Mycosphere Five trillion in a fruiting body of gigantea

De-Wei Li

The Connecticut Agricultural Experiment Station, Valley Laboratory, 153 Cook Hill Road, Windsor, CT 06095, USA

De-Wei Li. 2011 – Five trillion basidiospores in a fruiting body of . Mycosphere 2(4), 457–462.

Basidiospores in a fruiting body (38.5×37×22.5 cm in size) of Calvatia gigantea were quantified to be 5.1 trillion (5.1×1012). The results suggested that Buller’s study on a smaller fruiting body of 40×28×20 cm in size over-estimated the number.

Key words – basidioma – puff ball – Lycoperdaceae – – sterigmal remnant

Article Information Received 8 June 2011 Accepted 14 June 2011 Published online 21 September 2011 *Corresponding author: De-Wei Li – e-mail – [email protected]

Introduction this giant is the question people often Calvatia Fr. is a cosmopolitan of ask. To hypersensitive individuals the spores of medium- to large-sized with >35 this species in the air may become a health species (Coetzee & van Wyk 2007). The genus concern, as Calvatia is one of the most promi- is characterized by fruiting bodies that dehisce nent genera of the responsible by irregular fragmentation of the peridium for the induction of fungal type I allergy without a defined ostiolum (apical pore) (Gube (Simon-Nobbe et al 2008). C. gigantea does 2007). Calvatia gigantea (Batsch) Lloyd, com- not have the bellows mechanism of basidio- monly called giant puffball, is widespread in liberation (Ingold 1965), rather it is by the temperate zone (Kreisel 1994). It develops irregular fragmentation of the peridium, one of the world’s largest basidiomata from a exposing large spore masses to the air. Its single primordium (Clémençon 1990). It is a basidiospores are freely blown away by wind saprobe on pastures, fertile meadows or in open (Ingold 1965) and, subsequently, this woodland (Gube 2007). C. gigantea develops releases a large quantity of basidiospores in to fruiting bodies singly, gregariously, or in fairy the atmosphere. Its basidiospores were rings. The fairy rings were reported to associate frequently detected from air samples in the fall with Agrostis capillaris L., Festuca nigrescens (personal observation). Lam. (Festuca rubra L. subsp. commutata A.H. Reginald Buller was the first person Gaudin), or Lolium perenne L. on greens and to quantify the number of basidiospores fairways in golf courses (Arnold & Brien 1961) produced in a large fruiting body of C. gigan- and also have been observed on pasture land tea. He determined the number to be seven and low maintenance turfgrasses. It is edible trillion by counting the basidiospores from a when its is still white. single sample taken from the internal gleba People are often fascinated by the un- (Buller 1909). In the past 100 years, Buller’s usually large size and edibility of its fruiting study has not been repeated and his estimate bodies. It is a trophy species to hun- remains to be verified due to the rarity of natu- ters. However, the number of basidiospores rally mature and, more importantly, still intact produced and released by the fruiting bodies of fruiting bodies in the field. Usually fully ma-

