Fungal trunk diseases causing decline of apricot and plum trees in the Czech Republic.

Fungal trunk diseases (FTDs) have been a significant threat to the global stone fruit industry. FTDs are caused by a consortium of wood-decaying fungi. These fungi colonize woody tissues, causing cankers, dieback, and other decline-related symptoms in host plants. In this study, a detailed screening of the fungal microbiota associated with the decline of stone fruit trees in the Czech Republic was performed. The wood fragments of plum and apricot trees showing symptoms of FTDs were subjected to fungal isolation. The partial internal transcribed spacer (ITS) region, partial beta-tubulin (tub2) and translation elongation factor 1-α (tef) genes were amplified from genomic DNA extracted from fungal cultures. All isolates were classified, and the taxonomic placement of pathogenic strains was illustrated in phylogenetic trees. The most abundant pathogenic genus was Dactylonectria (31 %), followed by Biscogniauxia (13 %), Thelonectria (10 %), Eutypa (9 %), Dothiorella (7 %), Diplodia (6 %), and Diaporthe (6 %). The most frequent endophytic genus was Aposphaeria (17 %). The pathogenicity of six fungal spp. (Cadophora daguensis, Collophorina africana, Cytospora sorbicola, Dothiorella sarmentorum, Eutypa lata, and Eutypa petrakii var. petrakii to four Prunus spp. was evaluated and the Koch's postulates were fulfilled. All tested isolates caused lesions on at least one Prunus sp. The most aggressive species was E. lata, which caused the largest lesions on all four tested Prunus spp., followed by E. petrakii var. petrakii, and D. sarmentorum. Japanese plum (Prunus salicina) and almond (P. amygdalus) were the most susceptible hosts while apricot (P. armeniaca) was the least susceptible host in the pathogenicity trial.

Two new species of Trichocomaceae (Eurotiales), accommodated in Rasamsonia and Talaromyces section Bacillispori, from the Czech Republic.

During a previous study on microfungi associated with clematis roots, Penicillium-like fungi were isolated and identified based on morphology. In this study, we subjected those strains to a detailed examination which led to the proposal of two taxonomic novelties, named Rasamsonia chlamydospora and Talaromyces clematidis. The first taxon is characterized by rough-walled mycelium, acerose to flask shaped phialides, cylindrical conidia and by production of chlamydospore-like structures. The four-loci-based phylogeny analysis delineated the taxon as a taxonomic novelty in Rasamsonia. Talaromyces clematidis is characterized by restricted growth on Czapek yeast extract agar, dichloran 18% glycerol agar and yeast extract sucrose agar, and production of yellow ascomata on oatmeal agar. Phylogenetic analyses placed this taxon as a taxonomic novelty in Talaromyces sect. Bacillispori. Both taxa are introduced here with detailed descriptions, photoplates and information on their phylogenetic relationship with related species.

First report of Neofusicoccum parvum causing stem blight and dieback of highbush blueberry in the Czech Republic.

Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips is a cosmopolitan pathogen causing dieback of multiple diverse woody hosts including highbush blueberry (Vaccinium corymbosum L.). This fungus can survive inside colonized plants without causing any symptoms for several years. Once the endophytic lifestyle is switched to the parasitic one, the symptoms of dieback can rapidly occur (bronze leaves, necroses under the bark, apoplexy) and the plant usually declines within a few weeks (Slipper and Wingfield 2007). In August 2022, blueberry plants displaying symptoms described above were observed in a production orchard located in Hovorany, the Czech Republic. Around 3 % of 1000 observed plants were symptomatic. In order to identify the pathogen, leaves, stems and roots of three diseased plants were collected, sectioned into small pieces (5 × 5 mm), surface sterilized (60 s in 75% ethanol, followed by 60 s in 1% sodium hypochlorite and rinsed three times using sterile distilled water), plated on potato dextrose agar (PDA) supplemented with 0.5 g/liter of streptomycin sulfate (PDAS) (Biosynth, Staad, Switzerland) and incubated at 25°C for 2 weeks at dark. Newly developed mycelia were immediately transferred to fresh PDA plates and purified by single-spore or hyphal-tip isolation. In total 33 fungal isolates were obtained. All the 33 isolates shared similar morphology and resembled Botryosphaeriaceae spp. Colonies on PDA (7 d at 25°C) were felty, white to iron grey in the centre. Conidiomata were observed on sterile pine needles on 2 % water agar (WA) at 25°C under near-UV light after 2 wks (110-220 × 60-175 µm). Conidia (n=30) were cylindrical to ellipsoidal, hyaline, 0(-1)-septate, (3.8-8.1 × 2-3 µm). Two representative isolates were deposited at the Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands (CBS 149846 and CBS 149847). The partial internal transcribed spacer (ITS) regions, beta-tubulin gene (tub2) and translation elongation factor 1-alpha (tef) gene were amplified from genomic DNA of both isolates following primers and protocols previously described (Eichmeier et al. 2020). Newly generated sequences were deposited in NCBI GenBank (acc. nos. ITS: OQ376566, OQ376567; tub2: OQ401701, OQ401702 and tef: OQ401699, OQ401700), being >99% identical (ITS 483/484 nt, tub2 426/430 nt and tef 230/232 nt) with the ex-type ITS (AY236943), tub2 (AY236888) and tef (AY236917) sequences of N. parvum strain CMW9081. Phylogenetically, newly obtained isolates grouped with ex-type and another three cultures of N. parvum in the three gene-based phylogenetic tree with strong 98/1.0 (BP/PP) support. To confirm pathogenicity, one-year-old canes of ten two-year-old V. corymbosum plants grown in pots were wounded by a 5 mm diam cork borer, and a 5-mm mycelial plug of a 7-day-old culture of both (CBS 149846 and CBS 149847) strains (five plants per strain) was inserted into the wound. Ten plants were inoculated with sterile PDA plugs and served as controls. Wounds were covered by sterile wet cotton, sealed with Parafilm® and inoculated plants were maintained in a growth chamber at 20 °C with 12/12 h light/dark period. Within two weeks, inoculated shoots changed colour from green to dark brown and exhibited dark necroses under the bark; after one month inoculated plants declined, while controls remained symptomless. The pathogen was reisolated from the inoculated plants with 100 % re-isolation rate, and its identity confirmed by sequencing ITS region. The experiment was repeated. Neofusicoccum parvum causing dieback of highbush blueberry was already reported from Australia, California, Chile, China, Italy, Mexico, Portugal and Uruguay (Rossman et al. 2023). Pecenka et al. (2021) reported a presence of another pathogen - Lasiodiplodia theobromae (Pat.) Griffon & Maubl. from the same plantation. This suggests that stem blight and dieback of highbush blueberry is caused by more than one Botryosphaeriaceae spp. as it was previously proved by Xu et al. (2015). To the best of our knowledge, this is the first report of stem blight and dieback of highbush blueberry caused by N. parvum in the Czech Republic.

Calophoma clematidina causing leaf spot and wilt on Clematis plants in the Czech Republic.

