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Department of Plant Pathology, 1453 Fifield Hall, University of Florida, Gainesville, Florida 32611-0680
Meredith Blackwell
Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803-1715
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ABSTRACT |
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Lobosporangium is proposed as a new name for Echinosporangium Malloch, a later homonym of Echinosporangium Kylin. Lobosporangium transversale was isolated from arid soils on three occasions between 1964 and 1968 but has not been reported again. Observations on sporangium development in culture revealed rapid sporangiospore germination, rapidly growing hyphae forming anastomoses, threefold dichotomously branching aerial sporangiophores and formation of clusters of eight sporangia. The sporangia of L. transversale are illustrated, and the placement of Lobosporangium in the Mortierellaceae is discussed. A new genus, Gamsiella, is proposed that is based on Mortierella multidivaricata. Sporangial ontogeny of Gamsiella is compared with that presented here for Lobosporangium.
Key words: Echinosporangium, Gamsiella, Lobosporangium, Mortierella, Mortierellaceae, Mortierellales, sporangium, Zygomycetes
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSTAXONOMYRESULTSDISCUSSIONLITERATURE CITED |
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, was isolated from soil collected near Virginia City, Nevada. One year later it was found in soil collected about 50 miles south of Hermosillo, Sonora, Mexico (Ranzoni 1968). A third isolate was made in 1966 from dry soil collected behind the Biological Laboratories Building on the campus of the University of Texas at Austin (Ellis and Hesseltine 1974, Blackwell unpubl). Despite the three isolations in a two-year span, the fungus has never been isolated again.
Echinosporangium originally was described as a member of the Mucorales with unknown affinities (Malloch 1967). E. transversale initially was included in the Mucoraceae (Pidoplichko and Mil'ko 1971, Mil'ko 1974). After a few years, Echinosporangium was transferred to the Saksenaeaceae Hesseltine & Ellis (Ellis and Hesseltine 1974). Malloch's new genus shared several characteristics with Saksenaea Saksena (1953), including the formation of rapidly growing, hyaline, aerial hyphae that can be branched dichotomously or stoloniferous below the multispored sporangia that are not globose or obpiriform. Other characters of both Echinosporangium and Saksenaea cited by Ellis and Hesseltine were that no sporangiola were formed and that both fungi were found mostly in soil. Other mycologists (Hesseltine and Ellis 1973, O'Donnell 1979, Benny 1982, Hawksworth et al 1983) also included E. transversale in the Saksenaeaceae. The characters cited by Ellis and Hesseltine (1974) did not suggest a close relationship between Echinosporangium transversale and Saksenaea vasiformis Saksena (1953), the only fungi to have been included in the Saksenaeaceae. Benjamin (1979) thought that the inclusion of the two genera with unusual sporangia in the Saksenaeaceae was provisional.
Zycha et al (1969) placed E. transversale in the Mortierellaceae. This disposition of Echinosporangium has been followed by Alexopoulos et al (1996), Benny et al (2001), Hawksworth et al (1995) and Kirk et al (2001). A phylogenetic study based on the comparison of multigene data sets more recently has provided strong support for the position of E. transversale in the Mortierellaceae (O'Donnell et al 2001).
Echinosporangium Malloch (1967), however, is a later homonym for Echinosporangium Kylin (1956), a red alga, described without a Latin diagnosis but still valid according to the International Code of Botanical Nomenclature (Greuter et al 2000; Article 36.2). Latin was not required for the valid description of living algae until 1 Jan 1958 (Greuter et al 2000).
A second mortierellaceous fungus that forms dichotomously branched sporangiophores is Mortierella multidivaricata R.K. Benjamin (Benjamin 1978), which was isolated from debris collected under a rotting log in Solkolniki Park, Moscow, Russia. The similarity in the sporangiophore branching pattern between E. transversale and M. multidivaricata was noted by W. Gams (pers comm 2002) and Wrzosek (2002). In addition colonies of both of these mortierellaceous fungi produce a garlic-like odor.
The purpose of this study is to: (i) discuss the ontogeny, morphology and classification of Echinosporangium transversale; (ii) rename Echinosporangium Malloch; and (iii) describe a new genus for Mortierella multidivaricata.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSTAXONOMYRESULTSDISCUSSIONLITERATURE CITED |
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The fungal specimen used for Fig. 12 was grown on Leonian's agar + 1 g yeast extract (LYE; Malloch and Cain 1971) agar at 25 C (O'Donnell 1979). The specimen was processed for scanning electron microscopy using the procedure of O'Donnell and Hooper (1974).
