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This article was submitted to Plant Biotic Interactions, a section of the journal Frontiers in Microbiology
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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.
The increasing interest in endophyte communities of plants, including those of forest trees, is fueled by the apparent potential of the endophytes to shape and modulate the stress tolerance in host plants, directly by priming or elevating defence responses in the plant (
The functional traits of organisms are expressed as phenotypic attributes, jointly defined by the genome and the environment (cf.
An emerging method that seems to have a realistic potential to provide at high throughput information about the phenotypes of microbial isolates is the Phenotype MicroArray (PM) technique. This technique relies on microtiter-plate-based substrate utilization assays (
The aim of this method paper is to illustrate how PM methods can be applied in studies on the ecology and utilization potential of forest tree endophytes, and validate the performance and reliability of these methods. In particular, we report experiences from two procedures, one where we used pre-configured, commercially available PM arrays to evaluate nutritional niches of endophytes and pathogens sharing the same host plant (Procedure I; part of the results modified from
We employed PM technique in studies where the aim was to experimentally explore the competitive interactions between pathogens and endophytes that co-exist in time and space in trees. For this purpose, the carbon and nitrogen substrate utilization patterns of two pathogens (causal agents of Dutch elm disease,
For preparation of homogenous inoculum, we developed the following protocol that is carried out under sterile conditions. In order to obtain pure fungal mass and to avoid any contamination of agar in the inoculation fluid (IF), fungi were cultivated on semi-permeable cellophane membrane on MEA. After incubation at 26°C (10–15 days depending on species’ individual growth rate), the fungal mass was lifted from the cellophane membrane with a cotton swab. Material from 2 to 5 agar plates per isolate was found to contain sufficient fungal material for the tests. The material was transferred into 2 mL Eppendorf vials and manually homogenized with a pestle together with 500 μL of Biolog FF-IF. When a thick suspension was obtained, it was poured over cotton wool on a metal sieve placed over a beaker. By adding 1–3 mL FF-IF, the material was flushed through the cotton. The longer fungal hyphae and bigger cell aggregates were collected into the cotton wool, and a dense, homogenous solution containing fungal spores and small aggregates of mycelial cells was collected underneath the sieve. The viability of the cells in the suspension immediately after the collection procedure was tested by spreading 200 μL aliquots of the cell stock suspension on MEA plates. The viability of the cells in the suspension immediately after the cell collection procedure was tested by spreading 200 μL aliquots of the suspension on MEA plates. After 3–4 days incubation, outgrowing mycelium was visually checked for development and purity.
Using a turbidimeter, the optical density of the inoculum was adjusted to 62% by adding small amounts from the cell suspension. Depending on the species, 400 μL-1 mL of cell stock suspension per 17 mL FF-IF was used. Then, following the protocol from the manufacturer, solutions of glucose, sodium sulfate, and potassium phosphate were added. The final inoculum was transferred into a sterile reservoir for multichannel pipettes, and 100 μL of suspension was pipetted into each PM array well. The suspension was added to plates on the same day than it was prepared.
In order to count the colony forming units (CFU) of the inoculum, an aliquot of 100 μL was pipetted into Petri dishes containing MEA, gently tilting the dish with sterile glass beads to evenly distribute the fluid on the agar surface. After 3–4 days incubation, the CFU was determined.
