Preferential colonization of Solanum tuberosum L. roots by the fungus Glomus intraradices in arable soil of a potato farming area

Abstract

The symbiosis between plant roots and arbuscular mycorrhizal (AM) fungi has been shown to affect both the diversity and productivity of agricultural communities. In this study, we characterized the AM fungal communities of Solanum tuberosum L. (potato) roots and of the bulk soil in two nearby areas of northern Italy, in order to verify if land use practices had selected any particular AM fungus with specificity to potato plants. The AM fungal large-subunit (LSU) rRNA genes were subjected to nested PCR, cloning, sequencing, and phylogenetic analyses. One hundred eighty-three LSU rRNA sequences were analyzed, and eight monophyletic ribotypes, belonging to Glomus groups A and B, were identified. AM fungal communities differed between bulk soil and potato roots, as one AM fungal ribotype, corresponding to Glomus intraradices, was much more frequent in potato roots than in soils (accounting for more than 90% of sequences from potato samples and less than 10% of sequences from soil samples). A semiquantitative heminested PCR with specific primers was used to confirm and quantify the AM fungal abundance observed by cloning. Overall results concerning the biodiversity of AM fungal communities in roots and in bulk soils from the two studied areas suggested that potato roots were preferentially colonized by one AM fungal species, G. intraradices.

FIG. 1.

Diagram of work flow to prepare the PCR fragment pools used in this study. To prepare PCR fragments from soil, one aliquot of soil for each of the four selected fields per area was used for genomic DNA extraction. Four genomic DNA samples per area (CSN-B1, CSN-B2, CSN-B3, and CSN-B4 and CSS-B1, CSS-B2, CSS-B3, and CSS-B4) were obtained and used in a heminested PCR in which the primer pairs LR1 and FLR2 for the first amplification step and LR1 and FLR4 for the second step were applied. The four LR1/FLR4 heminested PCR products (CSN-B1, CSN-B2, CSN-B3, and CSN-B4 and CSS-B1, CSS-B2, CSS-B3, and CSS-B4) were pooled two by two in equal molar quantities as follows: CSN-B1 and CSN-B2 were pooled to yield CSN-Ba, CSN-B3 and CSN-B4 were pooled to yield CSN-Bb, CSS-B1 and CSS-B3 were pooled to yield CSS-Ba, and CSS-B2 and CSS-B4 were pooled to yield CSS-Bb. The PCR products were then cloned into the pCR4-TOPO vector. To prepare PCR fragments from potato roots, a pooled root sample from each area was obtained by mixing potato roots randomly selected from each field. One root genomic DNA sample (CSN-R or CSS-R) per area was assessed, and one LR1/FLR4 heminested PCR product (CSN-R or CSS-R) per area was cloned into the pCR4-TOPO vector. Finally, inserts from randomly selected clones were sequenced and analyzed by using bioinformatics tools.

FIG. 2.

NJ tree representing AM fungal sequences isolated from CSN and CSS soils and potato roots in this study (designations in bold) in comparison to known sequences (*). Bootstrap values were estimated from 1,000 replicates. Ribotypes, defined as sequence groups showing bootstrap values of ≥980‰, are indicated.

FIG. 3.

Bar plot showing the relative proportions of monophyletic groups originating from soils and from potato roots, as identified by the NJ algorithm and the bootstrap method with 1,000 replicates. G. claroi, G. claroideum; G. intra, G. intraradices; G. moss, G. mosseae.

FIG. 4.

Rarefaction curves for LSU rRNA gene libraries from CSN soil (closed triangles), CSS soil (closed squares), CSN potato roots (open triangles), and CSS potato roots (open squares) determined using the Analytical Rarefaction program version 1.3 (http://www.uga.edu/∼strata/software/anRareReadme.html).

FIG. 5.

Optical density measurements obtained for the PCR fragments during the exponential phase of semiquantitative PCR to detect G. intraradices (G. intra; primer pair LR1/8.24), G. geosporum (G. geosp; primer pair LR1/53.12), G. etunicatum/G. claroideum (G. etun/claroi; primer pair LR1/Getunsp2), and G. mosseae (primer pair FLR4/5.25) in the soil and potato root samples. The data shown are representative of results for at least three replicas.