Visualization of an Endophytic Streptomyces Species in Wheat Seed

Abstract

Endophytic filamentous actinobacteria were isolated from surface-sterilized roots of wheat plants. Endophytic colonization of germinating wheat seed was examined using one of these endophytes, Streptomyces sp. strain EN27, tagged with the egfp gene. Endophytic colonization was observed from a very early stage of plant development with colonization of the embryo, endosperm, and emerging radicle.In order to find effective biocontrol agents for fungal plant pathogens of cereal crops, we isolated actinobacterial strains from surface-sterilized roots of healthy wheat plants (3; J. T. Coombs and C. M. M. Franco, submitted for publication). There have been reports of actinobacteria, other than Frankia spp., inhabiting the tissues of healthy plants (5,15,16). However, spatial colonization of the root tissue of cereals by actinobacteria has not been reported, and evidence of their distribution as endophytes is required to prove endorhizosphere competence. A representative endophytic strain, Streptomyces sp. strain EN27, was tagged with green fluorescent protein (GFP) to study early colonization events after it was applied to wheat (Triticum aestivum L.) seeds. Expression of GFP has been used to visualize a number of unicellular bacteria-plant interactions (6,9,10,14,21), but this is the first report on a filamentous actinobacterial endophyte. For actinobacteria, gfp expression has been optimized (4, 20) and coupled to a constitutively expressed promoter, ermEp (17).Streptomyces sp. strain EN27, identified by 16S ribosomal DNA (rDNA) sequence analysis to be closely related to Streptomyces caviscabies, was isolated from surface-sterilized wheat root tissue (3; Coombs and Franco, submitted). Control experiments to validate the sterilization procedure were done by subjecting five individual actinobacterial endophytes and two endophytic pseudomonads, at 10 7 to 10 9 CFU per ml or per g of seed, to the sterilization protocol. They were tested as coatings on wheat seeds and as suspensions. Simple washing steps with sterile water do not easily remove the microbial coatings, but the sterilization protocol was effective in removing all surface-adhering microorganisms.Streptomyces sp. strain EN27 was selected as a model organism with which to investigate the colonization of germinating seeds of the wheat host due to its wide distribution among wheat plants in the field and its ability to promote plant growth and control a number of root-infective phytopathogenic fungi (Coombs and Franco, submitted; J. T. Coombs, P. P. Michelsen, and C. M. M. Franco, submitted for publication). Streptomyces sp. strain EN27 was transformed with egfp by using an 8.0-kb vector, pIJ8641 (20), containing the egfp gene downstream of a strong constitutive ErmE promoter, an apramycinresistant marker (aac(3)IV), an oriT/RK2 region, and a lambda phage chromosomal integration sequence (IntC31). Competent Escherichia coli S17.1 transformed with pIJ8641 DNA was used for intergeneric recombination with Streptomyces sp. strain EN27 by the intergeneric recombinatio...

Endophytic actinomycetes: promising source of novel bioactive compounds

“…7,8,10 Furthermore, in comparison of the colony formation from outer and inner part of tissues using two kinds of plant roots, 10 7 CFU g − 1 (dried sample) from near the exterior and 10 3 -10 5 CFU g À 1 (dried sample) from the inside tissue of both samples were detected. The data supported the results detected by FISH.…”

“…6 Bacteria exist on the surface and inside of plant roots, as demonstrated by fluorescence in situ hybridization, scanning electron microscopy or detection of target genes. [7][8][9][10] Numerous bacteria live in rhizospheres, up to 10 6 cells per mm 3 , including filamentous bacteria with actinomycetes accounting for an estimated 4% of the total. 9 This shows the relationship that exists between actinomycetes and plants.…”

Long-term agrichemical use leads to alterations in bacterial community diversity

“…Indeed the rhizosphere usually selects some specific bacterial populations and therefore decreases the bacterial diversity in comparison to bulk soils (Normander andProsser 2000, Kleineidam et al 2010). This effect observed in this study could be partially explained by possible introduction of endophytic bacteria associated to the barley seeds (Coombs and Franco 2003).…”

Biocontrol Potential of Endophytic Bacteria Isolated from Healthy Rice Plant against Rice Blast Disease (Pyricularia oryzae Cav.)

“…Bacteria are able to invade the plant tissue through the roots opening before started to colonize the root. It is then move upward toward stem, leaf sheath and finally leaf blade [16]. Therefore, it was not surprising to find out that the isolated endophytic bacteria mostly came from root.…”

“…Thus, the validation of the surface sterilization method is very important to confirm the success of the surface sterilization method and that the isolated microbes are truly endophytes. Sodium hypochlorite and ethanol have known as an effective sterilization agent [16,17]. No microbes present on the imprint path of the rice plant tissues which were surface sterilized earlier (Fig 1).…”