The commercially available, pre-configured Biolog Phenotype MicroArrays (Biolog Inc., Harvard, CA, USA) are composed of microtiter plates with one negative control well and 95 wells pre-filled with a nutrient source (e.g., C,N,P,S, amino acids) or substrates leading to inhibitory conditions (pH, NaCl, antibiotics) in a dried state. The substrate rehydrates after the target cell suspension, mixed with an IF at a standardized cell density, is inserted in each well. The IF provided by Biolog contains nutrients or chemicals (e.g., C, N, P, S, K, Na, Mg, Ca, Fe, amino acids, purines, pyrimidines, and vitamins) at sufficient levels to maintain cell viability. Through this combination (a nutrient source or an inhibitory compound and IF), unique culture conditions are created for the inoculated cells (
The phenotypic response, i.e., how the cells respond to the conditions, is monitored by the change of color or turbidity in each well. The IF contains a tetrazolium salt, which is reduced by the action of dehydrogenases and reductases of the prokaryote and yeast cells, yielding a purple formazan dye. This color reaction is irreversible, and thus the more intensive the stronger the organism is able to catabolize the provided substrate in the well. In other words, a color reaction indicates that the inoculated cells are actively metabolizing a substrate in the well, while the lack of color change implies that the cells are not able to utilize the substrate. The rate and extent of color formation in each well can be monitored at 490 nm and recorded by the OMNILOG instrument (
In our Procedure I, we examined the nutritional niche of endophytes and pathogens with the Biolog plates: PM1 and 2A, that represent 190 carbon sources (95 on each plate) (
Data for fungal activity was obtained through measurements of OD at 590 nm (PM1 and PM 2A,
Differences in OD for specific substrates were tested using standard ANOVA analyses. Global differences in substrate use were compared by implementing multivariate statistics or by calculation of a niche overlap index (NOI) which compares the number of substrates used and the intensity by which they are used between two strains of fungi (
Pre-configured PM plates were used to examine whether the carbon-substrate utilization profiles of elm endophytes differ from those of the Dutch elm disease pathogen (
In order to explore the role of fungal endophytes in the early stages of wood degradation, we employed a combination of NGS and PM approaches. Endophytic fungi were isolated from the wood (including phloem and xylem, but excluding external bark) of
The selected isolates were cultured in Petri dishes on an autoclaved cellophane sheet over MEA medium (darkness, 25°C). After a week, the fungal biomass was harvested by rubbing with a sterilized scalpel and transferring the fungal tissue into a sterile centrifuge tube (15 mL) with a known volume of sterile distilled water. Centrifuge tubes were weighed before and after introduction of the tissue to calculate the weight of the added biomass. Then, the content of the centrifuge tube was homogenized using a sterilized tissue grinder, first with a large clearance pestle and then with a small clearance one (~20 strokes with each). The homogenate was inserted back into the centrifuge tube and stored at 4°C until use. The concentration of fungal tissue in the suspension was calculated and the suspension was diluted to 1 g/L before pipetting into the PM plates.
Optical 96-well, round-bottom, sterile polystyrene plates (Deltalab, Barcelona, Spain) were used in the modified PM tests. Each well was first filled with 60 μL of liquid basal culture media (35 μL for inhibition tests; see below), composed by autoclaved Murashige and Skoog (MS) salts (1x; ref. n. 0926230; MP Biochemicals; Santa Ana, CA, USA), Biolog Redox Dye E (2x; ref. n. 74225; Biolog Inc., Hayward, CA, USA), and filtered 1-metoxy-5-methylphenazine methosulfate (1.5 mg/l; ref n. A3799; Applichem, Darmstadt, Germany). We prepared the plates by pouring into each well 50 μL of MS salt solution (2x; i.e., two times as concentrated as the standard recipe; 25 μL of MS salt solution (4x) and glucose 1 M of C atoms for inhibition tests; see below), 10 μL of dye mix, which contained Biolog Redox Dye E (20x; provided by the manufacturer at 100x; this reagent’s final concentration was 2x) and of 1-metoxy-5-methylphenazine methosulfate (15 mg/L). MS salts are normally used to plant tissue
After adding these components, each well was supplemented with selected substances (see the details below). Combinations of two groups of substances were tested: carbon sources and inhibitors (phenolic compounds). For testing the effect of seven different carbon sources (cellobiose, galactose, glucose, sucrose, xylose, pectin, and starch) on the growth of the fungi, we supplemented the media with 20 μL of carbon source solution, to reach a final concentration 0.25 M of C atoms in each well. For testing the possible inhibitory effect on the fungi by 10 phenolic compounds that have been associated to tree resistance as metabolites or external treatments (
After addition of the test substances, water was added to fill the volume in each cell to 90 μL. Finally, from a suspension of 1 g/L of homogenized fungal biomass (see above), 10 μL was added into each well, making up a final concentration of 0.1 mg of fungal biomass per mL.
