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Detection of arbuscular mycorrhizal fungi in an east Texas forest by analysis of SSU rRNA gene sequence.

Abstract. -- PCR amplification of a region of the small subunit ribosomal RNA gene sequence with Glomeromycota-specific primers was used to detect arbuscular mycorrhizal fungi (AMF) in the roots of Chasmanthium sessiliforum (Poir.) Yates (longleaf woodoats) from the east Texas Pineywoods. Nineteen amplified sequences along with sequences from forty other AMF species reported from previous studies outside Texas with >97% pairwise sequence identity were used to build a phylogenetic tree. At least four AMF types, several of which do not appear to be closely related to previously described taxa or sequences, were identified from east Texas clones.


Arbuscular mycorrhizal fungi (AMF, Phylum Glomeromycota, Helgason et al. 2003) form mutualistic symbiotic associations with the roots of 80-90% of all terrestrial plant species, acting as an extension of the plant root system, increasing the uptake of nutrients, improving soil stability and protecting plants against soil pathogens (Jakobsen & Nielsen 1983; Newsham et al. 1995a; & 1995b; Budi et al. 1999).

Because of their significance to plants, knowledge of AMF could be considered a prerequisite for sustainable agriculture, effective forest management (Smith & Read 1997), or conservation. While AMF communities have been described for British croplands and forests (Daniel et al. 2001), Panama tropical forest (Husband et al. 2002), Danish croplands (Kjoller & Rosendahl 2001), Minnesota experimental fields (Burrows & Pfleger 2002), various Southern Ontario and Quebec fields (Klironomos 2002), and others, they have not been described for east Texas forests. This study attempts to isolate and identify AMF from a natural forest in the Pineywoods region (Hatch et al. 1990) of east Texas with the goal of sampling local AMF diversity by isolating AMF gene sequences from roots of a widespread host growing in a typical east Texas forest habitat. The results provide preliminary baseline data that can then be compared to other habitats, hosts, and seasons.

Although AMF associated with plant roots are extremely difficult to identify using traditional morphological methods, recent molecular techniques (Simon et al. 1992; Daniel et al. 2001) show promise in detecting and identifying AMF (Hahn et al. 1993; Gadkar et al. 1997; Longato & Bonfante 1997). The small subunit ribosomal RNA (SSU rRNA) gene was chosen for PCR because it has variable regions that have been proven to provide phylogenetic information (Cedergren et al. 1988; Hamby & Zimmer 1988; Amann et al. 1991; Simon et al. 1992; 1993; Gehrig et al. 1996; Simon 1996; Redeker et al. 2000; Schussler et al. 2001), while also possessing highly conserved regions that enable the use of universal primers to amplify DNA (Bousquet et al. 1990; White et al. 1990; Clapp et al. 1995). While the phenomenon of variable nuclear ribosomal repeats even within single individuals of Glomeromycota may obscure differences between taxa, the region of rRNA used in this study is fairly conserved and was successfully used by De Souza et al. (2004).

Designed by Helgason et al. (1998), the reverse primer AM1 excludes both plant and non-Glomalian fungal SSU sequences while amplifying SSU rRNA genes from the three traditional Glomeromycota families of Acaulosporaceae, Gigasporaceae, and Glomaceae. While it also excludes several AMF types, particularly the newly-discovered ancestral lineages (Morton & Redecker 2001; Schwarzott & Schussler 2001), AMI in combination with forward primer NS31 appears to detect a large portion of the AMF community and has been used successfully in several ecological studies including those of Helgason et al. (1998), Helgason et al. (1999), and Daniell et al. (2001).


Study area and host plant. -- The study site was a moderately-steep south-facing slope adjacent to the floodplain of the Angelina River located in the Stephen F. Austin Experimental Forest in Nacogdoches County, Texas. The topsoil was a brown (7.5YR 5/4) sandy loam. Vegetated with common native species including Pinus taeda L., Pinus echinata p. Mill, Liquidambar styraciflua L., Toxicodendron radicans (L.) Kuntze, Quercus alba. L., Dichanthelium comutatum (J.A. Schultes) Gould, Smilax bonanox L., Callicarpa Americana L., Cornus florida L., Vitis rotundifolia Michx., Quercus nigra, L. Parthenocissus quinquefolia (L.) Planch, Arisaema triphyllum, (L.) Schott, Elephantopus tomentosus L., Ulmus alata Michx., Ilex opaca Ait., and Sanicula Canadensis L., the site represents a widespread east Texas forest community type and is classified as a white oak-loblolly pine/Callicarpa loamy mesic slope under the US Forest Service ecological classification system (Turner et al. 1999).

