TY - JOUR

T1 - Real-time PCR and microscopy

T2 - are the two methods measuring the same unit of arbuscular mycorrhizal fungal abundance?

AU - Gamper, Hannes A

AU - Young, J Peter W

AU - Hodge, Angela

AU - Jones, Davey L.

PY - 2008/5/1

Y1 - 2008/5/1

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.

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.

KW - Actins/genetics

KW - Colony Count, Microbial/methods

KW - DNA, Fungal/genetics

KW - Fungal Proteins/genetics

KW - Microscopy, Fluorescence

KW - Mycelium/genetics

KW - Mycorrhizae/cytology

KW - Plant Roots/microbiology

KW - Polymerase Chain Reaction/methods

KW - RNA, Fungal/genetics

KW - RNA, Ribosomal, 18S/genetics

KW - Reproducibility of Results

KW - Sensitivity and Specificity

KW - Soil Microbiology

KW - Spores/genetics

U2 - 10.1016/j.fgb.2007.09.007

DO - 10.1016/j.fgb.2007.09.007

M3 - Article

C2 - 17964831

VL - 45

SP - 581

EP - 596

JO - Fungal genetics and biology : FG & B

JF - Fungal genetics and biology : FG & B

SN - 1087-1845

IS - 5

ER -