The thermotolerant solutions (carbon sources, tannic, salicylic, and gallic acids) were sterilized by autoclaving and the thermolabile or volatile substances and the substances dissolved in alcoholic solution (chlorogenic acid, flavonoids, and simple phenols) were filtered through disposable, sterile cellulose acetate syringe filters of 0.2 μm pore size. Water was always deionized and autoclaved prior to use. All the operations were done under axenic conditions in a laminar flow chamber.
The PM plates (a total of 30) were composed following four general principles. First, each combination of carbon source or secondary metabolite with a fungal strain was replicated in three separate wells. Second, with few justified exceptions (see Unintentional Chemical Interactions), all wells of a single plate had the same concentration of inhibitory substances, ethanol, or methanol, whenever present. Third, all the treatments included one negative control with the relevant conditions, but fungal inoculum substituted by water, and another one containing inoculum, but the carbon source/secondary metabolite substituted by water. The first was used to calculate the net absorbance (see below), while the second was used as a reference to compare between different endophytic strains. Fourth, all plates were cultivated in the dark at 25°C.
The following aspects that have relevance for the applicability of PMs in our studies on tree endophytes were evaluated from the in-house configurated PMs.
To evaluate the stability and repeatability of the designed configuration, we repeated assays with carbon sources (cellobiose, galactose, glucose, sucrose, xylose, pectin, and starch) with a six months interval, using the freshly prepared inocula of five eucalypt endophyte strains and a model fungus (
Our preliminary tests indicated that certain volatile metabolites might affect cells in neighboring wells in a plate where no such substance had been added. This unwanted effect was evaluated in a plate as described above, where the first three columns were supplemented with 1 g/L (final concentration) of the simple phenol
Preliminary tests also indicated that in some inhibitory compound tests the culture media unexpectedly changed color to orange (note that dye should change to violet) when in contact with certain inhibitory substances and certain strains. To explore this phenomenon, we performed a test to infer if this change of color could be because the strains used certain phenolic chemicals as carbon sources. We tested thirteen endophyte strains (selected from the Spanish tree endophyte collection) and the two Polyporales model fungi in media with chlorogenic acid, gallic acid, and tannic acid (1 g/L final concentration, solved in water), salicylic acid (0.02 g/L, in water) and catechins (1 g/L final concentration, solved in ethanol) with glucose. Absorbance was later measured at λ = 405 nm and λ = 630 nm.
Because some of the phenolic compounds had to be dissolved in solvents (see above), we wanted to test if these affected fungal activity. Thus, we tested the same set of strains as in the preceding assay, in four solvents (ethanol, methanol, NaOH+HCl as described above, and water) with added sugar in the form of either glucose or sucrose (0.25 M of carbon atoms). To the basal culture media (MS salts+dye mix) we supplemented with glucose and 5% ethanol or water or with sucrose (0.25 M of carbon atoms) and methanol 5%, NaOH+HCl 0.002 M (i.e., saline solution) or water. Growth in the media with ethanol, methanol, and saline solution were compared to the growth in water.
We defined and calculated the following parameters from the absorbance reads: (i) gross absorbance (
Phenolic compounds have been identified as potential plant internal defenses and as external inducers of plant defenses (
Optical densities were measured in a microplate absorbance reader ELx808 (BIOTEK, Winooski, VT, USA). We measured at λ = 405, 490, and 630 nm, and at 0, 1, 2, 3, 4, 5, 7, 9, and 11 days after inoculation (dai). Wavelengths were selected in order to detect if the absorbance shifts were due to an increase in the turbidity, a change of color due to Biolog Redox Dye, or a change of color by other causes. Single measurement was considered sufficient, because variation between repeated, consecutive measurements were found to be negligible in preliminary tests (Macaya-Sanz, personal observation) Absorbance measurements were stored using the software KCjunior provided by the plate reader’s manufacturer. The plates were also photodocumented at 0, 5, and 11 dai.
