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Application of high-throughput dna sequencing in phytopathology

Following sequencing of the plasmid inserts, comparisons were made with published sequence for RoCV1.

Introduction

The results led us to conclude that the samples were infected, giving positive results with the RT-qPCR and negative results with the conventional RT-PCR most likely due to lower sensitivity. Based on the provenance of samples, these results suggest that RoCV1 has been in the UK at least since Following the genus demarcation criteria from the International Committee on Taxonomy of Viruses Vainio et al.

There is no known natural vector for RoCV1; it is assumed to be pollen and seed transmitted like other cryptoviruses. In New Zealand, infected roses were associated with mottling, leaf spots and necrosis and also one with flower break but it was also found in asymptomatic samples Milleza et al. In this study, several samples were asymptomatic, others had vein banding, mottling or leaf distortion symptoms. It is possible that these symptoms could be caused by another, as yet unidentified virus or pathogen , by herbicide damage or adverse cultural conditions.

A sample with symptoms similar to those caused by rose rosette virus was positive for RoCV1; further studies will be undertaken to investigate if those symptoms were due to mixed infections with other, as yet undescribed viruses. Cryptoviruses are not thought to cause direct economic losses in their plant host, although it is not clear what impact they have in mixed infections. This work was carried out as part of a joint studentship funded by the Fera Science Ltd.

This article does not contain any studies with human participants or animals performed by any of the authors. It is original and has not been submitted or published elsewhere. Informed consent was obtained from all individual participants included in the study. All the authors have seen and approved the manuscript, and all have taken a valid role through either study design, data generation or manuscript preparation. Skip to main content Skip to sections. Advertisement Hide. Download PDF. Journal of Plant Pathology pp 1—5 Cite as. Open Access.

First Online: 06 May Further analysis is required to confirm these results. Open image in new window. Acknowledgements This work was carried out as part of a joint studentship funded by the Fera Science Ltd. Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest.

Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. Informed consent Informed consent was obtained from all individual participants included in the study. Fungal-classification results showed that the dominant phylum was Ascomycota , accounting for The next most dominant phyla were Zygomycota Relative abundances of the dominant bacterial a and fungal b taxa in 1-year and year continuous-cropping ramie soil samples at the phylum level.

Venn diagrams were generated using the Mothur program, based on the shared OTU tables from 4 different soil samples Fig 4. The total number of unique bacterial OTUs was 51,, of which were associated only with 1-year healthy cropping ramie 1YN, 1YR , were associated only with year continuously cropped ramie 10YN, 10YR , and were shared by all samples Fig 4A.

In terms of fungi, 8, different OTUs were identified, of which were associated only with 1-year healthy-cropping ramie soil, were associated only with year continuous-cropping ramie soil, and were shared by all samples Fig 4B. Among the fungal genera, Cortinarius , Malassezia , Mortierella , Ceratobasidium , Guehomyces , Rhizophydium , Fusarium , and Trichoderma were detected only in the 1-year healthy-cropping ramie soil, whereas Leucosporidium , Sporobolomyces , Cryptococcus , Humicola , and Trichosporon were detected only in year continuous-cropping ramie soil.

Venn diagram showing the number of unique bacterial a and fungal OTUs b detected in ramie soil samples. Heatmap clustering analysis results for the 30 dominant genera from the different samples are shown in Fig 5. The Methylovirgula genera had high abundances in the RS of 1-year and year ramie, Alkaliphilus and Carnobacterium genera had relatively high abundances in the RS of year continuous-cropping ramie, but the Methylibium genus had a relatively high abundance in the RS of all 1-year and year ramie.


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For fungi, heatmap clustering analysis showed that the Mortierella , Candida , Humicola , Fusarium , and Cryptococcus genera had relatively high abundances, and the Bulleribasidium , Rhizophydium , and Gongronella genera differed between 1-year healthy-cropping and year continuous-cropping ramie samples. The Ceratobasidium and Colletotrichum genera had high abundances in the RS of 1-year and year ramie, whereas the Trichoderma , Lectera and Chalara genera had low abundances in year ramie. Heatmap analysis of bacteria a and fungi b based on the relative abundances of dominant genera from different Ramie soil samples.

