Assessment of fluorescein-based fluorescent dyes for tracing ...

Mycologia, 105(1), 2013, pp. 221–229. DOI: 10.3852/12-062 # 2013 by The Mycological Society of America, Lawrence, KS 66044-8897

Assessment of fluorescein-based fluorescent dyes for tracing Neotyphodium endophytes in planta Stuart D. Card1 Brian A. Tapper

Neotyphodium (formerly Acremonium) endophytes initially were considered a serious problem in agriculture because some natural endophyte-grass associations produce secondary metabolites, in particular certain alkaloids, which are detrimental to livestock. Particularly notable are the lolitrems produced in endophyte-infected perennial ryegrass (Lolium perenne) that cause the disease ryegrass staggers (Fletcher and Harvey 1981) and the ergot alkaloids in perennial ryegrass and tall fescue (Festuca arundinacea) that lead to circulatory disorders resulting in heat stress and fescue-foot syndromes (Bacon et al. 1977). Elimination of these strains from forage grasses often is not practical because the endophytes also produce bioactive compounds that protect plants against insect damage. However, strains of Neotyphodium endophytes have been identified that are less toxic to livestock while maintaining certain advantageous traits. Selection and transfer of these endophytes, such as strain AR1 in L. perenne (Fletcher 1999) and MaxP/MaxQ (strain AR542) in F. arundinacea (Bouton et al. 2002), into improved grass cultivars can result in nontoxic pastures with effective insect deterrence. Thus the presence of novel Neotyphodium endophytes in pasture grasses now are seen by many as a necessary component of the sward (Hill et al. 2005). Neotyphodium species, the anamorphs of Epichloe¨ species, are strictly seed-transmitted, spending their entire lives in their host plants (Chung and Schardl 1997). Endophytes are the first patented technology to be placed in grass cultivars (Bouton and Hopkins 2003), and this has led to an expansion in the number of products available from the pasture seed industry. As one part of the commercial grass seed product, the viability of the endophyte is distinct from the viability of the seed, which can remain viable long after the endophyte has died (Siegel et al. 1984, Welty et al. 1987). Many methods for detecting fungal endophytes in Pooid grasses have been described. These include direct observational techniques, such as staining of hyphae in plant material with aniline blue followed by bright field microscopy (Card et al. 2011, Latch and Christensen 1982, Latch et al. 1987) or direct isolation of the fungus from surface-disinfected plant material (Bacon and White 1994). On the other hand, indirect detection techniques, such as the tissue print-immunoblot (TPIB) (Gwinn et al. 1991), enzyme-linked immunosorbent assay (ELISA) (Reddick and Collins 1988) or the polymerase chain

Forage Improvement, AgResearch Ltd, Grasslands Research Centre, Private Bag 11008, Palmerston North, New Zealand

Catherine Lloyd-West Bioinformatics and Statistics Group, AgResearch Ltd, Grasslands Research Centre, Private Bag 11008, Palmerston North, New Zealand

Kathryn M. Wright Cell Biology and Imaging Group, The James Hutton Institute, Invergowrie, Dundee, Scotland

Abstract: Fluorescent dyes were assessed for their ability to stain viable hyphae of the fungi Neotyphodium lolii and N. coenophialum, symbiotic endophytes of the Pooideae grasses Lolium perenne and Festuca arundinacea respectively. The fluorescein-based fluorophores; fluorescein diacetate (FDA), 5(6)-carboxyfluorescein diacetate (CFDA), 5-chloromethylfluorescein diacetate (CMFDA) and the chitin-binding stain, Calcofluor white M2R, were assessed for staining of endophyte hyphae in vitro from axenic fungal cultures and in planta, including epidermal leaf sheath peels, nodes, ovaries, embryos and meristems. CMFDA produced the greatest intensity of staining of fungal hyphae and gave excellent contrast in planta compared to the plant cells. Compared to the other dyes, CMFDA was also the least affected by photo bleaching and continued to fluoresce up to 2 h after initial excitation. None of the fluorescent dyes stained fungal hyphae in seed. Key words: Epichloe¨, fluorescence microscopy, vital stain INTRODUCTION Members of the Clavicipitaceae form a range of associations with cool season grasses of the Poaceae. The relationships range from pathogenic, for example, Claviceps purpurea on grasses and cereals, to mutualistic, as with Neotyphodium species, that remain asymptomatic within their grass hosts (Christensen et al. 2001, Selosse and Schardl 2007).

