Vol. 129, No. 1 Printed in U.S.A.
JOURNAL OF BACTERIOLOGY, Jan. 1977, p. 525-526 Copyright © 1977 American Society for Microbiology
NOTES Basidiospore Numbers in Agaricus bisporus (Lange) Imbacfi T. J. ELLIOTT
Glasshouse Crops Research Institute, Littlehampton, Sussex BN16 3PU, England
Received for publication 31 August 1976
The number of spores per basidium in the cultivated mushroom Agaricus bisporus can be readily determined using the light microscope.
Saksena and colleagues (6) have recently suggested that studies of spore number per basidium in Agaricus bisporus (Lange) Imbach "based on light microscopy may not be quantitatively accurate in view of the limitations on resolution of structures on gill surfaces at low magnifications." Although the cultivated mushroom is predominantly two-spored, many authors have reported basidia with one, three, four, or even more spores. It is important that spore number per basidium should not have been misinterpreted in the past, since our knowledge of the life-cycle and sexuality of this commercial species is based on studies of cultures derived from four-spored basidia (1, 3, 5). Aberrant multispored basidia are the only ready source of homokaryons in A. bisporus. Mushrooms are normally harvested for 6 weeks and they are produced in four waves or "flushes," which occur at 10-day intervals. The arbitrary classification of Hammond and Nichols (2) is used throughout this communication to describe the stages of sporophore development. Mushroom basidia do not mature synchronously, and mature basidia are found even in immature sporophores. Counts of basidial types therefore relate only to a specific preparation at a specific time and not to overall frequencies. Pelham (4), using light microscopy, made extensive counts of the different basidial types, and the frequencies he found are shown in modified form with those of Saksena et al. (6) (Table 1). Pelham's data show that the frequency of aberrant basidia depends on the maturity ofthe individual sporophore and its time of origin in the cropping period. This finding has since been confirmed by Song et al. (7). Immature mushrooms (stages 2, 3, and 4), produced late in the cropping period, had the highest numbers of three- and four-spored basidia. The differences in frequency between immature (stages 2, 3, and 4) and mature (stages 6 and 7) mushrooms are highly significant (P < 0.001, based on x2
value for 2 x 3 contingency table). Saksena et al. scored 1,503 basidia on gill fragments from sporophores at different stages of development but did not indicate at what time in the cropping cycle they were collected (6). They did not record any four-spored basidia in the two strains they examined, but Pelham's data suggest that such a sample size might not include any. All the strains of A. bisporus that we have examined in this laboratory have four-spored basidia, although their overall frequency is generally low and they may be absent from certain areas of the gills. The region near the gill edge in immature mushrooms, however, is particularly productive of aberrant basidia. It is simplicity itself to prepare gill tissue for examination using the light microscope. A strip of agar gel is cut from a petri dish and placed on a glass slide. Pieces of gill are taken from the cut edge of a segment of pileus and laid flat on the surface of the agar. This helps to keep them moist. The preparations are routinely examined by using transmitted light at low magnification (x 225). The frequency of four-spored basidia varies with genotype and environment, and Fig. 1 shows a gill fragment of a single-spore strain, B131, which produces many three- and fourspored basidia. Their relative frequencies are given in Table 1. Experience suggests that it is unlikely that the strains described by Saksena et al., which produce aberrant basidia with one or three spores, should fail to produce fourspored basidia. Saksena et al. also report that in "several instances apparent four-spored basidia were seen at low magnifications (x 1,000) which could be resolved clearly through the scanning electron microscope either by tilting the specimen or increasing the magnification." It is clear from Fig. 1 that the different basidial types can be readily distinguished using the light microscope and that errors of interpretation are improbable. Problem basidia can be 525
526
J. BACTERIOL.
NOTES
TABLE 1. Frequencies (percent) of basidial types in A. bisporus Authority
Pelham (4)
Strain
Commercial white
Stages (Hammond and Nichols [2])
Flush(es) from which sporophores taken
2, 3, and 4 2, 3, and 4 6 and 7
Saksena et al. (6)
D-26 70-2
Elliott
B131
a
? ?