To further compare the microbiota among different samples, we performed principal component analysis PCA to study the relative abundances of bacterial and fungal genera, using Canoco software, version 4. The data are presented as a 2-dimensional plot to better illustrate the relationship among these soil samples.

In bacteria except for 1YN. The PCA results agreed with those from the heatmap analysis, indicating that all samples exhibited different characteristic bacterial and fungal communities. PCA of the OTUs detected major variations in the bacterial a and fungal b communities in healthy and continuous Ramie soil samples. We used Pearson correlation coefficients to evaluate the relationships between abundant phyla bacterial and fungal and the yield properties of continuous-cropping ramie samples Table 2. The relative abundances of the Firmicutes , Bacteroidetes , and Glomeromycota phyla correlated positively with the yield properties of continuous ramie.

Other genera did not display any significant correlation Table 2. Thus, Actinobacteria , Chloroflexi , and Zygomycota appear to have exerted the largest impact on yields of continuous-cropping ramie. Ramie is a perennial, herbaceous plant. Long-term continuous cropping of ramie results in reduced yield and even death. This phenomenon has been observed also for many other plants, including cotton [ 39 ], apple [ 25 ], and sugarcane [ 7 ], whose yields are significantly lower after long-term continuous cropping. Ramie fiber extracted from stem bark is a vegetative tissue whose yield is determined by stem growth.

In this study, continuous cropping for 10 years severely inhibited stem growth and led to a decrease in fiber yield Fig 1. Hence, a stable and healthy long-term growth environment is essential for maintaining high ramie production. Recently, as the planting area of ramie has decreased greatly, the economic benefit has become reduced.

Problems linked to continuous cropping represent some of the main hindrances to the development of the ramie industry. Therefore, elucidating potential microbial community-structure mechanisms that could improve continuous cropping tolerance are highly important for ramie production in China. Illumina NGS technology is a powerful tool in many research areas, including re-sequencing, micro-RNA expression profiling, DNA methylation, de novo transcriptome sequencing, and whole-genome sequencing [ 40 — 42 ].

In this study, NGS was used to identify the microbial community associated with ramie plants grown under continuous field conditions and to elucidate differences in community structure based on the health status of plants. Limited studies on the interactions between continuous cropping ramie and the rhizospheric microbial community have been conducted in natural ecosystems. To the best of our knowledge, this study represents the first implementation of Illumina sequencing technology to investigate the microbial diversity associated with healthy and continuous RS samples from ramie plants grown under field conditions.

Interestingly, we analyzed the diversity of ramie rhizospheric soil according to richness Chao 1 and diversity Shannon indices, which showed marked changes between 1-year healthy cropping ramie and year continuous-cropping ramie, whereas no obvious change in fungi was observed Fig 2 and Table 1. The fiber yield of year continuous ramie decreased significantly compared to that of 1-year ramie. Thus, we speculated that changes in the bacterial composition and diversity were more related to the continuous cropping of ramie.

Moreover, these properties varied according to the planting year and health status of the ramie plants.


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  • The community structure of soil microorganisms underwent changes during different years of ramie cultivation, resulting mainly in reduced microbial diversity. This trend was analogous to that reported for Picea mariana based on Sanger sequencing, which showed that healthy seedlings grown in a nursery had greater bacterial diversity than that of diseased seedlings [ 43 ]. Healthy greenhouse tomato RS samples have been reported to harbor higher bacterial diversities than RS samples from diseased tomatoes, according to pyrosequencing results [ 44 ].

    However, in studies based on Illumina sequencing, healthy Lanzhou lily Lilium davidii plants exhibited lower bacterial diversity than diseased plants [ 45 ]. Differences in plant-growth conditions, plant disease-causing agents, microbe-identification methods, and plant genotypes may explain these contrasting results.

