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

Johnston-Monje D, Raizada MN - PLoS ONE (2011)

Bottom Line: 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.Conservation and diversity in Zea-microbe relationships are discussed in the context of ecology, crop domestication, selection and migration.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Agriculture, University of Guelph, Guelph, Ontario, Canada.

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.

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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.
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pone-0020396-g004: 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.

Mentions: The Generation 1 seed came from a diversity of Zea species and subspecies from parents grown in different geographic locations across North America and Europe (Table 1). In Generation 2, these seed all came from parents growing in a new field in Canada. In spite of these differences, TRFLP analysis showed that there were four 16S peaks conserved across Zea groups in both Generation 1 and Generation 2 seeds (Figure 3A, 3B). Two of these peaks with 512 and 521 bp sizes were not often observed in stem tissue (Figure 4, 5). Cultured isolates and 16S PCR clones from seed DNA were sequenced and used to predict taxonomic identities which were then matched in silico to TRFLP fragment sizes (Figure 6, 7). Sequences were submitted to Genbank and received accession numbers JF753273–JF753552. When neither clone nor culture 16S information was available, forward and reverse TRFLP fragments (Table S1) were submitted to the APLAUS+ bacterial TRFLP prediction program to predict microbial identity [40]. The peak sizes of the conserved set and their predicted taxonomic identities were: 27 bp (unidentified), 86 bp (unidentified), 511/512 bp (99% to Clostridium beijerinckii) and 521 bp (100% to Paenibacillus sp. IHB B 2257). Their frequencies amongst Zea genotypes were as follows: 27 bp (Seed 1, 11/14 Zea genotypes; Seed 2, 7/9 Zea genotypes), 86 bp (Seed 1, 14/14; Seed 2, 6/9)(Figure 5A), 511/512 bp (Seed 1, 14/14; Seed 2, 5/9)(Figure 5H) and 521 bp (Seed 1, 11/14; Seed 2, 6/9)(Figure 5I). TRFLP peak 726 bp was also conserved across Zea subgroups in Generation 1 seed (8/14) and stems (13/13) but less so in Generation 2 seed (5/13)(Figure 5J). Peak 726 bp is predicted to represent Burkholderia or Herbaspirillum spp. based on APLAUS+. Culturing also showed that Methylobacteria, Pantoea and Pseudomonas were conserved across all Zea groups in Generation 1 seed, while only Enterobacter species were isolated from all groups of Zea seed in Generation 2 (Figure 7); there was also a predicted Methylobacteria/Psuedomonas TRFLP peak (338/339 bp) which was conserved in stems (but not seeds) across Zea genotypes (Figure 4, 5G). We conclude based on TRFLP evidence that there is a heritable seed core microbiota in Zea that is conserved across boundaries of evolution, human selection and ecology.


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

Johnston-Monje D, Raizada MN - PLoS ONE (2011)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3108599&req=5

pone-0020396-g004: 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.
Mentions: The Generation 1 seed came from a diversity of Zea species and subspecies from parents grown in different geographic locations across North America and Europe (Table 1). In Generation 2, these seed all came from parents growing in a new field in Canada. In spite of these differences, TRFLP analysis showed that there were four 16S peaks conserved across Zea groups in both Generation 1 and Generation 2 seeds (Figure 3A, 3B). Two of these peaks with 512 and 521 bp sizes were not often observed in stem tissue (Figure 4, 5). Cultured isolates and 16S PCR clones from seed DNA were sequenced and used to predict taxonomic identities which were then matched in silico to TRFLP fragment sizes (Figure 6, 7). Sequences were submitted to Genbank and received accession numbers JF753273–JF753552. When neither clone nor culture 16S information was available, forward and reverse TRFLP fragments (Table S1) were submitted to the APLAUS+ bacterial TRFLP prediction program to predict microbial identity [40]. The peak sizes of the conserved set and their predicted taxonomic identities were: 27 bp (unidentified), 86 bp (unidentified), 511/512 bp (99% to Clostridium beijerinckii) and 521 bp (100% to Paenibacillus sp. IHB B 2257). Their frequencies amongst Zea genotypes were as follows: 27 bp (Seed 1, 11/14 Zea genotypes; Seed 2, 7/9 Zea genotypes), 86 bp (Seed 1, 14/14; Seed 2, 6/9)(Figure 5A), 511/512 bp (Seed 1, 14/14; Seed 2, 5/9)(Figure 5H) and 521 bp (Seed 1, 11/14; Seed 2, 6/9)(Figure 5I). TRFLP peak 726 bp was also conserved across Zea subgroups in Generation 1 seed (8/14) and stems (13/13) but less so in Generation 2 seed (5/13)(Figure 5J). Peak 726 bp is predicted to represent Burkholderia or Herbaspirillum spp. based on APLAUS+. Culturing also showed that Methylobacteria, Pantoea and Pseudomonas were conserved across all Zea groups in Generation 1 seed, while only Enterobacter species were isolated from all groups of Zea seed in Generation 2 (Figure 7); there was also a predicted Methylobacteria/Psuedomonas TRFLP peak (338/339 bp) which was conserved in stems (but not seeds) across Zea genotypes (Figure 4, 5G). We conclude based on TRFLP evidence that there is a heritable seed core microbiota in Zea that is conserved across boundaries of evolution, human selection and ecology.

Bottom Line: 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.Conservation and diversity in Zea-microbe relationships are discussed in the context of ecology, crop domestication, selection and migration.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Agriculture, University of Guelph, Guelph, Ontario, Canada.

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.

Show MeSH
Related in: MedlinePlus