Standardisation of instrumentation in plant DNA image cytometry

DNAimage cytometry is a relatively new technique for densitometric measurement of nuclear DNAcontent, which has only rarely been used in botany and thus no methodological standards exist for this method to be applied to the measurement of plant material. In the present paper we address several problems related to standardisation of DNA image cytometry, such as stability of the measuring system, linearity of optical density measurements, correction of uneven illumination of the field of view, and uniformity of integrated optical density measurement over the entire field of view. Furthermore, image processing procedures are described for mitigation of the effects of electronic noise (image averaging) and for densitometric calibration of the measuring system. We have developed a macro for plant DNAimage cytometry, using a general image analysis software package. The described quality control procedures, adopted from international medical standards for diagnostic DNAimage cytometry, were used during software development to test performance of our measurement system. Until a specific botanical consensus is reached, we recommend that the quality control standards for instrumentation described in the present paper are considered when DNA image cytometry is used for measurement of plant genome size as well as for any other image analysis-related densitometric measurement based on light microscopy

Isolation of diverse phytoplasmas from symptomatic grapevine samples

Phytoplasmas from different ribosomal groups were isolated from grapevine samples in which “flavescence dorée” or “bois noir” phytoplasmas had been identified. The results indicate that the developed medium is not phytoplasma specific and supports the growth of phytoplasmas which cannot be detected by other molecular methods and are very likely present in a very low titre in the endobiome of the plants. The applied method discriminates the presence of bacterial contamination already in the liquid phase, and the colony purification technique allows excluding the contamination

Diagnostics of fruit trees phytoplasmas – the importance of latent infections

In the period 2000-2008 more than 1300 fruit trees from different regions of Slovenia were tested for the quarantine phytoplasmas Apple proliferation (AP), Pear decline (PD), and European stone fruit yellows (ESFY). The majority of samples were collected within systematic official surveys, which was conducted for assessing the presence of these phytoplasma in Slovenia in production and mother plant orchards. Samples were taken from trees with and without expressed symptoms. DNA was extracted from the symptomatic shoots. In addition some roots from asymptomatic trees were sampled for the evaluation of latent infections. The presence of phytoplasmas was analyzed with a nested PCR, RFLP and a real time PCR (Hren et al., 2007). AP, PD and ESFY were confirmed as being present in several areas in Slovenia where fruit trees are cultivated. AP was found not only in apple, but also in stone fruit trees such as cherry, apricot and plum (Mehle et al., 2007). By using highly sensitive diagnostic methods, such as real time PCR, some latent infections were detected and they were confirmed next year also by less sensitive methods.Keywords: Phytoplasma, AP, PD, ESFY, latent infection, fruit tree

LAMP assay and rapid sample preparation method for on-site detection of flavescence dorée phytoplasma in grapevine

In Europe the most devastating phytoplasma associated with grapevine yellows (GY) diseases is a quarantine pest, flavescence dorée (FDp), from the 16SrV taxonomic group. The on-site detection of FDp with an affordable device would contribute to faster and more efficient decisions on the control measures for FDp. Therefore, a real-time isothermal LAMP assay for detection of FDp was validated according to the EPPO standards and MIQE guidelines. The LAMP assay was shown to be specific and extremely sensitive, because it detected FDp in all leaf samples that were determined to be FDp infected using quantitative real-time PCR. The whole procedure of sample preparation and testing was designed and optimized for on-site detection and can be completed in one hour. The homogenization procedure of the grapevine samples (leaf vein, flower or berry) was optimized to allow direct testing of crude homogenates with the LAMP assay, without the need for DNA extraction, and was shown to be extremely sensitive

Grapevine yellows diseases and their phytoplasma agents - Biology and detection.

Phytoplasmas were discovered almost 50 years ago and initially they were named mycoplasma-like organisms. These cell-wall lacking bacteria and members of the class Mollicutes inhabit plant phloem sieve elements and are transmitted and spread primarily by leafhoppers, plant hoppers and psyllids that feed on infected plants phloem. Phytoplasmas interact with their hosts in a strong manner, through manipulation of the morphological features of the plants, and in several cases, also of the biology of their insect vectors. Molecular genetics analyses have improved the understanding of phytoplasma taxonomy, and also enhanced the ability to identify phytoplasmas that are detected in hosts and insect vectors. In particular, it has been determined that among the plant species infected by phytoplasmas, grapevine is one of those that are most severely affected, at a worldwide level. Molecular studies have provided considerable insights into phytoplasma molecular diversity and genetic interrelationships, taxonomic ranking has been achieved by using 16S ribosomal gene classification and other phytoplasma genes as epidemiologic molecular markers. On the other hand, the inability to fulfil Koch\u2019s postulates severely restricts the understanding of the real roles of phytoplasmas in diseases and in plant\u2013insect interaction. Together with the new possibility to cultivate phytoplasmas in artificial media, molecular genetics studies are now opening new possibilities for studying the best management of these bacteria that severely threaten worldwide agriculture, and in particular viticulture

First detection of \u201cCandidatus Phytoplasma asteris\u201d - and \u201cCandidatus Phytoplasma solani\u201d-related strains in fig trees

In July 2017, a survey was conducted in a fig collection plot at Locorotondo (south of Italy) to investigate the possible presence of phytoplasmas in plants showing yellowing, deformed leaves, short internodes, mottling and mosaic. Samples were collected from symptomatic plants and tested by nested PCR assays using universal and specific primers to amplify the 16S rDNA of these prokaryotes. PCR results detected the presence of phytoplasma sequences in twenty plant samples that resulted clustering two phylogenetically distinct phytoplasmas, i.e., \u201cCandidatus Phytoplasma asteris\u201d and \u201cCandidatus Phytoplasma solani\u201d affiliated to 16SrI and 16SrXII ribosomal groups, respectively. The presence of phytoplasmas belonging to both ribosomal groups was confirmed with group specific quantitative PCR and RFLP assays on 16S ribosomal amplicons. Results of this study indicate for the first time the occurrence of phytoplasmas in fig; however, more work should be carried out to verify their association with the symptoms observed on diseased fig plants