The absorbance values at all the wavelengths, but especially at shorter ones, were due to increase of turbidity of the medium and the cumulative quantity of redox reactions (reflected in the change of color of the Redox Dye), i.e., two interrelated processes, and were thus considered a proxy of the catabolic activity and the vegetative growth of the fungi.
Adequate quality inoculum for PM tests was achieved from the studied endophytes through both procedures. In Procedure I, the inoculum concentration was determined by transmittance, whereas in Procedure II, the inoculum was standardized by biomass. Standard culture conditions were used for studied fungi in both Procedures (I and II), resulting in adequate amount of viable fungal biomass.
In Procedure II studies, we found that fungal inocula lost vitality after a month storage at 4°C, showing clearly reduced growth rate (Macaya-Sanz, personal observation). All the inocula were, however, alive after the storage period.
In Procedure I, the test for the CFU in the inoculum gave varying results for the different species. For instance, CFU for
The repeated assays showed that the precision of the in-house configurated PMs was moderate. In the experiment which tested 15 strains in glucose (
The repeated assays where six strains were tested on seven carbohydrates, the precision (measured as correlation of cumulated growth) was extremely high (
Correlations between the growth of six fungal strains on seven different carbon sources at 9 days after inoculation (dai; λ = 405 nm) in two independent assays with identical conditions.
Carbon source | σ | |||||
---|---|---|---|---|---|---|
Cellobiose | 1,258 | 0,830 | 0.433 | 0.179 | 0.403 | 0.5555 |
Galactose | 0,747 | 0,849 | 0.165 | 0.002 | 0.940 | –0.0361 |
Glucose | 0,916 | 0,859 | 0.275 | 0.039 | 0.708 | 0.1372 |
Sucrose | 0,646 | 0,658 | 0.381 | 0.986 | 0.0001 | 1.1845 |
Xylose | 1,073 | 0,723 | 0.385 | 0.009 | 0.857 | 0.0681 |
Pectin | 0,219 | 0,474 | 0.359 | 0.910 | 0.012 | 1.0413 |
Starch | 0,860 | 0,341 | 0.441 | 0.977 | 0.001 | 0.6013 |
Our tests with
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 (
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 (
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 (
Phenotype MicroArray technique also allowed us to observe the effect of substrates on the morphology of the tested fungi. In particular, nitrogen sources seemed to induce varying morphological responses. For instance, only little fungal mass was produced when
The measurements of the inhibitory effects of eleven substances on fifteen fungal strains (13 of them endophytes) were analyzed by means of Principal component analysis. The two main principal components collated the fungi following its phylogenetic relations (
Our studies demonstrate that the PM approach is a useful tool to investigate the cellular phenotypes of forest tree endophytes at semi-high throughput rate and in a standardized manner, and to functionally interpret the taxonomic data generated by NGS. For instance, in a recent study exploring the role of endophytes in the Dutch elm disease complex, we were able to identify nutritional niche overlap as a potential mechanism of interaction between the pathogen and potential antagonist endophytes (
Based on our experiences, we conclude that the general benefits of PM approach include its great versatility that allows various research questions to be addressed in a same experiment (e.g., testing of competitive relationships between fungal strains along with gaining information about their sensitivity to individual chemical compounds), testing of a broad array of different compounds and concentrations, and a higher throughput of samples, as compared with earlier methods that have been used in studies of fungal phenotypes (
One of the fundamental challenges when working with fungi in the PM procedure is to prepare a representative, homogenous and viable inoculum. Part of this challenge is because the external growth conditions can strongly modulate the quality and quantity of inoculum. Fungi are known to show great phenotypic plasticity in their responses to their immediate growth environment (see, e.g.,
Standardization of growth conditions may, however, also add bias to the analysis, given the strain-specific preferences for optimal growth. Indeed, in both procedures (I and II), we found evidence for strain-specificity in fungal responses. For instance, the above mentioned conditioning effects of pre-inoculum preparation growth environment could be highly genotype-specific because the nutritional niches of the strains differ (
Intriguingly, we also observed (Procedure II) how complex strain-specific interactions with the chemical environment were expressed as unexpected color changes in the wells (
The second essential step in use of PM approach is to establish an inoculum preparation routine that ensures good viability and accurate and repeatable quantification of inoculum. Our method for preparing a homogenous inoculum (Procedure I) resulted in dense emergence of hyphae on the Petri dishes within 2–4 days, indicating that the process did not negatively affect the viability of the cells. According to the manufacturer’s protocol, the density of the IF should be set to 62% transmittance, conveniently measured from inoculum fluid tubes with the original Biolog turbidimeter.