    In this study, sequence analyses revealed that the relative abundances of bacterial and fungal phyla Fig 3. For bacteria, we observed that Firmicutes , Proteobacteria , and Acidobacteria were the most abundant bacterial phyla in 1-year healthy and year continuous ramie Fig 3A. The most dominant phylum was Firmicutes. Data from numerous studies have shown that most rhizosphere species belong to the Firmicutes phylum [ 46 — 48 ] Moreover, Firmicutes correlated positively with the stem length, stem diameter, and fiber yield of continuous ramie Table 2 , and obvious differences were observed in the Firmicutes phylum in the RS and NRS between 1-year healthy and year continuous ramie.

    This change may represent the main factor causing soil weakness after continuous ramie cropping, as reported previously [ 45 , 49 ]. Firmicutes was implicated as serving a very important role in the growth and yield of ramie, but the underlying mechanism needs to be determined in a future study. The second-most dominant phylum was Proteobacteria.

    Although no obvious differences were observed in RSs between 1-year healthy and year continuous ramie Fig 3A , an obvious difference was found between RS and NRS from ramie. Proteobacteria have been previously shown to be enriched in cucumber rhizosphere [ 50 , 6 ]. Recent studies of maize [ 51 ], soybeans [ 52 ], oak trees [ 53 ], and poplar trees [ 54 ] have also revealed an enrichment of Proteobacteria in the rhizosphere. The other independent studies have shown that Proteobacteria are dominant members of the rhizosphere microbiota [ 55 — 56 ].

    These findings are in line with Proteobacteria having a fast-growth phenotype among rhizosphere bacteria and being capable of utilizing a broad range of root-derived carbon substrates [ 57 ]. We found that the relative abundance of Proteobacteria was elevated in ramie rhizosphere. Thus, we speculated that some species of Proteobacteria may serve as beneficial bacteria agents in the rhizosphere of ramie, some ones may beneficial for ramie growth. Our results showed that Actinobacteria were the most sensitive in the major distribution zone of ramie roots, and a significant negative correlation was found between Actinobacteria abundance and the stem length, stem diameter, and fiber yield.

    Some Actinobacteria showed reduced microbial diversity and, thus, may correlate with the adverse effects of continuous ramie cropping. This finding suggests that ramie cultivation strongly influences the community structure of soil Actinomycetes. Previous findings have suggested that changes in the soil microflora may be responsible for impairing the growth of some continuously cropped plants [ 57 ]. Among microorganisms, some fungi have been found to play important roles in soil ecosystems and contribute to plant diseases [ 58 ].

    Among fungi, Ascomycota , Zygomycota , and Basidiomycota were identified as the main phyla of ramie soil, which was consistent with previous continuous-cropping studies of peanut and soybean soils [ 22 , 59 ]. Moreover, these phyla may be involved in the degradation of simple root exudates, as well as the more complex compounds present in sloughed root cells [ 60 ]. With an increase in the number of continuous planting years, reduced crop production and other barriers may appear, and a substantial body of research has shown that rhizosphere microbial diversity can be partially influenced by the cropping history [ 61 ].

    In our study, the abundance of Ascomycota decreased in the RS of year ramie, and the opposite results were found in NRS.

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    Ascomycota also changed with increasing numbers of years of ramie cropping, and the microbial population densities in the rhizosphere were much higher than in the surrounding bulk soil. We speculate that it is likely that the rhizosphere effect led to a decrease of some beneficial Ascomycota genera or increased the abundance of some harmful genera in the year RS of ramie. Ascomycota have function in the decay of organic substrates and act as mutualists [ 63 ]. Thus, we speculate that the Ascomycota genera may also function in the decay of organic substrates in the ramie rhizosphere.

    In soil ecosystems, rhizosphere microorganisms interact with plants [ 64 ] and are escorted by a myriad of microorganisms living freely or in intimate association with their roots, which leads to root and stem development, growth stimulation, or crown rot diseases [ 65 ]. Some Glomeromycota members have been considered generally as obligate symbiotic fungi, and the Ascomycota and Glomeromycota phyla can respond rapidly to rhizodeposits [ 66 , 67 ].

    Previous data showed that members of the Glomeromycota phylum depend on carbon and energy derived from plant synthesis to survive, and shares a symbiotic relationship with the roots of plants [ 68 , 69 ]. Therefore, we propose that the Glomeromycota phylum may also affect symbiosis and interactions between ramie roots and soil microbes, considering that it was highly enriched in the ramie rhizosphere.

