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© 2013 Plant Management Network.
Accepted for publication 3 January 2013. Published 9 May 2013.


A Quick and Simple Method to Evaluate Anisogramma anomala Ascospore Viability


Stephanie Heckert, Jay W. Pscheidt, and Jeff K. Stone, Department of Botany & Plant Pathology, Oregon State University, Corvallis, OR 97331


Corresponding author: S. Heckert. heckerts@science.oregonstate.edu


Heckert, S., Pscheidt, J. W., and Stone, J. K. 2013. A quick and simple method to evaluate Anisogramma anomala ascospore viability. Online. Plant Health Progress doi:10.1094/PHP-2013-0509-09-RS.


Abstract

Viability of ascospores of Anisogramma anomala, cause of eastern filbert blight on European hazelnut, was assessed using the vital stain trypan blue (working solution of 0.05% in lactoglycerol). Viable ascospores only had faint blue staining around their cell walls while non-viable ascospores absorbed the stain and turned dark blue. The number of viable (non-stained) ascospores as determined by trypan blue was similar to the proportion of ascospores germinating on culture media. Viability of field collected ascospores from rainwater spore traps ranged from 41 to 68%. Disease incidence of hazelnut seedlings was more closely related to differences in ascospore abundance than to differences in ascospore viability.


Introduction

The fungus Anisogramma anomala is the causal agent of eastern filbert blight (EFB) on European hazelnut (3). A perennial branch canker with a sunken appearance is the most common symptom of this disease (3). Fruiting bodies (stromata) of the pathogen develop in the cankers and release ascospores from early winter and throughout the spring during rain events (9). Hazelnut trees, however, are susceptible to infection only in the spring during bud break and early shoot expansion (11); this fact has prompted interest ascertaining the abundance and viability of ascospores released during the spring when hosts are susceptible. Initial signs of the disease (incipient stromata) are generally observed twelve to fourteen months after infection (3).

Rainwater [or gravity-type (4)] spore traps have been used to monitor A. anomala ascospores in epidemiological studies (9). However, the proportion of viable ascospores found in these traps has never been evaluated due to lack of a technique and/or expense of a culture-based viability assay (12). Trap trees have been used to determine the relationship between host phenology and susceptibility and ultimate viability and infectivity of ascospores during rain events. But due to the latent period, disease evaluation occurred after months of incubation (11). Although ascospore inoculum is easily produced from symptomatic tissue, the proportion of viable ascospores released from stromata when hosts are susceptible has not been investigated.

A simple, quickly applied technique such as vital staining would be helpful to assess ascospore viability to aid in estimating inoculum concentration in controlled inoculation experiments or field studies. This study was conducted to evaluate a vital staining method for determining viability of ascospores of A. anomala.


Vital Stains

Assessing the viability of cells by using differential stains has been practiced since the early 1900s (2). A vital stain is able to distinguish viable cells (cells that are capable of performing all cell functions necessary for survival) from non-viable cells (cells that have lost the ability of performing cell functions necessary for survival) either by staining active metabolites in living cells (such as using fluorescein diacetate) or by exclusion of stain by live cells (5). Samples, after proper preparation, are exposed to vital stains and then examined microscopically. Many different stains and techniques have been used including but not limited to fluorescein dicetate, acridine orange, and trypan blue.

The trypan blue dye exclusion method uses membrane integrity to determine a cell’s viability (6). Evaluation only requires a compound microscope and thus several samples can be processed in a short amount of time (13). The stain has been used to assess viability of plant, animal, and fungal cells with a majority of vitality assays being on mammalian cells (6,7,8).


Assessment of Trypan Blue

Ascospores were collected from hazelnut branches bearing mature stromata of A. anomala. Ascospore suspensions of 1 × 106 spores/ml were prepared by extracting perithecia from stromata using a scalpel, hydrating the extracted perithecia in deionized water and crushing it to liberate ascospores that were then pipetted into deionized water (11). This method reliably results in disease when inoculated onto European hazelnut (11). A trypan blue stain working solution of 0.05% in lactoglycerol (1:1:1 of water, lactic acid, and glycerol) was used in all of the following experiments. The ascospores were used in experiments within two hours after extraction from the stromata or stored in a refrigerator after extraction at 6°C until needed.