457 ture fruiting bodies are either damaged by The spore suspension in a flask was vor- animals or have already dehisced by fragmen- texed five times, each lasted 10s, to suspend tation. the spores into alcohol. A mini vortexer (Fisher A large fruiting body of C. gigantea de- Scientific) at a speed setting of 5 was used. veloped on low maintenance turfgrass about 5 One mL of the spore suspension was pipetted m from a mixed forest to the south and 3 m from the flask and was serially diluted 100× in from a newly planted peach orchard to the two test tubes, each containing 9 mL distilled north in a research farm of the Connecticut water. Prior to each pipetting, the spore suspen- Agricultural Experiment Station, Valley Labo- sion in a test tube was vortexed for 10s. ratory in Windsor, Connecticut, USA (Figs 1– The spore suspension in the second test 2). When it was fully mature in the field and tube was pipetted onto an Improved Neubauer still unopened, the basidiospores in the fruiting Hemacytometer (CA Hausser & Son, Philadel- body were quantified with a method to allow phia, USA). Basidiospores in one mm2 area statistical analysis to be performed and to were counted. For each sample, spore counting veryfy Buller’s estimate. was replicated eight times. Formulas for calculating the total basi- Materials and Methods diospores in the fruiting body are listed as: The fruiting body had been observed daily in the field since it was spotted on 12 Spores/g = spore count mm-2 × 104 × 20 × 102 October 2011. The fruiting body was left in the (dilution factor)/sample weight = spore count field untouched to allow it to mature naturally mm-2 × 2 × 107/sample weight. so that the basidiospores produced in it would reach full potential. When the fruiting body Total basidiospores = spore count mm-2 × 2 × was picked on 15 December 2010 it had be- 107/sample weight × 278.6g (gleba weight). come completely air-dried, dark brown in colour, but the peridium was still intact without Data were analyzed using NCSS software any fragmentation or opening. Soil was gently (NCSS, 329 North 1000 East, Kaysville, Utah brushed off from the base of the fruiting body. 84037) for ANOVA and Kukey-Kramer Multi- The fruiting body was measured as 38.5 × 37 × ple Comparison Test. 22.5 cm in size, and 112 cm, 97 cm, and 90.5 cm in perimeter along X, Y, Z axes. The whole Results fruiting body weighed 298.4 g. The peridium Calvatia gigantea produced 5.1×1012 was carefully peeled off from ¼ of the fruiting basidiospores in this fruiting body. Basidio- body and weighed 4.95 g. Thus, the whole spore number estimates from outer, interme- peridium was 19.8 g. The gleba was olive in diate, and center glebal areas were significantly colour and weighed 278.6 g (fruiting body different due to the differences in spore densi- minus peridium = 298.4 g minus 19.8 g). The ties in the different areas of the fruiting body residual basidiospores were gently brushed off (Table 1). Basidiospore densities (spores/g) from the peridium pieces using a painting from outer, intermediate, and center glebal brush in a Hume hood prior to weighing the areas were also significantly different (Table peridium pieces. 2). The basidiospore density in the intermediate The fruiting body was cut in half. Nine area was significantly higher than in the outer samples were taken from the fruiting body, and center areas. There were significant diffe- three from the outer area (0.069, 0.072, 0.074 rences among samples collected from within a g) (0–5 cm from peridium), three from an inter- similar area also. The basidiospore density of mediate area (0.063, 0.1, 0.044 g), and three the gleba in the fruiting body was not evenly from the center (0.039, 0.055, 0.07 g) with a distributed. pair of tweezers. Each sample was soaked in 20 mL 70% isopropyl alcohol in a 150 mL flask. Discussion The flasks were sealed with a piece of 5×5 cm Buller (1909) quantified the number of parafilm. basidiospores produced in a large fruiting body

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1 2

Figs 1–2 – Calvatia gigantea. 1 Immature fruiting body, 2 Mature fruiting body.

Table 1 Kukey-Kramer multiple comparison of basidiospore number estimates of Calvatia gigan- tea based on the samples collected from different glebal areas.

Gleba area Basidiospore numbers ( x ) SD1 Outer 4.97×1012 a* 1.41×1012 Intermediate 6.16×1012 b 1.59×1012 Center 4.16×1012 a 1.82×1012 *Treatments with different letters mean statistically significant difference. 1SD: standard deviation.

Table 2 Kukey-Kramer multiple comparison of Calvatia gigantea spore density in different glebal areas.

