The Impact of Climate Change on Vegetable Crop Diseases and Their Management: The Value of Phytotron Studies for the Agricultural Industry and Associated Stakeholders.

Climate change is having a significant impact on global agriculture, particularly on vegetable crops, which play a critical role in global nutrition. Recently, increasing research has concentrated on the impact of climate change on vegetable crop diseases, with several studies being conducted in phytotrons, which have been used to explore the effects of increased temperatures and CO2 concentrations to simulate future scenarios. This review focuses on the combined effects of temperature and carbon dioxide increases on foliar and soilborne vegetable diseases, as evaluated under phytotron conditions. The influence of climate change on mycotoxin production and disease management strategies is also explored through case studies. The results offer valuable information that can be used to guide both seed and agrochemical industries, as well as to develop disease-resistant varieties and innovative control measures, including biocontrol agents, considering the diseases that are likely to become prevalent under future climatic scenarios. Recommendations on how to manage vegetable diseases under ongoing climate change are proposed to facilitate plants' adaptation to and enhanced against the changing conditions. A proactive and comprehensive response to climate-induced challenges in vegetable farming is imperative to ensure food security and sustainability.

Characterization and Pathogenicity of Fungal Species Associated with Dieback of Apple Trees in Northern Italy.

Severe dieback symptoms were recently observed on apple (Malus × domestica) trees in Northern Italy, representing a growing concern for producers. Surveys were conducted over a 3-year period (2019 to 2021), and five apple orchards, from 5 to 12 years old, were monitored. A total of 33 fungal isolates isolated from symptomatic plants was selected for characterization. The species identification was achieved through multilocus phylogenetic analyses performed on sequences of three genomic loci (ITS, tub2, and tef1). Morphological features were assessed, and the average growth rate at different temperatures was determined. Seven species were identified in association with dieback of apple trees: Botryosphaeria dothidea, Cadophora luteo-olivacea, Diaporthe rudis, Diplodia seriata, Eutypa lata, Kalmusia longispora, and Paraconiothyrium brasiliense. All the species were pathogenic when inoculated on healthy apple plants. B. dothidea resulted in the most aggressive infections. This study provides an insight into the fungal species diversity associated with apple dieback and provides basis for further investigations to assess the phytosanitary status of plant materials to recommend and implement effective management strategies.

First Report of Gnomoniopsis fragrariae causing leaf spots on strawberry in Italy.

