Comparison of Spore Lengths in Crimini and White Button Mushrooms

Abstract
The spore lengths of two commercial edible mushroom strains, white button and crimini, were compared. Since these strains are of the same species (Agaricus bisporus) their spore lengths were expected to be similar. However, the crimini spores were longer and more variable in length that those of the white button mushroom. Although commercial Agaricus mushrooms are the same species, different strains have different inherited characteristics. Spore length (as well as other spore parameters such as width, length-to-width ratio and volume) could be used to assist in identifying specific strains of commercial mushrooms.

Introduction
Some people like mushrooms (mycophiles) and some hate them (mycophobes). Plenty of people like them though and mushroom growing is big business. They are generally pretty good for you too.

The most popular mushroom is still the standard white button mushroom (Agaricus bisporus) although others such as shiitake, oyster, and a few others, have been gaining on them in recent years. What about portabello and crimini? Crimini is a just a fancy name given to browner varieties of the white button mushroom and portabellos are simply Agaricus mushrooms allowed to grow much larger instead of harvesting them at the button stage. They are both the same species as the white button mushroom! Like other agricultural products, mushroom varieties (strains) have been developed to make them more interesting and to meet demand for novelty and variety.

This experiment had two goals. One was to see if there was a morphological difference in the spores of locally available crimini and white button mushrooms. In doing so, it also provided an opportunity to test an Exolabs digital camera system to see if it was suitable for analytical microscopy of this sort.

Null hypothesis: There is no difference in the average length of the crimini and white button mushroom spores.

The mushrooms. Specimens of crimini and white button mushrooms were obtained from a local grocery store and stored in a refrigerator for several days before use (Figure 1).

Microscopy. An Exolabs (Exolabs, Seattle, WA) digital iPad camera system and software was used to capture and analyze images of spores. The camera was attached to a Swift M3200 student microscope (Figure 2). An initial attempt at calibration was successful, but resulted in poor measuring resolution. The spores lengths could only be determined to be 7 or 8 micrometers in length and nothing in between (Figure 4). This was improved by intentionally calibrating the software measurement system with a 10x error and then dividing the resulting measurements by 10 to obtain sub-micrometer resolution (Figure 5).

Spore measurements. When studying fungus spores, it is standard practice to record both the lengths and widths of the spores. For this experiment, the lengths alone were deemed good enough for accomplishing the goal of evaluating the camera system and testing the hypothesis. Spores were scraped off the gills of sample mushrooms and stirred into a drop of 0.9% NaCl solution placed on a slide. A cover slip was put on and the spores were imaged at 400x magnification. The spores were brought into focus in low-resolution mode and images for analysis were captured with the system in high-resolution mode. The software measuring tool was used to measure the lengths of spores along their longest axis after zooming the image to maximum size for each individual spore. For the white button mushroom, 35 spores were measured and for the crimini, 30 were measured.

Data analysis. Data analysis was done using Kaleidagraph 4.1 for Macintosh (Synergy Software, Reading, PA). Student's T-test was performed on the data to test for differences in the mean spore lengths of the two mushroom strains.

A typical spore measurement is shown in Figure 6. A typical microscope field showing a number of measured white button mushroom spores is shown in Figure 7. In appearance the spores of both strains were essentially the same in overall shape and color (brown).

Summary statistics for the two data sets (crimini and white button) are shown in Figure 8. The average spore length of the crimini mushroom was 7.9 micrometers. The average spore length of the white button mushroom was 6.8 micrometers. Figures 9 and 10 show histograms of the measurement distributions for each sample and Figure 11 presents box plots illustrating the clear difference in the two spore sample populations.

Student's t-test results (data not shown) suggests that the null hypothesis that the mean spore lengths were equal should be rejected (p < 0.0001) and the F-test indicated that the variances were also not equal (p < 0.0001).

The experiment has shown that the crimini strain and the white button strain have significantly different spore lengths as shown by Student's t-test. The variability of the spore lengths was also significantly different based on the F-test. The spore lengths are within range of the lengths found for a number of other spores of Agaricus bisporus species [1]. Clearly, these two mushrooms are different strains and the crimini is not the same variety as the white button, even though they are quite similar in just about every respect except cap color. From these results, it seems feasible that spore length, or other spore dimensional measurements, could be used to differentiate between highly similar mushroom strains.

The Exolabs camera and software system seems quite capable of this kind of work, although it took some thought and additional work to figure out a calibration procedure that would provide the desired measurement resolution in the micron range. The microscope used was not research grade and it would be interesting to see what kind of results could be obtained with the Exolabs system when coupled with an instrument having better optics.

[1] Callac, P., de Haut, I. J., Imbernon, M. and Guinberteau, J. A novel homothallic variety of Agaricus bisporus comprises rare tetrasporic isolates from Europe. Mycologia, March/April 2003 vol. 95 no. 2, 222-231