    Soil fungi show high functional diversity. Chytridiomycota was abundant in the RS of year ramie soil, which may be a result of continuous cropping, single nutrient supply, and conditions that augment the growth of harmful soil microbes [ 70 ]. At the genus level Fig 5 , among the bacteria identified in this study, Bacillus , Aquicella , Lactococcus , and Paracoccus were more abundant in 1-year healthy ramie, whereas Cytophaga , Burkholderia , Flavobacterium , TM , and Phenylobacterium were more frequent in year continuously cropped ramie.

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    Among these genera, Bacillus is one of the most common plant growth-promoting rhizobacteria biocontrol agents [ 71 ]. Members of the Flavobacterium genus participate in mineralizing various types of organic matter. Bacillus and Lactococcus are members of the Firmicutes phylum, which was more abundant in healthy 1-year ramie soil. The abundance of the Bacillus genus decreased with the number of years of continuous cropping, and this genus was not observed after 10 years.

    Many previous findings have demonstrated that Bacillus plays important roles in the health of vanilla plants [ 72 ] and in the suppression of soil-borne diseases [ 73 ], indicating that the decrease in beneficial bacterial species may cause soil weakness after long-term ramie monoculture. Accordingly, we believe that the relatively high abundance of Bacillus among Firmicutes in healthy RS may contribute to disease suppression, thereby promoting ramie growth. Results from this study demonstrated that Mortierella , Fusarium , and Phoma were the major harmful genera in the year continuously cropped ramie Fig 5 , further illustrating that the accumulation of harmful microorganisms could impair continuous ramie cropping, which is consistent with previous results [ 74 ].

    For example, the main pathogens of wilt diseases in the Lanzhou lily field were members of the Fusarium genus [ 75 ]. The community structure of soil microorganisms is mainly determined by environmental factors such as soil properties [ 76 ], as well as the crop type and tillage-management measures [ 22 , 23 ]. Analysis of ramie RS and NRS showed that the contents of Alkaliphilus and Carnobacterium genera were relatively high in year ramie and that the Methylovirgula and Methylibium generas were more abundant in 1-year and year ramie.

    Therefore, these genera may play relatively important roles for ramie roots. Among the fungi identified in our samples, Cortinarius , Fusarium , and Trichoderma were relatively more abundant in healthy ramie, whereas Cryptococcus and Trichosporon were more abundant in continuously cropped ramie. The latter are major soil pathogens and may be one of the main causes of the adverse growth effects induced by continuous ramie cropping. In conclusion, continuous ramie cropping led to a significant decline in stem length, stem diameter, and fiber yield.

    The soil microbial community composition was altered following long-term continuous cropping of ramie. Soil bacterial diversity decreased with the time span of ramie monoculture, whereas no significant differences were observed for fungi Fig 2.

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    We speculate that the relatively high abundance of Bacillus among Firmicutes in healthy RS may contribute to disease suppression, thereby promoting ramie growth. In general, soil weakness and ramie disease increased after long-term continuous cropping, and both conditions can be attributed to alterations in soil microbes, a reduction of beneficial microbes, and the accumulation of harmful microbes.

    Furthermore, this is the first report to provide evidence of major differences between the microbial communities in the soil of healthy and continuously cropped ramie plants. Our results demonstrate that Illumina NGS technology was a useful and effective approach for characterizing the microbial communities of ramie RS.

    These differences in microbes provide important information regarding beneficial ramie rhizosphere microbial species, which can contribute to future improvements in the microbial community structure and decreasing pathogenic microbial diseases associated with continuously cropped ramie soil. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Abstract Ramie Boehmeria nivea L. Introduction Ramie Boehmeria nivea L.

    Application of high-throughput DNA sequencing in phytopathology.

    Materials and methods Ethics statement This work was conducted in our scientific research field for ramie cultivation studies, which is owned by our institution. Statistical analysis For all parameters, multiple comparisons were performed using one-way analysis of variance with Turkey's honest significant difference multiple-range test.

