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 Brown Rot Caused by Monilinia polystroma on Sweet Cherry and Almond in Italy.

Brown rot decay is an important disease of pome and stone fruits. In Italy, the main pathogens on stone fruits are Monilinia laxa, M. fructigena, and M. fructicola (Spitaler et al. 2022a). In addition, Monilinia polystroma (G. Leeuwen) L. M. Kohn (van Leeuwen et al. 2002), was recently found in Italy on peach (Martini et al. 2014), pear (Martini et al. 2015), plum (Abate et al. 2018), apple (Rosati et al. 2021), and quince (Spitaler et al. 2022b). In South Tyrol province, sweet cherry (Prunus avium L.) and almond (Prunus dulcis Mill. D. A. Webb), plants of the Rosaceae family and belonging to stone fruits, were observed to be frequently affected by brown rot. Affected cherries as well as almonds showed brown lesions, covered by yellowish or buff-colored stroma in concentric rings. Symptomatic cherries became shriveled, while symptomatic almonds remained firm. To determine the pathogen, single spore isolates were obtained from five symptomatic fruits, each from a cherry orchard of the cultivar Kordia in July 2021 and almond trees of the variety Dulcis in August 2021. Both sample sites were situated in Vadena/Pfatten. Infestation in the cherry orchard, covered by a rain-protection foil, was determined to be about 1 %. In almond, over 50 % of the fruits of various ripening stages showed brown rot symptoms. On potato dextrose agar (PDA) at 22 °C and a photoperiod of 16 h, isolates from both fruits matched the morphological characteristics of M. polystroma (Vasic et al. 2016) within 14 days. DNA was extracted from mycelium and the rRNA encoding gene region using ITS4 and ITS6 primers as well as a genomic sequence of unknown function using the primers UniMon_Forw and UniMon_Rev (Petróczy et al. 2012), were amplified and sequenced. MegaBLAST analysis revealed 100 % identity with M. polystroma sequences of the NCBI GenBank (rRNA encoding region: NR_154198; genomic region: JN128836). Sequences were deposited in GenBank under the accession numbers OP642545/OP654171 (cherry) and OP642546/OP654172 (almond). Pathogenicity was confirmed with mature cherries cultivar Duroncino or almost mature almond fruit of the variety Dulcis, respectively: 16 samples each for both fruits were surface-sterilized by dipping in 75 % ethanol for 10 s and subsequent rinsing with sterile water for 10 s. Mycelial plugs (1 mm) were dislodged from a 7-day old colony and inserted in a 1 mm hole into the fruits. Incubation was performed in plastic boxes under the conditions described above. PDA-inoculated fruit were used as controls. All cherries and all almonds were completely covered by brown rot lesions 7 days and 15 days post inoculation, respectively. Control fruits remained symptomless. Conidia were produced in branched chains on mycelium-inoculated fruit. Conidia were one-celled, limoniform, hyaline, measuring 13.1 to 22.2 × 9.7 to 14.8 µm (cherry) and 14.1 to 20.8 × 10.7 to 15.3 µm (almond). Additionally, 16 fruits each were inoculated with 20 µL conidial suspension (5 x 10^3 spores/mL) from mycelium-inoculated fruits. All cherries as well as all almonds were completely covered by brown rot lesions 7 days and 15 days post inoculation. Control fruits remained symptomless. To confirm identity, the fungus was isolated from five spore-inoculated fruits each for cherry and almond. The isolates showed identical morphological features and sequence identity as the original isolates. To our knowledge, this is also the first report of M. polystroma on almond, while the pathogen has already been reported on sweet cherry in Poland (Poniatowska et al. 2016). These additional host plants identified in this study suggest a broad impact of M. polystroma on Italian stone fruit production. Due to the economically important cultivation of stone fruit, further knowledge about the pathogens' host range will help to assign brown rot symptoms to M. polystroma and to improve targeted control strategies.

First report of Preharvest Decay caused by Colletotrichum chrysophilum on apples in Italy (South Tyrol).