Chasmanthium sessiliflorum (Poir.) Yates, (Poaceae), a native forest understory grass, was selected as the mycorrhizal host for the study. It is a tufted, summer-blooming, shade-tolerant perennial up to 1 m tall which bears nearly sessile, laterally-compressed, wedge-shaped spikelets arranged in a slender, erect, spike-like inflorescence (Correll & Johnston 1979). It is present across a wide range of east Texas forest habitat types ranging from wet-mesic stream bottoms to dry-mesic uplands on various soils. A significant component of most any east Texas community in which it is found, it is characteristic of the pine-broadleaf deciduous forests that cover much of eastern Texas and in many such stands is a dominant ground layer plant (Turner et al. 1999).

Field sampling and sample processing. -- Three random sample points were located along a transect established parallel to the direction of the slope, one from the upper third of the transect and one each from the middle and lower thirds. During July 2003, root samples were obtained from the three colonies of C. sessiliflorum found nearest to each sample point. Samples were transported to the laboratory at 4[degrees]C, washed free of soil, dried on tissue paper and kept at -70[degrees]C until analysis. Sub samples of each root sample were stained with ink (Vierheilig et al. 1998) to verify AMF infection prior to DNA analysis. Roots were sonicated two times for five minutes each, first in 10mM sodium pyrophosphate and then in distilled water (De Rooij Van Der Goes 1995) to remove root surface fungi.

DNA isolation & amplification. -- DNA was isolated from a 20 mg portion of ground root material from three replicates of each of the nine original root samples by the modified potassium ethyl xanthate (PEX) extraction method of Edwards et al. (1997). Fragments of SSU DNA ([approximately equal to]520bp) were amplified by PCR with Pfu DNA polymerase (Stratagene Inc.) using the universal eukaryotic primer NS31 (Simon et al. 1992) in combination with the primer AM1 (Helgason et al. 1998), which is specific to many AM species but excludes plant and non-glomalian fungal species. The conditions described by Helgason et al. (1999) were used for PCR.

DNA sequencing. -- The PCR products were cloned into pCR-Script Amp SK(+) (Stratagene Inc.) and transformed into Escherichia coli (XL1-blue MRF', Stratagene Inc.) following Helgason et al. (1999). Colonies were screened using the blue-white screening technique. Ten putative positive transformants from each of the three slope positions were selected for sequencing of plasmid inserts. Sequencing was performed by Amplicon Express Inc. (Pullman, Washington).

Sequence analysis of 18S rRNA sequenced from the clones.-Forward and reverse sequences were aligned and assembled using the CAP-Contig Assembly Program (Huang 1992). To verify glomalean origin and identify sequences with a high degree of similarity, BLAST search (Altschul ct al. 1990) was used with the sequences obtained to search the GenBank at NCBI ( Multiple sequence alignment was performed using ClustalX (Thompson et al. 1994). The alignment is available from the corresponding author upon request.

All phylogenetic trees were computed with MEGA version 3.0 (Kumar et al. 2004) using 422 sites certain to be in alignment. Two separate trees were generated: the first using maximum parsimony analysis with the initial tree generated by random addition, and the second using neighbor-joining analysis (Saitou & Nei 1987) with the Kimura two-parameter model (Kimura 1980). The robustness of the inferred trees was evaluated after 1000 bootstrap resampling. Only values >70% are shown on the trees. Sequences were deposited in the NCBI database and GenBank accession numbers were received.


Staining revealed the presence of AMF infection in all root samples (Figure 1). Three clones were not able to be sequenced and eight clones showed low similarity to AM fungal sequences and were removed from the analysis. These may have resulted from contaminating organisms, nonspecific binding, or PCR errors. The 19 remaining sequences, 528-548 bp in length, as expected, were undoubtedly of glomalean origin.

