Improvement in the sensitivity of viroid detection by adapting the reverse transcription step in one-step RT-qPCR assays.

Over the course of developing and applying a new real-time PCR assay for the detection of the newly described apple chlorotic fruit spot viroid (ACFSVd), slight modifications of the reverse transcription (RT) step were found to improve significantly the detection limit of the assay. To prove this hypothesis, three different one-step RT-qPCR kits for the detection of three plant viroids and three plant viruses were compared. The results showed both extension of the RT reaction time from 10 or 15 min-30 min or the increase in reaction temperature from 49 to 52 °C for the cDNA synthesis step results in a 10 times higher sensitivity for potato spindle tuber viroid and apple scar skin viroid one-step RT-qPCR assay and 45 higher sensitivity for ACFSVd one-step RT-qPCR assay. No variation in the detection limit was observed when the modifications were tested on tomato brown rugose fruit virus, plum pox virus and tomato ringspot virus assays. This finding is highly valuable for the investigation of viroids in general and could contribute to enhance sensitivity in their detection and to benefit regulatory outcomes for national plant protection organisations.

Resilience of agricultural soils to antibiotic resistance genes introduced by agricultural management practices.

Antimicrobial resistance (AR) represents a global threat in human and veterinary medicine. In that regard, AR proliferation and dissemination in agricultural soils after manure application raises concerns on the enrichment of endogenous soil bacterial population with allochthonous antibiotic resistance genes (ARGs). Natural resilience of agricultural soils and background concentrations of ARGs play key roles in the mitigation of AR propagation in natural environments. In the present study, we carried out a longitudinal sampling campaign for two crop vegetation periods to monitor spatial and temporal changes in the abundance of seven clinically relevant ARGs (sul1, ermB, vanA, aph(3')-IIa, aph(3')-IIIa, blaTEM-1 and tet(W)) and ribosomal 16S RNA. The absolute and relative abundances of the selected ARGs were quantified in total community DNA extracted from agricultural (manured and non-manured) and forest soils, fresh pig faeces and manure slurry. We observed that ARG concentrations return to background levels after manure-induced exposure within a crop growing season, highlighting the resilience capacity of soil. Naturally occurring high background concentrations of ARGs can be found in forest soil in due distance under low anthropogenic influences. It was observed that pesticide application increases the concentrations of three out of seven ARGs tested (ermB, aph(3')-IIIa and tet(W)). Moreover, we noticed that the absolute abundances of sul1, vanA, ermB and blaTEM-1 resistance genes show an increase by 100- to 10,000- fold, from maturation of fresh pig faeces to manure. Outcomes of our study suggest that agricultural soil environments show a strong capacity to alleviate externally induced disturbances in endogenous ARG concentrations. Naturally occurring high concentrations of ARGs are present also in low human impacted environments represented by the indigenous resistome.

Correction to: Transmission studies of the newly described apple chlorotic fruit spot viroid using a combined RT-qPCR and droplet digital PCR approach.

Authors would like to update the given name and family name of authors which were incorrect in the original version.

Transmission studies of the newly described apple chlorotic fruit spot viroid using a combined RT-qPCR and droplet digital PCR approach.

The transmission of the apscaviroid tentatively named apple chlorotic fruit spot viroid (ACFSVd) was investigated using a one-step reverse-transcription (RT) droplet digital PCR assay for absolute quantification of the viroid, followed by quantification of relative standard curves by RT-qPCR. Our results indicate that ACFSVd is effectively transmitted by grafting, budding and seeds. No transmission has yet been observed to the viroid-inoculated pome fruit species Pyrus sp. and Cydonia sp. ACFSVd was detected in viruliferous aphids (Myzus persicae, Dysaphis plantaginea) and in codling moths (Cydia pomonella). The viroid was also detected systemically in the infected hemiparasitic plant Viscum album subsp. album (mistletoe).

Apple chlorotic fruit spot viroid: a putative new pathogenic viroid on apple characterized by next-generation sequencing.

Viroid-like symptoms were observed in 2016 on apple fruits of the cultivar "Ilzer Rose" in southern Burgenland, Austria. Preliminary molecular biological investigations indicated that the symptoms were caused by a new unknown viroid. Therefore, new primers were designed, and the whole genome sequence of the viroid (354 nt) was determined by next-generation amplicon sequencing using the Illumina MiSeq® platform (San Diego, California, USA). The viroid secondary structure has a rod-like conformation and contains conserved regions (the TCR, CCR upper strand, and CCR lower strand) that are characteristic of members of the genus Apscaviroid. Based on our results and the demarcation criteria for viroids, the tentatively named "apple chlorotic fruit spot viroid" should be considered a putative new member of the genus Apscaviroid.

