PCR-Denaturing Gradient Gel Electrophoresis Profiling of Inter- and Intraspecies 18S rRNA Gene Sequence Heterogeneity Is an Accurate and Sensitive Method To Assess Species Diversity of Arbuscular Mycorrhizal Fungi of the GenusGigaspora

Abstract

Despite the importance of arbuscular mycorrhizal fungi in the majority of terrestrial ecosystems, their ecology, genetics, and evolution are poorly understood, partly due to difficulties associated with detecting and identifying species. We explored the inter-and intraspecies variations of the 18S rRNA genes of the genus Gigaspora to assess the use of this marker for the discrimination of Gigaspora isolates and of Gigasporaceae populations from environmental samples. Screening of 48 Gigaspora isolates by PCR-denaturing gradient gel electrophoresis (DGGE) revealed that the V3-V4 region of the 18S rRNA gene contained insufficient variation to discriminate between different Gigaspora species. In contrast, the patterns of 18S ribosomal DNA (rDNA) heterogeneity within the V9 region of this marker could be used for reliable identification of all recognized species within this genus. PCR-DGGE patterns provided insight into some putative misidentifications and could be used to differentiate geographic isolates of G. albida, G. gigantea, and G. margarita but not G. rosea. Two major clusters were apparent based upon PCR-DGGE ribotype patterns, one containing G. albida, G. candida, G. ramisporophora, and G. rosea and the other containing G. decipiens and G. margarita. Dissection of the DGGE patterns by cloning, DGGE screening, and sequencing confirmed these groupings and revealed that some ribotypes were shared across species boundaries. Of the 48 isolates examined, only two displayed any spore-to-spore variation, and these exceptions may be indicative of coisolation of more than one species or subspecies within these cultures. Two Brazilian agricultural soils were also analyzed with a Gigasporaceaespecific nested PCR approach, revealing a dominance of G. margarita within this family.

Discrimination of Gigaspora species by PCR specific primers and phylogenetic analysis

“…The PCR amplification tests with speciesspecific primers in this study also indicated that these two isolates should be reclassified as G. albida and G. rosea. Nevertheless recently Msiska & Morton Using PCR-DGGE analysis, Souza et al (2004) already observed, as we confirmed here, that the FL 927 isolate, originally identified as G. albida, should be ascribed to G. rosea, indicating identification problems. Endobacteria are usually observed in glomerospores of Gigaspora species, with the exception of G. rosea isolates.…”

“…Due to the low morphological variation amongst Gigaspora species (Bentivenga & Morton 1995), some papers have reported species misidentification (Bago et al 1998, Souza et al 2004, Lanfranco et al 2001. These studies also report the usefulness of molecular approach as an identification tool.…”

“…Gigaspora has five species (Bentivenga & Morton 1995) in which the morphological variation is low (Bago et al 1998, Souza et al 2004, Lanfranco et al 2001, and only diameter, color and spore wall thickness of glomerospores have been used for species identification (Bentivenga & Morton 1995). Species-specific primers designed for G. margarita (Lanfranco et al 1999) and G. rosea (Lanfranco et al 2001) have shown to be effective in solving such morphological conflicts.…”

A DGGE-cloning method to characterize arbuscular mycorrhizal community structure in soil

“…Parasitized and degraded spores were removed during weekly checks. Intact spores were further cleaned by ultrasonication and rinsing (De Souza et al, 2004). Individual clean spores were collected and transferred to a 1.5 ml microcentrifuge tube (30-100 spores each) and stored at −80 °C until DNA extraction.…”

“…Kowalchuk et al (2002) was the first to apply DGGE to assess AMF diversity in sand dune soil and root samples. Other studies used DGGE to detect AMF in forest and grassland soil (Opik et al, 2003), agricultural ecosystems (Ma et al, 2005), and for the discrimination of AMF species (De Souza et al, 2004).…”