Submitted 26 Feb 2012; accepted for publication 25 May 2012. 1 Corresponding author. E-mail: [email protected]

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reaction (PCR) (Doss and Welty 1995), also have been used. However, all these techniques have specific disadvantages. Staining with aniline blue, especially in seed squashes (SS), can give an estimate of endophyte presence but it is laborious. Furthermore, it does not assess endophyte viability in seed (Siegel et al. 1985), an important limitation because endophytes can die within seed that is still able to germinate. Although the TPIB technique does account for endophyte viability in grass shoots, the whole process from seed sowing to determination of results can take as long as 4– 6 wk; ELISA can give false reactions (Hill et al. 2002, Welty et al. 1986), while PCR can suffer from a lack in specificity if the DNA sequence of the endophytes being tested is not known; PCR also is not able to discriminate between viable and nonviable endophytes in certain material (Dombrowski et al. 2006). Our aim was to develop a direct observational technique using fluorescent microscopy that specifically could detect viable endophytes in infected plant material. Two types of fluorescent probe or fluorochrome exist, auto-fluorescent proteins, such as the commonly used green fluorescent protein (GFP), and fluorescent compounds that are chemically linked (conjugated) to an active substance, such as a protein, antibody or nucleic acid. Many researchers have documented the use of transformed strains of Epichloe¨ spp. endophytes expressing GFP (Christensen et al. 2008, Dombrowski et al. 2011, Mikkelsen et al. 2001), and although in many systems GFP-type insertions have been described as being less harmful to their host microorganisms than the conjugated compounds, they require the genetic transformation of the whole organism. This methodology therefore is considered a useful research tool but is unlikely to be deployed in commercial products due to strict containment issues. Conjugated fluorescent probes have been used extensively to observe fungal propagules (Barak and Chet 1986, Stewart and Deacon 1995, Gold et al. 2001, Hoch et al. 2005, Svircev et al. 2007, Hua et al. 2010, Thirugnanasambandam et al. 2011). However, few compounds have been used in the study of plantmicrobial interactions in planta, probably due to a lack of specificity in staining fungal cells. The objective of this study was to screen a number of fluorescent dyes to identify one that could be used to accurately detect viable endophyte in living plant tissues with commonly available fluorescent filters and imaging software. MATERIALS AND METHODS Calcofluor white M2R (Sigma-Aldrich Co, St Louis, Missouri) was prepared as a 180 mM stock solution in water and

used undiluted. Stock solutions of fluorescein diacetate (FDA) and 5(6)-carboxyfluorescein diacetate (CFDA) (Sigma-Aldrich Co, St Louis, Missouri) were dissolved in acetone at 8 mM and 10 mM respectively. 5-chloromethylfluorescein diacetate (CMFDA) (CellTrackerTM green, Life Technologies Corp, Carlsbad, California) was dissolved in dimethyl sulfoxide (DMSO) to 10 mM. Stock solutions were stored in 2 mL plastic screw-cap tubes (Axygen Inc., Union City, California) at 220 C in the dark until use. On the day of use, working solutions were prepared by diluting stock solutions in water, 1 : 200 for FDA, 1 : 10 for CFDA, 1 : 400 for CMFDA, and adding the Silwet detergent (Vac-In-Stuff L-77, Lehle Seeds, Round Rock, Texas) to aid in the wetting of plant material (according to Thirugnanasambandam et al. 2011). The dyes were tested on fungal hyphae from axenic cultures of N. lolii (commercial strains AR1, AR37) and N. coenophialum (wild-type strain AR582 and commercial strain AR584) symbiotic endophytes from perennial ryegrass and tall fescue. The dyes were assessed further on the same endophytes in planta from epidermal leaf sheath strips (Christensen et al. 2002), transverse sections of nodes of reproductive tillers, cut with a razorblade, ovaries/ovules from dissected flowers, seed squashes (Clark et al. 1983), embryos from dissected seeds and meristems from 6–7 d old seedlings. For embryo extraction, seed was imbibed in water overnight before dissection. With forceps and a scalpel, the glumes were removed and the embryos extracted under a stereomicroscope. Meristems were prepared by germinating seed in Petri plates on moist sterile filter papers (LabServH, East Tamaki, New Zealand) at 20 C for 5–7 d. The lower and upper parts of the seedling were removed and discarded, leaving a central zone consisting of the meristematic tissue covered by the outer leaf sheath. All plant tissues were prepared and soaked in each fluorescent dye for 30 min. Tissue pieces were rinsed in water and mounted on a glass slide. Fluorescence was observed with an Olympus BX50 epi-fluorescence microscope (Olympus New Zealand Pty Ltd) using the U-MWU filter cube containing a 330–385 nm excitation filter and 420 nm barrier filter for the Calcofluor white M2R material and a U-MGFPHQ filter cube containing a 460–480 nm excitation filter and a 485 nm barrier for the fluorescein-stained tissue, together with a mercury source. Images were captured with an Olympus ColorView II digital camera and AnalySIS 3.00 image-analysis software and subsequently transferred to ImageJ (Abramoff et al. 2004), a public imaging program, for further processing. Each of the fluorophores was assessed on a 0–5 scale that was composed of points awarded for intensity, contrast and longevity of the fluorescence under excitation. For intensity, images were converted to grayscale in ImageJ and corrected total cell fluorescence was calculated in Excel (Microsoft, USA) by averaging the intensity from 10 pixels per image (encompassing only the stained hyphae) for each of three replicated preparations. Images that had the highest intensity reading scored a maximum of two points, while a negative intensity reading scored zero points. For contrast, a maximum of one point was awarded for visualization of hyphae compared to the fluorescence emitted by the plant tissue. For longevity, one point was awarded for visualization of hyphae past 1 min and a second point if hyphae could be