Viability as determined by trypan blue staining was compared with germination of ascospores on culture media (12). Half of an ascospore suspension was placed in boiling water for five minutes and allowed to cool to room temperature to heat kill ascospores (heated solution). The other half of the ascospore suspension remained unaltered (non-heated solution). Trypan blue stain was added to each ascospore suspension at a 1:10 ratio (30 μl stain solution to 300 μl spore suspension) and allowed to stand for one minute. A drop of each solution was placed on a hemocytometer and examined to determine the proportion of viable spores under a compound microscope with brightfield illumination at 400× magnification (1). Ascospores with and without staining were counted (between 50 to 200 ascospores). Each subsample of the two solutions was prepared and examined microscopically three times. The entire procedure was repeated a total of three times.

Subsamples (30 μl) of non-heated and heated solutions were also plated on a 0.05% activated charcoal (AC) agar media to determine viability of ascospores (12). Anisogramma anomala is an obligate biotroph that does not grow well in standard culture, however, ascospores swell two to three times their initial size prior to germination and infrequently produce a short germ hypha, but typically lyse within a few days on agar media (12). Ascospore swelling was considered evidence of ascospore viability and was assessed after three to five days incubation at 20°C. The surface of agar was scanned under a compound microscope at 100× magnification and total ascospores and percent swollen ascospores determined for both solutions (12). Each subsample of the two solutions was plated out three times and later examined microscopically. The entire procedure was repeated a total of three times and data were analyzed in Excel.

A majority of the ascospores in the non-heated solution excluded the stain and only had a faint blue staining around their cell walls (Fig. 1). These ascospores were considered viable. The ascospores that were in the heated solution absorbed the stain and dyed dark blue, these were considered non-viable (Fig. 1). The overall percentage of non-stained ascospores in the non-heated solution was 74% (ranging from 63 to 100%) and germinated ascospores on AC media was 80% (63 to 100%). The proportion of stained ascospores in the heated solution and the non-germinated ascospores on AC media was 100%.


 

Fig. 1. A photo of a viable ascospores (stained faint blue) and a non-viable ascospores (stained dark blue) of Anisogramma anomala, the causal agent of eastern filbert blight on hazelnuts (400×).

 

In another set of experiments, the non-heated and heated solutions were mixed together in measured ratios to prepare a set of standard dilutions. Two mixtures were prepared, one with 1:1 mixture of the non-heated and heated spore suspensions, and another 1:3 mixture of the non-heated to the heated spore suspension. The two mixtures were homogenized, diluted, and filtered through a gridded, cellulose nitrate filter and then stained with trypan blue and repeated twice for each mixture. A 100% non-heated solution was treated similarly. Aliquots from the three treatments were examined microscopically as described above and the proportion of stained spores was determined for 50 to 100 ascospores from each mixture. Mixtures were also plated out on AC media (50 μl of each mixture) and then incubated for three to five days at 20°C and replicated twice for each mixture. The entire process was repeated three times and data were analyzed with Welch two-sample t-test in R.

The mean percentage of non-stained ascospores in the 100% non-heated solution was 86% (74 to 94%), 45% (39 to 49%) for the 1:1 dilution, 26% (19 to 34%) for the 1:3 dilution. Ascospore germination on AC media for 100% non-heated solution was 84% (82 to 85%), 1:1 mixture was 36% (33 to 42%), and 1:3 mixture was 21% (17 to 25%) (Fig. 2). No significant differences were found between viability determined by trypan blue or by germination on AC media (Fig. 2). The slightly lower germination rates on the AC media may have been due to A. anomala ascospores germination inconsistencies and germination rates were comparable to results found by Stone et al. (12). Based on this evidence it was determined that trypan blue could be used as vital stain for A. anomala ascospores.


