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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.


Venn-diagrams show the utilization patterns of carbon sources (divided in chemical groups) of three fungi as indicated by the color development data on BIOLOG plates Phenotype MicroArray (PM) 1 and 2A. All available substrates divided in chemical groups (number of individual compounds after the semicolon; A). Substrate utilization pattern by the endophyte Monographella nivalis var neglecta – a strong in vitro competitor of the pathogen, occupying the same niches in the tree and utilizing carbon sources more efficient than the pathogen (B), the pathogen causing Dutch elm disease, O. novo-ulmi(C) and Aureobasidium pullulans(D), a ubiquitous fungus with no detected competitive relation to the pathogen in vitro (Data modified from Blumenstein et al., 2015).
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Figure 6: Venn-diagrams show the utilization patterns of carbon sources (divided in chemical groups) of three fungi as indicated by the color development data on BIOLOG plates Phenotype MicroArray (PM) 1 and 2A. All available substrates divided in chemical groups (number of individual compounds after the semicolon; A). Substrate utilization pattern by the endophyte Monographella nivalis var neglecta – a strong in vitro competitor of the pathogen, occupying the same niches in the tree and utilizing carbon sources more efficient than the pathogen (B), the pathogen causing Dutch elm disease, O. novo-ulmi(C) and Aureobasidium pullulans(D), a ubiquitous fungus with no detected competitive relation to the pathogen in vitro (Data modified from Blumenstein et al., 2015).

Mentions: With the goal of studying the potential of endophytes in biocontrol, we used PM data to compare the nutritional preferences of a pathogen and endophytes that co-colonize the same host (Figure 6, data modified from Blumenstein et al., 2015). The comparison showed that all tested fungi were able to use all of the four tri-and tetrasaccharides tested (Figure 6). On the other hand, the endophyte M. nivalis was able to use a broader array of available amino acids (96%) and other acids (69%) as compared to the pathogen (56 and 49%, correspondingly) and A. pullulans had generally low preference for acids, utilizing 15% of the available amino acids and less than 1% of the other acids (Figure 6). Moreover, the endophytes were able to use all available phenolics (5 out of 5) while the pathogen could only use 60% of them (3 out of 5).


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)

Venn-diagrams show the utilization patterns of carbon sources (divided in chemical groups) of three fungi as indicated by the color development data on BIOLOG plates Phenotype MicroArray (PM) 1 and 2A. All available substrates divided in chemical groups (number of individual compounds after the semicolon; A). Substrate utilization pattern by the endophyte Monographella nivalis var neglecta – a strong in vitro competitor of the pathogen, occupying the same niches in the tree and utilizing carbon sources more efficient than the pathogen (B), the pathogen causing Dutch elm disease, O. novo-ulmi(C) and Aureobasidium pullulans(D), a ubiquitous fungus with no detected competitive relation to the pathogen in vitro (Data modified from Blumenstein et al., 2015).
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Related In: Results  -  Collection

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

Figure 6: Venn-diagrams show the utilization patterns of carbon sources (divided in chemical groups) of three fungi as indicated by the color development data on BIOLOG plates Phenotype MicroArray (PM) 1 and 2A. All available substrates divided in chemical groups (number of individual compounds after the semicolon; A). Substrate utilization pattern by the endophyte Monographella nivalis var neglecta – a strong in vitro competitor of the pathogen, occupying the same niches in the tree and utilizing carbon sources more efficient than the pathogen (B), the pathogen causing Dutch elm disease, O. novo-ulmi(C) and Aureobasidium pullulans(D), a ubiquitous fungus with no detected competitive relation to the pathogen in vitro (Data modified from Blumenstein et al., 2015).
Mentions: With the goal of studying the potential of endophytes in biocontrol, we used PM data to compare the nutritional preferences of a pathogen and endophytes that co-colonize the same host (Figure 6, data modified from Blumenstein et al., 2015). The comparison showed that all tested fungi were able to use all of the four tri-and tetrasaccharides tested (Figure 6). On the other hand, the endophyte M. nivalis was able to use a broader array of available amino acids (96%) and other acids (69%) as compared to the pathogen (56 and 49%, correspondingly) and A. pullulans had generally low preference for acids, utilizing 15% of the available amino acids and less than 1% of the other acids (Figure 6). Moreover, the endophytes were able to use all available phenolics (5 out of 5) while the pathogen could only use 60% of them (3 out of 5).

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.