Real-time PCR and microscopy: Are the two methods measuring the same unit of arbuscular mycorrhizal fungal abundance?

TY - JOUR

T1 - Real-time PCR and microscopy: Are the two methods measuring the same unit of arbuscular mycorrhizal fungal abundance?

AU - Gamper, Hannes A.

AU - Young, J. Peter W.

AU - Jones, David L.

AU - Hodge, Angela

PY - 2008/5

Y1 - 2008/5

N2 - To enable quantification of mycelial abundance in mixed-species environments, eight new TaqMan (R) real-time PCR assays were developed for five arbuscular mycorrhizal fungal (AMF, Glomeromycota) taxa. The assays targeted genes encoding 18S rRNA or actin, and were tested on DNA from cloned gene fragments, from spores, mycelia, and from root-free soil, and on reverse-transcribed rRNA templates from entire mycelia and from colonized roots. The assays showed high specificity, sensitivity, and reproducibility, enabling reliable quantitation over broad ranges of template molecules. From cultured mycelia, DNA and RNA measures both correlated with spore number rather than extraradical hyphal length, and epifluorescence microscopy identified pronounced heterogeneity in vitality and nuclear distribution in hyphae. Root colonization was also spatially heterogeneous, as shown by a mixing experiment with root fragments of different length. Therefore, although real-time PCR can reproducibly and accurately quantify AMF nucleic acids, these are poorly correlated with visual measures because of spatial heterogeneity. (c) 2007 Elsevier Inc. All rights reserved.

AB - To enable quantification of mycelial abundance in mixed-species environments, eight new TaqMan (R) real-time PCR assays were developed for five arbuscular mycorrhizal fungal (AMF, Glomeromycota) taxa. The assays targeted genes encoding 18S rRNA or actin, and were tested on DNA from cloned gene fragments, from spores, mycelia, and from root-free soil, and on reverse-transcribed rRNA templates from entire mycelia and from colonized roots. The assays showed high specificity, sensitivity, and reproducibility, enabling reliable quantitation over broad ranges of template molecules. From cultured mycelia, DNA and RNA measures both correlated with spore number rather than extraradical hyphal length, and epifluorescence microscopy identified pronounced heterogeneity in vitality and nuclear distribution in hyphae. Root colonization was also spatially heterogeneous, as shown by a mixing experiment with root fragments of different length. Therefore, although real-time PCR can reproducibly and accurately quantify AMF nucleic acids, these are poorly correlated with visual measures because of spatial heterogeneity. (c) 2007 Elsevier Inc. All rights reserved.

KW - Glomus

KW - Glomeromycota

KW - hyphae

KW - multi- and low copy genes

KW - population size

KW - quantification

KW - root colonization

KW - Scutellospora

KW - specific primers

KW - spores

KW - POLYMERASE-CHAIN-REACTION

KW - GLOMUS-INTRARADICES

KW - QUANTITATIVE PCR

KW - TRANSFORMED ROOTS

KW - GENE-EXPRESSION

KW - FATTY-ACIDS

KW - QUANTIFICATION

KW - SOIL

KW - DNA

KW - MYCELIUM

UR - http://www.scopus.com/inward/record.url?scp=41649095297&partnerID=8YFLogxK

U2 - 10.1016/j.fgb.2007.09.007

DO - 10.1016/j.fgb.2007.09.007

M3 - Article

SN - 1087-1845

VL - 45

SP - 581

EP - 596

JO - FUNGAL GENETICS AND BIOLOGY

JF - FUNGAL GENETICS AND BIOLOGY

IS - 5

ER -