CARD ET AL.: FLUOROPHORES FOR TRACING ENDOPHYTES visualized after 2 min excitation. The assessment was repeated once. The fluorophore with the highest number of points at the end of the assessment was ranked the most effective. The hypothesis of interest was that there is a significant difference between at least two techniques. The nonparametric statistical test, Friedman’s rank test, was selected because no assumptions are required about the distribution of the score data (Petrucelli et al. 1999) and was carried out in R statistical software (2011). Means of the scores from the two assessments were used in Friedman’s rank test. The most successful fluorophore, CMFDA, was optimized further by increasing the infiltration of the stain into fungal hyphae in planta by creating a partial vacuum. Plant tissue was prepared as mentioned above and immersed in 40 mL of stain within a 1.5 mL plastic screw-capped vial 1–2 h at room temperature. The vial then was placed in a 100 mL glass chamber and the vial cap loosened. The chamber was sealed and connected to a V-700 diaphragm vacuum pump (Bu¨chi, Switzerland) via a VacMasterTM vacuum control unit (VCU) and a partial vacuum created for 5 min. Three freshly harvested pasture grass seed accessions, of L. perenne and F. arundinacea infected with either N. lolii (AR5 or AR37) or N. coenophialum (AR584) respectively, were chosen for comparison studies. The proportion of endophyte-infected seeds within selected accessions was determined with the fluorescent technique and either the TPIB (Hahn et al. 2003) or SS (Latch and Vaughn 1995) for 100 seed/ seedlings per accession. To test for false positive results produced by the CMFDA stain fluorescing when the endophyte was present but dead, one of the seed accessions was exposed to high humidity (100% RH) and temperature (40 C) 4 wk to kill the fungus (Rolston et al. 1991). The proportion of endophyte-infected seedlings within this accession was re-analyzed with the TPIB and the CMFDA fluorescent stain. The hypothesis of interest was that for each accession there was no difference between the proportion of infected seed detected by the CMFDA technique when compared with the proportion detected by the SS or TPIB method. Fisher’s exact test was used to compare the proportions for the two techniques for each of the accessions because this test is suitable for small samples (Petrucelli et al. 1999). The test was performed with the statistical software package MinitabH 16 (2010).

RESULTS To identify an effective method for staining viable endophyte hyphae in different grass hosts, tissue segments from endophyte infected plant material and from fungal strains growing in axenic culture were subjected to four fluorescent dyes (FIG. 1), and the effectiveness of each dye ranked 0–5 for intensity, contrast of hyphae compared to plant tissue and longevity of the stain under excitation (TABLE I). Because the results from both studies followed a similar trend, only data from the first study are presented.

