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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.


Correlation plot of two independent assays of cumulative growth with glucose as the only carbon source (time point 9 days after inoculation (dai), λ = 405 nm) with 15 fungal strains, of which 13 were isolated as endophytes (r2 = 0.485; P = 0.004). The dotted line represents the bisector of slope b = 1. Dots close to this line produced even results in both assays (filled dots). Empty dots indicate deviating strains.
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Figure 2: Correlation plot of two independent assays of cumulative growth with glucose as the only carbon source (time point 9 days after inoculation (dai), λ = 405 nm) with 15 fungal strains, of which 13 were isolated as endophytes (r2 = 0.485; P = 0.004). The dotted line represents the bisector of slope b = 1. Dots close to this line produced even results in both assays (filled dots). Empty dots indicate deviating strains.

Mentions: The repeated assays showed that the precision of the in-house configurated PMs was moderate. In the experiment which tested 15 strains in glucose (Figure 2), the correlation was moderate (r2 = 0.485) but significant (P = 0.004), and the slope of the regression line was close to the unity (b = 1.17). Nevertheless, ignoring the results of three strongly deviating strains (marked with blank dots in Figure 2), the correlation grew to r2 = 0.889 and the regression slope shifted slightly toward one (b = 1.10).


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)

Correlation plot of two independent assays of cumulative growth with glucose as the only carbon source (time point 9 days after inoculation (dai), λ = 405 nm) with 15 fungal strains, of which 13 were isolated as endophytes (r2 = 0.485; P = 0.004). The dotted line represents the bisector of slope b = 1. Dots close to this line produced even results in both assays (filled dots). Empty dots indicate deviating strains.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Correlation plot of two independent assays of cumulative growth with glucose as the only carbon source (time point 9 days after inoculation (dai), λ = 405 nm) with 15 fungal strains, of which 13 were isolated as endophytes (r2 = 0.485; P = 0.004). The dotted line represents the bisector of slope b = 1. Dots close to this line produced even results in both assays (filled dots). Empty dots indicate deviating strains.
Mentions: The repeated assays showed that the precision of the in-house configurated PMs was moderate. In the experiment which tested 15 strains in glucose (Figure 2), the correlation was moderate (r2 = 0.485) but significant (P = 0.004), and the slope of the regression line was close to the unity (b = 1.17). Nevertheless, ignoring the results of three strongly deviating strains (marked with blank dots in Figure 2), the correlation grew to r2 = 0.889 and the regression slope shifted slightly toward one (b = 1.10).

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.