First report of Colletotrichum grossum causing apple bitter rot worldwide (Italy).

Apple bitter rot is a globally widespread disease that is observed on fruits both pre-harvest and post-harvest, contributing to considerable economic losses. While the Colletotrichum acutatum species complex are predominant in Europe (Baroncelli et al. 2014; Amaral Carneiro and Baric 2021), in recent years, the Colletotrichum gloeosporioides species complex are emerging, raising many concerns (Amaral Carneiro et al. 2023). Circular, slightly sunken, brown lesions with acervuli produced in concentric spots were observed on 'Story® Inored' cultivar harvested in September 2022 from an organic orchard in Masi (Padova province, Italy), with a disease incidence close to 30%. From ten diseased apples, tissue samples were excised under aseptic conditions from surface-cleaned fruit at the margin between healthy and diseased pulp tissue, transferred to potato dextrose agar medium and incubated in the dark at 25 °C for 7 days, whereafter five single-spore cultures were obtained. Pure colonies grown at 25 °C for 7 days appeared light gray-white on the upper side with floccose aerial mycelium, while the reverse side was dark gray with a distinct margin. Conidia were hyaline, cylindrical in shape with both ends rounded or one end acute and measured 16.6 ± 1.4 × 6.1 ± 0.5 µm [mean ± SD] (n=50) as described by Diao et al. 2017. To identify the species, genomic DNA of a representative isolate (C38) was extracted, beta-tubulin (TUB2), calmodulin (CAL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutamine synthetase (GS), Apn2-Mat1-2 intergenic spacer (ApnMat) genes and the internal transcribed spacer (ITS) region, were amplified by PCR and Sanger sequenced (Rojas et al. 2010; Weir et al. 2012). The obtained DNA sequences of, TUB2, CAL, GAPDH, GS, ApnMat and ITS were submitted to GenBank under the accession numbers OR025589, OR025586, OR025587, OR025588, OR025585 and OR004800, respectively. A MegaBLAST analysis resulted 100 % identity to the epitype CAUG7 of Colletotrichum grossum (Diao et al. 2017) for GAPDH (KP890159), for TUB2 (KP890171), 99.85% for CAL (KP890147) and 99,5 % for ITS (KP890165). The phylogenetic tree constructed by concatenation with the obtained sequences, as well as references, revealed that the C38 isolate clustered within C. grossum, confirming the BLAST approach. Pathogenicity tests were performed on 40 'Story® Inored' apples cleaned and wounded with a sterilized needle and exposed to two different conditions: 20 apples (10 inoculated with 20 µl of spore suspension (105 ml-1) and 10 with sterile water as control) were incubated at 20°C with a 12-hour photoperiod for 14 days, while the remaining 20 apples, prepared with same approach, were placed at 1°C for 3 months, then at room temperature for 14 days. Symptoms appeared after 6 days on apples incubated at 20°C, whereas those stored at 1°C displayed symptoms at 11 days after being placed at room temperature. In both conditions, lesions were similar to those observed on the original fruits; while the controls remained asymptomatic. Identity of reisolated fungal colonies was confirmed by CAL, GAPDH and GS region sequence analysis. C. grossum has been reported rarely: in 2017 on Capsicum annuum var. grossum in China, in 2018 on Mangifera indica leaves in Cuba, and in 2021 on Rhyncospermum jasminoides in Italy (Diao et al. 2017; Manzano León et al. 2018; Guarnaccia et al. 2021). To the best of our knowledge, this is the first report of apple bitter rot caused by Colletotrichum grossum worldwide.

Phylogenetic Diversity and Phenotypic Characterization of Phlyctema vagabunda (syn. Neofabraea alba) and Neofabraea kienholzii Causing Postharvest Bull's Eye Rot of Apple in Northern Italy.