 

Fig. 2. Percent of non-stained ascospores of Anisogramma anomala treated with trypan blue and germination of ascospores on AC (activated charcoal) culture media in various ascospore solutions. The percent of non-heated ascospore solution: 100 = all non-heated ascospore suspension; 50 = 1:1 ratio of non-heated ascospore suspension to heated ascospore suspension; 25 = 1:3 ratio non-heated ascospore suspension to heated ascospore suspension. The vertical line on each bar represents the standard error.


Length of time that viable ascospores were able to exclude trypan blue stain was also determined. An ascospore suspension was prepared, diluted, and filtered through a gridded, cellulose nitrate filter. The filter was then placed on a slide and a drop of trypan blue stain (60 μl) was added and allowed to stand for one minute and then one grid of the filter was examined under a compound microscope (400× magnitude). The proportion of stained ascospores was determined and recorded, as well as the time it took to read the first grid. A second grid was then read the same way. The difference in proportion of stained ascospores between pairs of grids was determined. This procedure was repeated five times and data were analyzed with Welch two-sample t-test in R.

The first grid on average was read in four minutes and the second grid was read before nine minutes. The average similarity in percentages of non-stained ascospores in first grid compared to second grid was 87% (73 to 97%). No significant differences were found between the percentage of non-stained ascospores zero to five minutes after application of the vital stain and the percentage of non-stained ascospores at five to ten minutes (Fig. 3). Preliminary observation into the length that non-stained ascospores excluded trypan blue showed that ascospores initially non-stained after preparation were completely stained after 20 minutes. Due to the high recovery rate at nine minutes an arbitrary ten minute cut off point was set to ensure consistent results.


 

Fig. 3. Duration of viability of Anisogramma anomala ascospores in the vital stain trypan blue. Above shows comparisons between ascospore viability (%) 0-5 minutes (dark gray bar) after being stained with trypan blue to ascospore viability (%) 5-10 minutes (light gray bar) after being stained with trypan blue over 5 trials.

 

Field Study

In the spring of 2010 and 2011, infected hazelnut branches bearing mature stromata of A. anomala were placed in six piles at the Botany and Plant Pathology Field Laboratory in Corvallis, OR. Each infected brush pile had a rainwater spore trap placed in the middle of the pile (9). Rainwater spore traps were constructed from a -inch PVC pipe leaving 20.3 cm intact on one end and sawing longitudinally for 218.4 cm making a semi-circle shaped trough where the rainwater collected. The altered PVC pipe was fastened with wire to a board making a completed rainwater spore trap that was mounted on metal posts and were orientated west to east with west end at a height of 81.3 cm above ground and the east end 48.3 cm above ground at an angle of 65°to facilitate the run-off of water into a bucket that collected the rainwater. During four major rainstorms in 2010 and three major rainstorms in 2011, five hazelnut seedlings were placed at each of the spore trap locations (29 May 2011 only four seedlings per spore trap) (Fig. 4). These three to four month old seedlings were grown from nuts collected from the highly susceptible cultivar ‘Ennis’ openly pollinated in the same orchard with the cultivar ‘Butler. Rainwater samples were also collected from rainwater spore traps after each of these major rainstorms. The total amount of rainwater collected was recorded and a subsample (0.5 liter) was brought back to the laboratory for viability staining.


 

Fig. 4. Photo of a pile of pruned hazelnut branches bearing mature stromata of A. anomala, the causal agent of eastern filbert blight and a rainwater spore trap at the Oregon State University Botany and Plant Pathology Field Laboratory near Corvallis, OR. Hazelnut seedlings cv. ‘Ennis’ were placed at each pile during four major rainstorms in 2010 and three major rainstorms in 2011.

