Conservation and diversity of seed associated endophytes in Zea across boundaries of evolution, ethnography and ecology

Abstract

Endophytes are non-pathogenic microbes living inside plants. We asked whether endophytic species were conserved in the agriculturally important plant genus Zea as it became domesticated from its wild ancestors (teosinte) to modern maize (corn) and moved from Mexico to Canada. Kernels from populations of four different teosintes and 10 different maize varieties were screened for endophytic bacteria by culturing, cloning and DNA fingerprinting using terminal restriction fragment length polymorphism (TRFLP) of 16S rDNA. Principle component analysis of TRFLP data showed that seed endophyte community composition varied in relation to plant host phylogeny. However, there was a core microbiota of endophytes that was conserved in Zea seeds across boundaries of evolution, ethnography and ecology. The majority of seed endophytes in the wild ancestor persist today in domesticated maize, though ancient selection against the hard fruitcase surrounding seeds may have altered the abundance of endophytes. Four TRFLP signals including two predicted to represent Clostridium and Paenibacillus species were conserved across all Zea genotypes, while culturing showed that Enterobacter, Methylobacteria, Pantoea and Pseudomonas species were widespread, with γ-proteobacteria being the prevalent class. Twenty-six different genera were cultured, and these were evaluated for their ability to stimulate plant growth, grow on nitrogen-free media, solubilize phosphate, sequester iron, secrete RNAse, antagonize pathogens, catabolize the precursor of ethylene, produce auxin and acetoin/butanediol. Of these traits, phosphate solubilization and production of acetoin/butanediol were the most commonly observed. An isolate from the giant Mexican landrace Mixteco, with 100% identity to Burkholderia phytofirmans, significantly promoted shoot potato biomass. GFP tagging and maize stem injection confirmed that several seed endophytes could spread systemically through the plant. One seed isolate, Enterobacter asburiae, was able to exit the root and colonize the rhizosphere. Conservation and diversity in Zea-microbe relationships are discussed in the context of ecology, crop domestication, selection and migration.

Figure 1. Genetic and geographic relationships of Zea genotypes used in this study.

A microsatellite based dendogram shows the known genetic relationship between genotypes, (adapted from [13]), while dotted lines show where seed originate. Group 1 maize landraces grow in semi-hot temperatures of 14–21°C under either semi-dry conditions (540 to 640 mm)(1A) or semi-wet conditions (over 650 mm)(1B). Group 2 landraces grow in hot temperatures (20 to 27°C) and semi-wet growing seasons (500 to 870 mm of precipitation). Group 3 landraces grow in very hot (24.5–27.5°C) and wet (990–1360 mm) growing seasons. Group 4 plants are found mostly at mid elevations in Western Mexico (1200–1800 m) and form very large and numerous kernels. Group 5 plants are temperate landraces. The asterisk indicates seed used were not actually grown at this location; see Table 1 for details.

Figure 2. Biplot diagrams to show the relatedness of endophytic microbial communities between Zea genotypes.

The analysis is based on principle component analysis (PCA) of bacterial 16S rDNA terminal fragment length polymorphism (TRFLP) fingerprints. Shown are PCA analysis for (A) first generation seeds, (B) second generation seeds, and (C) stems. Vectors are drawn in red and represent the different Zea genotype samples. Diagrams are biplots of the first and second principle components, and are based on covariance between samples for differently sized forward and reverse terminal fragments (not shown). Angles between vectors represent the degree of covariance between samples, and are summarized on the vertical bars next to each biplot. The genotypes Nal-Tel, Tuxpeno, Jala, Pioneer 3751, and B73, lack phylogenetic support in this study, so they are omitted from vector angle bars on the right.

Figure 3. Profiles of endophytic microbial communities present in seeds of diverse Zea genotypes.

Shown are profiles of (A) first generation seeds and (B) second generation seeds using bacterial DNA fingerprinting (16S rDNA TRFLP). Each peak is the fluorescence intensity average of 3 TRFLP amplifications from 15 pooled seed, a semi-quantitative indicator of microbial titre. The immediate parents of Generation 1 seeds were grown in diverse geographic locations as indicated in Figure 1 and Table 1, while all Generation 2 seeds came from Generation 1 seeds planted in a common field in Guelph, Canada. 16S rDNA amplicons were first generated using forward primers 799f/1492rh and then were restricted using DdeI. Small fragments and those corresponding to 16S chloroplast rDNA or 18S rDNA were removed. In Generation 2, a few genotypes did not produce mature seed and were not included.

Figure 4. Profiles of endophytic microbial communities present in stems of diverse Zea genotypes using bacterial DNA fingerprinting (16S rDNA TRFLP).