Strawberry (Fragaria × ananassa Duch.) is widely cultivated in Italy. During May-June 2022, mild symptoms of an unknown leaf spot disease appeared on 5-10% of June-bearing strawberry (cv. Elodì) plants transplanted in July 2021 in a commercial farm located in the province of Cuneo, North Italy. During September-November 2022, the symptoms appeared also on 10-15% of the plants transplanted in July 2022. The disease was scattered throughout the field, large 600 m2, both on new and senescent leaves. Fungicides (sulphur, Tiovit Jet; penconazole, Topas 10 EC) were applied to the plants according to integrated pest management during the growing period. The disease symptoms were purplish to brown necrotic leaf spots up to 1-3 mm in diameter and chlorotic leaf margins. Black lesions were occasionally observed on the petioles, appearing as small necrotic or larger elongated lesions causing leaf death. Peritechia were observed in planta after about 4 months from sampling and measured (144 to 239 µm and 200 to 291 µm, n = 10). Diseased leaves and petioles from about 10 plants were surface disinfested for 1 min in 1% NaClO, rinsed with sterile water and plated on potato dextrose agar (PDA) amended with 25 mg streptomycin sulphate/liter. A fungus with white cottony colonies was repeatedly recovered and maintained in pure culture on PDA. Biguttulate conidia with rounded ends were measured (4.3 to 8.0 µm and 1.2 to 2.9 µm, average 6.1× 2.3 µm, n = 50) from 21-day old colonies grown in PDA at 22°C and 12 h photoperiod. According to colony and conidia morphology, the isolate was identified as Gnomoniopsis sp. (Walker et al., 2010). The fungal DNA was extracted from a pure culture of one isolate selected as a representative (code FR2-22), by using the E.Z.N.A. Fungal DNA Mini Kit (Omega Bio-Tek, Darmstadt, Germany). The identification was carried out by amplifying and sequencing the internal transcribed spacer (ITS) region and the partial translation elongation factor 1-α (TEF) gene using the primers ITS1/ITS4 and EF-728F/EF2 (Udayanga et al., 2021), respectively. The purified PCR products were sequenced at the BMR Genomics Centre (Padova, Italy) obtaining 551bp (ITS) and 652bp (TEF) sequences deposited in GenBank (Accession nos. OQ179950 and OQ190173, respectively). A BLASTn search of both sequences revealed to be 100% identical to the ITS and TEF loci of Gnomoniopsis fructicola sequences of the isolates VPRI_15547 and CBS 275.51 deposited in GenBank with accession Nos. MT378345 and MT383092. The pathogenicity of the isolate FR2-22 was assessed in two trials by biological tests (3 replicates with 1 plant per replicate/pot) in two greenhouse compartments, kept at temperature 20-24°C and at humidity 80-90%. Healthy leaves of forty-day-old strawberry plants (cv. Elodì) were sprayed with 1-5 x106 conidia/ml obtained from the FR2-22 isolate grown on PDA at 25°C for 20 days. The control (water-sprayed plants) was kept in the same conditions. Small leaf spots similar to the symptoms previously observed in the farm were observed 15 days post inoculation. Furthermore, 30 to 40% of leaves developed symptoms similar to those observed in the field after 25-40 days, while the control remained health. The same fungal isolate was repeatedly reisolated from the affected leaves and petioles and identified based on TEF sequencing. Gnomoniopsis fragariae comb. nov., designed as new name for Gnomoniopsis fructicola (Udayanga et al., 2021), has previously been reported on Fragaria × ananassa plants in Australia and in the USA (Farr and Rossman, 2023). To the best of our knowledge, this is the first report of G. fragariae on strawberry in Italy. The impact of the disease caused by this pathogen could be of high importance in the future of strawberry production in Italy. Healthy propagation material and strict disease management practices in nurseries is a requirement to avoid disease epidemics.

Do the ends justify the means? Impact of drought progression rate on stress response and recovery in Vitis vinifera.

Plants are frequently exposed to prolonged and intense drought events. To survive, species must implement strategies to overcome progressive drought while maintaining sufficient resources to sustain the recovery of functions. Our objective was to understand how stress rate development modulates energy reserves and affects the recovery process. Grenache Vitis vinifera cultivar was exposed to either fast-developing drought (within few days; FDD), typical of pot experiments, or slow-developing drought (few weeks, SDD), more typical for natural conditions. FDD was characterized by fast (2-3 days) stomatal closure in response to increased stress level, high abscisic acid (ABA) accumulation in xylem sap (>400 µg L-1 ) without the substantial changes associated with stem priming for recovery (no accumulation of sugar or drop in xylem sap pH). In contrast, SDD was characterized by gradual stomatal closure, low ABA accumulation (<100 µg L-1 ) and changes that primed the stem for recovery (xylem sap acidification from 6 to 5.5 pH and sugar accumulation from 1 to 3 g L-1 ). Despite FDD and SDD demonstrating similar trends over time in the recovery of stomatal conductance, they differed in their sensitivity to xylem ABA. Grenache showed near-isohydric and near-anisohydric behavior depending on the rate of drought progression, gauging the risk between hydraulic integrity and photosynthetic gain. The isohydry observed during FDD could potentially provide protection from large sudden swings in tension, while transitioning to anisohydry during SDD could prioritize the maintenance of photosynthetic activity over hydraulic security.

First report of Diaporthe eres causing stem blight and dieback on highbush blueberry (Vaccinium corymbosum) in Italy.

