molds and A. flavus reached higher values than 8.00 and 4.00 log10 cfu/g, respectively, thus determining the ex novo production of AFB1 during aerobic deterioration, regardless of treatments. The analysis of gene pattern showed that 64% of the selected colonies of A. flavus showed the presence of all 4 AF gene patterns, and 43% of the selected colonies were able to produce AF in vitro. During air exposure, after 1,000°C·h have been cumulated, starch content decreased (below 10% DM) and concentration of neutral detergent fiber, acid detergent fiber, hemicelluloses, crude protein, and ash increased. The inoculation with LB and LB+LH increased the aerobic stability of the silages and delayed the onset of aerobic microbial degradation, which in turn indirectly reduced the risk of A. flavus outgrowth and AFB1 production after silage opening.

Rapid Detection of Monilinia fructicola and Monilinia laxa on Peach and Nectarine using Loop-Mediated Isothermal Amplification.

Monilinia laxa and M. fructicola are two causal agents of brown rot, one of the most important diseases in stone fruit. Two species cause blight on blossoms and twigs and brown rot on fruit in pre- and postharvest. Both species are distributed worldwide in North and South America, Australia, and Japan. In Europe, M. laxa is endemic, while M. fructicola was introduced in 2001 and it is now widespread in several countries. Currently, both species coexist in European stone fruit orchards. Monilinia spp. overwinter in cankers and mummified fruit. Mummy monitoring during winter permits growers to understand which species of Monilinia will be prevalent in an orchard during the following season, permitting planning of an appropriate crop protection. Traditionally, the identification has been carried out using morphological features and even with polymerase chain reaction (PCR)-based assays that requires time and well-equipped laboratories. In this study, two isothermal-based methods were designed to identify these pathogens in a faster way than using traditional methods. The loop-mediated amplification (LAMP) assays were validated on some isolates of Monilinia spp. coming from the mummy monitoring according to the international European and Mediterranean Plant Protection Organization standard (PM7/98), taking into account specificity, sensitivity, repeatability, and reproducibility. The sensitivity of both assays was checked by monitoring (at different time points) two nectarine varieties artificially inoculated and stored at two different temperatures. The reliability of both LAMP assays against the quantification of the inoculum was compared with previously published quantitative PCR assays. Both LAMP methods were able to detect a low number of cells. These LAMP methods could be a useful tool for monitoring brown rot causal agents in the field and during postharvest.

Native soils with their microbiotas elicit a state of alert in tomato plants.

Several studies have investigated soil microbial biodiversity, but understanding of the mechanisms underlying plant responses to soil microbiota remains in its infancy. Here, we focused on tomato (Solanum lycopersicum), testing the hypothesis that plants grown on native soils display different responses to soil microbiotas. Using transcriptomics, proteomics, and biochemistry, we describe the responses of two tomato genotypes (susceptible or resistant to Fusarium oxysporum f. sp. lycopersici) grown on an artificial growth substrate and two native soils (conducive and suppressive to Fusarium). Native soils affected tomato responses by modulating pathways involved in responses to oxidative stress, phenol biosynthesis, lignin deposition, and innate immunity, particularly in the suppressive soil. In tomato plants grown on steam-disinfected soils, total phenols and lignin decreased significantly. The inoculation of a mycorrhizal fungus partly rescued this response locally and systemically. Plants inoculated with the fungal pathogen showed reduced disease symptoms in the resistant genotype in both soils, but the susceptible genotype was partially protected from the pathogen only when grown on the suppressive soil. The 'state of alert' detected in tomatoes reveals novel mechanisms operating in plants in native soils and the soil microbiota appears to be one of the drivers of these plant responses.