Each peak is the fluorescence intensity average of 3 TRFLP amplifications from 10 pooled samples, a semi-quantitative indicator of microbial titre. All samples were taken from basal stem region of plants of Generation 1 seeds grown in a common field in Guelph, Canada. 16S rDNA amplicons were generated using forward primers 799f/1492rh followed by restriction using DdeI. Small fragments and those corresponding to 16S chloroplast rDNA or 18S rDNA were removed. Landrace Gaspe Flint stems died before harvest and were not included.

Figure 5. Select Zea seed endophyte TRFLP profiles demonstrating the range of inheritance patterns observed across samples.

Each panel shows the TRFLP fluorescence intensities for a given bacterial 16S rDNA forward size fragment in pooled Generation 1 seeds (black), their pooled stems (green) and subsequent pooled Generation 2 self/sib pollinated seeds (red). Corresponding 16S rDNA clones were sequenced and the predicted taxonomic identifications are indicated. Shown are transmission patterns for the following TRFLP forward size fragments: (A) 86 bp, (B) 92 bp, (C) 187 bp, (D) 239 bp, (E) 255 bp, (F) 258 bp, (G) 338 bp, (H) 512 bp, (I) 521 bp, (J) 726 bp.

Figure 6. Summary of forward labelled 16S rDNA TRFLP fragments in seeds and stems displayed as presence or absence data.

Fragment sizes are listed on the left side in base pairs, and fragments are noted as being present if amplified in at least 1 of the 3 PCR trials but not the water control. Potential fragment identities were determined by sequencing of isolates or clones, or by submitting raw TRFLP data to APLAUS+. Microbial presence is indicated by coloured shading depending on which plant samples it was observed in, with grey being Generation 1 seed, horizontal black bars being Generation 2 seed, vertical green bars being stem tissue, black being Generation 1 and 2 seed, green being Generation 1 seed and stems, blue being Generation 2 seed and stems, and red being Generation 1 and 2 seed plus stems. Fragments smaller than 25 bp and those representing mitochondrial 18S (536–538 bp) were excluded from the display.

Figure 7. Examples of microbes cultured on diverse media (LGI, R2A, and PDA) from Zea seed pools followed by genus level taxonomic identification of all unique colonies.

For each genus (row), a yellow box indicates successful culturing of that genus from Generation 1 seed, blue indicates culturing from Generation 2 seed, and green indicates culturing from both generations. Taxonomic identification was based on sequencing of 16S rDNA. Predicted 16S rDNA DdeI forward cleavage product fragment sizes are indicated for each genus from both cultures (black text) and from PCR clone libraries (red text).

Figure 8. Analysis of functional traits of endophytes cultured from Zea seed.

Shown are (A–F) select examples of trait assays and (G) the complete summary grouped by Zea genotype. Shown are assays for (A) antagonism to E. coli; (B) growth in nitrogren free LGI media with only ACC as a nitrogen source; (C) growth promotion of tissue cultured potato one month after inoculation with (from L–R) Enterobacter cloacae, Cellulomonas denverensis, sterile buffer, or Burkholderia phytofirmans; (D) ability to solubilise tricalcium phosphate; (E) acetoin and butanediol production; and (F) extracellular digestion of cellulose. For panel (G), light yellow shading indicates that <25% of isolates from the Zea genotype indicated exhibited the trait, deep yellow indicates 25–50%, orange indicates 50–75%, and red indicates 75–100%.

Figure 9. Summary of functional traits exhibited by cultured seed endophytes grouped by bacterial genus.

Light yellow shading indicates that <25% of isolates from the Zea genotype indicated exhibited the trait, deep yellow indicates 25–50%, orange indicates 50–75%, and red indicates 75–100%. A more detailed listing by isolate is included in Table S4.

Figure 10. Persistence and migration of Zea seed endophytes in stems, roots and the rhizosphere.

The 11 endophytes indicated were successfully tagged with GFP (pDSK-GFPuv, Kan^R) (out of 124 isolates attempted) and injected into maize stems. The 6 endophytes indicated migrated to roots and persisted for >5 days as shown by fluorescence microscopy and culturing from macerated root tissues onto R2A Kanamycin media. (A) Panteoa agglomerans shown spilling out of a metaxylem vessel. (B) Enterobacter asburiae spilling out of root vascular tissue. (C) Culturing confirmed that E. asburiae was present in the roots of two plants (top two quandrants) as well as in their rhizospheres (bottom two quadrants).

Figure 11. A phylogenetic tree of bacterial 16S rDNA sequences from Zea seed endophyte clones and cultured isolates.

A multisequence alignment of the 16S region bounded by basepairs 867–1458 on an E. coli K12 reference sequence was used to generate a UPGMA tree. Included are sequences from clones (UnculturedbacteriumDJMX) and cultured isolates (Genus<StrainDJM-Plate#>) which are identified in Tables S2 and S3. Bacterial classes are labelled in red letters at major branch points.