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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 plots of the cumulative growth (measured at λ = 405 nm vs. λ = 630 nm at time point 9 dai) of 15 fungi in media supplemented with four different phenolic compounds. (A) Chlorogenic acid (r2 = 0.683; b = 0.430); (B) Gallic acid (r2 = 0.788; b = 0.321); (C) Salicylic acid (r2 = 0.992; b = 0.460); and (D) (+)-catechin (r2 = 0.407; b = 0.240). The dotted line represents the bisector of slope b = 1. Dots close to this line produced even results when measured with both wavelengths (filled dots). Empty dots indicate strains in which an unexpected change of color to yellow–orange was visually evident. Note that it is not expected that points aggregate to the bisector, because the measurements at different wavelengths need not to be alike, regardless of the undesired color change.
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Figure 4: Correlation plots of the cumulative growth (measured at λ = 405 nm vs. λ = 630 nm at time point 9 dai) of 15 fungi in media supplemented with four different phenolic compounds. (A) Chlorogenic acid (r2 = 0.683; b = 0.430); (B) Gallic acid (r2 = 0.788; b = 0.321); (C) Salicylic acid (r2 = 0.992; b = 0.460); and (D) (+)-catechin (r2 = 0.407; b = 0.240). The dotted line represents the bisector of slope b = 1. Dots close to this line produced even results when measured with both wavelengths (filled dots). Empty dots indicate strains in which an unexpected change of color to yellow–orange was visually evident. Note that it is not expected that points aggregate to the bisector, because the measurements at different wavelengths need not to be alike, regardless of the undesired color change.

Mentions: Our tests confirmed that unexpected color change (to orange) occurred only in certain combinations of strain and inhibitory substances. The combination of certain strains with the four tested secondary metabolites (salicylic acid, tannic acid, chlorogenic acid, gallic acid, and catechins) resulted in change of color to yellow–orange in last three of them. Occasional change of color was also found in tannic acid assays. This change of color was measurable as a shift in the ratio between absorbance at wavelength λ = 405 nm and at λ = 630 nm. In the cases were a change of color occurred, the absorbance at λ = 405 nm increased abnormally, and the ratio λ = 405 to λ = 630 was not conserved (Figure 4). Such color change did not occur when other strains were combined with these metabolites or when the strains were growing without these substrates.


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 plots of the cumulative growth (measured at λ = 405 nm vs. λ = 630 nm at time point 9 dai) of 15 fungi in media supplemented with four different phenolic compounds. (A) Chlorogenic acid (r2 = 0.683; b = 0.430); (B) Gallic acid (r2 = 0.788; b = 0.321); (C) Salicylic acid (r2 = 0.992; b = 0.460); and (D) (+)-catechin (r2 = 0.407; b = 0.240). The dotted line represents the bisector of slope b = 1. Dots close to this line produced even results when measured with both wavelengths (filled dots). Empty dots indicate strains in which an unexpected change of color to yellow–orange was visually evident. Note that it is not expected that points aggregate to the bisector, because the measurements at different wavelengths need not to be alike, regardless of the undesired color change.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Correlation plots of the cumulative growth (measured at λ = 405 nm vs. λ = 630 nm at time point 9 dai) of 15 fungi in media supplemented with four different phenolic compounds. (A) Chlorogenic acid (r2 = 0.683; b = 0.430); (B) Gallic acid (r2 = 0.788; b = 0.321); (C) Salicylic acid (r2 = 0.992; b = 0.460); and (D) (+)-catechin (r2 = 0.407; b = 0.240). The dotted line represents the bisector of slope b = 1. Dots close to this line produced even results when measured with both wavelengths (filled dots). Empty dots indicate strains in which an unexpected change of color to yellow–orange was visually evident. Note that it is not expected that points aggregate to the bisector, because the measurements at different wavelengths need not to be alike, regardless of the undesired color change.
Mentions: Our tests confirmed that unexpected color change (to orange) occurred only in certain combinations of strain and inhibitory substances. The combination of certain strains with the four tested secondary metabolites (salicylic acid, tannic acid, chlorogenic acid, gallic acid, and catechins) resulted in change of color to yellow–orange in last three of them. Occasional change of color was also found in tannic acid assays. This change of color was measurable as a shift in the ratio between absorbance at wavelength λ = 405 nm and at λ = 630 nm. In the cases were a change of color occurred, the absorbance at λ = 405 nm increased abnormally, and the ratio λ = 405 to λ = 630 was not conserved (Figure 4). Such color change did not occur when other strains were combined with these metabolites or when the strains were growing without these substrates.

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.