Gleba area Basidiospore numbers ( x )basidiospores/g SD1 Outer 1.78×1010 a* 5.1×109 Intermediate 2.21×1010 b 5.7×109 Center 1.49×1010 a 6.6×109 *Treatments with different letters mean statistically significant difference. 1SD: standard deviation. of C. gigantea when he was teaching at the of quantifications of the basidiospores between University of Birmingham, UK. He estimated the two fruiting bodies. The sample in Buller’s that the fruit body of C. gigantea (40 × 28 × 20 study might be on the higher side of analytical cm, weighed 232 g) contained over seven variation or his sample came from a high spore trillion basidiospores by taking a single 0.1 g density area, since basidiospore density in a sample from the internal gleba and suspending gleba is not constant. The variation among the it in 250 mL methylated spirit. Buller’s fruiting nine samples in the present study ranged from body was smaller by 30% in size and lighter by 2.98–7.66×1012 basidiospores. The two fruiting 22% in weight than the specimen studied by bodies may have grown on different substrates, the author. Buller’s estimate of 7×1012 basi- which led to different basidiospore producti- diospores (3×1010/g) is higher than the 5.1× vities and densities in the fruiting bodies. In 1012 basidiospores (1.7×1010/g) calculated from Buller’s study, the peridium was not weighed the present study. It seems that Buller (1909) and excluded from the fruiting weight. When over-estimated the number within the fruiting taking the weight of the peridium into conside- body. Considering the time at which Buller’s ration, the size of the fruiting body, and with a study was conducted, it is understandable that statistical analysis of multiple samples, Buller’s limitations existed in his study and this led to estimate would be much lower. No doubt, C. an over-estimation. Nobody should or can deny gigantea is the most prolific fungal species for that Buller’s study is instrumental. Several spore production in each fruiting body accor- reasons might be responsible for the difference ding to the reports available to date. 459 The exoperidium of Calvatia species is cording to the result of the present study. Murat only one layer and it does not have the et al (2008) stated that a Guinness World pseudoparenchymatous endostratum typical of Record as the largest edible fungus is Calvatia all other Lycoperdaceae (Gube 2007). This is gigantea and provided a web link (http://www. the reason that peridia of C. gigantea are thin guinnessworldrecords.com/content_pages/recor and light in weight, only taking less than 10% d.asp?recordid=47431). However, the record of of the whole fruiting body weight. Also, it does this fungus cannot be found by following this not have a sterile base. These factors make link. Searching the webpage of the Guinness quantification of basidiospores in fruiting bo- World Records using different key words dies relatively easy and Buller’s study possible. yielded nothing. In responding to my inquiry It is intriguing to find out that spore den- about the record to website_inquiry@guin sity was not evenly distributed in the fruiting nessworldrecords.com, the Guinness World body. When the gleba was cut open, it looked Records confirmed their record “The world’s uniform in texture and colour. The unevenness largest edible fungi (us) was a giant puffball of spore density is a crucial factor which can (Calvatia gigantea) measuring 2.64 m (8 ft 8 interfere with accuracy for the estimation of in) in circumference and weighing 22 kg (48 lb total spores, if sampling was not properly 8 oz). It was found by Jean-Guy Richard of conducted. The unevenness might be due to Montreal, Quebec, Canada in 1987” (personal several possible factors: uneven dehydration of communication 2011). According to aforemen- the fruiting body, unstable spore development tioned information, this fruiting body is not the rate, irregular cellular/tissue structure etc. Gube record holder. Carlyon (2011) cited a fruiting (2007) studied the gleba development of seve- body of C. gigantea >150 cm in diameter, 45 ral species of Lycoperdaceae and found that the cm in height and weighing more than 100 kg gleba of Calvatia gigantea develops in a when fresh in her webpage of Giant Puffball. unique and previously unknown manner. The However, Carlyon’s report cannot be verified -forming palisade structures borne by photo, specimens or collection information. on fan-like branches of hyphae are different If verifiable, this fruiting body could be the re- from those of species of Lycoperdaceae inclu- cord. World Records Academy (2010) claimed ding other species of Calvatia. It is not clear that the world record of the largest puffball is whether the glebal development is playing any the one found by Finley O’Neill in Slaithwaite, role in the unevenness of spore densities. What West Yorkshire, UK in October 2010 measu- is the biological advantage of the unevenness ring 169 cm. However, according to the photo to the species? Further study is necessary to posted on the webpage, the measurement ap- determine the true reason for the unevenness. pears to be the circumference, rather than the Buller’s fruiting body and the present one diameter. Clearly this fruiting body is unus- of C. gigantea are far from the biggest fruiting ually large, but far from the world record of body ever observed. The largest one that the giant puffball. Thus, the verifiable record of author observed in 1990’s was the size of a this species is still the one found by Professor small sheep. Apparently this size is not the re- RE Call in New York State in 1877. cord one. Bessey (1884) reported a specimen as Airborne basidiospores of Calvatia are 1.63 × 1.37 × 0.24 m, which was observed, difficult to identify based on morphological photographed, and measured by Professor RE characters under optical microscopy. Levetin et Call in New York State in 1877. This specimen al (1992) conducted a morphological study on was too big to collect and preserve in the 19th the basidiospores of century. Bessey (1950) extrapolated that this (Schwein.) Fr., C. cyathiformis (Bosc) Morgan, fruiting body would produce approximately C. rubroflava (Cragin) Lloyd, and C. gigantea. 160 trillion basidiospores. Bessey (1950) did They found that each species showed unique not indicate how he did his calculation. It was ornamentation on spore surface under scanning quite possible that his extrapolation was based electron microscopy (SEM), but only C. cyathi- on Buller’s study, since it was the only formis basidiospores were identifiable with quantitative study of C. gigantea basidio- sufficiently distinctive characters on spore trap spores. Bessey’s extrapolation is too high ac- slides under compound microscopy. Baseia