Antibiotic resistance marker genes as environmental pollutants in GMO-pristine agricultural soils in Austria.

Antibiotic resistance genes may be considered as environmental pollutants if anthropogenic emission and manipulations increase their prevalence above usually occurring background levels. The prevalence of aph(3')-IIa/nptII and aph(3')-IIIa/nptIII - frequent marker genes in plant biotechnology conferring resistance to certain aminoglycosides - was determined in Austrian soils from 100 maize and potato fields not yet exposed to but eligible for GMO crop cultivation. Total soil DNA extracts were analysed by nptII/nptIII-specific TaqMan real time PCR. Of all fields 6% were positive for nptII (median: 150 copies/g soil; range: 31-856) and 85% for nptIII (1190 copies/g soil; 13-61600). The copy-number deduced prevalence of nptIII carriers was 14-fold higher compared to nptII. Of the cultivable kanamycin-resistant soil bacteria 1.8% (95% confidence interval: 0-3.3%) were positive for nptIII, none for nptII (0-0.8%). The nptII-load of the studied soils was low rendering nptII a typical candidate as environmental pollutant upon anthropogenic release into these ecosystems.

PCR-based specific techniques used for detecting the most important pathogens on strawberry: a systematic review.

BACKGROUND: Strawberry diseases are a major limiting factor that severely impact plant agronomic performance. Regarding limitations of traditional techniques for detection of pathogens, researchers have developed specific DNA-based tests as sensitive and specific techniques. The aim of this review is to provide an overview of polymerase chain reaction (PCR)-based methods used for detection or quantification of the most widespread strawberry pathogens, such as Fusarium oxysporum f.sp. fragariae, Phytophthora fragariae, Colletotrichum acutatum, Verticillium dahliae, Botrytis cinerea, Macrophomina phaseolina, and Xanthomonas fragariae. An updated and detailed list of published PCR protocols is presented and discussed, aimed at facilitating access to information that could be particularly useful for diagnostic laboratories in order to develop a rapid, cost-effective, and reliable monitoring technique. METHODS: The study design was a systematic review of PCR-based techniques used for detection and quantification of strawberry pathogens. Using appropriate subject headings, AGRICOLA, AGRIS, BASE, Biological Abstracts, CAB Abstracts, Google Scholar, Scopus, Web of Knowledge, and SpringerLink databases were searched from their inception up to April 2014. Two assessors independently reviewed the titles, abstracts, and full articles of all identified citations. Selected articles were included if one of the mentioned strawberry pathogens was investigated based on PCR methods, and a summary of pre-analytical requirements for PCR was provided. RESULTS: A total of 259 titles and abstracts were reviewed, of which 22 full texts met all the inclusion criteria. Our systematic review identified ten different protocols for X. fragariae, eight for P. fragariae, four for B. cinerea, six for C. acutatum, three for V. dahlia, and only one for F. oxysporum. The accuracy and sensitivity of PCR diagnostic methods is the focus of most studies included in this review. However, a large proportion of errors in laboratories occur in the pre-analytical phase of the testing process. Due to heterogeneity, results could not be meta-analyzed. CONCLUSIONS: From a systematic review of the currently available published literature, effective detection assays to detect the major strawberry pathogens have been developed. These assays can function as a basis for clinical labs, regulatory personnel, and other diagnosticians to adapt or implement for detection of these six important strawberry pathogens.

Molecular diversity of fungal communities in agricultural soils from Lower Austria.

A culture-independent survey of fungal diversity in four arable soils and one grassland in Lower Austria was conducted by RFLP and sequence analysis of clone libraries of the partial ITS/LSU-region. All soils were dominated by the ascomycetous orders Sordariales, Hypocreales and Helotiales, taxa that are known from traditional cultivation approaches to occur in agricultural soils. The most abundant genus in the investigated soils was Tetracladium, a hyphomycete which has been described as occurring predominantly in aquatic habitats, but was also found in agricultural soils. Additionally, soil clone group I (SCGI), a subphylum at the base of the Ascomycota with so far no cultivated members, was identified at high frequency in the grassland soil but was below detection limit in the four arable fields. In addition to this striking difference, general fungal community parameters like richness, diversity and evenness were similar between cropland and grassland soils. The presented data provide a fungal community inventory of agricultural soils and reveal the most prominent species.