Development and validation of an oligonucleotide microarray to characterise ectomycorrhizal fungal communities

Abstract

BackgroundIn forest ecosystems, communities of ectomycorrhizal fungi (ECM) are influenced by several biotic and abiotic factors. To understand their underlying dynamics, ECM communities have been surveyed with ribosomal DNA-based sequencing methods. However, most identification methods are both time-consuming and limited by the number of samples that can be treated in a realistic time frame. As a result of ongoing implementation, the array technique has gained throughput capacity in terms of the number of samples and the capacity for parallel identification of several species. Thus far, although phylochips (microarrays that are used to detect species) have been mostly developed to trace bacterial communities or groups of specific fungi, no phylochip has been developed to carry oligonucleotides for several ectomycorrhizal species that belong to different genera.ResultsWe have constructed a custom ribosomal DNA phylochip to identify ECM fungi. Specific oligonucleotide probes were targeted to the nuclear internal transcribed spacer (ITS) regions from 95 fungal species belonging to 21 ECM fungal genera. The phylochip was first validated using PCR amplicons of reference species. Ninety-nine percent of the tested oligonucleotides generated positive hybridisation signals with their corresponding amplicons. Cross-hybridisation was mainly restricted at the genus level, particularly for Cortinarius and Lactarius species. The phylochip was subsequently tested with environmental samples that were composed of ECM fungal DNA from spruce and beech plantation fungal communities. The results were in concordance with the ITS sequencing of morphotypes and the ITS clone library sequencing results that were obtained using the same PCR products.ConclusionFor the first time, we developed a custom phylochip that is specific for several ectomycorrhizal fungi. To overcome cross-hybridisation problems, specific filter and evaluation strategies that used spot signal intensity were applied. Evaluation of the phylochip by hybridising environmental samples confirmed the possible application of this technology for detecting and monitoring ectomycorrhizal fungi at specific sites in a routine and reproducible manner.

Host preferences and differential contributions of deciduous tree species shape mycorrhizal species richness in a mixed Central European forest

“…), and beech ( Fagus sylvatica ). High-throughput sequencing revealed extremely high fungal species diversity in soils of these forests (Reich et al 2009 ). Meta-analysis across ecosystems indicated that each of these tree species can be colonized by 160 to 226 different EM fungal taxa (De Roman et al 2005 ).…”

Advantages and disadvantages of microarrays to study microbial population dynamics a minireview

“…Macro-and microarrays have been used for studying populations of microorganisms in clinical samples (Wang et al 2002a), the human intestine (Wang et al 2002b), human sputum (Fukushima et al 2003), air filtrates (Wilson et al 2002), soil, compost or sludge (Stralis-Pavese et al 2004;Lievens et al 2005;Kyselkova et al 2009b), ectomycorrhizal fungal communities in soil (Reich et al 2009), human pathogens or other bacteria in water (Peplies et al 2004;Urakawa et al 2007), and human pathogens in milk (Giannino et al 2009). Using microarrays to study microbial populations on plant surfaces is less common, and only one example of a study of microbial populations on ready-to-eat vegetable salads could be found (Rudi et al 2002).…”

Systematic Internal Transcribed Spacer Sequence Analysis for Identification of Clinical Mold Isolates in Diagnostic Mycology: a 5-Year Study

“…Analysis of the ITS regions is the most commonly used molecular method for identification of molds (3,4,29). Studies reporting on the use of molecularly based identification procedures in diagnostic mycology have mainly focused on method development (12,25,30,40) and include anecdotal case reports (15,17,20). There are few studies addressing the use of systematic ITS sequencing implemented as a routine tool according to a defined work flow and its impact on diagnostic performance in a medical mycology laboratory (21).…”