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There was no evidence of autofluorescence from either fungal species tested with any of the dyes. There was also no difference between the intensity or longevity of the fluorophores between each Neotyphodium species or strain tested. However, none of the fluorescent dyes were fungal specific and all stained the plant material to some degree. Friedman’s rank test showed that CMFDA scored significantly higher than Calcofluor white M2R and FDA but not significantly different from CFDA. CFDA and CMFDA were by far the most successful probes for visualizing viable Neotyphodium hyphae in leaf sheath peels, nodes, ovaries and meristematic zones from seedlings and scored 17 and 22 points respectively (FIG. 1, TABLE I). None of the fluorescent dyes stained hyphae in the seed tissues, either in seed squashes or the embryo. CMFDA providing good contrast against the fluorescing background of plant material and the longest duration or least affected by photo bleaching. Hyphae in meristems continued to fluoresce under the microscope up to 2 h after the initial excitation, for example. In pure axenic cultures and leaf sheath peels of both N. coenophialum and N. lolii, Calcofluor white M2R had intense fluorescence, longevity, and relatively good contrast. However this was not found for hyphae in any of the other plant tissues stained with this compound (FIG. 1, TABLE I). Similarly FDA produced satisfactorily intense fluorescence and high contrast between hyphae and plant tissue in leaf sheath peels and axenic cultures (TABLE I). However, a very high rate of photobleaching occurred with the dye and hyphae of neither fungal strain could be observed after 5–10 s of excitation. The process of staining meristem tissue from plant grass seedlings with CMFDA was optimized by the use of a vacuum infiltration system. This greatly improved the visibility of stained hyphae within epidermal leaf sheath peels (FIG. 2A), excised ovaries, transverse sections of nodes (FIG. 2B) and apical meristems (FIG. 2C), although hyphae still could not be visualized in seed tissue. Fisher’s exact test showed that for the selected seed accessions CMFDA did not significantly differ from the SS or TPIB when assessing the proportion of endophyte-infected seed in three of the four accessions tested (TABLE II). The fluorophore equally stained hyphae of two strains of N. lolii (AR5 and AR37) in two cultivars of L. perenne. However there was a significant difference between the TPIB and the fluorophore when analyzing a strain of N. coenophialum (AR584) in a cultivar of F. arundinacea (TABLE II). This was due to the high infection (100%) of this accession, as assessed by the TPIB, and not due to the grass cultivar, endophyte species or endophyte strain. CMFDA also was tested on an accession of L.

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FIG. 1. Fluorescence microscopy of Neotyphodium hyphae axenic culture (A–D) in leaf sheath peels (E–H), nodes (I–L) and ovules (M–P) stained with Calcofluor white M2R, fluorescein diacetate (FDA), 5(6)-carboxyfluorescein diacetate (CFDA) or 5-chloromethylfluorescein diacetate (CMFDA).

perenne that had been heat treated to remove viable endophyte. No hyphae were observed in any of the 100 seedlings assessed, demonstrating that the fluorophore stains only viable fungal cells. DISCUSSION FDA, CFDA and CMFDA are similar fluorescent probes that produce fluorescein after being activated within a cell by esterase enzymes. These enzymes are found widely in the cells of mammals, plants and microorganisms (Bornscheuer 2002), and their activity is commonly used as an indicator of cell viability (Brul et al. 1997).

CMFDA produced the most intense staining of fungal hyphae in planta, including leaf sheath peels, nodes, ovaries and meristematic zones from seedlings, and gave excellent contrast compared to the plant cells. The fluorescent dye produced satisfactory staining of hyphae from axenic cultures but did not stain hyphae associated with embryos or seed. These plant tissues however did fluoresce with great intensity due to their metabolic activity during germination. All other fluorescein-based fluorophores caused this effect with varying intensity, and we speculate that this masked the fluorescence from the intended target of fungal hyphae. Therefore the limitation of these fluorescent dyes in association with

22

4 4 4 5 0 0 5 2 2 2 2 0 0 2 1 1 1 1 0 0 1

17

1 1 1 2 0 0 2 3 4 4 3 0 0 3 1 1 1 1 0 0 1 1 1 1 1 0 0 1 10

1 2 2 1 0 0 1 4 3 0 3 0 0 0 1 1 0 1 0 0 0 1 1 0 1 0 0 0 8

2 1 0 1 0 0 0 4 4 0 0 0 0 0 2 2 0 0 0 0 0 1 1 0 0 0 0 0 1 1 0 0 0 0 0 Mycelia Leaf sheath Node Ovule Embryo Seed squash Meristem Total points from all tissues

Total L C I

C

L

Total

I

C

L

Total

I

C

L

Total

I

5-chloromethylfluorescein diacetate (CMFDA) 5(6)-carboxyfluorescein diacetate (CFDA) Fluorescein diacetate (FDA) Calcofluor white M2R

TABLE I. Assessment of fluorescent dyes on staining of mycelia in axenic culture and in planta within a range of grass tissues. (0–5 scale where 0 5 no fluorescence). A maximum of two points each was awarded for intensity (I) and longevity (L), while one point was awarded for contrast (C)