2, 3, and 4
No. of spores/basidium 1
2
3
4
Total Tored scored
1 4 1 to 4 1 to 4
-a -
95.1 82.0 90.3 95.4
4.7 16.8 9.1 4.4
0.2 1.2 0.6 0.2
10,980 6,387 36,610 36,897
? ?
2.0 4.6
93.6 95.0
4.4 0.4
0 0
772 731
1
-
24.4
58.8
16.8
721
-
-_, Not scored.
FIG. 1. High incidence of three- and four-spored basidia in a strain ofA. bisporus. Surface view ofgill by transmitted light.
resolved by judicious focusing. Light microscopy has advantages compared with the scanning electron microscope in the ease with which preparations can be made and in that large areas of the gill surface can be quickly searched for aberrant basidia. Accurate counts can be made from such preparations, and it is not necessary to resort to the scanning electron microscope. LITERATURE CITED 1. Elliott, T. J. 1972. Sex and the single spore. Mushroom Sci. 8:11-18. 2. Hammond, J. B. W., and R. Nichols. 1975. Changes in respiration and soluble carbohydrate during the post-
3. 4.
5. 6.
7.
harvest storage of mushrooms (Agaricus bisporus). J. Sci. Food. Agric. 26:835-842. Miller, R. E. 1971. Evidence of sexuality in the cultivated mushroom, Agaricus bisporus. Mycologia 63:630-634. Pelham, J. 1965. Techniques for mushroom genetics. Mushroom Sci. 6:49-64. Raper, C. A., J. R. Raper, and R. E. Miller. 1972. Genetic analysis of the life cycle of Agaricus bisporus. Mycologia 74:1088-1117. Saksena, K. N., R. Marino, M. N. Haller, and P. A. Lemke. 1976. Study on development of Agaricus bisporus by fluorescent microscopy and scanning electron microscopy. J. Bacteriol. 126:417-428. Song, S. F., K. J. Hu, and Y. L. Hsieh. 1972. Observations on the spored-basidium in the cultivated mushroom (Agaricus bisporus). Mushroom Sci. 8:295-303.
JOURNAL OF BACTERIOLOGY, Jan. 1977, p. 525-526 Copyright © 1977 American Society for Microbiology
NOTES Basidiospore Numbers in Agaricus bisporus (Lange) Imbacfi T. J. ELLIOTT
Glasshouse Crops Research Institute, Littlehampton, Sussex BN16 3PU, England
Received for publication 31 August 1976
The number of spores per basidium in the cultivated mushroom Agaricus bisporus can be readily determined using the light microscope.
Saksena and colleagues (6) have recently suggested that studies of spore number per basidium in Agaricus bisporus (Lange) Imbach "based on light microscopy may not be quantitatively accurate in view of the limitations on resolution of structures on gill surfaces at low magnifications." Although the cultivated mushroom is predominantly two-spored, many authors have reported basidia with one, three, four, or even more spores. It is important that spore number per basidium should not have been misinterpreted in the past, since our knowledge of the life-cycle and sexuality of this commercial species is based on studies of cultures derived from four-spored basidia (1, 3, 5). Aberrant multispored basidia are the only ready source of homokaryons in A. bisporus. Mushrooms are normally harvested for 6 weeks and they are produced in four waves or "flushes," which occur at 10-day intervals. The arbitrary classification of Hammond and Nichols (2) is used throughout this communication to describe the stages of sporophore development. Mushroom basidia do not mature synchronously, and mature basidia are found even in immature sporophores. Counts of basidial types therefore relate only to a specific preparation at a specific time and not to overall frequencies. Pelham (4), using light microscopy, made extensive counts of the different basidial types, and the frequencies he found are shown in modified form with those of Saksena et al. (6) (Table 1). Pelham's data show that the frequency of aberrant basidia depends on the maturity ofthe individual sporophore and its time of origin in the cropping period. This finding has since been confirmed by Song et al. (7). Immature mushrooms (stages 2, 3, and 4), produced late in the cropping period, had the highest numbers of three- and four-spored basidia. The differences in frequency between immature (stages 2, 3, and 4) and mature (stages 6 and 7) mushrooms are highly significant (P < 0.001, based on x2