The third crucial step in PM analyses, in particular when working without the OMNILOG instrumentation and software, is to decide the time points of interest for data collection. There is a temporal dynamic in the substrate use by the cells, which is a fundamental to the evaluation of cell phenotypes. The reaction in the wells is often characterized by a lag-period that can last up to 2–4 days. After that the reaction develops and finally tends to saturate. In the case of the fungi that we have studied the saturation often started after 96 h from the start of the inoculation period. Hierarchical cluster analysis proved to be a useful tool for determination of the appropriate time for downstream analyses of the substrate use by the fungi (
For the in-house configured PMs, we also identify a fourth crucial step: Our Procedure II studies demonstrated that the repeatability and reliability of the in-house arrays can only be ensured through a careful design that acknowledges the specific characters of the studied chemicals. For instance, the volatility of a compound dispensed in a certain well could affect fungal growth in surrounding wells (e.g., observed in adjacent wells to inhibitory, volatile
The PM technique is a powerful tool to estimate the relative speed of substrate use for particular fungi. However, the interpretation of the between-species difference in the speed of substrate use is not straightforward. A change is absorbance values can be interpreted as a proxy of metabolic activity, but it is risky to propose narrower views. The change of color could be produced by a shift in the tetrazolium dye due to respiration, by an increase of turbidity through fungal body proliferation or even by change of the medium color after production of metabolites by the fungi. Such mélange of processes makes it challenging to contrast different organisms in a fine scale. The inconsistency of results in the repeated assays where six strains were tested on seven carbohydrates (
Despite the uncertainty, the proxy of metabolic activity can be valuable additional information, e.g., in studies addressing the potential endophyte-based applications (see Benefits of PM Approach in Endophyte Studies). In our recent study (Protocol I;
The obviously artificial growth environment in PM plate wells, and the distant resemblance of preconfigured plates with the substrate availability under natural conditions, may obviously obscure the interpretations. We only see the response to one isolated substrate in each well on a PM plate. In nature, however, fungal cells inside the plant tissues will exist in a chemical environment that is likely to constantly and gradually change. This dynamicity will be caused both by the metabolism of the plant and by the endophytic inhabitants, and the composition of the substrate, as well as the fungal community, will continuously be altered. Similar successions have been studied for macro-organisms including fungi and insects in decaying leaf litter (
In natural conditions, variations in species-specific infection mode and presence of specific endophytes are also determining the use of substrates by a fungus, which, in turn, will affect the succession of endophytes (
The technical challenges of the PM method are multifaceted and the interpretation of PM data is not straightforward. Thus, ideally, extensive validation through carefully standardized pre-conditions for the fungal growth and careful replication and control well strategies are needed for successful PM analyses, whether the studies use preconfigured or in-house designed PM plates. However, it is evident that the PM technique may significantly help to bridge the genotype–phenotype gaps for the culturable fraction of endophytic fungi. Despite the above-mentioned challenges, PM analyses can provide unique knowledge about functional properties of individual strains and species, and thereby contribute to the knowledge pool that is needed for a more comprehensive understanding of the associations between the NGS-profiles and functional fungal biodiversity.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
This work was supported by the research project CTQ2011-28503-C02-02 (Ministerio de Economía y Competitividad, Spain) to DM-S and JM, Erasmus Mundus Joint Doctoral Programme FONASO fellowship to KB, and VINNMER Marie Curie International Qualification grant (2011-01350) to JW.