Vaccinium corymbosum L. is a woody deciduous shrub belonging to the Ericaceae family. During the last decade, Northern Italy has become a major area for blueberry cultivation and production in Europe, supplying other countries with high quality fruit (FAOSTAT 2020). In June 2020, plant stem and branch wilting with brownish necrotic internal lesions were observed on 20% of around 250 plants of the cultivar (cv.) Draper cultivated in a soilless culture system located in Peveragno (Piedmont, Cuneo Province, 44°21'05.6''N, 07°37'23.2''). Dieback and death of the plants also occurred. Fifteen symptomatic plants were collected. Wood pieces (5-10 mm) excised from diseased stems and branches were sterilized in 1% sodium hypochlorite for 1 min, rinsed in sterile distilled water and dried on sterile absorbent paper. Small fragments (2-3 mm), obtained from the edge of the necrotic tissues, were plated on potato dextrose agar (PDA) amended with 25 mg/L of streptomycin sulfate. Plates were incubated at 25 ± 1°C under a 12 h photoperiod and, five days after, colonies with the same characteristics of Diaporthe spp. were established from single hyphal tip transfers. Two representative strains (CVG1130 and CVG1131) were grown on PDA from single hyphal-tip transfers. After 7 days, white colonies with fluffy, aerial, mycelium reaching 8.3 cm were observed. colonies turned gray after three weeks producing dark brown pycnidia. Aseptate, hyaline, fusiform to ellipsoidal, measuring 6.3 to 8.4 × 2.2 to 3.0 µm alpha conidia were produced. No beta conidia were observed. The DNA of the same strains was extracted and a polymerase chain reaction (PCR) was performed for the ITS (primer set: ITS1-ITS4) (GenBank Accession no. ON834528; ON834529), tef (EF1-728/EF1-986) (GenBank Accession no. ON843715; ON843716) and tub2 (T1/Bt2b) (GenBank Accession no. ON843713; ON843714) regions, in accordance with previous studies (Gomes et al. 2013, Bezerra et al. 2021). Sequences analysed with BLASTn algorithm (Altschul et al. 1997) exhibited 98% identity with the ex-type strain CBS 138594 of Diaporthe eres for ITS (GenBank Accession no. KJ210529), 100% identity for tub2 (GenBank Accession no. KJ420799) and 99% identity for tef (GenBank Accession no. KJ210550). The maximum likelihood method based on combined sequences of ITS, tef and tub2 loci was performed, and the isolates CVG1130 and CVG1131 clustered with several reference strains of D. eres (Supplementary figure). To fulfil Koch's postulates, pathogenicity tests were performed on 1-year-old blueberry potted plants cv. Draper and Duke. A small piece of the bark tissue was removed with a sterile-bladed scalpel to expose the cambium. Mycelium plugs (5 mm diameter), obtained from 10-day-old cultures of the strains CVG1130 and CVG1131, were placed with the mycelium in contact with the internal plant tissues. Three plants were used for each isolate and the inoculation points were sealed with Parafilm®. The same number of plants treated with sterile PDA plugs were used as control. Plants were placed in a growth chamber at 25°C for 3 weeks. After this period, symptoms similar to those observed in the field appeared on the inoculated plants, while control plants remained healthy. A fungus with the same morphological characteristics of D. eres was reisolated from inoculated plants and identified by sequencing the tub2 gene to confirm Koch's postulates. Diaporthe eres was previously reported on different Vaccinium spp. in Chile, Lithuania, the Netherlands and the U.S.A. (Farr and Rossman, 2022). To our knowledge, this is the first report of D. eres associated with stem blight and dieback of highbush blueberry in Italy. Duke and Draper are among the most cultivated blueberry cultivars in Piedmont, where the spread of D. eres could represent a serious threat.

First Report of Gray Leaf Spot Caused by Pyricularia oryzae on Common Millet in Italy.