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Colony growth and morphology, and sporangium and zygospore formation in Echinosporangium transversale grown at either 18 C or 25 C were determined on several culture media. These culture media included: cornmeal agar (CMA; BBL-Ranzoni 1968), Czapek-Dox agar (CZA; Ranzoni 1968), Emerson YpSs agar (Y; Difco-Ranzoni 1968), hempseed agar (HSA; Kuhlman 1972), Leonian's agar (LA; Leonian 1924), malt-extract agar (MEA; Ranzoni 1968), malt extract-yeast extract agar (MEYE; Benjamin 1959), Pablum agar (PAB; Benjamin 1959), Sabouraud synthetic medium (SAB-S; Gams and Williams 1963), shrimp agar (ShA; Degawa and Tokumasu 1998), sodium caseinate agar (SCA; BBL-Ranzoni 1968), Thornton's standardized medium (TSM; Thornton 1922), wheat germ agar (Wg; Benny 1972) and modified V8 Juice agar (V8; based on Miller 1955). The components of TSM and V8 are: TSM (K2HPO4, 1.0 g; MgSO4·7H2O, 0.2 g; CaCl2, 0.1 g; NaCl, 0.1 g; FeCl3, 0.002 g; KNO3, 0.5 g; asparagine, 0.5 g; mannitol, 1.0 g; agar, 15 g; distilled water, 1000 mL), and V8 (V8 juice, 163 mL; powdered CaCl3, 3 gm; agar, 15 g; distilled water, to make 1000 mL). We also used the media suggested by Ranzoni (1968) when the fungi he isolated did not sporulate: potato-dextrose agar (PDA; Difco), Alphacel medium (AM; Sloan et al 1960), Modified Alphacel medium (MAM; Sloan et al 1960), and potato-glucose agar (PGA; Sloan et al 1960).
Mortierella multidivaricata sporulated on most culture media when grown at room temperature (ca 25 C) in a room with a south-facing window. The sporangia of M. multidivaricata and E. transversale were compared when grown on CMA at room temperature with light from a south-facing window.
The relative abundance of sporangia was graded by eye from the maximum number to none being produced using this scale: maximum (4+), excellent (3+), good (2+), and poor (+), to none (0).
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TAXONOMY |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSTAXONOMYRESULTSDISCUSSIONLITERATURE CITED |
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Echinosporangium Malloch, Mycologia 59:327. 1967 (nom. illeg., Art. 53.1 of the ICBN, Greuter et al [2000]), non Echinosporangium Kylin, 1956 (Die Gattungen der Rhodophyceen, p. 537). Mycelium coenocytic, anastomosing, producing lateral branches that form 3–4 dichotomies, with the final branch becoming a cylindrical sporangium with rounded apices bearing several apical spines.
Type species: Lobosporangium transversale (Malloch) M. Blackwell & Benny
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Mycelium coenocytic when young, irregular in outline, anastomosing, hyaline to very light yellow, producing a garlic-like odor; giving rise to lateral hyphae that branch three to four or more times dichotomously with each successive branch becoming longer and wider, with the ultimate branch elongating laterally resulting in the sporangia being formed in pairs and attached medially. Sporangia bacilliform with rounded apices, or reniform to allantoid and often medially constricted, 230–400 µm in length x 43–80 µm in diam, hyaline to light yellow, filled with sporangiospores and often containing a pseudocolumella; wall relatively thick except in spines, smooth, apices bearing 1–6 spines, or composed of a three-sided structure that is proximally constricted. Spines smooth, conical, straight to somewhat curved, 3.8–19 µm in diam basally x 7.4–64 µm in length, with a hollow core. Sporangiospores globose to irregular in shape, 6.4–22 x 7.6–25 µm, hyaline, smooth-walled, completely filling a sporangium, or a pseudocolumella produced and spores almost completely filling the sporangium. Pseudocolumella, when formed, more or less hemispherical, partially or completely filling the medial area of sporangium, consisting of uncleaved, transparent cytoplasm, usually wider at side bearing nutritive hypha. Zygospores were not observed.