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these particular plant tissues was the lack of specificity to fungal cells. With FDA and CFDA, on axenic fungal cultures, within epidermal leaf sheath peels and grass ovaries, we found the emission of the fluorescence also faded quickly, indicating a photo-bleaching effect. However, this was experienced only with CMFDA after 20–30 min excitation, giving a clear advantage over the other fluorescein stains investigated. Calcofluor white originally was designed for the fabric and paper industries as an optical brightener to enhance the appearance of color, causing a perceived whitening effect. In this study, the compound was successful only when staining hyphae in leaf sheath peels, where it produced good contrast between the hyphae and plant cells. However, this compound is not a vital stain and binds to cellulose, chitin and other B-1,4-linked carbohydrates (Fischer et al. 1985) regardless of their metabolic state. Although researchers have documented the successful use of FDA on Neotyphodium mycelia in seed squashes (Dapprich et al. 1994), we were unable to repeat this for any of the strains used in this study. Possible reasons could be that FDA works only for certain fungal genotypes (Hahn et al. 2000), the fluorescence fades too fast to be visualized (Stewart and Deacon 1995), dormancy of the fungus in host tissues or some other unknown parameter. Because the fluorophores require an actively metabolizing cell to carry out esterase-catalyzed hydrolysis, the hyphae within the seed may not be sufficiently metabolically active and the seed may need to imbibe a few days to produce the enzymes required to activate the probe. Indications from our work suggest that the fungus, although viable, may remain metabolically inactive under some circumstances, such as in dormant seed, and may not be detectable by CFMDA staining until active fungal metabolism begins after the seed has broken dormancy and started germination. These results indicate that FDA, CFDA and CFMDA cannot be used as a vital dye for staining endophytes in ryegrass and tall fescue seed. The majority of the published research on observing microorganisms with the aid of fluorophores has concentrated primarily on using pure axenic cultures of fungi/yeasts and bacteria with no interaction from a host, whether mammalian or plant. The current study has shown that, although none of the fluorescent dyes were specific to fungi, one compound in particular, CMFDA, can provide excellent contrast between hyphae and plant cells, as well as indicating that the hyphae are viable. The TPIB technique, which detects mycelial proteins of epichloid fungi, is commonly employed by the New Zealand seed industry to provide an estimation of the proportion of viable endophytes. This procedure is simple and

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FIG. 2. Hyphae of Neotyphodium strains stained with CMFDA after optimization. Hyphae of Neotyphodium coenophialum AR584 within an epidermal leaf sheath peel (A) and a transverse section of a node (B) and hyphae of N. lolii AR5 within an apical meristem of a young seedling (C).

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TABLE II. Grass endophyte associations selected for comparison of endophyte proportions in seed lots as determined by SS or TPIB to the fluorophore CMFDA using Fisher’s exact test

Grass species

Grass cultivar

Endophyte species

Endophyte strain

Technique 1: proportion of seed infected with endophyte (%) as determined by SS or TPIB (95% CI)

AR37

51b(40.8–61.1)

56(45.7–65.9)

0.571

AR37 AR5

0c(0–3.6) 93 (86.1–97.1)

0(0–3.6) 95(88.7–98.4)

1.000 0.767

AR584

100 (96.4–100)

94(87.4–97.8)

0.029

Lolium perenne

Samson

L. perenne a L. perenne Festuca arundinacea

Samson Banquet II

Neotyphodium lolii N. lolii N. lolii

Fortuna

N. coenophialum

c

c

Technique 2: proportion of seed infected with endophyte (%) as determined by CMFDA (95% CI)

P value from Fisher’s exact test

a

Seed was heat treated 4 wk at 40 C, 100% relative humidity. As determined by SS on 100 seed. c As determined by a TPIB on 100 seedlings. b

does not require specialist mycological skills (Gwinn et al. 1991). However, this technique can take from 3 wk in L. perenne to 4 wk in F. arundinacea to complete, because the seeds have to be sown and the resulting tillers from the young plants blotted onto a nitrocellulose membrane, processed and assessed. The time between seed harvest to retail chain and end user is currently very tight because seed has to be cleaned and packaged before the final product is available for sale. The introduction of novel endophytes into commercial seed production adds a layer of complexity and quality assurance (Rolston and Agee 2006). Quality of the seed products must be upheld, which includes high endophyte viability (high proportion of seeds infected with viable endophytes, with an alkaloid profile that is true to type). Although researchers have concluded that the most accurate method for detection of viable Neotyphodium endophytes in seed is TPIB (Olivier et al. 2005), we think that our method with the fluorophore CMFDA could be equally accurate and provide results within 5–7 d of germinating. At this stage we see the technique being used as a powerful research tool rather than a technique to replace the TPIB because a seed technician would not only have to be retrained in microscopy and staining but also the seed testing laboratory would have to invest in a microscope with fluorescent capabilities. ACKNOWLEDGMENTS We thank Mike Christensen and Christine Voisey for helpful comments and Alishea Woodhead and Tamara Bejakovich for valuable technical support. We thank Grasslanz Technology Ltd for financial support.

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