value for 2 x 3 contingency table). Saksena et al. scored 1,503 basidia on gill fragments from sporophores at different stages of development but did not indicate at what time in the cropping cycle they were collected (6). They did not record any four-spored basidia in the two strains they examined, but Pelham's data suggest that such a sample size might not include any. All the strains of A. bisporus that we have examined in this laboratory have four-spored basidia, although their overall frequency is generally low and they may be absent from certain areas of the gills. The region near the gill edge in immature mushrooms, however, is particularly productive of aberrant basidia. It is simplicity itself to prepare gill tissue for examination using the light microscope. A strip of agar gel is cut from a petri dish and placed on a glass slide. Pieces of gill are taken from the cut edge of a segment of pileus and laid flat on the surface of the agar. This helps to keep them moist. The preparations are routinely examined by using transmitted light at low magnification (x 225). The frequency of four-spored basidia varies with genotype and environment, and Fig. 1 shows a gill fragment of a single-spore strain, B131, which produces many three- and fourspored basidia. Their relative frequencies are given in Table 1. Experience suggests that it is unlikely that the strains described by Saksena et al., which produce aberrant basidia with one or three spores, should fail to produce fourspored basidia. Saksena et al. also report that in "several instances apparent four-spored basidia were seen at low magnifications (x 1,000) which could be resolved clearly through the scanning electron microscope either by tilting the specimen or increasing the magnification." It is clear from Fig. 1 that the different basidial types can be readily distinguished using the light microscope and that errors of interpretation are improbable. Problem basidia can be 525
526
J. BACTERIOL.
NOTES
TABLE 1. Frequencies (percent) of basidial types in A. bisporus Authority
Pelham (4)
Strain
Commercial white
Stages (Hammond and Nichols [2])
Flush(es) from which sporophores taken
2, 3, and 4 2, 3, and 4 6 and 7
Saksena et al. (6)
D-26 70-2
Elliott
B131
a
? ?
2, 3, and 4
No. of spores/basidium 1
2
3
4
Total Tored scored
1 4 1 to 4 1 to 4
-a -
95.1 82.0 90.3 95.4
4.7 16.8 9.1 4.4
0.2 1.2 0.6 0.2
10,980 6,387 36,610 36,897
? ?
2.0 4.6
93.6 95.0
4.4 0.4
0 0
772 731
1
-
24.4
58.8
16.8
721
-
-_, Not scored.
FIG. 1. High incidence of three- and four-spored basidia in a strain ofA. bisporus. Surface view ofgill by transmitted light.
resolved by judicious focusing. Light microscopy has advantages compared with the scanning electron microscope in the ease with which preparations can be made and in that large areas of the gill surface can be quickly searched for aberrant basidia. Accurate counts can be made from such preparations, and it is not necessary to resort to the scanning electron microscope. LITERATURE CITED 1. Elliott, T. J. 1972. Sex and the single spore. Mushroom Sci. 8:11-18. 2. Hammond, J. B. W., and R. Nichols. 1975. Changes in respiration and soluble carbohydrate during the post-
3. 4.
5. 6.
7.
harvest storage of mushrooms (Agaricus bisporus). J. Sci. Food. Agric. 26:835-842. Miller, R. E. 1971. Evidence of sexuality in the cultivated mushroom, Agaricus bisporus. Mycologia 63:630-634. Pelham, J. 1965. Techniques for mushroom genetics. Mushroom Sci. 6:49-64. Raper, C. A., J. R. Raper, and R. E. Miller. 1972. Genetic analysis of the life cycle of Agaricus bisporus. Mycologia 74:1088-1117. Saksena, K. N., R. Marino, M. N. Haller, and P. A. Lemke. 1976. Study on development of Agaricus bisporus by fluorescent microscopy and scanning electron microscopy. J. Bacteriol. 126:417-428. Song, S. F., K. J. Hu, and Y. L. Hsieh. 1972. Observations on the spored-basidium in the cultivated mushroom (Agaricus bisporus). Mushroom Sci. 8:295-303.