    Results Ramie stem traits and fiber yields in response to continuous cropping In 1-year cultivated ramie, the stem length, diameter, and bark thickness were Download: PPT. Fig 1. Systematic studies on repeatability and reproducibility have yet to be published, although resampling of data to explore the impact of sequencing depth indicated a high degree of reproducibility, with qualitatively different results occurring only when reducing sequence depth negatively impacted sensitivity Visser et al.

    This again highlighted a link between appropriate depth of coverage and the repeatability of the test. Harnessing the diagnostic power and flexibility of HTS for screening applications also brings the inherent tension of how to deal with inadvertent non-target findings. Dealing with these findings can only be done on a case by case basis and will depend upon the virus detected and the purpose of testing Massart et al. For vegetatively propagated crops it is conceivable that mother plants could be accompanied by a HTS sequence metadata passport obtained according to recognized standards Saldarelli et al.

    To support such advances there is a need to understand the virome of a given crop MacDiarmid et al. Contamination, potentially leading to erroneous reporting is recognized as a significant issue in HTS Dickins et al. In common with other molecular techniques contamination of samples with nucleic acid from other samples can occur at a number of steps in the HTS protocols. Appropriate use of negative controls during the process and introduction of cut-offs based on signal-to-noise is a solution used in many routine testing laboratories deploying other, similarly sensitive techniques.

    In the longer term, the advent of more diagnostic-focused platforms may improve this situation. Use of adequate and appropriate first-line controls is essential for demonstrating that a given assay is performing within acceptable criteria, allowing for the correct interpretation of results Roenhorst et al.

    To address this, two positive control strategies have been developed, using known nucleic acids as an internal control. One uses leaf discs from a plant infected with a known virus Kesanakurti et al. Both strategies are considered adequate in current virus discovery applications, however this may not be the case when trying to detect a range of known viruses and other strategies, such as including a suite of non-target viruses as a positive control, could be investigated. Alongside the appropriate use of controls, expertise in bioinformatics and plant virology has been identified as critical for the effective interpretation of diagnostic data Roenhorst et al.

    The advancement in HTS technologies undoubtedly brought great potential for virus detection and discovery.

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    However, like any new technology, HTS-based approaches should be validated for sensitivity, specificity, reproducibility and repeatability before their routine implementation. Solutions need to be brought to deal with HTS specific challenges, such as the use of controls appropriate for the diagnostic workflow in which the method is implemented or the handling of the detection of novel or non-target viruses. Application-specific validation will ensure that the performance of HTS methods is equivalent or better than those of current targeted approaches.

    As more laboratories access HTS and apply it to routine virus detection there will be an increasing need for both test performance studies and regular proficiency tests to evaluate the methods themselves and the capabilities of diagnostics laboratories. Such studies will require access to a range of well characterized virus isolates and data sets and should address both the competence of the laboratory to perform all the steps of the diagnostic process, including the bioinformatics analysis, as well as its expertise in interpretation of results.

    As a diagnostic tool, HTS is perhaps more broad-spectrum than any previously used assay. Whilst the technique is powerful, the available frameworks of validation e. Expanding its use to include current as well as future advancements in HTS applications requires integration and validation steps that are all well known to diagnosticians and should not be a cause for concern.

    All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Al Rwahnih, M. Comparison of next-generation sequencing versus biological indexing for the optimal detection of viral pathogens in grapevine. Phytopathology , — Pokorski Cham: Springer International Publishing , 53— PubMed Abstract Google Scholar.

    Dickins, B. Controlling for contamination in re-sequencing studies with a reproducible web-based phylogenetic approach. BioTechniques 56, — Golino, D. Google Scholar. Hagen, C. Accurate and sensitive diagnosis of geminiviruses through enrichment, high-throughput sequencing and automated sequence identification. Jiang, L. Synthetic spike-in standards for RNA-seq experiments. Genome Res. Kesanakurti, P. Screening for plant viruses by next generation sequencing using a modified double strand RNA extraction protocol with an internal amplification control.

    MacDiarmid, R. Biosecurity implications of new technology and discovery in plant virus research.