Bull's eye rot, caused by Phlyctema vagabunda and Neofabraea species, is one of the most important postharvest diseases of apple. South Tyrol (northern Italy) is the largest continuous apple-producing area in Europe, with approximately 1 million tons being produced yearly and conserved in technologically advanced storage facilities for several months. Still, studies on the pathogen species causing postharvest bull's eye rot of apple, as well as their diversity and biology, are lacking for this region. Therefore, the main purpose of the present work was to identify and characterize fungal isolates obtained from decayed apple fruit with symptoms of bull's eye rot that were collected in 2018 and 2019 in different packinghouses in South Tyrol. Among more than 1,000 fungal isolates that were obtained, 419 could be assigned to the genera Phlyctema and/or Neofabraea based on rot symptoms on apple fruit and colony morphology on potato dextrose agar. A smaller subset of 101 representative isolates was further analyzed by DNA sequencing of the internal transcribed spacer region. Furthermore, partial segments of the ß-tubulin gene, the translation elongation factor 1α gene, and the 16S mitochondrial ribosomal RNA gene were studied. The phylogenetic analyses, including sequences of reference species, showed that P. vagabunda is the dominant species associated with bull's eye rot of apple in the study area, whereas Neofabraea kienholzii was found only on a small number of apple fruit samples. The combination of multilocus sequence data revealed 11 unique genotypes that belonged to P. vagabunda and four to N. kienholzii. To the best of our knowledge, this study is the first to report N. kienholzii as a postharvest pathogen of apple in Italy. Finally, a pathogenicity test demonstrated different degrees of virulence among selected isolates of P. vagabunda and N. kienholzii on the cultivar Golden Delicious. The present study emphasizes the importance of accurate species identification, because different species may vary in their biological and pathogenic characteristics, and consequently require distinct disease management strategies, both in the field and during the postharvest stages.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

First Report of Colletotrichum salicis Causing Bitter Rot of Apple in Italy.

Apple (Malus domestica Borkh.) is the most important fruit crop in South Tyrol (northern Italy), with a total production of 905.089 tons in 2019 (Chamber of Commerce, Industry, Crafts and Agriculture of Bozen-Bolzano 2020). Symptoms of bitter rot were observed on organic apples of the cultivar 'Roho 3615'/Evelina® collected in a packinghouse in South Tyrol at the end of March 2018 after approximately six months of storage in controlled atmosphere. Lesions were circular and light brown with orange conidial masses. Tissue samples were removed under aseptic conditions from surface-cleaned fruit at the margin between healthy and diseased pulp tissue, transferred to Petri dishes with potato dextrose agar (PDA), and incubated in the dark at room temperature for two weeks. Single spore cultures were obtained by adapting the procedure of Choi et al. (1999). Pure cultures were grown in quadruplicate on PDA at 20°C in the dark for two weeks. The colony appearance on the upper side was mostly flat with a distinct margin, the surface was covered with short, floccose aerial mycelium, and the color ranged from light gray to dark gray, while the reverse side appeared yellow. From each replicate culture, conidia were harvested, and the length and width of 50 randomly selected conidia were measured using a compound light microscope coupled to a digital camera (Leica DMLS, Leica Microsystems, Wetzlar, Germany). Conidia were cylindrical to fusiform, pointed at one end, and measured 10.0 to 19.5 × 2.5 to 5.0 µm (14.5 ± 1.9 × 3.9 ± 0.7 µm [mean ± SD]) in consistency with Damm et al. (2012). In order to determine the species of isolate 18-DSS-BS-EL-1-012, a multi-locus DNA sequence analysis was performed. Genomic DNA was extracted by following the protocol described by Cassago et al. (2002). Four loci, actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone H3 (HIS3) and the internal transcribed spacer (ITS) region of the rRNA operon were amplified by PCR and Sanger sequenced (Damm et al. 2012; Lévesque and de Cock 2004). The obtained DNA sequences of ACT, GAPDH, HIS3 and ITS were 186, 150, 317 and 505 bp long and were submitted to GenBank under the accession numbers MT347599, MT347600, MT347598 and MT337388, respectively. A MegaBLAST analysis resulted in 100% sequence identity at all four loci with a type culture of Colletotrichum salicis (CBS 607.94; GenBank accession numbers: JQ949781, JQ948791, JQ949451 and JQ948460), which belongs to the C. acutatum species complex (Damm et al. 2012). A pathogenicity test was performed with twelve 'Golden Delicious' apples by wounding the fruit with a sterile piercing tool and inoculating 20 µl of spore suspension (104 conidia per ml) from a 21-day-old PDA culture. Inoculated fruits were incubated for 21 days in a moist chamber at 20°C in the dark. The symptoms were recorded at 3, 5, 7, 10, 14 and 21 days post-inoculation (dpi). Symptoms appeared after 7 days on all inoculated fruits and resembled those observed on the original fruit, while mock-inoculated controls with sterile water remained symptomless. Fungal colonies resembling the original culture were re-isolated from lesions on the apple and plated on PDA. Their identity was confirmed by DNA sequence analysis of the ITS region, thereby proving Koch's postulates. Bitter rot occurs globally and is considered one of the most important diseases of apple that has the potential to cause significant crop losses (Sutton et al. 2014). Other Colletotrichum species have been commonly reported from apple in Europe, such as C. godetiae and C. fioriniae, whereas C. salicis has been reported solely in New Zealand and Belgium (Damm et al. 2012; Grammen et al. 2019). To the best of our knowledge, this is the first report of C. salicis causing bitter rot of apple in Italy.