 

The method for processing rainwater samples from rainwater spore traps was similar to the procedure used by Pinkerton et al. (9). Samples from spore traps were filtered through a 20-μm sieve, to remove any excess debris (still allowing the ascospores of A. anomala, ~5 × 10 μm, to go through) and then diluted depending on the ascospore counts with deionized water. A 30 to 50 ml volume of this subsample was filtered through a gridded, filter that then was positioned on a glass microscope slide. A drop of the stain (~45 μL) was placed on the filter for one minute and then a cover slip was added on top of the filter and the slide was put under a compound microscope (×100-400). The number of ascospores in the four corners of the gridded filter and the middle grid were counted. Viability was ascertained at the same time by counting the ascospores that were excluding the trypan blue stain as viable, and the ascospores that took up the stain as non-viable and a cut off point for viability counting was set at ten minutes at which time a fresh subsample was filtered through a new filter and the procedure was repeated.

Seedlings were returned to the greenhouse immediately after each rainstorm and transplanted into one-gallon plastic pots with media and fertilizer. Hazelnut seedlings were destructively sampled within three months of exposure. Freehand sections one to two cells thick were cut in the region that was susceptible to infection at the time of exposure (11). These sections were transferred to glass slides, stained with a drop (~60 μl) of 0.05% trypan blue (1:1 water to lactic acid), left overnight for the dye to soak into the plant tissue, and examined microscopically (100-400× magnification) for the presences of A. anomala hyphae (11). Data were analyzed using multiple regression in R.

Viability of ascospores for rainwater spore traps in 2010 ranged from 52% on 20 May to 68% on 21 April (Table 1). Viability of ascospores found in traps during 2011 was slightly lower ranging from 41% on 29 May to 46% on 17 April. Larger differences in ascospore abundance were observed between the various rainstorms ranging from 160 ascospores/m²/h on 12 May 2011 to 4270 ascospores/m²/h on 28 April 2010.


Table 1. Incidence of eastern filbert blight in European hazelnut seedlings placed adjacent to rainwater spore traps near infected brush piles on seven major rain events during the spring of 2010 and 2011.

Ascospore Viability and Disease Incidence in Seedlings
2010 Season
Datesu Ascospore
counts

(spores
/m/h)v
Ascospore
viability

(%)w
Disease
incidence
of seedlings

(%)x
Dur
(hr)y
Rainfall
(cm)z
30 Mar 2760 57 93 57 6.1
21 Apr 363 68 7 18 0.8
28 Apr 4270 66 80 40 2.8
20 May 849 52 3 25 0.8
2011 Season
17 Apr 3654 46 70 91 2.8
12 May 160 41 0 9 0.3
29 May 560 41 0 46 2.1

 u Dates = major rain event in which seedlings were placed adjacent to spore traps.

 v Ascospore Counts (spores/m²/hr) = average ascospore count for major rain event.

 w Ascospore Viability (%) = average ascospore viability for major rain event.

 x Disease Incidence of Seedlings = average disease incidence of hazelnut seedlings in major rain event.

 y Dur (hr) = time in hours from beginning of rainfall event until bark wetness was < 4 U (U - measurement of electrical resistance from the weather station’s readout) or collection of samples for major rain event.

 z Rainfall (cm) = amount of rainfall for major rain event.


There was evidence that disease incidence in hazelnut seedlings was associated with differences in ascospore abundance (P ≤ 0.01, multiple regression) rather than differences in ascospore viability for both years. Disease incidence was high (70-90%) when ascospore counts were over 1000 ascospores/m²/h and low (0-7 %) when ascospore counts were less than 1000 ascospores/m²/h (Table 1). Other weather variables are also likely involved with seedling infection such as wetness duration and temperature.


Conclusion

Trypan blue stain can be used as a vital stain for A. anomala ascospores as a working solution of 0.05% in lactoglycerol. A staining time of one minute and evaluation within ten minutes of staining are recommended. Hazelnut seedlings became infected with observed ascospore viability between 41 to 68%. High ascospore counts, favorable environmental conditions, and observed ascospore viability around 50% can lead to infection on susceptible hazelnut tissue. Trypan blue as a vital stain for A. anomala ascospores can be useful in future studies to monitor ascospore viability in field trials and also to help standardize inoculum concentrations in future inoculation trials.


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