Results from a BLAST search yielded 35 sequences from the NCBI nucleotide data bank with at least 97% pairwise sequence identity with at least one of the east Texas clones. Each belonged to the genus Glomus. Moreover, those sequences in the search results which were included in Schussler et al. (2001) belonged to the group "GlGrA" (Glomus Group A) described in that publication. A preliminary neighbor-joining tree (not shown) constructed using only the east Texas sequences and those of Schussler et al. (2001) also showed that the Texas sequences occurred within a monophyletic group of the genus Glomus and belonged to Schussler's group "G1GrA". Accordingly, neighbor-joining and maximum parsimony trees were constructed using the best-matching NCBI sequences (>97% sequence identity), the east Texas clones, and five sequences from groups "G1GrB" (Glomus Group B) and "GlGrC" (Glomus Group C) of Schussler et al. (2001) which were used as an outgroup (Figure 2).

Both trees resulted in similar topologies. Definitions of groups were based on tree topology and pairwise distance. Pairwise distance between groups was > 0.03. The 19 east Texas sequences fell within four well-supported groups with bootstrap values of at least 70% (Figure 2). The first group received 99% (neighbor joining analysis) and 94% (Parsimony analysis) bootstrap support and included the sequences ET2, ET9, ET15 and ET5 whose closest neighbors on the tree included Glomus sp. ZJ (GenBank accession number AB076271) and uncultured Glomus clone p3740 (GenBank accession number AJ563879, Figure 2). The second group consisted of 11 east Texas clones (Figure2) and received 85%/86% bootstrap support. The third group, which had 94%/90% bootstrap support, included the east Texas clone (ET13) along with two additional Glomus species clones reported from the GenBank database: Glomus sp. Gloll isolate Dd2.14 (AF131052) and Glomus sp. Gloll isolate Dd2.6 (AF131053). The fourth group, ET6 and ET10 belonged to a branch with 98%/90% bootstrap support that included G. fasciculatum, G. interadices, and G. vesiculiferum, but was not closely related to them. A single east Texas clone, ET11, appeared to form an additional separate lineage but was not well supported statistically. No formally named species closely matched any of the four groups of east Texas clones.



The AMF types detected were not uniformly distributed across the sample site. Group III was found in a sample from the lower portion of the hill slope. Group IV was restricted to midslope samples while Group I occurred in both lower slope and midslope samples. Group II was found on all three landscape positions; however, the majority of sequences (7/11) were isolated from the upper slope. Moreover, several AMF types (Groups I, II, and III for the lower slope and groups I, II, and IV for the mid-slope) were isolated from the root samples taken from the lower and middle slopes, while only Group II types were found in upper slope samples.


While it is not possible to directly infer numbers of species from this limited study, it is clear that at least four AMF taxa were detected--including several not reported elsewhere. By comparison, Daniell et al. (2001) and Husband et al. (2002) in far more extensive studies involving multiple hosts and sites isolated eight and 30 fungal types from British cropland and tropical forest respectively. The results of this current study support the emerging consensus that there are many undiscovered species of AM fungi and that there is significant localized diversity (Husband et al. 2002; Helgason et al. 2002). Future data from different hosts, habitats, and seasons will enable us to further understand AMF diversity in east Texas ecosystems.

Small sample size does not permit a definitive statement as to whether the uneven distribution of AMF types is the result of habitat differences associated with the hill slope gradient or the result of chance distributions of fungal taxa across the landscape. However, it is evident that different combinations of local AMF species may occur on the same species of host plant in the natural environment and that these differences can occur across a relatively small spatial scale (<150m in the case of the current study). This suggests that future ecological studies relating differences in east Texas AMF composition to local differences in habitat or vascular plant species composition will be a fruitful area of inquiry.


We wish to thank Dr. Josephine Taylor for assistance with microscopy and photography and Dr. Don Pratt for useful advice on phylogenetic analysis. Two anonymous reviewers provided helpful comments on the manuscript. This research was supported by a Stephen F. Austin State University faculty research grant.


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Alexandra Martynova-Van Kley, Hailun Wang, Armen Nalian and James Van Kley

Science Research Center, Stephen F. Austin State University

Nacogdoches, Texas 75965
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Author:Martynova-Van Kley, Alexandra; Wang, Hailun; Nalian, Armen; Van Kley, James
Publication:The Texas Journal of Science
Geographic Code:1U7TX
Date:Aug 1, 2006
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