Clematis L. is one of the largest genera of Ranunculaceae, accommodating over 300 plant species (Wang & Li 2005). They are mostly flowering creepers commonly grown as ornamentals. Clematis leaf spot and wilt is a fungal disease caused by Calophoma clematidina (Thüm.) Q. Chen & L. Cai. Infected plants initially show irregular brown to black leaf spots which later turn into large necroses, usually leading to wilt disease. In June 2021, Clematis plants displaying symptoms described above were observed in three independent nurseries located in three counties (Brno-venkov, Breclav and Nymburk) in the Czech Republic. Around 60% of 120 inspected plants were symptomatic, including both mother plants and young plants. Leaves, stems and roots of 43 diseased plants originating from the three nurseries were collected, sectioned into small pieces (5 × 5 mm), surface sterilized (60 sec in 75% ethanol, followed by 60 sec in 1% sodium hypochlorite and rinsed three times using sterile distilled water), plated on potato dextrose agar (PDA) and incubated at 25°C for 5 weeks. Newly developed mycelia were immediately transferred to a fresh PDA plates and purified by single-spore isolation. A total of 21 strains morphologically resembled the genus Calophoma. Colonies on PDA (7 d at 25°C) were felty, white to olivaceous/iron grey in the centre. Conidiomata were dark brown, pycnidial, solitary or in groups, (117-220 × 65-170 µm). Conidia were cylindrical to ellipsoidal, hyaline, 0(-1)-septate, (4-8 × 2-3 µm). Two representative isolates were deposited at the Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands (CBS 149230 and CBS 149231). The partial internal transcribed spacer (ITS) regions, large ribosomal subunit of the nrRNA gene (LSU), beta-tubulin gene (tub2) and RNA polymerase II second largest subunit gene (rpb2) were amplified from genomic DNA of both isolates following protocols previously described (Spetik et al. 2022). Sequences were deposited in NCBI GenBank (accession nos. ITS: ON107539, ON107540; LSU: ON108575, ON108576; tub2: ON314832, ON314833; rpb2: ON125007, ON125008), being 100% identical with that of the ex-type strain of C. clematidina (CBS 108.79), ITS (NR_135964), LSU (FJ515632), tub2 (FJ427100), and rpb2 (KT389588). Phylogenetically, the two representative isolates formed a fully supported clade with sequences of the ex-type and another culture of C. clematidina in the multigene phylogeny. To confirm Koch's postulates, leaves of ten two-month-old Clematis plants grown in pots were wounded by a needle and inoculated with a conidial suspension (1.0 × 106 conidia ml-1) of both strains (five plants per strain) following Golazar et al. (2011). Ten plants were mock-inoculated with sterile distilled water and served as controls. Within one month, inoculated plants exhibited dark necrotic leaf spots similar to the symptoms observed in the nurseries, while controls remained symptomless. Calophoma clematidina was reisolated from the inoculated plants, and its identity confirmed (ITS, GenBank OP363927). The experiment was repeated. Although known from Europe, this is the first report of Clematis leaf spot and wilt caused by C. clematidina in the Czech Republic. Clematis leaf spot and wilt represents a serious disease in Czech nurseries, with the pathogen present in leaves, stems and roots of Clematis spp.

Exploring the Temporal Dynamics of the Fungal Microbiome in Rootstocks, the Lesser-Known Half of the Grapevine Crop.

Rootstocks are the link between the soil and scion in grapevines, can provide tolerance to abiotic and biotic stresses, and regulate yield and grape quality. The vascular system of grapevine rootstocks in nurseries is still an underexplored niche for research, despite its potential for hosting beneficial and pathogenic microorganisms. The purpose of this study was to investigate the changes in the composition of fungal communities in 110 Richter and 41 Berlandieri rootstocks at four stages of the grapevine propagation process. Taxonomic analysis revealed that the fungal community predominantly consisted of phylum Ascomycota in all stages of the propagation process. The alpha-diversity of fungal communities differed among sampling times for both rootstocks, with richness and fungal diversity in the vascular system decreasing through the propagation process. The core microbiome was composed of the genera Cadophora, Cladosporium, Penicillium and Alternaria in both rootstocks, while the pathogenic genus Neofusicoccum was identified as a persistent taxon throughout the propagation process. FUNguild analysis showed that the relative abundance of plant pathogens associated with trunk diseases increased towards the last stage in nurseries. Fungal communities in the vascular system of grapevine rootstocks differed between the different stages of the propagation process in nurseries. Numerous genera associated with potential biocontrol activity and grapevine trunk diseases were identified. Understanding the large diversity of fungi in the rootstock vascular tissue and the interactions between fungal microbiota and grapevine will help to develop sustainable strategies for grapevine protection.

Lignans Extract from Knotwood of Norway Spruce-A Possible New Weapon against GTDs.

Grapevine trunk diseases (GTDs) pose a major threat to the wine industry worldwide. Currently, efficient biological methods or chemical compounds are not available for the treatment of infected grapevines. In the present study, we used an extract from the knotwood of spruce trees as a biological control against GTDs. Our in vitro trial was focused on the antifungal effects of the extract against the most common GTD pathogens-Cadophora luteo-olivacea, Dactylonectria torresensis, Diaporthe ampelina, Diaporthe bohemiae, Diplodia seriata, Eutypa lata, and Phaeoacremonium minimum. Our in vitro trial revealed a high antifungal effect of the extract against all tested fungi. The inhibition rates varied among the different species from 30% to 100% using 1 mg·mL-1 extract. Subsequently, the efficiency of the extract was supported by an in planta experiment. Commercial grafts of Vitis vinifera were treated with the extract and planted. The total genomic DNA of grapevines was extracted 10 days and 180 days after the treatment. The fungal microbial diversities of the treated/untreated plants were compared using high-throughput amplicon sequencing (HTAS). Treated plants showed 76.9% lower relative abundance of the genus Diaporthe and 70% lower relative abundance of the genus Phaeoacremonium 10 days after treatment. A similar scenario was observed for the genus Cadophora 180 days after treatment, where treated plants showed 76% lower relative abundance of this genus compared with untreated grapevines.

