Use of bromodeoxyuridine immunocapture to identify active bacteria associated with arbuscular mycorrhizal hyphae

Abstract

Arbuscular mycorrhizae are beneficial for crops grown under low-till management systems. Increasingly, it is becoming apparent that bacteria associated with mycorrhizae can enhance the beneficial relationship between mycorrhizae and plants. However, it has been difficult to study these relationships by conventional techniques. In this study actively growing bacteria were identified in soil from an undisturbed fallow field known to contain arbuscular mycorrhizae by using molecular tools to eliminate the need for cultivation. A thymidine analog, bromodeoxyuridine (BrdU), was added to the soil and incubated for 2 days. DNA was extracted, and the newly synthesized DNA was isolated by immunocapture of the BrdU-containing DNA. The active bacteria in the community were identified by 16S rRNA gene PCR amplification and DNA sequence analysis. Based on 16S rRNA gene sequence information, a selective medium was chosen to isolate the corresponding active bacteria. Bacillus cereus strain VA1, one of the bacteria identified by the BrdU method, was isolated from the soil and tagged with green fluorescent protein. By using confocal microscopy, this bacterium was shown to clearly attach to arbuscular mycorrhizal hyphae. This study was the first to use this combination of molecular and traditional approaches to isolate, identify, and visualize a specific bacterium that is active in fallow soil and associates with arbuscular mycorrhizal hyphae.

FIG. 1.

RFLP profiles of 16S rRNA gene fragments amplified from BrdU-labeled DNA extracted from field soil. The letters at the top correspond to distinct RFLP patterns. The preparations were digested with HaeIII (top) and RsaI (bottom). (A) Clones obtained from the sample collected on 1 July 2002. (B) Clones obtained from the sample collected on 30 August 2002.

FIG. 2.

Association of gfp-tagged B. cereus strain VA1 and controls with AM hyphae. (a) B. cereus strain VA1(pnf8) on mycorrhizal hyphae from an AM inoculum (G. dussii). (b) B. cereus strain VA1(pnf8) on a hyphal fragment from field soil. (c) B. cereus ATCC 14579(pnf8) on mycorrhizal hyphae (G. dussii). (d) E. coli DH5α::gfp/lux applied to mycorrhizal hyphae (G. dussii). Bars = 4 μm. Green cells are the result of pseudocolor rendering in the Adobe Photoshop 7.0 software.

FIG. 3.

Outline of the approach used in the present study. rDNA, ribosomal DNA.