Common millet (Panicum miliaceum L.) is a niche crop cultivated in Italy for its high nutritional and gluten-free characteristics. These aspects combined with its sustainability and adaptability to paddy soils make it relevant in crop rotation with rice in some organic farms in Northwestern Italy. In September 2021, in a farm producing organic rice located in Rovasenda (VC), Italy (GPS: 45°54'82"N, 8°.31'96"E), plants of millet (var. San Marino), sown on July 1st and almost at physiological maturity, were observed with gray leaf spot symptoms with an incidence of 50% in a 12.5 ha field cultivated in a yearly rotation with rice. Oval shape lesions (between 1 and 5 cm long and 0.5 to1 cm wide) with a light gray colored centers and dark reddish-brown edges were observed on the length of the leaf blade. Gray colonies producing conidia morphologically like Pyricularia developed from 140 symptomatic leaf fragments with a 2% frequency. Monosporic cultures were obtained from five colonies and were grown on Potato Dextrose Agar for morphological and molecular identification. The colonies showed gray, fluffy mycelium, with a smooth margin, with a radius of 28 to 30 mm after 10 days at 28°C. The isolates produced hyaline, pyriform, and 2-septate conidia 17.9 to 33.5 µm (average 25.7 µm) long and 8.3 to 11.7 µm (average 10. µm) wide (n=50) on PDA. Conidiophores were solitary, erect, straight, or curved, septate, and measured 80.2 to 221.3 µm (average 150.7 µm) long and 3.6 to 5.8 µm (average 4.7 µm) wide (n=50). The molecular identification of one single-conidia isolate was confirmed by PCR and sequence comparison of the region of the transposon Pot2 (Harmon et al., 2003). BLASTn searches of GenBank using transposon Pot2 (ON843711) representative isolate 21-03-C, revealed 99.79% identity to P. oryzae (Syn. Magnaporthe oryzae) isolate MD1 (MK608664). Pathogenicity tests were carried out by suspending conidia from a 14-days old culture on PDA in sterile water at a concentration of 1×105 CFU/ml. Twenty seeds of P. miliaceum were sown in pots filled with a steamed mix of white peat and perlite, 80:20 v/v, and maintained at 28°C under a 12 hours day/night light cycle. Diseased leaves of 10 plants were inoculated by aspersion of 10 ml of the conidial suspension of isolate 21-03-C 2 weeks after the sowing (Harmon et al.; 2003). Plants were incubated in a near moisture-saturated environment in plastic bags at 28°C for 72h, after which symptoms were visually assessed. Ten control plants, inoculated with 10 ml of sterile distilled water remained healthy. P. oryzae was reisolated from leaves showing oblong, whitish lesions, surrounded by light brown margins and identified by resequencing of transposon Pot2. P. oryzae has been already reported on millet in South Korea as causal agent of blast (Klaubauf et al.; 2014). To our knowledge, this is the first report of P. oryzae as a pathogen of millet in Italy and in Europe. Further studies should be conducted to characterize these millet isolates by assessing their virulence on Oryza sativa and the implications in the adoption of P. miliaceum in rotation with rice, to prevent possible carry over of the pathogen on the main crop.

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.

First report of Fusarium commune causing root and crown rot on maize in Italy.

