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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.


Related in: MedlinePlus

Correlation plot reflecting the effects of different solvents on the cumulative fungal growth (time point 9 dai, λ = 405 nm) of 15 fungal strains. Each dot represents one strain. The dotted line represents the bisector of slope b = 1. Placement of dots under this line indicates inhibition by the tested solvent. (A) Growth in a saline solvent (NaOH+HCl) versus water with sucrose as carbon source (r2 = 0.634); (B) Growth in methanol versus water with sucrose as carbon source (r2 = 0.178); (C) Growth in ethanol or water with glucose as carbon source (r2 = 0.230).
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Figure 5: Correlation plot reflecting the effects of different solvents on the cumulative fungal growth (time point 9 dai, λ = 405 nm) of 15 fungal strains. Each dot represents one strain. The dotted line represents the bisector of slope b = 1. Placement of dots under this line indicates inhibition by the tested solvent. (A) Growth in a saline solvent (NaOH+HCl) versus water with sucrose as carbon source (r2 = 0.634); (B) Growth in methanol versus water with sucrose as carbon source (r2 = 0.178); (C) Growth in ethanol or water with glucose as carbon source (r2 = 0.230).

Mentions: Tests with different solvents showed that some of them have a strong effect on the activity of the strains. Alkaline solution, which was neutralized with an acid, did not affect the activity of the strains (Figure 5A), while 5% methanol in water (v/v) induced a general decrease in the growth of all the endophytic strains (Figure 5B). Ethanol (5% in water, v/v) had an inconsistent effect on strains, decreasing the growth in some of them, but promoting it in others (Figure 5C).


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 reflecting the effects of different solvents on the cumulative fungal growth (time point 9 dai, λ = 405 nm) of 15 fungal strains. Each dot represents one strain. The dotted line represents the bisector of slope b = 1. Placement of dots under this line indicates inhibition by the tested solvent. (A) Growth in a saline solvent (NaOH+HCl) versus water with sucrose as carbon source (r2 = 0.634); (B) Growth in methanol versus water with sucrose as carbon source (r2 = 0.178); (C) Growth in ethanol or water with glucose as carbon source (r2 = 0.230).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Correlation plot reflecting the effects of different solvents on the cumulative fungal growth (time point 9 dai, λ = 405 nm) of 15 fungal strains. Each dot represents one strain. The dotted line represents the bisector of slope b = 1. Placement of dots under this line indicates inhibition by the tested solvent. (A) Growth in a saline solvent (NaOH+HCl) versus water with sucrose as carbon source (r2 = 0.634); (B) Growth in methanol versus water with sucrose as carbon source (r2 = 0.178); (C) Growth in ethanol or water with glucose as carbon source (r2 = 0.230).
Mentions: Tests with different solvents showed that some of them have a strong effect on the activity of the strains. Alkaline solution, which was neutralized with an acid, did not affect the activity of the strains (Figure 5A), while 5% methanol in water (v/v) induced a general decrease in the growth of all the endophytic strains (Figure 5B). Ethanol (5% in water, v/v) had an inconsistent effect on strains, decreasing the growth in some of them, but promoting it in others (Figure 5C).

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.


Related in: MedlinePlus