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.

First report of Lasiodiplodia theobromae causing decline of blueberry (Vaccinium corymbosum L.) in the Czech Republic.

Popularity of blueberries (Vaccinium corymbosum L.) has been rising worldwide, due to their nutritional bioactive compounds such as flavonoids, anthocyanins and polyphenols (Sinelli et al. 2008). These factors led to an increase of the demand for fresh blueberries in the Czech market, which resulted in increased blueberry planted in the Czech Republic. In spring 2018, about 200,000 1-year-old blueberry plants originally from the Netherlands were planted on 47 ha in the South Moravian region. In September 2019, wilting of leaves and shoots, bark necrosis and dieback of the plant were observed. The occurrence of blueberry decline symptoms in the field was near 2% of 5,000 observed plants which resulted in significant yield losses at the beginning of the production. Twenty shoots of symptomatic plants were collected (one shoot from each plant, twenty plants in total) and surface-disinfected using 75% ethanol. Necrotic tissues of xylem were aseptically placed on potato dextrose agar (PDA) (Himedia, India) amended with streptomycin and incubated at 25°C in the dark for one week. Nine isolates of Botryosphaeriaceae-like fungi were obtained from twenty plants and were transferred on PDA. Aerial mycelium grew rapidly, initially white, and then became grey to black. To enhance the conidia production, the isolates were transferred on water agar (Himedia, India) with autoclaved poplar twigs, placed near day light and incubated at room temperature. Three weeks later, black pycnidia were observed on the poplar twigs. Mature pycnidia exuded white/grey coloured masses of conidiospores. Conidia were granular, subcylindrical to elliptical. Immature conidia were single-celled and hyaline measuring 9.73 ± 1.39 × 16.86 ± 1.81 µm (n=60). Mature conidia were thick-walled with a septum from 11.30 ± 1.17 × 16 ± 1.58 µm (n=60). Based on conidiogenous cells, paraphyses and longitudinal striations on mature conidia characteristics, the isolates were morphologically assigned to the Lasiodiplodia genus (Phillips et al. 2013). Isolates were single-spored and total DNA was extracted with NucleoSpin Tissue (Macherey-Nagel, Germany) following manufacturer's protocol. ITS region, including the 5.8S, and part of the translation elongation factor 1 alpha (TEF-1α) were amplified with ITS1/ITS4 and EF1-728F/EF1-986R primers, respectively (Carbone and Kohn 1999; White et al. 1990). Consensus sequences were blasted using NCBI nucleotide BLASTn search. The sequences of ITS region were in 468/469 nts (MEND-F-00167) and 486/490 nts (MEND-F-00168) similar to Lasiodiplodia theobromae isolate CBS 287.47 and 286/289 nts (MEND-F-00167), 287/290 nts (MEND-F-00168) in case of TEF-1α. Sequences of two representative isolates (MEND-F-00167 and MEND-F-00168) were deposited in GenBank under accession numbers MN983133 and MN983134 for ITS and MN989914 and MN989915 for TEF-1α. For pathogenicity tests, 6 mm agar plugs with mycelium obtained from ten day old mycelia of both fungal isolates were inoculated on ten 1-year-old blueberry shoots cv. Top Shelf collected from one plant, using a cork borer. Five shoots and pure agar plugs were used as a control. Plugs were covered with wet cotton wool to keep moisture and fixed with Parafilm. After three weeks, characteristic discoloration and lesions appeared on the inoculated shoots. Control shoots remained symptomless. Necrotic tissue was aseptically cut and L. theobromae was re-isolated on PDA. Identity of fungus was confirmed by morphological characterization and sequencing, fulfilling Koch´s postulates. Blueberry decline caused by Botryosphaeriaceae spp. has already been described around the world (Hilário et al. 2020; Tennakoon et al. 2018; Wiseman et al. 2017; Xu et al. 2015). To our knowledge, this is the first report of L. theobromae on blueberry in the Czech Republic.

Multiplex real-time PCR for the detection of Clavibacter michiganensis subsp. michiganensis, Pseudomonas syringae pv. tomato and pathogenic Xanthomonas species on tomato plants.

A multiplex real-time PCR method based on fluorescent TaqMan® probes was developed for the simultaneous detection of the tomato pathogenic bacteria Clavibacter michiganensis subsp. michiganensis, Pseudomonas syringae pv. tomato and bacterial spot-causing xanthomonads. The specificity of the multiplex assay was validated on 44 bacterial strains, including 32 target pathogen strains as well as closely related species and nontarget tomato pathogenic bacteria. The designed multiplex real-time PCR showed high sensitivity when positive amplification was observed for one pg of bacterial DNA in the cases of Clavibacter michiganensis subsp. michiganensis and Pseudomonas syringae pv. tomato bacteria and 100 pg for bacterial spot-causing xanthomonads. The reliability of the developed multiplex real-time PCR assay for in planta detection was verified by recognition of the target pathogens in 18 tomato plants artificially inoculated by each of the target bacteria and tomato samples from production greenhouses.

MicroRNAs in Vitis vinifera cv. Chardonnay Are Differentially Expressed in Response to Diaporthe Species.

Diaporthe species are important pathogens, saprobes, and endophytes on grapevines. Several species are known, either as agents of pre- or post-harvest infections, as causal agents of many relevant diseases, including swelling arm, trunk cankers, leaf spots, root and fruit rots, wilts, and cane bleaching. A growing body of evidence exists that a class of small non-coding endogenous RNAs, known as microRNAs (miRNAs), play an important role in post-transcriptional gene regulation, during plant development and responses to biotic and abiotic stresses. In this study, we explored differentially expressed miRNAs in response to Diaporthe eres and Diaporthe bohemiae infection in Vitis vinifera cv. Chardonnay under in vitro conditions. We used computational methods to predict putative miRNA targets in order to explore the involvement of possible pathogen response pathways. We identified 136 known and 41 new miRNA sequence variants, likely generated through post-transcriptional modifications. In the Diaporthe eres treatment, 61 known and 17 new miRNAs were identified while in the Diaporthe bohemiae treatment, 101 known and 21 new miRNAs were revealed. Our results contribute to further understanding the role miRNAs play during plant pathogenesis, which is possibly crucial in understanding disease symptom development in grapevines infected by D. eres and D. bohemiae.

High-throughput small RNA sequencing for evaluation of grapevine sanitation efficacy.

This study describes the application of high-throughput sequencing of small RNA analysis of the efficacy of using Ribavirin to eliminate Grapevine leafroll-associated virus 1, Grapevine fleck virus and Grapevine rupestris stem pitting-associated virus from Vitis vinifera cv. Riesling. The original plant used for sanitation by Ribavirin treatment was one naturally infected with all the viruses mentioned above as confirmed by RT-PCR. A tissue cultures of the plant were established and plantlets obtained were sanitized using Ribavirin. Three years after sanitation, a small RNA sequencing method for virus detection, targeting 21, 22 and 24 nt-long viral small RNAs (vsRNAs), was used to analyze both the mother plant and the sanitized plants. The results showed that the mother plant was infected by the three mentioned viruses and additionally by two viroids - Hop stunt viroid and Grapevine yellow speckle viroid 1. After Ribavirin treatment, the plants contained only the two viroids, with the complete elimination of all the viruses previously present.