Maize (Zea mays L.) is a cereal crop of great economic importance in Italy; production is currently of 62,587,469 t, with an area that covers 628,801 ha, concentrated in northern Italy (ISTAT 2020). Fusarium species are associated with root and crown rot causing failures in crop establishment under high soil moisture. In 2019 maize seedlings collected in a farm located in San Zenone degli Ezzelini (VI, Italy) showed root and crown rot symptoms with browning of the stem tissues, wilting of the seedling, and collapsing due to the rotting tissues at the base of the stem. The incidence of diseased plants was approximately 15%. Seedlings were cleaned thoroughly from soil residues under tap water. Portions (about 3-5 mm) of tissue from roots and crowns of the diseased plants were cut and surface disinfected with a water solution of NaClO at 0.5% for 2 minutes and rinsed in sterile H20. The tissue fragments were plated on Potato Dextrose Agar (PDA) amended with 50 mg/l of streptomycin sulfate and incubated for 48-72 hours at 25oC. Over the 80 tissue fragments plated, 5% were identified as Fusarium verticillioides, 60% as Fusarium spp., 35% developed saprophytes. Fusarium spp. isolates that showed morphological characteristics not belonging to known pathogenic species on maize were selected and used for further investigation while species belonging to F. oxysporum were discarded. Single conidia of the Fusarium spp. colonies were cultured on PDA and Carnation Leaf Agar (CLA) for pathogenicity tests, morphological and molecular identification. The colonies showed white to pink, abundant, densely floccose to fluffy aerial mycelium. Colony reverse showed light violet pigmentation, in rings on PDA. On CLA the isolates produced slightly curved macronidia with 3 septa 28.1 - 65.5 µm long and 2.8-6.3 µm wide (n=50). Microconidia were cylindrical, aseptate, 4.5 -14.0 µm long and 1.5-3.9 µm wide (n=50). Spherical clamydospores were 8.8 ± 2.5 µm size (n=30), produced singly or in pairs on the mycelium, according to the description by Skovgaard et al. (2003) for F. commune. The identity of two single-conidia strains was confirmed by sequence comparison of the translation elongation factor-1α (tef-1α), and RNA polymerase II subunit (rpb2) gene fragments (O'Donnell et al. 2010). BLASTn searches of GenBank, and Fusarium-ID database, using the partial tef-1α (MW419921, MW419922) and rpb2 (MW419923, MW419924) sequences of representative isolate DB19lug07 and DB19lug20, revealed 99% identity for tef-1α and 100% identity to F. commune NRRL 28387(AF246832, AF250560). Pathogenicity tests were carried out by suspending conidia from a 10-days old culture on PDA in sterile H2O to 5×104 CFU/ml. Fifty seeds were immersed in 50 ml of the conidial suspension of each isolate for 24 hours and in sterile water (Koch et al. 2020). The seeds were drained, dried at room temperature, and sown in trays filled with a steamed mix of white peat and perlite, 80:20 v/v, and maintained at 25°C and RH of 80-85% for 14 days with 12 hours photoperiod. Seedlings were extracted from the substrate, washed under tap water, and observed for the presence of root and crown rots like the symptoms observed on the seedlings collected in the field. Control seedlings were healthy and F. commune was reisolated from the symptomatic ones and identified by resequencing of tef-1α gene. F. commune has been already reported on maize (Xi et al. 2019) and other plant species, like soybean (Ellis et al. 2013), sugarcane (Wang et al. 2018), potato (Osawa et al. 2020), indicating that some attention must be paid in crop rotation and residue management strategies. To our knowledge this is the first report of F. commune as a pathogen of maize in Italy. References Ellis M L et al. 2013. Plant Disease, 97, doi: 10.1094/PDIS-07-12-0644-PDN. ISTAT. 2020. http://dati.istat.it/Index.aspx?QueryId=33702. Accessed December 28, 2020. Koch, E. et al. 2020. Journal of Plant Diseases and Protection. 127, 883-893 doi: 10.1007/s41348-020-00350-w O'Donnell K et al. 2010. J. Clin. Microbiol. 48:3708. https://doi.org/10.1128/JCM.00989-10 Osawa H et al. 2020. Journal of General Plant Pathology, doi.org/10.1007/s10327-020-00969-5. Skovgaard K 2003. Mycologia, 95:4, 630-636, DOI: 10.1080/15572536.2004.11833067. Wang J et al. 2018. Plant Disease, 102, doi/10.1094/PDIS-07-17-1011-PDN Xi K et al. 2019. Plant Disease, 103, doi/10.1094/PDIS-09-18-1674-PDN.

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.

Alternaria Leaf Spot Caused by Alternaria Species: An Emerging Problem on Ornamental Plants in Italy.

Serious outbreaks of Alternaria leaf spot and plant decay have recently been recorded on several ornamental plants in the Biella Province (Northern Italy). Twenty-two fungal isolates were obtained from Alternaria infected plant tissues from 13 ornamental hosts. All the isolates were identified morphologically as small-spored Alternaria species. Multilocus sequence typing, carried out by means of ITS, rpb2, tef1, endoPG, Alt a 1, and OPA10-2, assigned 19 isolates as Alternaria alternata, two isolates as belonging to the Alternaria arborescens species complex, and one isolate as an unknown Alternaria sp. Haplotype analyses of ornamental and reference A. alternata isolates from 12 countries identified 14 OPA10-2 and 11 endoPG haplotypes showing a relatively high haplotype diversity. A lack of host specialization or geographic distribution was observed. The host range of the studied A. alternata isolates expanded in cross-pathogenicity assays, and more aggressiveness was frequently observed on the experimental plants than on the host plants from which the fungal isolates were originally isolated. High disease severity, population expansion, intraspecies diversity, and increased range of experimental hosts were seen in the emergence of Alternaria disease on ornamentals. More epidemiological and molecular studies should be performed to better understand these diseases, taking into consideration factors such as seed transmission and ongoing climate changes.

The Molecular Priming of Defense Responses is Differently Regulated in Grapevine Genotypes Following Elicitor Application against Powdery Mildew.