Conserved MicroRNAs in Human Nasopharynx Tissue Samples from Swabs Are Differentially Expressed in Response to SARS-CoV-2.

The use of high-throughput small RNA sequencing is well established as a technique to unveil the miRNAs in various tissues. The miRNA profiles are different between infected and non-infected tissues. We compare the SARS-CoV-2 positive and SARS-CoV-2 negative RNA samples extracted from human nasopharynx tissue samples to show different miRNA profiles. We explored differentially expressed miRNAs in response to SARS-CoV-2 in the RNA extracted from nasopharynx tissues of 10 SARS-CoV-2-positive and 10 SARS-CoV-2-negative patients. miRNAs were identified by small RNA sequencing, and the expression levels of selected miRNAs were validated by real-time RT-PCR. We identified 943 conserved miRNAs, likely generated through posttranscriptional modifications. The identified miRNAs were expressed in both RNA groups, NegS and PosS: miR-148a, miR-21, miR-34c, miR-34b, and miR-342. The most differentially expressed miRNA was miR-21, which is likely closely linked to the presence of SARS-CoV-2 in nasopharynx tissues. Our results contribute to further understanding the role of miRNAs in SARS-CoV-2 pathogenesis, which may be crucial for understanding disease symptom development in humans.

Epigenetic Modulating Chemicals Significantly Affect the Virulence and Genetic Characteristics of the Bacterial Plant Pathogen Xanthomonas campestris pv. campestris.

Epigenetics is the study of heritable alterations in phenotypes that are not caused by changes in DNA sequence. In the present study, we characterized the genetic and phenotypic alterations of the bacterial plant pathogen Xanthomonas campestris pv. campestris (Xcc) under different treatments with several epigenetic modulating chemicals. The use of DNA demethylating chemicals unambiguously caused a durable decrease in Xcc bacterial virulence, even after its reisolation from infected plants. The first-time use of chemicals to modify the activity of sirtuins also showed some noticeable results in terms of increasing bacterial virulence, but this effect was not typically stable. Changes in treated strains were also confirmed by using methylation sensitive amplification (MSAP), but with respect to registered SNPs induction, it was necessary to consider their contribution to the observed polymorphism. The molecular basis of the altered virulence was deciphered by using dualRNA-seq analysis of treated Xcc strains infecting Brassica rapa plants. The results of the present study should promote more intensive research in the generally understudied field of bacterial epigenetics, where artificially induced modification by epigenetic modulating chemicals can significantly increase the diversity of bacterial properties and potentially contribute to the further development of the fields, such as bacterial ecology and adaptation.

Lasiodiplodia mitidjana sp. nov. and other Botryosphaeriaceae species causing branch canker and dieback of Citrus sinensis in Algeria.

Several Botryosphaeriaceae species are known to occur worldwide, causing dieback, canker and fruit rot on various hosts. Surveys conducted in ten commercial citrus orchards in the northern region of Algeria revealed five species of Botryosphaeriaceae belonging to three genera associated with diseased trees. Morphological and cultural characteristics as well as phylogenetic analyses of the internal transcribed spacer (ITS) region and the translation elongation factor 1-alpha (tef1-α) identified Diplodia mutila, Diplodia seriata, Dothiorella viticola, Lasiodiplodia mediterranea and a novel species which is here described as Lasiodiplodia mithidjana sp. nov.. Of these, L. mithidjana (14.1% of the samples) and L. mediterranea (13% of the samples) were the most widespread and abundant species. Pathogenicity tests revealed that L. mediterranea and D. seriata were the most aggressive species on citrus shoots. This study highlights the importance of Botryosphaeriaceae species as agents of canker and dieback of citrus trees in Algeria.