Cultures examined. U.S.A. NEVADA. Washoe County, Virginia Range near Virginia City, soil collected by D. Malloch, Sep 1964 (culture derived from the holotype: ATCC 16960, CBS 357.67, IMI 130776, NRRL 3116)(HOLOTYPE TRTC 43983); TEXAS. Travis County, Austin, University of Texas, behind the Biological Laboratories Building, soil collected by M. Blackwell, Feb 1966 (ATCC 18036, NRRL 5525).
Literature report: MEXICO. SONORA. 50 miles south of Hermosillo, soil collected by F.V. Ranzoni, 23 Mar 1965 (Ranzoni 1968).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSTAXONOMYRESULTSDISCUSSIONLITERATURE CITED |
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Isolation – NRRL 5525 was isolated by sprinkling dry soil on the surface of CMA. Within a week the mycelium had grown to the periphery of the Petri plate and numerous clusters of sporangia were visible to the unaided eye. The fast-growing mycelium was easily isolated into pure culture because it outgrew other fungi in the soil.
Spore dehiscence – Sporangia did not release spores after being placed on moist agar for 3 wk. Flooding cultures with distilled water or 0.7% or 1.7% sodium chloride solutions also did not induce spore release. Spores were released only when sporangium walls were ruptured from applied pressure.
Spore germination – Sporangiospores from broken sporangia germinated in 2–3 h after being placed on agar. One to five germ tubes emerged from each spore and developed into a profuse, rapidly growing mycelium. The resulting colony covered the surface of an 8.5 cm diam Petri dish 3–4 days after it was inoculated with a single spore.
Hyphae – Rapidly growing mycelium consisted of hyphae with irregular swellings formed at intervals. Anastomoses also were produced (Fig. 1). The hyphae varied from 0.5 to 4.0 µm in diam. The mycelium was coenocytic for the first 3–4 days, and protoplasmic streaming was directed toward the bluntly tapered hyphal tips. Over the next 48 h, clusters of aerial hyphae appeared, usually near the periphery of the colony. This coincided with the evacuation of cytoplasm from the older mycelium
Sporulation – Soon after the appearance of the aerial hyphae some of this mycelium began to enlarge and a septum was formed. A portion of the mycelium on one side of the septum disintegrated, whereas the other part continued to grow into a dichotomously branched sporophore (Figs. 2–5). Growth and branching usually continued until three dichotomies (Figs. 5–7) were formed, the last of which produced sporangia. Sporangia were elongate, often centrally constricted, and each was centrally attached to one of the eight sporangiophore branches. Enlargement of the sporangia soon obscured the relatively small sporangiophores, and this resulted in the presence of clusters of bi- or trilobed smooth-walled sporangia on the surface of the agar (Figs. 7–12). The sporangia were connected to each other by tubular hyphae (Figs. 6, 9) and were connected to the hyphal system. When the sporangia attained approximately 55 µm in length, 1–6, usually 4–6, hornlike papillae formed at the apex of each sporangial lobe. Sporangiospore formation commenced soon thereafter. Often a small uncleaved piece of cytoplasm remained—the pseudocolumella (Fig. 10)—but in other cases the entire sporangium protoplast was cleaved into spores. The sporangia remained in clusters when mature (Figs. 11, 12).
Effect of light on sporulation – Preliminary experiments conducted in duplicate indicated that light had a stimulatory effect on sporulation. Two cultures incubated in continuous light or under a 12 h light/12 h dark photoperiod produced sporangia within 6 d after inoculation. None of the cultures incubated in the dark produced sporangia even 3 wk after inoculation. When these cultures were transferred to a lighted incubator maintained at the same temperature, sporangial formation was initiated within 12 h.
Sporangium formation – Sporangium formation occurred on these media at room temperature (ca 25 C): it was maximum (4+) on LA and HSA: excellent (3+) on CMA, CZA, and TSM; good (2+) on MEA, PAB, and ShA; and poor (+) on MEYE, Wg, and V8. Sporangia were not formed (0) on AM, MAM, PDA, PGA, SAB-S and Y. At 18 C sporangium formation was good (2+) on LA, and poor (+) on HSA, PAB, and TSM. Sporangia were not formed on SAB-S, V8, and Wg.
Zygospore formation – Both isolates of L. transversale (NRRL 3116 and NRRL 5525) were crossed on all of the available culture media listed above except AM, MAM, PDA and PGA. These fungi then were grown at both 18 C and room temperature (ca 25 C), but zygospores were never observed.