Molecular changes associated with response to powdery mildew (PM) caused by Erysiphe necator have been largely explored in Vitis vinifera cultivars, but little is known on transcriptional and metabolic modifications following application of resistance elicitors against this disease. In this study, the whole transcriptome sequencing, and hormone and metabolite analyses were combined to dissect long-term defense mechanisms induced by molecular reprogramming events in PM-infected 'Moscato' and 'Nebbiolo' leaves treated with three resistance inducers: acibenzolar-S-methyl, potassium phosphonate, and laminarin. Although all compounds were effective in counteracting the disease, acibenzolar-S-methyl caused the most intense transcriptional modifications in both cultivars. These involved a strong down-regulation of photosynthesis and energy metabolism and changes in carbohydrate accumulation and partitioning that most likely shifted the plant growth-defense trade-off towards the establishment of disease resistance processes. It was also shown that genotype-associated metabolic signals significantly affected the cultivar defense machinery. Indeed, 'Nebbiolo' and 'Moscato' built up different defense strategies, often enhanced by the application of a specific elicitor, which resulted in either reinforcement of early defense mechanisms (e.g., epicuticular wax deposition and overexpression of pathogenesis-related genes in 'Nebbiolo'), or accumulation of endogenous hormones and antimicrobial compounds (e.g., high content of abscisic acid, jasmonic acid, and viniferin in 'Moscato').

Priming xylem for stress recovery depends on coordinated activity of sugar metabolic pathways and changes in xylem sap pH.

Some plant species are capable of significant reduction of xylem embolism during recovery from drought despite stem water potential remains negative. However, the functional biology underlying this process is elusive. We subjected poplar trees to drought stress followed by a period of recovery. Water potential, hydraulic conductivity, gas exchange, xylem sap pH, and carbohydrate content in sap and woody stems were monitored in combination with an analysis of carbohydrate metabolism, enzyme activity, and expression of genes involved in sugar metabolic and transport pathways. Drought resulted in an alteration of differential partitioning between starch and soluble sugars. Upon stress, an increase in the starch degradation rate and the overexpression of sugar symporter genes promoted the efflux of disaccharides (mostly maltose and sucrose) to the apoplast. In turn, the efflux activity of the sugar-proton cotransporters caused a drop in xylem pH. The newly acidic environment induced the activity of apoplastic invertases leading to the accumulation of monosaccharides in the apoplast, thus providing the main osmoticum necessary for recovery. During drought and recovery, a complex network of coordinated molecular and biochemical signals was activated at the interface between xylem and parenchyma cells that appeared to prime the xylem for hydraulic recovery.

Emergence of Leaf Spot Disease on Leafy Vegetable and Ornamental Crops Caused by Paramyrothecium and Albifimbria Species.

The genera Paramyrothecium and Albifimbria have been established from the former genus Myrothecium and they generally comprise common soil-inhabiting and saprophytic fungi. Within these genera, only two fungi have been recognized as phytopathogenic thus far: P. roridum and A. verrucaria, both of which cause necrotic leaf spots and plant collapse. Severe leaf necrosis and plant decay have been observed in Northern and Southern Italy on leafy vegetable crops. Thirty-six strains of Paramyrothecium- and Albifimbria-like fungi were isolated from affected plants belonging to eight different species. Based on morphological characteristics, 19 strains were assigned to A. verrucaria, whereas the remaining strains, which mostly resembled Paramyrothecium-like fungi, could not be identified precisely. Molecular characterization of six loci (internal transcribed spacer [ITS], ß-tubulin [tub2], calmodulin [cmdA], translation elongation factor 1-alpha [tef1], large subunit ribosomal RNA [LSU], and mitochondrial ATP 6synthase 6 [ATP6]) of the 36 new isolates and three previously ITS-characterized isolates assigned all strains to four species: A. verrucaria, P. roridum, P. foliicola, and P. nigrum. Single and concatenated phylogenetic analyses were conducted, and they clearly distinguished the isolated fungi into four different groups. A. verrucaria, P. roridum, P. foliicola, and P. nigrum were able to induce leaf necrosis singly, and they were confirmed to be the causal agents of the leaf spot disease through pathogenicity assays. The involvement of fungi previously considered saprophytic (i.e., P. foliicola and P. nigrum) in the development of plant disease for the first time deserves particular attention because of the possibility of their transmission by seeds and the limited knowledge of their management with chemicals.

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.

