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Research was conducted to infections of a (PB1-) potato crop grown from minitubers with blackleg causing soft rot Pectobacteriaceae (BL-SRP). For this, an enrichment multiplex TaqMan assay (EM-TaqMan assay) was developed, allowing to detect the various BL-SRP variants simultaneously with a sensitivity of 10-100 cells per gram plant material. During four growing seasons (2019- 2022) PB1 crops were surveyed. Plant tissues (leaves, stems and tubers) were collected and analyzed using the EM-TaqMan assay. During wet growing seasons relatively high infection incidences were found both in leaves (8%) and tubers (≤ 42%), in particular with a virulent haplotype of P. brasiliense. During dry seasons, only low incidences were found. It was found that P. brasiliense is able to enter a VBNC-state in the presence of cupper ions (CuSO4). However, we were unable to find conditions to let the cells multiply again. Therefore we have no indications that VBNC play a role in the epidemiology of P. brasiliense. The role of potential infection sources was studied. BL-SRP were not found in rain water, but 2.3% of the sampled insects at a PB crop were contaminated with BL-SRP, indicating that insects may be important in the transmission of BL-SRP from an infection source to a PB1 crop. Inoculum deposited on haulms poses a high risk for tuber infections. This was studied by spray inoculation of haulms of young potato plants with different densities (102 – 109 cfu per plant) of a virulent, rifampicin resistant mutant of P. brasiliense. In a wet growing season, low densities could already result in haulm infections while all plants yielded infected tubers. In a dry year only at the highest inoculum densities haulm infections were found, but tuber infections even at the lowest densities. Analysis conducted at the NAK to the role of environmental factors on the infection level of seed potatoes were done using data of seed lots tested in high classes in the period 2015-2018. Characteristics point more...

In total 58 Xanthomonas strains isolated from Araceae worldwide, together with 13 other phylogenetically-related Xanthomonas strains, were characterized using multilocus sequence analysis based on concatenated sequences of seven single copy orthologous genes, extracted from whole genome sequences. The analysis revealed a monophyletic clade of 48 strains, 44 isolated from Anthurium, identified as X. phaseoli pv. dieffenbachiae (Xpd) confirmed by nucleotide identity analysis. The other strains from aroids were identified as Xanthomonas euvesicatoria (2 strains), X citri (5 strains) and Xanthomonas sacchari (3 strains). Two TaqMan assays were designed for specific detection of Xpd, one targeting sequences of a hypothetical protein and one targeting a type I restriction endonuclease subunit S. The two assays showed similar reaction kinetics and were merged with an assay comprising an amplification and extraction control into a triplex assay. The assay was able to detect minimally 100 copies of a target sequence delivered as a gBlock, 100 fg of genomic DNA and 104 cells per mL in an Anthurium leaf extract. more...

A potential cause of the dissemination of the potato ring rot bacterium Clavibacter sepedonicus (Cs) is the use of automated seed potato cutters. The present study focuses on the question of whether disinfection practices are sufficient to prevent the transmission of Cs from contaminated machine parts to a new tuber lot. The disinfection efficacy was determined by establishing the culturability of Cs that remained after spray application of sodium-p-toluenesulfochloramide solution on clean and fouled specimens of machinery material that had been provided with an imprint of Cs biofilm.Although conventional spraying, with the authorized concentration of sodium-p-toluenesulfochloramide, of inoculated rubber, PVC and lacquered steel led to a substantial decrease of colony forming units, the treatment was insufficient for complete eradication of Cs. The presence of dirt negatively affected the efficacy of the disinfectant. more...

The objective of the present study was to establish whether exposure to temperatures of 55-70 °C results in eradication of the pathogen Clavibacter sepedonicus (Cs) in colonised potato tissue, in order to evaluate the efficiency of composting for disinfection of Cs-infected potato waste. Pre-sprouted potato tubers were inoculated and planted to produce Cs-colonised stem and tuber material. After incubation in temperature-controlled water baths, the infected potato tissue was analysed for the presence of culturable and pathogenic Cs. Additional experiments were performed with Cs-colonised potato stem tissue crushed and deaerated, thus simulating macerated stem tissue in the compost heap. To enable a comparison with bacterial cells that are not enclosed by their natural organic matrix, temperature treatments were applied to noninfected stem tissue crushed and deaerated, and spiked with freshly prepared Cssuspensions. Cs settled inside colonised potato tissue, as well as Cs present as Cssuspensions supplemented to potato tissue, was eradicated by exposure to heat, even after a treatment of 1 h at 55 °C, with the exception of one case in which the pathogen present in intact stem material escaped a treatment of 6 h at 60 °C, indicating that incidentally stems may provide niches in which the pathogen is protected against heat. more...