Mortierella multidivaricata – Sporangium formation in M. multidivaricata was induced on these media at room temperature (ca 25 C): sporulation was maximum (4+) on SCA; excellent (3+) on Y; good (2+) on CMA; and poor (+) on AM, CZA, MA, MAM, and PGA; no other media were tried.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSTAXONOMYRESULTSDISCUSSIONLITERATURE CITED |
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was able to recognize Lobosporangium transversale because the fungus sporulated on three of the five culture media that he routinely used to isolate soil fungi: malt-extract (Difco) agar, Czapek-Dox (Difco), and cornmeal agar (BBL®).
O'Donnell (1979) published scanning electron micrographs of Lobosporangium sporangial clusters. These micrographs showed that the sporangial clusters formed on a hyphal mat. Individual sporangia are smooth-walled and bear three or four spines. Lobosporangium transversale has been isolated three times, but it might be more common but unnoticed because sporangium formation occurs only on certain culture media when grown in the light.
Sporulation in Lobosporangium transversale can be compared with that of another member of the Mortierellales, Mortierella multidivaricata R.K. Benjamin (Benjamin 1978). Mortierella multidivaricata would have been described as the only species in the genus Gamsiella R.K. Benjamin except that 1 yr earlier Gams (1977) described a new subgenus of Mortierella, Micromucor, for species close to Mortierella ramanniana (Möller) Linnemann and M. isabellina Oudemanns. As a result, M. multidivaricata was described as the only member of the third subgenus, Gamsiella of Mortierella, instead of being in a new genus (R.K. Benjamin, pers comm 1985). Micromucor was placed in the second subgenus of Mortierella (Gams 1977). Members of Micromucor often have a distinct columella but the colony lacks a distinct garlic- or onion-like odor, whereas the taxa in the genus Mortierella lack a distinct columella but often produce a garlic- or onion-like odor (Gams 1977). Later, von Arx (1982) elevated the subgenus Micromucor to generic status, as Micromucor (Gams) von Arx. Recently, Micromucor was transferred to Umbelopsis Amos & Barnett (Meyer and Gams 2003) and the latter taxon is the only genus included in the Umbelopsidaceae W. Gams & W. Meyer (Mucorales).
Mortierella subgenus Gamsiella should be elevated to the rank of genus because of these unique characteristics: the sporangiophores are repeatedly divaricately branched, 2-spored sporangia are formed simultaneously on long, slender pedicels, and the globose, ornamented chlamydospores are mostly terminal.
Basionym: Mortierella Coemans subgenus Gamsiella R.K. Benjamin, Aliso 9:157. 1978.
Basionym: Mortierella multidivaricata R.K. Benjamin, Aliso 9:158. 1978.
The fertile branches or sporangiophores of G. multidivaricata initially arise laterally from aerial hyphae and then form irregularly lobed hyphae that become branched three to five times. The terminal branches are ovoid to clavate, and they give rise to two or three simple, long, tapered and attenuated branches that bear two-spored sporangia. Chlamydospores are usually globose and arise terminally. The asexual spores and associated structures of G. multidivaricata also have been observed using electron microscopy (O'Donnell 1979, Ansell and Young 1982); zygospores are unknown.
A comparison of sporangium formation in Gamsiella multidivaricata (Benjamin 1978) and Lobosporangium transversale demonstrates that the early stages of fertile-branch formation are similar. Each fertile branch of Lobosporangium appears to be shorter than those of Gamsiella because the sporangia of L. transversale are much longer than the sporangia and pedicels of G. multidivaricata (230–400 µm long versus 31–88 µm long). As a result, the fertile heads of Lobosporangium are 300–530 µm in diam whereas these structures in Gamsiella are 250–425 µm wide.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Accepted for publication June 23, 2003.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSTAXONOMYRESULTSDISCUSSIONLITERATURE CITED |
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Alexopoulos CJ, Mims CW, Blackwell M., 1996 Introductory mycology. 4th ed. New York: John Wiley & Sons Inc. 868 p
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———, Cain RF., 1971 New genera of Onygenaceae. Canad J Bot 49:839-846
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———, Hooper GR., 1974 Scanning ultrastructural ontogeny of paragymnohymenial apothecia in the operculate discomycete Peziza quelepidotia. Canad J Bot 52:873-876
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