Comparative transcriptome profiling of resistant and susceptible rice genotypes in response to the seedborne pathogen Fusarium fujikuroi.

BACKGROUND: Fusarium fujikuroi is the causal agent of bakanae, the most significant seed-borne disease of rice. Molecular mechanisms regulating defence responses of rice towards this fungus are not yet fully known. To identify transcriptional mechanisms underpinning rice resistance, a RNA-seq comparative transcriptome profiling was conducted on infected seedlings of selected rice genotypes at one and three weeks post germination (wpg). RESULTS: Twelve rice genotypes were screened against bakanae disease leading to the identification of Selenio and Dorella as the most resistant and susceptible cultivars, respectively. Transcriptional changes were more appreciable at 3 wpg, suggesting that this infection stage is essential to study the resistance mechanisms: 3,119 DEGs were found in Selenio and 5,095 in Dorella. PR1, germin-like proteins, glycoside hydrolases, MAP kinases, and WRKY transcriptional factors were up-regulated in the resistant genotype upon infection with F. fujikuroi. Up-regulation of chitinases and down-regulation of MAP kinases and WRKY transcriptional factors were observed in the susceptible genotype. Gene ontology (GO) enrichment analyses detected in Selenio GO terms specific to response to F. fujikuroi: 'response to chitin', 'jasmonic acid biosynthetic process', and 'plant-type hypersensitive response', while Dorella activated different mechanisms, such as 'response to salicylic acid stimulus' and 'gibberellin metabolic process', which was in agreement with the production of gibberellin A3 in Dorella plants. CONCLUSIONS: RNA-seq profiling was performed for the first time to analyse response of rice to F. fujikuroi infection. Our findings allowed the identification of genes activated in one- and three- week-old rice seedlings of two genotypes infected with F. fujikuroi. Furthermore, we found the pathways involved in bakanae resistance, such as response to chitin, JA-dependent signalling and hypersensitive response. Collectively, this provides important information to elucidate the molecular and cellular processes occurring in rice during F. fujikuroi infection and to develop bakanae resistant rice germplasm.

Jasmonic Acid, Abscisic Acid, and Salicylic Acid Are Involved in the Phytoalexin Responses of Rice to Fusarium fujikuroi, a High Gibberellin Producer Pathogen.

Fusarium fujikuroi, the causal agent of bakanae disease, is the main seedborne pathogen on rice. To understand the basis of rice resistance, a quantitative method to simultaneously detect phytohormones and phytoalexins was developed by using HPLC-MS/MS. With this method dynamic profiles and possible interactions of defense-related phytohormones and phytoalexins were investigated on two rice cultivars, inoculated or not with F. fujikuroi. In the resistant cultivar Selenio, the presence of pathogen induced high production of phytoalexins, mainly sakuranetin, and symptoms of bakanae were not observed. On the contrary, in the susceptible genotype Dorella, the pathogen induced the production of gibberellin and abscisic acid and inhibited jasmonic acid production, phytoalexins were very low, and bakanae symptoms were observed. The results suggested that a wide range of secondary metabolites are involved in plant defense against pathogens and phytoalexin synthesis could be an important factor for rice resistance against bakanae disease.