Angiosperm DNA contamination by endophytic fungi: Detection and methods of avoidance

Saar, Dayle E.; Polans, Neil O.; Sørensen, Paul D.; Duvall, Melvin R.

doi:10.1007/bf02772897

Cited by 29 publications

(13 citation statements)

References 26 publications

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“…Storage of some of the specimens for many years in herbaria did not compromise DNA extraction or the quality of AFLP profiles. We have also demonstrated the utility in this organism of using ITS-2 PCR to screen for fungal contamination, a method reviewed and advocated by Saar et al (32) for angiosperm DNA. Our use of this PCR screen resulted in the exclusion of five contaminated individuals but also provided the useful information that G. laevigata does not harbor obligate endophytic fungi of appreciable quantity to interfere with future molecular studies.…”

Section: Discussionmentioning

confidence: 80%

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Cryptic species within the cosmopolitan desiccation-tolerant moss Grimmia laevigata

Fernandez

Shevock

Glazer

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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The common cushion moss Grimmia laevigata (Bridel) Bridel grows on bare rock in a broad range of environments on every continent except Antarctica. As such, it must harbor adaptations to a remarkably broad set of environmental stresses, the extremes of which can include very high temperatures, prolonged nearly complete desiccation, and high ultraviolet B (UVB) exposure. Yet, like many mosses, G. laevigata shows very little morphological variability across its cosmopolitan range. This presents an evolutionary puzzle, the solution to which lies in understanding the phylogeographic structure of this morphologically simple organism. Here we report the results of an analysis of amplified fragment length polymorphisms (AFLPs) in G. laevigata, focusing on individuals from the California Floristic Province. We found evidence that populations within California constitute two distinct geographically overlapping cryptic species. Each clade harbors multiple private alleles, indicating they have been genetically isolated for some time. We suggest that the existence of cryptic species within G. laevigata, in combination with its life history, growth habits, and extreme desiccation tolerance, makes this moss an ideal research tool and a candidate for a biological indicator of climate change and pollution.amplified fragment length polymorphisms ͉ bioindication ͉ California Floristic Province ͉ Grimmiaceae ͉ phylogeography

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Section: Discussionmentioning

confidence: 80%

“…The ITS-2 region was amplified with the primers ITS4 and ITS3 (32). We labeled the ITS3 primer with the blue fluorophore 6-FAM on the 5Ј end.…”

Section: Distribution Of Samplesmentioning

confidence: 99%

Cryptic species within the cosmopolitan desiccation-tolerant moss Grimmia laevigata

Fernandez

Shevock

Glazer

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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“…Therefore, it should not be surprising then that E+ plant tissues have been found to exhibit different chemical profiles from E-plant tissues (Petrini et al 1992, Saikkonen et al 1998, Yue et al 2000, 2001; L. C. Mejia, T. Gianfagna, and E. A. Herre, unpublished manuscript). Even genetic content that has been attributed to being of plant origin sometimes turns out to be derived from the fungi (Camacho et al 1997, Chiang et al 2001, Saar et al 2001.…”

Section: Discussionmentioning

confidence: 99%

Ecological Implications of Anti-Pathogen Effects of Tropical Fungal Endophytes and Mycorrhizae

Herre

Mejía

Kyllo

et al. 2007

Ecology

262

185

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We discuss studies of foliar endophytic fungi (FEF) and arbuscular mycorrhizal fungi (AMF) associated with Theobroma cacao in Panama. Direct, experimentally controlled comparisons of endophyte free (E−) and endophyte containing (E+) plant tissues in T. cacao show that foliar endophytes (FEF) that commonly occur in healthy host leaves enhance host defenses against foliar damage due to the pathogen (Phytophthora palmivora). Similarly, root inoculations with commonly occurring AMF also reduce foliar damage due to the same pathogen. These results suggest that endophytic fungi can play a potentially important mutualistic role by augmenting host defensive responses against pathogens. There are two broad classes of potential mechanisms by which endophytes could contribute to host protection: (1) inducing or increasing the expression of intrinsic host defense mechanisms and (2) providing additional sources of defense, extrinsic to those of the host (e.g., endophyte‐based chemical antibiosis). The degree to which either of these mechanisms predominates holds distinct consequences for the evolutionary ecology of host–endophyte–pathogen relationships. More generally, the growing recognition that plants are composed of a mosaic of plant and fungal tissues holds a series of implications for the study of plant defense, physiology, and genetics.