Apple (Malus × domestica Borkh.) is economically the most important fruit crop in South Tyrol (Italy). At the end of the growing season 2020, necrotic lesions and chlorosis developed on leaves and premature leaf dropping was observed on the cultivars Gala, Granny Smith and Cripps Pink(cov)/Rosy Glow(cov) in the Etsch/Adige valley. After the appearance of these symptoms, small circular brownish spots were observed on above 90 % of apples in the respective orchards. Fungal isolates were obtained from symptomatic apples by culturing small portions of fruit flesh from the lesion margin on potato dextrose agar (PDA) at 25°C in the dark. The colonies showed a white to peachy color on the upper surface and were greyish on the reverse side. Conidia were cylindrical, predominantly rounded and averaged 16.39 ± 1.37 µm and 5.75 ± 0.81 µm (n = 33), consistent morphological characteristics as described recently in Fuentes-Aragón et al. (2021). A multi-locus sequence analysis according to Astolfi et al. (2022) and Weir et al. (2022) was conducted based on the internal transcribed spacer (ITS) region and on fragments of actin (ACT), DNA-(apurinic or apyrimidinic site) lyase (APN2), calmodulin (CAL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutamine synthetase (GS), beta-tubulin (TUB2), and intergenic spacer and partial mating type (Mat1-2) genes. MegaBlast analysis revealed 100 % identity to the reference genome of C. chrysophilum for ITS (NR_160821), for ACT (KX093982), APN2 (KX094018), CAL (KX094063), GAPDH (KX094183), 99.29 % for TUB2 (KX094285), 99.85 % for Mat1-2 (KX094325) and 99.87 % for GS (KX094204). Sequences were deposited in GenBank (ITS: OK485032; ACT: OK539650; APN2: ON624100; CAL: ON624104; GAPDH: OK539654; GS: ON624108; TUB2: OK539658; Mat1-2: ON645973). Pathogenicity assays were performed on wounded and unwounded apples of cvs. Gala and Rosy Glow(cov). Apples of both cvs. were wounded with a sterile needle. For each variant, 8 fruits were inoculated with 10 µL spore suspension (1 × 106 spores mL-1) in 0.05 % Tween®20 or with 10 µL 0.05 % Tween®20 as control. The apples were put into plastic boxes containing moist tissue and incubated at 25°C, 100 % relative humidity and 12 h photoperiod. First lesions appeared on inoculated wounded fruits after 5 days, whereas unwounded and control fruits remained asymptomatic. After 15 days, symptoms could be observed only on inoculated wounded apples with spore suspension: on 95 % of individual wounds of Rosy Glow(cov) and on 77.5 % of Gala. Koch's postulates were fulfilled by re-isolating the fungus and by identifying the re-isolates as C. chrysophilum (ITS: OK485033-OK485035; ACT: OK539651-OK539653; APN2: ON624101-ON624103; CAL: ON624105-ON624107; GAPDH: OK539655-OK539657; GS: ON624109; TUB2: OK539659-OK539661; Mat1-2: ON645974-ON645976). To date, only one study confirmed C. chrysophilum as causal agent of apple bitter rot in Europe (Cabrefiga et al. 2022). Recently, Astolfi et al. (2022) reclassified C. chrysophilum as the main causal agent of GLS on apples in Southern Brazil and Uruguay. The authors stressed that C. chrysophilum might also be the potential agent of GLS in Europe (Astolfi et al. 2022). This confirms the observations made in 2020 in South Tyrol, where massive leaf spots preceded the symptoms on fruit. To the best of our knowledge, this is the first report of a preharvest decay on apples caused by C. chrysophilum in Italy (South Tyrol).

Assessment of Cu applications in two contrasting soils-effects on soil microbial activity and the fungal community structure.

Copper (Cu)-based fungicides have been used in viticulture to prevent downy mildew since the end of the 19th century, and are still used today to reduce fungal diseases. Consequently, Cu has built up in many vineyard soils, and it is still unclear how this affects soil functioning. The present study aimed to assess the short and medium-term effects of Cu contamination on the soil fungal community. Two contrasting agricultural soils, an acidic sandy loam and an alkaline silt loam, were used for an eco-toxicological greenhouse pot experiment. The soils were spiked with a Cu-based fungicide in seven concentrations (0-5000 mg Cu kg-1 soil) and alfalfa was grown in the pots for 3 months. Sampling was conducted at the beginning and at the end of the study period to test Cu toxicity effects on total microbial biomass, basal respiration and enzyme activities. Fungal abundance was analysed by ergosterol at both samplings, and for the second sampling, fungal community structure was evaluated via ITS amplicon sequences. Soil microbial biomass C as well as microbial respiration rate decreased with increasing Cu concentrations, with EC50 ranging from 76 to 187 mg EDTA-extractable Cu kg-1 soil. Oxidative enzymes showed a trend of increasing activity at the first sampling, but a decline in peroxidase activity was observed for the second sampling. We found remarkable Cu-induced changes in fungal community abundance (EC50 ranging from 9.2 to 94 mg EDTA-extractable Cu kg-1 soil) and composition, but not in diversity. A large number of diverse fungi were able to thrive under elevated Cu concentrations, though within the order of Hypocreales several species declined. A remarkable Cu-induced change in the community composition was found, which depended on the soil properties and, hence, on Cu availability.

Effect of the nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) on N-turnover, the N2O reductase-gene nosZ and N2O:N2 partitioning from agricultural soils.

Nitrification inhibitors (NIs) have been shown to reduce emissions of the greenhouse gas nitrous oxide (N2O) from agricultural soils. However, their N2O reduction efficacy varies widely across different agro-ecosystems, and underlying mechanisms remain poorly understood. To investigate effects of the NI 3,4-dimethylpyrazole-phosphate (DMPP) on N-turnover from a pasture and a horticultural soil, we combined the quantification of N2 and N2O emissions with 15N tracing analysis and the quantification of the N2O-reductase gene (nosZ) in a soil microcosm study. Nitrogen fertilization suppressed nosZ abundance in both soils, showing that high nitrate availability and the preferential reduction of nitrate over N2O is responsible for large pulses of N2O after the fertilization of agricultural soils. DMPP attenuated this effect only in the horticultural soil, reducing nitrification while increasing nosZ abundance. DMPP reduced N2O emissions from the horticultural soil by >50% but did not affect overall N2 + N2O losses, demonstrating the shift in the N2O:N2 ratio towards N2 as a key mechanism of N2O mitigation by NIs. Under non-limiting NO3- availability, the efficacy of NIs to mitigate N2O emissions therefore depends on their ability to reduce the suppression of the N2O reductase by high NO3- concentrations in the soil, enabling complete denitrification to N2.