460 Mycosphere (2003) studied the ornamentations on basidio- References spores of four species of Calvatia in Brazil. Bates et al (2009) conducted an ultrastructural Arnold EH, Brien RM. 1961 – Diseases of turf. study on 28 species in Lycoperdaceae inclu- Turf Culture. Palmerston North, New ding eight species of Calvatia using SEM. Zealand Institute for Turf Culture. 71–76. These ultrastructural studies on basidiospores Baseia IG. 2003 – Contribution to the study of of a total of 13 species of Calvatia showed that the genus Calvatia (Lycoperdaceae) in ornamentation characters of basidiospores are Brazil. Mycotaxon 88, 107–112. taxonomically significant. Basidiospore pedi- Bates ST. 2004 – Arizona members of the Gea- cels and sterigmal remnants are taxonomically straceae and Lycoperdaceae (Basidiomy- important in distinguishing certain Lycoperdon cota, Fungi). M.Sc. Thesis, Arizona State species (Bates 2004). However, the signifi- University, Tempe. cance of these characters for differentiating Bates ST, Roberson RW, Desjardin DE. 2009 – Calvatia species is not fully studied. It is the Arizona I: Lycoperdaceae author’s opinion that sterigmal remnant charac- (, Basidiomycota). Fungal Di- ters, such as length, width, and detaching versity 37, 153–207. patterns are very useful for identification of air- Bessey CE. 1884 – An enormous puffball. borne spores of Calvatia in combination with American Naturalist 18, 530. the characters of spore ornamentation, size, and Bessey EA. 1950 – Gasteromyceteae. In: Mor- colour. phology and of Fungi. The Quantitative real time polymerase chain Blakiston Company, Philadelphia, USA. reaction might be an alternative method for de- 530–571. tecting and quantifying Calvatia spp. However, Buller AHR. 1909 – Researches on Fungi. Vol. up to date, primers and probes for quantita- I, Longmans, Green and Co. London. 287 tively detecting Calvatia species have not been pp. developed. Also, due to lack of study on the Carlyon K. 2011 – Giant puffball http://bioweb allergenic properties of C. gigantea, the health .uwlax.edu/bio203/2010/carlyon_kath/fac effects of its airborne spores are not fully un- ts.html. Accessed 8 January 2011. derstood. In the study by Levetin et al. (1992), Clémençon H. 1990 – Fixierung, Einbettung allergen could not be extracted from basidio- und Schnittfär-bungen für die plectolo- spores of C. gigantea in several attempts. gische Untersuchung von Hymenomy- Calvatia is currently conserved against ceten mit dem Lichtmikroskop. Myco- the earlier synonyms Omalycus, Langermannia logia Helvetica 3, 451–466. and Hippoperdon (McNeill et al. 2006). How- Coetzee JC, van Wyk AE. 2007 – Proposal to ever, it is not protected against Lanopila, conserve Calvatia nom. cons. (Basidio- another older synonym. Coetzee & van Wyk mycota, Lycoperdaceae) against an addi- (2007) proposed to conserve Calvatia against tional name, Lanopila. Taxon 56, 598– Lanopila. Their proposal has been passed by 599. the Nomenclature Committee for Fungi Gube M. 2007 – The gleba development of (Norvell 2010) and will obtain the final Langermannia gigantea (Batsch: Pers.) approval of XVIII International Botanical Rostk. (Basidiomycetes) compared to o- Congress in July, 2011 in Melbourne, ther Lycoperdaceae, and some systematic Australia. Therefore, the valid scientific name implications Mycologia 99, 396–405. for giant puffball should remain as Calvatia Ingold CT. 1965 – Spore Liberation. Clarendon gigantea (Batsch) Lloyd, which has 12 Press, Oxford. 210 pp. synonyms. Kreisel H. 1994 – Studies in the Calvatia com- plex (Basidiomycetes) 2. Feddes Repert Acknowledgements 105, 369–376. I am very grateful to Dr James LaMondia Levetin E, Horner WE, Lehrer SB. 1992 – and Mr James Preste for bringing my attention Morphology and allergenic properties of to the fruiting body. basidiospores from four Calvatia species. Mycologia 84, 759–767.

461 McNeill J, Barrie FR, Burdet HM, Demoulin Norvell L. 2010 – Report of the Nomenclature V, Hawksworth DL, Marhold K, Committee for Fungi: 15. Taxon 59, Nicolson DH, Prado J, Silva PC, Skog 291–293. JE, Wiersema JH, Turland NJ. 2006 – Simon-Nobbe B, Denk U, Pöll V, Rid R, International Code of Botanical Nomen- Breitenbach M. 2008 – The spectrum of clature (Vienna Code). Regnum Vegeta- fungal allergy. International Archives of bile 146. A.R.G. Gantner Verlag KG. Allergy and Immunology 145, 58–86. (ibot.sav.sk/icbn/main.htm). World Records Academy. 2010 – Largest puff- Murat C, Mello A, Abbà A, Vizzini A, ball-world record found by Finley Bonfante P. 2008 – Edible mycorrhizal O’Neill. http://www.worldrecordsacade fungi: identification, life cycle and mor- my.org/nature/largest_puffball-world_re phogenesis. In: Mycorrhiza: State of the cord_found_by_Finley_ONeill_101919.h art, genetics and molecular biology, eco- tml. Accessed 4 January 2011. function, biotechnology, eco-physiology, structure and systematic 3rd edn (ed. A Varma). Springer, Berlin, 707–732.

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