A one-step multiplex real-time reverse transcription polymerase chain reaction (RT-qPCR) based on TaqMan probes was developed for the simultaneous detection of Apple mosaic virus (ApMV), Apple stem ...pitting virus (ASPV) and Apple stem grooving virus (ASGV) in total RNA of pome trees extracted with a CTAB method. The sensitivity of the method was established using in vitro synthesized viral transcripts serially diluted in RNA from healthy, virus-tested (negative) pome trees. The three viruses were simultaneously detected up to a 10-4 dilution of total RNA from a naturally triple-infected apple tree prepared in total RNA of healthy apple tissue. The newly developed RT-qPCR assay was at least one hundred times more sensitive than conventional single RT-PCRs. The assay was validated with 36 field samples for which nine triple and 11 double infections were detected. All viruses were detected simultaneously in composite samples at least up to the ratio of 1:150 triple-infected to healthy pear tissue, suggesting the assay has the capacity to examine rapidly a large number of samples in pome tree certification programs and surveys for virus presence.
Verticillium wilt is a devastating disease of a wide range of herbaceous and woody plant hosts, incited by the soilborne fungus
Verticillium dahliae. Since there are no chemical treatments to control ...the pathogen, management strategies are focused on preventive measures. In the present study, the microbial nature involved in the suppressiveness of a compost amendment (GR6, fabricated of horse manure, unbroken bedding hay
+
wood shavings and municipal green waste) against
V. dahliae was investigated. For this purpose, eggplants grown in sterilised or non-sterilised compost were transplanted in soil infested with
V. dahliae microsclerotia, amended or not with sterilised or non-sterilised compost. The most efficient treatments were those that included non-sterilised compost; therefore, the observed suppressiveness could be attributed to microbial agents. Several microbes were isolated from the root system of eggplants grown in the compost and tested
in vitro against
V. dahliae. Two bacterial strains identified as members of the
Pseudomonas fluorescens complex and two fungal isolates belonging to
Fusarium oxysporum were selected for further evaluation under glasshouse conditions. The ability of the microbial agents to reduce the percentage of diseased leaves compared to the control treatment was demonstrated. Moreover, it was shown that the most efficient bacterial strain was a rhizosphere and endophyte inhabitant. In a field experiment, the treatment where compost was applied both in the nursery and the field and treatments with
F. oxysporum and
P. fluorescens strains exhibited reduced disease severity; however, an increase in yield compared to the untreated control was not observed.
A one-step multiplex quantitative reverse transcription polymerase chain reaction protocol is described, for the detection in pome trees of Pear blister canker viroid and Apple scar skin viroid, ...together with universal detection of phytoplasmas. Total nucleic acids extraction is performed according to a modified CTAB protocol and TaqMan MGB probes are used to surpass high genetic variability of viroids. The multiplex real-time assay is at least ten times more sensitive than conventional protocols and its features make it suitable for rapid and massive screening of pome fruit trees phytoplasmas and viroids in certification schemes and surveys.
Citrus impietratura disease (CID) is a graft transmissible, virus-like disease observed in old-line citrus trees; its characteristic symptom is the appearance of gum in the albedo of the affected ...fruits. To identify the causal agent of the disease, high-throughput sequencing (HTS) was performed on symptomatic orange fruits. The analysis of the obtained data revealed in all samples mixed infections of viroids commonly found in citrus trees together with the recently described citrus virus A (CiVA). Examination of additional symptomatic fruits with conventional reverse transcription PCR led to the identification of a single CiVA infection in one tree, which was verified by HTS. Indexing of the single CiVA-infected tree on indicator plants resulted in the appearance of characteristic symptoms in the leaves that were correlated with virus accumulation. Moreover, a comparative analysis among symptomatic and asymptomatic fruits derived from the same trees was performed and included the single CiVA-infected orange tree. The analysis revealed a positive correlation between the appearance of symptoms and the accumulation of CiVA RNAs. To facilitate CiVA detection during certification programs of propagation material, a quantitative RT-PCR targeting the movement protein of the virus was developed and evaluated for reliable and sensitive detection of the virus. To the best of our knowledge this is the first study that associates CiVA with the appearance of CID symptoms.
•Single-tube multiplex RT-qPCR for detection of pome viroids and phytoplasmas.•A modified CTAB protocol for total nucleic acids extraction.•Assay's sensitivity and reliability higher than those of ...conventional protocols.•Three target detection in mixtures of 1:100 ratio of infected to healthy tissue.
A one-step multiplex real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) based on TaqMan chemistry was developed for the simultaneous detection of Pear blister canker viroid and Apple scar skin viroid along with universal detection of phytoplasmas, in pome trees. Total nucleic acids (TNAs) extraction was performed according to a modified CTAB protocol. Primers and TaqMan MGB probes for specific detection of the two viroids were designed in this study, whereas for phytoplasma detection published universal primers and probe were used, with the difference that the later was modified to carry a MGB quencher. The pathogens were detected simultaneously in 10-fold serial dilutions of TNAs from infected plant material into TNAs of healthy plant up to dilutions 10−5 for viroids and 10−4 for phytoplasmas. The multiplex real-time assay was at least 10 times more sensitive than conventional protocols for viroid and phytoplasma detection. Simultaneous detection of the three targets was achieved in composite samples at least up to a ratio of 1:100 triple-infected to healthy tissue, demonstrating that the developed assay has the potential to be used for rapid and massive screening of viroids and phytoplasmas of pome fruit trees in the frame of certification schemes and surveys.
We have examined the Wind/3DP/SST electron and Wind/EPACT/LEMT ion data to investigate the path length difference between solar electrons and ions in the ground-level enhancement (GLE) events in ...solar cycle 23. Assuming that the onset time of metric type II or decameter-hectometric (DH) type III radio bursts is the solar release time of non-relativistic electrons, we have found that within an error range of + or -10% the deduced path length of low-energy (~27 keV) electrons from their release site near the Sun to the 1 AU observer is consistent with the ion path length deduced by Reames from the onset time analysis. In addition, the solar longitude distribution and IMF topology of the GLE events examined are in favor of the coronal mass ejection-driven shock acceleration origin of observed non-relativistic electrons. We have also found an increase of electron path lengths with increasing electron energies. The increasing rate of path lengths is correlated with the pitch angle distribution (PAD) of peak electron intensities locally measured, with a higher rate corresponding to a broader PAD. The correlation indicates that the path length enhancement is due to the interplanetary scattering experienced by first arriving electrons. The observed path length consistency implies that the maximum stable time of magnetic flux tubes, along which particles transport, could reach 4.8 hr.
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