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.

Characterisation of microbial communities colonising the hyphal surfaces of arbuscular mycorrhizal fungi

“…In addition to B. cereus, also Paenibacillus spp. have been mentioned in a number of reports to attach to AMF hyphae, to stimulate mycorrhiza formation and to proliferate in the presence of hyphae (Budi et al, 1999;Mansfeld-Giese et al, 2002;Artursson and Jansson, 2003). In our experiments, we did find genetic signature evidence for Paenibacillus spp., notably from T-RF 303, but only in two cloned inserts.…”

“…This is in marked contrast to what has been found earlier. Artursson and Jansson (2003) reported good attachment of pure cultures of B. cereus to hyphae of Glomus dussii. Our data did not specifically suggest presence of B. cereus in the communities isolated from soil and therefore we cannot refute or confirm their findings.…”

Linking Species Richness, Biodiversity and Ecosystem Function in Soil Systems

“…6). This study by Artursson and Jansson (2003) is a pioneering attempt, using molecular and traditional approaches to isolate, identify and visualize a specific bacterium that is active in fallow soil and associates with AM hyphae. …”

“…The field of mycorrhizosphere research (Andrade et al, 1998) has taken a quantum leap forward with elegant microscopic methods i n conjunction with molecular tools to pinpoint organisms that are co-associates. Artursson and Jansson (2003) used the thymidine analog bromodeoxyuridine (BrdU) to identify active bacteria associated with arbuscular mycorrhizal (AM) hyphae. After adding BrdU to the soil and incubating for 2 days, DNA was extracted, and the newly synthesized DNA was isolated by immunocapture of the BrdU-containing DNA.…”

Closing the Global Energy and Nutrient Cycles through Application of Biogas Residue to Agricultural Land – Potential Benefits and Drawback

“…However, there is an imminent risk of overlooking the short-term effects of residue application when relying on solely these analyses. Therefore, to distinguish short-term changes (including the resulting long-term effects) in soil quality, it is essential to analyze the structure, function and/or activity of microbial communities present in the soil [37,[118][119][120][121], which present a more rapid response to environmental changes [58]. An accurate picture of the early changes in soil quality can be obtained by evaluating microbial properties, preferably in combination with chemical characteristics, since perturbations affecting the function of soil are reflected at an early stage.…”