The Promise and Pitfalls of Sequence-Based Identification of Plant-Pathogenic Fungi and Oomycetes

Kang, Seogchan; Mansfield, Michele A.; Park, Bong Soo; Geiser, David M.; Ivors, Kelly; Coffey, Michael D.; Grünwald, Niklaus J.; Martin, Frank N.; Lévesque, C. André; Blair, Jaime E.

doi:10.1094/phyto-100-8-0732

Cited by 72 publications

(51 citation statements)

References 43 publications

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“…A few nucleotide differences in the ITS region, thus a variation similar to what was detected in this work in single powdery mildew samples, is often considered as a reliable marker in the identification of certain fungal species (Bridge et al 2003;Nilsson et al 2008) especially if this is supported by sequences retrieved from GenBank. However, GenBank data have frequently been criticized for inaccuracies from several points of view (e.g., Bridge et al 2003;Kang et al 2010) including the means of obtaining the genetic information deposited in this database. This work showed that the ITS sequences determined in powdery mildew samples, and eventually deposited at GenBank, might not describe all the existing variation in this DNA region and the routine direct sequencing of the ITS region might mask diversity of these important pathogens.…”

Section: Resultsmentioning

confidence: 99%

Variation in the nrDNA ITS sequences of some powdery mildew species: do routine molecular identification procedures hide valuable information?

2011

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During the past years, nrDNA ITS sequences have supported the identification of many powdery mildew fungi because comprehensive analyses showed that differences in these sequences have always correlated with the delimitation of different species and formae speciales of the Erysiphales. Published data, obtained using direct sequencing of the PCR products, suggested that even one to five nucleotide differences in the ITS sequences delimit different, albeit closely related, species, and/or indicate differences in host range patterns. Here we show that such differences in the ITS sequences can be detected even in a single sample of a powdery mildew fungus. We sequenced the ITS region in 17 samples, representing six powdery mildew species, both directly and after cloning the PCR products. Among these, samples of O. longipes exhibited two or three, samples of O. neolycopersici three or four, those of an Oidium sp. from Chelidonium majus up to seven, and a sample of another Oidium sp. from Passiflora caerulea two different ITS types determined after cloning. No ITS nucleotide polymorphisms were found in samples of O. lycopersici and Erysiphe aquilegiae. This suggests that some powdery mildew taxa are more variable at the ITS level than others. Thus, although the ITS sequences determined by direct sequencing represent robust data useful in delimitation and phylogenetic analysis of distinct species of the Erysiphales, these need to be used with precaution, and preferably determined after cloning, especially when dealing with closely related taxa at species and sub-species levels. With this method a hitherto undetected genetic diversity of powdery mildews can be revealed.

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Section: Resultsmentioning

confidence: 99%

Variation in the nrDNA ITS sequences of some powdery mildew species: do routine molecular identification procedures hide valuable information?

2011

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“…Alternatively they could be false positives in that they lacked explicit taxonomic annotation but nevertheless were easy to assign to a known taxonomic lineage. To minimize these concerns, we examined the 50 largest lineages at the phylum, class, and order levels (as ordered by the number of constituent studies) through BLAST searches in UNITE and the INSDC following Kang et al (2010) and Nilsson et al (2012). The full length of the sequences as they were deposited in INSDC/UNITE, as well as the ITS2 and 5.8S separately, were used for these searches.…”

Section: Sorting Of the List Of Taxa (Sequence Or Study Count)mentioning

confidence: 99%

Top 50 most wanted fungi

Nilsson¹,

Wurzbacher²,

Bahram³

et al. 2016

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Environmental sequencing regularly recovers fungi that cannot be classified to any meaningful taxonomic level beyond "Fungi". There are several examples where evidence of such lineages has been sitting in public sequence databases for up to ten years before receiving scientific attention and formal recognition. In order to highlight these unidentified lineages for taxonomic scrutiny, a search function is presented that produces updated lists of approximately genus-level clusters of fungal ITS sequences that remain unidentified at the phylum, class, and order levels, respectively. The search function (https://unite.ut.ee/top50.php) is implemented in the UNITE database for molecular identification of fungi, such that the underlying sequences and fungal lineages are open to third-party annotation. We invite researchers to examine these enigmatic fungal lineages in the hope that their taxonomic resolution will not have to wait another ten years or more. Key words

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“…a) BLAST is sensitive to the length and level of sequence conservation of the query and reference sequences, and the user is advised to prune any large parts of the SSU and LSU from the ITS sequences before doing BLAST searches (cf. Kang et al 2010). It sometimes pays off to use only the ITS1 or ITS2 for the searches.…”

Section: Guideline 4 Sequences Can Be Broken In Other Puzzling Waysmentioning

confidence: 99%

Five simple guidelines for establishing basic authenticity and reliability of newly generated fungal ITS sequences

Nilsson¹,

Tedersoo²,

Abarenkov³

et al. 2012

167

125

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Larsson K-H, Larsson E, Kõljalg U (2012) Five simple guidelines for establishing basic authenticity and reliability of newly generated fungal ITS sequences. MycoKeys 4: 37-63. doi: 10.3897/mycokeys.4.3606 Abstract Molecular data form an important research tool in most branches of mycology. A non-trivial proportion of the public fungal DNA sequences are, however, compromised in terms of quality and reliability, contribut- Nilsson et al. / MycoKeys 4: 37-63 (2012) 38 ing noise and bias to sequence-borne inferences such as phylogenetic analysis, diversity assessment, and barcoding. In this paper we discuss various aspects and pitfalls of sequence quality assessment. Based on our observations, we provide a set of guidelines to assist in manual quality management of newly generated, near-full-length (Sanger-derived) fungal ITS sequences and to some extent also sequences of shorter read lengths, other genes or markers, and groups of organisms. The guidelines are intentionally non-technical and do not require substantial bioinformatics skills or significant computational power. Despite their simple nature, we feel they would have caught the vast majority of the severely compromised ITS sequences in the public corpus. Our guidelines are nevertheless not infallible, and common sense and intuition remain important elements in the pursuit of compromised sequence data. The guidelines focus on basic sequence authenticity and reliability of the newly generated sequences, and the user may want to consider additional resources and steps to accomplish the best possible quality control. A discussion on the technical resources for further sequence quality management is therefore provided in the supplementary material.

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The Promise and Pitfalls of Sequence-Based Identification of Plant-Pathogenic Fungi and Oomycetes

Cited by 72 publications

References 43 publications

Variation in the nrDNA ITS sequences of some powdery mildew species: do routine molecular identification procedures hide valuable information?

Variation in the nrDNA ITS sequences of some powdery mildew species: do routine molecular identification procedures hide valuable information?

Top 50 most wanted fungi

Five simple guidelines for establishing basic authenticity and reliability of newly generated fungal ITS sequences

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