Limits...
Phenotype MicroArrays as a complementary tool to next generation sequencing for characterization of tree endophytes.

Blumenstein K, Macaya-Sanz D, Martín JA, Albrectsen BR, Witzell J - Front Microbiol (2015)

Bottom Line: Here, we present detailed descriptions of two different PM protocols used in our recent studies on fungal endophytes of forest trees, and highlight the benefits and limitations of this technique.We found that the PM approach enables effective screening of substrate utilization by endophytes.For the best result, we recommend that the growth conditions for the fungi are carefully standardized.

View Article: PubMed Central - PubMed

Affiliation: Southern Swedish Forest Research Centre, Swedish University of Agricultural Sciences, Alnarp Sweden.

ABSTRACT
There is an increasing need to calibrate microbial community profiles obtained through next generation sequencing (NGS) with relevant taxonomic identities of the microbes, and to further associate these identities with phenotypic attributes. Phenotype MicroArray (PM) techniques provide a semi-high throughput assay for characterization and monitoring the microbial cellular phenotypes. Here, we present detailed descriptions of two different PM protocols used in our recent studies on fungal endophytes of forest trees, and highlight the benefits and limitations of this technique. We found that the PM approach enables effective screening of substrate utilization by endophytes. However, the technical limitations are multifaceted and the interpretation of the PM data challenging. For the best result, we recommend that the growth conditions for the fungi are carefully standardized. In addition, rigorous replication and control strategies should be employed whether using pre-configured, commercial microwell-plates or in-house designed PM plates for targeted substrate analyses. With these precautions, the PM technique is a valuable tool to characterize the metabolic capabilities of individual endophyte isolates, or successional endophyte communities identified by NGS, allowing a functional interpretation of the taxonomic data. Thus, PM approaches can provide valuable complementary information for NGS studies of fungal endophytes in forest trees.

No MeSH data available.


Principal Component analysis of the cumulative growth (time point 9 dai, λ = 630 nm) of 15 fungal strains in an array of in-house PM supplemented with 11 inhibitory substances and glucose. Green triangles indicate three strains of the family Dothioraceae. Orange squares represent members of the order Pleosporales (families Pleosporaceae and Phaeosphaeriaceae). Blue diamonds for the basidiomycetes assayed. Crosses represent a variety of ascomycetous endophytic fungi (Davidiellaceae, Lophiostomataceae, Botryosphaeriaceae, two sordariomycetes, and one incertae sedis). Empty circle represents the negative control without inoculum. Principal component 1 explains 42.76% and principal component 2 does 21.50%.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4585013&req=5

Figure 8: Principal Component analysis of the cumulative growth (time point 9 dai, λ = 630 nm) of 15 fungal strains in an array of in-house PM supplemented with 11 inhibitory substances and glucose. Green triangles indicate three strains of the family Dothioraceae. Orange squares represent members of the order Pleosporales (families Pleosporaceae and Phaeosphaeriaceae). Blue diamonds for the basidiomycetes assayed. Crosses represent a variety of ascomycetous endophytic fungi (Davidiellaceae, Lophiostomataceae, Botryosphaeriaceae, two sordariomycetes, and one incertae sedis). Empty circle represents the negative control without inoculum. Principal component 1 explains 42.76% and principal component 2 does 21.50%.

Mentions: The measurements of the inhibitory effects of eleven substances on fifteen fungal strains (13 of them endophytes) were analyzed by means of Principal component analysis. The two main principal components collated the fungi following its phylogenetic relations (Figure 8).


Phenotype MicroArrays as a complementary tool to next generation sequencing for characterization of tree endophytes.

Blumenstein K, Macaya-Sanz D, Martín JA, Albrectsen BR, Witzell J - Front Microbiol (2015)

Principal Component analysis of the cumulative growth (time point 9 dai, λ = 630 nm) of 15 fungal strains in an array of in-house PM supplemented with 11 inhibitory substances and glucose. Green triangles indicate three strains of the family Dothioraceae. Orange squares represent members of the order Pleosporales (families Pleosporaceae and Phaeosphaeriaceae). Blue diamonds for the basidiomycetes assayed. Crosses represent a variety of ascomycetous endophytic fungi (Davidiellaceae, Lophiostomataceae, Botryosphaeriaceae, two sordariomycetes, and one incertae sedis). Empty circle represents the negative control without inoculum. Principal component 1 explains 42.76% and principal component 2 does 21.50%.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4585013&req=5

Figure 8: Principal Component analysis of the cumulative growth (time point 9 dai, λ = 630 nm) of 15 fungal strains in an array of in-house PM supplemented with 11 inhibitory substances and glucose. Green triangles indicate three strains of the family Dothioraceae. Orange squares represent members of the order Pleosporales (families Pleosporaceae and Phaeosphaeriaceae). Blue diamonds for the basidiomycetes assayed. Crosses represent a variety of ascomycetous endophytic fungi (Davidiellaceae, Lophiostomataceae, Botryosphaeriaceae, two sordariomycetes, and one incertae sedis). Empty circle represents the negative control without inoculum. Principal component 1 explains 42.76% and principal component 2 does 21.50%.
Mentions: The measurements of the inhibitory effects of eleven substances on fifteen fungal strains (13 of them endophytes) were analyzed by means of Principal component analysis. The two main principal components collated the fungi following its phylogenetic relations (Figure 8).

Bottom Line: Here, we present detailed descriptions of two different PM protocols used in our recent studies on fungal endophytes of forest trees, and highlight the benefits and limitations of this technique.We found that the PM approach enables effective screening of substrate utilization by endophytes.For the best result, we recommend that the growth conditions for the fungi are carefully standardized.

View Article: PubMed Central - PubMed

Affiliation: Southern Swedish Forest Research Centre, Swedish University of Agricultural Sciences, Alnarp Sweden.

ABSTRACT
There is an increasing need to calibrate microbial community profiles obtained through next generation sequencing (NGS) with relevant taxonomic identities of the microbes, and to further associate these identities with phenotypic attributes. Phenotype MicroArray (PM) techniques provide a semi-high throughput assay for characterization and monitoring the microbial cellular phenotypes. Here, we present detailed descriptions of two different PM protocols used in our recent studies on fungal endophytes of forest trees, and highlight the benefits and limitations of this technique. We found that the PM approach enables effective screening of substrate utilization by endophytes. However, the technical limitations are multifaceted and the interpretation of the PM data challenging. For the best result, we recommend that the growth conditions for the fungi are carefully standardized. In addition, rigorous replication and control strategies should be employed whether using pre-configured, commercial microwell-plates or in-house designed PM plates for targeted substrate analyses. With these precautions, the PM technique is a valuable tool to characterize the metabolic capabilities of individual endophyte isolates, or successional endophyte communities identified by NGS, allowing a functional interpretation of the taxonomic data. Thus, PM approaches can provide valuable complementary information for NGS studies of fungal endophytes in forest trees.

No MeSH data available.