Molecular characterization of Xanthomonas species pathogenic on Araceae and development of a triplex Taqman assay for detection of Xanthomonas phaseoli pv. dieffenbachiae, Molecular characterization of Xanthomonas species pathogenic on Araceae and development of a triplex Taqman assay for detection of Xanthomonas phaseoli pv. dieffenbachiae more...

The Ralstonia solanacearum species complex (RSSC) can cause bacterial wilt in a wide variety of plant species, including a number of ornamental glasshouse crops. Recently in Europe, ornamental rose plants for the production of cut flowers and propagation materials have been strongly affected by Ralstonia pseudosolanacearum , phylotype I, biovar 3. To test for the presence of the pathogen in the glasshouse, sampling of water from a drainage gutter or well may be an efficient strategy since it is known that RSSC can be released from infected root systems in the water. A protocol was developed to detect low densities of R. pseudosolanacearum in drain water collected from rose growers. Drain water was filtered through a bacterial filter, the filtrate was collected and target bacteria enriched for 48 h in Semi‐selective Medium South Africa (SMSA) broth supplemented with sterilized tomato plant extracts. DNA extracted from the enrichment broth was analysed using a TaqMan test in a duplex format, based on specific egl sequences of RSSC and the use of an extraction and amplification control. The optimized protocol had a detection level of ≤1–10 colony forming units of R. pseudosolanacearum in drain water. more...

The socio-economic impact of Fusarium odoratissimum, which is colloquially called tropical race 4 (TR4), is escalating as this fungal pathogen spreads to new banana-growing areas. Hence, the development of simple, reliable and rapid detection technologies is indispensable for implementing quarantine measures. Here, a versatile loop-mediated isothermal amplification (LAMP) assay has been developed that is applicable under field and laboratory conditions. DNA markers unique to TR4 isolates were obtained by diversity arrays technology sequencing (DArTseq), a genotyping by sequencing technology that was conducted on 27 genotypes, comprising 24 previously reported vegetative compatibility groups (VCGs) and three TR4 isolates. The developed LAMP TR4 assay was successfully tested using 22 TR4 isolates and 45 non-target fungal and bacterial isolates, as well as on infected plants under greenhouse and field conditions. The detection limit was 1 pg µL−1 pure TR4 DNA or 102 copies plasmid-localized TR4 unique sequence (SeqA) per reaction, which was not affected by background DNA in complex samples. The LAMP TR4 assay offers a powerful tool for the routine and unambiguous identification of banana plants infected with TR4, contributing to advanced diagnosis in field situations and monitoring of fusarium wilt. more...

A Monascus ruber strain was isolated that was able to grow on mineral medium at high sugar concentrations and 175 g/l lactic acid at pH 2.8.

Four commercially available disinfection products were tested for their efficacy against Clavibacter michiganensis subsp. sepedonicus (Cms), causative agent of bacterial ring rot, on wooden potato storage crates. Each of these products represented a different class of biocide, i.e. organic acids (benzoic acid), peroxygenous compounds (potassium peroxysulfate), quaternary ammonium compounds (didecyldimethylammoniumchloride) and hypochlorite-generating compounds (sodium-p-toluenesulfochloramide). The target objects consisted of small wooden panels that were smeared with potato tuber pulp homogenized with a high inoculum of Cms. By dipping the inoculated panels into the product solutions and subsequent determination of the densities of colony-forming units, the relative efficacy of the products was established. The two products that exhibited the highest relative efficacy were studied for their disinfection performance when applied as cleaning agent in a conventional automated crate washer. The results showed that jet cleaning in a crate washer for 2 min using the authorised dose of the product containing sodium-p-toluenesulfochloramide is an effective method for disinfecting Cms-contaminated wooden potato crates. more...