Isolation and identification of actinobacteria from surface-sterilized wheat roots

Abstract

This is the first report of filamentous actinobacteria isolated from surface-sterilized root tissues of healthy wheat plants (Triticum aestivum L.). Wheat roots from a range of sites across South Australia were used as the source material for the isolation of the endophytic actinobacteria. Roots were surface-sterilized by using ethanol and sodium hypochlorite prior to the isolation of the actinobacteria. Forty-nine of these isolates were identified by using 16S ribosomal DNA (rDNA) sequencing and found to belong to a small group of actinobacterial genera including Streptomyces, Microbispora, Micromonospora, and Nocardiodes spp. Many of the Streptomyces spp. were found to be similar, on the basis of their 16S rDNA gene sequence, to Streptomyces spp. that had been isolated from potato scabs. In particular, several isolates exhibited high 16S rDNA gene sequence homology to Streptomyces caviscabies and S. setonii. None of these isolates, nor the S. caviscabies and S. setonii type strains, were found to carry the nec1 pathogenicity-associated gene or to produce the toxin thaxtomin, indicating that they were nonpathogenic. These isolates were recovered from healthy plants over a range of geographically and temporally isolated sampling events and constitute an important plant-microbe interaction.

FIG. 1.

SEM images (×500 magnification) of Streptomyces aerial hyphae emerging from surface-sterilized wheat root fragments after 4 weeks of incubation on an isolation plate. The arrows indicate the Streptomyces mycelium.

FIG. 2.

NJ phylogenetic tree of the full 16S rDNA sequences from selected isolates. The sequence data for several closely related actinobacterial type cultures were recovered from GenBank and included in the tree. The accession numbers for the sequences are as follows: B. subtilis, NC_000964 (region 9809.0.11361); Microbispora amethystogenes, U48988; N. albus, X53211; S. scabies, D63862; S. galilaeus, AB045878; S. argenteolus, AB045872; S. setonii, D63872; S. caviscabies, AF112160; and Streptosporangiacae strain PA147, AF223347. The bootstrap values from 5,000 pseudoreplications are shown at each of the branch points on the tree.

FIG. 3.

nec1 screening in Streptomyces type strains: lane 1, molecular weight marker; lane 2, negative control; lane 3, S. acidiscabies; lane 4, S. caviscabies ATCC 51928; lane 5, S. scabies ATCC 49173; lane 6, S. setonii ATCC 25497.