High-throughput DNA isolation method for detection of Xylella fastidiosa in plant and insect samples

Brady, Jeff A.; Faske, Jennifer B.; Castañeda-Gill, Jessica M.; King, Jonathan L.; Mitchell, Forrest L.

doi:10.1016/j.mimet.2011.06.007

Cited by 12 publications

(8 citation statements)

References 11 publications

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“…Moreover, dissection of insects can generate cross-contamination. Thus, we tried to improve each steps of the DNA extraction protocols classically used 37 , 38 to reduce the impact of PCR inhibitors that may be contained in the insects 31 , 32 and increase yield in bacterial DNA. The complete protocol is available in Supplementary data (Appendix 1).…”

Section: Methodsmentioning

confidence: 99%

Using insects to detect, monitor and predict the distribution of Xylella fastidiosa: a case study in Corsica

Cruaud

Gonzalez

Martin

et al. 2018

Sci Rep

111

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We sampled ca 2500 specimens of Philaenus spumarius (Hemiptera: Aphrophoridae) throughout Corsica without a priori knowledge on the presence of symptoms on plants. We screened 448 specimens for the presence of Xylella fastidiosa (Xf) using qPCR and a custom nested PCR. qPCR appeared versatile and under-estimated the prevalence of Xf. Nested PCR showed that Xf was present in all populations. Molecular results were validated by prediction on the distribution of Xf made from tests conducted on plants, which shows the pertinence of using vectors in risk assessment studies. Xf was detected in tenerals and adults. Thus, P. spumarius could acquire Xf from its host plant, mostly Cistus monspeliensis in Corsica, which may act as reservoir for the next season. This contrasts with other observations and suggests that management strategies may have to be adapted on a case-by-case basis. At least two genetic entities and several variants of Xf not yet identified on plants were present in the insects, which suggests ancient introductions of Xf and a probable underestimation of the current diversity of the strains present in Corsica. Interestingly 6% of the specimens carried two subspecies of Xf. Studies are required to better characterize the strains present in Corsica and to determine how the disease was introduced, spread and why no sign of a potential epidemic was detected earlier. This study shows that, when sensitive enough methods are implemented, spittlebugs (and more specifically P. spumarius for which species distribution modelling shows it could be a good sentinel for Europe) can be used to predict and better assess the exact distribution of Xf. Furthermore, Xf multiply only in their foregut and does not become circulative, which facilitates its detection.

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Section: Methodsmentioning

confidence: 99%

Using insects to detect, monitor and predict the distribution of Xylella fastidiosa: a case study in Corsica

Cruaud

Gonzalez

Martin

et al. 2018

Sci Rep

111

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“…If such a cohabitation is occurring in landscape trees, alternative methods must be employed to further characterize the X. fastidiosa strains within the District. A probe-based real-time PCR method for multilocus melt typing of X. fastidiosa strains in plant samples was suggested to resolve any genotypic diversity without the need for culturing (4).…”

Section: Discussionmentioning

confidence: 99%

Bacterial Leaf Scorch in the District of Columbia: Distribution, Host Range, and Presence ofXylella fastidiosaAmong Urban Trees

Harris

Bello

Lear³

et al. 2014

Plant Disease

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“…DNA was extracted from each sample using a solid phase extraction protocol from Brady et al [19]. After extraction, DNA was amplified utilizing prokaryote-specific primers, 519F 5’-CAGCMGCCGCGGTAA-3’) and 785R (5’-TACNVGGGTATCTAATCC-3’), that target the V4 region of the 16Ss rRNA [20,21].…”

Section: Methodsmentioning

confidence: 99%

A new technique for use in the study of the microbiome: An evaluation of a three-dimensional cell culture technique in maintaining the gastrointestinal microbiome of four Balb/c female mice and implications for future studies

Castaneda

Brady²,

Speshock

2019

Preprint

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Fluctuations in oxygen, pH, nutrients, or other factors such as food or pharmaceuticals, may perturb the microbiota of the gastrointestinal (GI) tract. This environmental variation is a cause for concern given dysbiosis of the microbiome is correlated with disease states; thereby, model organisms are utilized to study microbial communities during, after, or before shifts in microbes since intact ex vivo microbiomes have historically been challenging to utilize. The objective of this study is to culture an explant microbiome of 4 Balb/c, laboratory bred mice to develop an ex vivo tool for future microbiome studies. We cultured homogenates of the distal colon of 4 mice in three dimensional, 24 well plate culture dishes. These dishes were incubated for 24 hours in two different oxygen concentration levels, 0% and 20%. The pH of the plate was tested before and after incubation. To analyze the integrity of the microbiome, we utilized 16S sequencing. Further, we utilized 16S metagenomics to characterize fecal samples and colon samples to speculate whether future studies may utilize feces in constructing an explant microbiome to spare animal lives. We found that pH and familial relationship had a profound impact on community structure while oxygen did not have a significant influence. The feces and the colon were similar in community profiles, which lends credence to utilizing feces in future studies. In addition, our efforts successfully cultured archaea, which included difficult to culture strains such as Miscellaneous Crenarchaeota group (MCG) and Methanobacteria. Ultimately, further attempts to culture and preserve an animal’s microbiome needs to control for and maintain stable pH.

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High-throughput DNA isolation method for detection of Xylella fastidiosa in plant and insect samples

Cited by 12 publications

References 11 publications

Using insects to detect, monitor and predict the distribution of Xylella fastidiosa: a case study in Corsica

Using insects to detect, monitor and predict the distribution of Xylella fastidiosa: a case study in Corsica

Bacterial Leaf Scorch in the District of Columbia: Distribution, Host Range, and Presence ofXylella fastidiosaAmong Urban Trees

A new technique for use in the study of the microbiome: An evaluation of a three-dimensional cell culture technique in maintaining the gastrointestinal microbiome of four Balb/c female mice and implications for future studies

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