Ready-to-Eat Salad Crops: A Plant Pathogen's Heaven.

The ready-to-eat salad sector, also called fresh-cut or bagged salads, is a fast-growing segment of the fresh-food industry. The dynamism and specialization of this sector, together with the lack of adequate crop rotation, the globalization of the seed market, and climate change, are the main causes of the development of many new diseases that cause severe production losses. Newly detected diseases of the most important crops grown (lettuce, wild and cultivated rocket, lamb's lettuce, chicory, endive, basil, spinach, and Swiss chard) are critically discussed. The management of these diseases represents a formidable challenge, since few fungicides are registered on these minor-use crops. An interesting feature of the ready-to-eat salad sector is that most crops are grown under protection, often in soilless systems, which provide an environment helpful to the implementation of innovative control methods. Current trends in disease management are discussed, with special focus on the most sustainable practices.

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.

Molecular phylogeny and characterization of secondary metabolite profile of plant pathogenic Alternaria species isolated from basil.

Alternaria leaf-spot is a new disease recently reported on basil in Italy. The correct identification of Alternaria species has suffered from many reclassifications in function of morphological features and molecular data. In our study, we performed an overall approach to obtain a better characterization of basil Alternaria isolates. Morphological characteristics, seven-genome region phylogenic analysis, and secondary metabolite profile differentiated the majority of the isolates as A. alternata. OPA 1-3 and OPA 10-2 were the best molecular regions to discriminate among the isolates. Morphological characteristics and sporulation groups helped to discriminate A. tenuissima from A. alternata isolates. All isolates in the A. sect. Alternaria were mycotoxigenic and pathogenic on basil, the production of mycotoxins was enhanced on basil compared to in vitro conditions used in this work.

Several species of Penicillium isolated from chestnut flour processing are pathogenic on fresh chestnuts and produce mycotoxins.

A collection of 124 isolates of Penicillium spp. was created by monitoring fresh chestnuts, dried chestnuts, chestnut granulates, chestnut flour and indoor chestnut mills. Sequencing of the ITS region, ß-tubulin and calmodulin, macro-morphology and secondary metabolite production made it possible to determine 20 species of Penicillium. P. bialowiezense was dominant in the fresh chestnuts, while P. crustosum was more frequent in the other sources. A pathogenicity test on chestnut showed that around 70% of the isolates were virulent. P. corylophilum and P. yezoense were not pathogenic, while the other 18 species had at least one virulent isolate. P. expansum and P. crustosum were the most virulent. The isolates were characterized to establish their ability to produce 14 toxic metabolites in vivo: 59% were able to produce at least one mycotoxin. P. expansum was able to produce patulin, chaetoglobosin A and roquefortine, while P. bialowiezense produced C. Mycophenolic acid. Cyclopenins and viridicatins were produced by most of the P. crustosum, P. polonicum, P. solitum and P. discolour isolates. Some of the P. crustosum isolates were also able to produce roquefortine C or penitrem A. Information about the occurrence of Penicillium spp. and their mycotoxins will help producers to set up management procedures that can help to control the fungal growth and the mycotoxin production of chestnuts.

Characterization of Aspergillus section Flavi isolated from fresh chestnuts and along the chestnut flour process.

An extensive sampling of Aspergillus section Flavi considered to be the main agent responsible for aflatoxin contamination, was carried out in the field and along the processing phases of chestnut flour production in 2015. Fifty-eight isolates were characterized by means of biological, molecular and chemical assays. The highest incidence of Aspergillus section Flavi was found in the field. The identification of the isolates was based on ß-tubulin and calmodulin gene sequences. A. flavus was found to be the dominant species, and this was followed by A. oryzae var effusus, A. tamarii, A. parasiticus and A. toxicarius. Nineteen percent of the strains produced aflatoxins in vitro and forty percent in vivo. The pathogenicity assay on chestnut showed 56 virulent strains out of 58. The molecular, morphological, chemical and biological analyses of A. flavus strains showed an intraspecific variability. These results confirm that a polyphasic approach is necessary to discriminate the species inside the Aspergillus section Flavi. The present research is the first monitoring and characterization of aflatoxigenic fungi from fresh chestnut and the chestnut flour process, and it highlights the risk of a potential contamination along the whole chestnut production chain.