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“…Instead, we designed a single species-specific primer that could be paired with the universal ITS5m primer (Saar et al 2001) in all PCR reactions. By doing this, we reduced the amount of unique sequence necessary for positive species identification to about 20 bp.…”

Section: Species Specific Primer Designmentioning

confidence: 99%

A PCR-based method for the identification of the roots of 10 co-occurring grassland species in mesocosm experiments

McNickle

2008

Botany

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An understanding of factors influencing the distribution of plant roots is intimately linked to our understanding of basic ecosystem functions such as nutrient flux and productivity. However, it is not usually possible to measure root distributions because it is difficult to identify the roots of different species when they are grown in mixture. This is because the roots of most species are not visually distinguishable. We designed a simple, PCR-based method for the identification of roots in mesocosm experiments, which we have applied to 10 co-occurring grassland species. Species-specific primers based on ITS sequences from GenBank were evaluated in PCR assays using either homogeneous or heterogeneous DNA templates, as well as DNA extracted from mixed-root samples from multiple combinations of species. The speciesspecific primers reported here produced accurate identifications, free from both false negatives and false positives, in 100% of our assays. We also evaluated the sensitivity of our system and demonstrated detection of species when they comprised as little as 0.05 ng of target DNA mixed in a total of 2.5 ng of multi-species template DNA. Our PCR-based method for root identification in mesocosms is more cost effective, and simpler to apply than previously described methods.Résumé : La compréhension des facteurs qui influencent la distribution des racines des plantes est intimement liée à notre perception de fonctions écosystémiques de base, comme le flux de nutriments et la productivité. Cependant, il demeure gé-néralement impossible de mesurer la distribution des racines parce qu'il est difficile de reconnaître celles des différentes espèces lorsqu'elles poussent en mélange, parce qu'on ne peut pas distinguer visuellement les racines de la plupart des espèces. Pour identifier les racines lors d'expériences en mésocosmes, les auteurs ont développé une méthode simple basée sur la PCR qu'ils ont appliquée à dix espèces de la prairie vivant ensemble. Ils ont évalué des amorces spécifiques à l'espèce, basées sur les séquences ITS provenant de GenBank dans des essais PCR en utilisant des modèles d'ADN homogènes ou hétérogènes, ainsi que de l'ADN extrait d'échantillons de racines provenant de multiples espèces combinées. Les amorces spécifiques à l'espèce développées ici conduisent à des identifications précises, sans fausses réactions négatives ni positives, dans 100 % des essais. Les auteurs ont également évalué la sensibilité du système et démontré la détection des espèces lorsqu'il comporte aussi peu que 0,05 ng d'ADN cible mélangé avec un total de 2,5 ng d'ADN modèle de plusieurs espèces. La méthode basée sur la PCR pour l'identification des racines en mésocosmes proposée ici est moins coû-teuse et plus simple que celles déjà décrites.

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Angiosperm DNA contamination by endophytic fungi: Detection and methods of avoidance

Cited by 29 publications

References 26 publications

Cryptic species within the cosmopolitan desiccation-tolerant moss Grimmia laevigata

Cryptic species within the cosmopolitan desiccation-tolerant moss Grimmia laevigata

Ecological Implications of Anti-Pathogen Effects of Tropical Fungal Endophytes and Mycorrhizae

A PCR-based method for the identification of the roots of 10 co-occurring grassland species in mesocosm experiments

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