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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

Effect of volatile inhibitory substances on adjacent cells. All the wells of the plate were supplemented with the standard basal medium. In each row, cell suspension of one endophyte strain was added, excluding the last row one where water was added. In the first three columns, the phenolic compound o-cresol was incorporated to a final concentration of 1 g/L. Note that the fungi in the fourth and fifth columns were partially inhibited in their growth. E = endophyte; W = water; S = standard basal medium; oC = standard basal medium supplemented with o-cresol.
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Figure 3: Effect of volatile inhibitory substances on adjacent cells. All the wells of the plate were supplemented with the standard basal medium. In each row, cell suspension of one endophyte strain was added, excluding the last row one where water was added. In the first three columns, the phenolic compound o-cresol was incorporated to a final concentration of 1 g/L. Note that the fungi in the fourth and fifth columns were partially inhibited in their growth. E = endophyte; W = water; S = standard basal medium; oC = standard basal medium supplemented with o-cresol.

Mentions: Our tests with o-cresol indicate that there is a risk that the volatile compounds cause unintentional effects in the neighboring wells: we found that the fungal growth was severely reduced in the adjacent wells and visibly limited in the next columns (Figure 3).


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)

Effect of volatile inhibitory substances on adjacent cells. All the wells of the plate were supplemented with the standard basal medium. In each row, cell suspension of one endophyte strain was added, excluding the last row one where water was added. In the first three columns, the phenolic compound o-cresol was incorporated to a final concentration of 1 g/L. Note that the fungi in the fourth and fifth columns were partially inhibited in their growth. E = endophyte; W = water; S = standard basal medium; oC = standard basal medium supplemented with o-cresol.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Effect of volatile inhibitory substances on adjacent cells. All the wells of the plate were supplemented with the standard basal medium. In each row, cell suspension of one endophyte strain was added, excluding the last row one where water was added. In the first three columns, the phenolic compound o-cresol was incorporated to a final concentration of 1 g/L. Note that the fungi in the fourth and fifth columns were partially inhibited in their growth. E = endophyte; W = water; S = standard basal medium; oC = standard basal medium supplemented with o-cresol.
Mentions: Our tests with o-cresol indicate that there is a risk that the volatile compounds cause unintentional effects in the neighboring wells: we found that the fungal growth was severely reduced in the adjacent wells and visibly limited in the next columns (Figure 3).

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