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Mycotoxin Research Unit, National Center for Agricultural Utilization Research, USDA, Agricultural Research Service, 1815 N. University Street, Peoria, Illinois 61604
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ABSTRACT |
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The ability of Aspergillus caelatus, a species in Aspergillus section Flavi, to produce synnemata and sclerotia was investigated. Forty-eight isolates of A. caelatus differed widely in their production of synnemata and sclerotia; 83% of the isolates produced varying numbers of synnemata and sclerotia, and 17% produced neither sclerotia nor synnemata. Most strains produced synnemata and mostly sessile and few stipitate sclerotia on the same Czapek agar (CZA) plate. Two strains of A. caelatus were selected for further study because of the contrasting morphology of their synnemata and sclerotia. Those strains are NRRL 25528, the type species and a representative of the synnema- and black sclerotium-forming isolates, and NRRL 26119, considered an atypical strain that produced numerous synnemata and few slightly melanized or tan sclerotia. The induction and maturation of sclerotia in A. caelatus were affected greatly by the type of media as well as the kind and concentration of the carbon and nitrogen sources. CZA induced synnema and sclerotium production in both strains, whereas other media did not. Production of abundant synnemata and sclerotia also occurred when the carbon source in CZA is replaced with dextrose, xylose, cellobiose, melibiose and trehalose. CZA amended with serine, threonine, KNO3 and NaNO3 induced the production of numerous sclerotia and synnemata. For both strains, the optimal levels of sucrose and NaNO3 for maximum production of synnemata or sclerotia were 3 and 0.9%, respectively. The production of synnemata and stipitate/sessile sclerotia by several wild-type strains of A. caelatus further substantiates previous suggestions for an evolutionary link between Aspergillus section Flavi and synnematal species A. togoensis, which also produces stipitate sclerotia.
Key words: amino acids, C:N ratio, carbohydrates, development, morphology, Stilbothamnium
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Raper and Fennell 1965, Raper and Thom 1949). Aspergillus togoensis (Henn.) Samson and Seifert is a tropical fungus that forms tall synnemata, large sessile or stipitate sclerotia/stromata and yellow to greenish yellow radiate conidial heads. Although Samson and Seifert (1985) placed this species in Aspergillus subgenus Stilbothamnium, it has been suggested as an ancestral form of Aspergillus section Flavi (Roquebert and Nicot 1985, Samson and Seifert 1985).
The ability to produce synnemata and stipitate sclerotia in Aspergillus section Flavi was not known until recently. McAlpin (2001) first described an A. flavus mutant (NRRL 29254) that produced synnemata and stipitate sclerotia on different media, on carbon- or nitrogen-amended Czapek agar (CZA) and on CZA with different concentrations of carbon and nitrogen. The development of these structures was modified by temperature, light and pH. A synnema (pl. synnemata) is "a conidioma composed of more or less compacted groups of erect and sometimes fused conidiophores bearing conidia at the apex only or on both apex and sides" (Hawksworth et al 1995). This description fits the synnema-like structures of the A. flavus mutant (McAlpin 2001), which produced white, erect, intricate stipe with conidia borne on both apex and sides, similar to the synnemata produced by the Aspergillus subgenus Stilbothamnium, although much smaller. The stipitate sclerotia resembled the teleomorphic stage of the genus Penicilliopsis according to descriptions by Samson and Seifert (1985).
Aspergillus caelatus B.W. Horn, a recently described species in Aspergillus section Flavi isolated from agricultural field soils and insect-damaged peanut seeds in the Southern United States (Horn 1997, Horn and Dorner 1998) as well as from tea field soils in Japan (Peterson et al 2000), was found to produce both synnemata and sessile and stipitate sclerotia on the same CZA plate (personal observation). As in the A. flavus mutant (McAlpin 2001), the synnemata and stipitate sclerotia produced by A. caelatus, resembled those of A. togoensis in miniature form (see FIG. 1A, B). Unlike the synnema- and stipitate sclerotium-producing mutant strain of A. flavus NRRL 29254 (McAlpin 2001), the A. caelatus strains described in this study are wild types.
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In this study, the nutritional factors critical to synnema/sclerotium initiation and maturation of some strains of A. caelatus were compared with those for the mutant A. flavus NRRL 29254 (McAlpin 2001) to find some commonality and/or differences in the formation of the synnemata and sclerotia between these two Aspergillus species. It is also imperative to affirm the significance of synnema and stipitate sclerotium production as morphological bases for evaluating the relationship among A. caelatus, A. flavus and A. togoensis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The relative humidity inside the incubation boxes was checked (Traceable Memory Hygrometer, Control Co., Friendswood, Texas) and maintained within 60–80%. After these conditions were determined, each of the 48 A. caelatus strains was inoculated centrally on four replicate CZA plates with 3 µL of spore suspension (1 x 105 spores/mL in 0.1% agar) obtained from 5 d old CZA slants, then placed in plastic boxes, covered with aluminum foil and incubated at 30 C. All 48 isolates also were grown on Murashige-Skoog agar (MSA) for comparison because this medium induced only synnema production in the A. flavus mutant NRRL 29254 (McAlpin 2001). Another preliminary study was conducted on the nutritional requirements of four representative A. caelatus strains with black sclerotia (NRRL 26114, 25528, 26105, 25577), using two replicate plates for each isolate. Their response to different media and different concentrations of carbon and nitrogen on CZA were similar regardless of the number of synnemata/sclerotia produced by each strain (data not shown). Two strains (NRRL 26108, 25568) that did not produce sclerotia on CZA also produced no sclerotia on CZA amended with different carbon or nitrogen sources. Therefore, only the type species (NRRL 25528), which is fairly representative of the black sclerotium-forming isolates, and an atypical isolate (NRRL 26119), which produced slightly melanized or tan sclerotia and numerous short synnemata and few sclerotia, were selected for further investigation because of their contrasting morphological characteristics.
These media were prepared for synnema and sclerotium production according to Booth (1971) and Atlas (1993) except where the commercial brand or source is indicated: Coon’s medium (CM), cornmeal agar (CMA) (Difco, Detroit, Michigan), Czapek agar (CZA), complete medium (CYM) (CZA + 0.25% yeast extract + 0.75% malt extract), malt-extract agar (MEA), Murashige and Skoog basal medium (Sigma Chemical Co., St. Louis, Missouri) plus 3% sucrose and 1.5% agar, oatmeal agar (OA), potato-dextrose agar (PDA), potato-dextrose agar + 0.5% yeast extract (PDAYE), and V8-juice agar (V8). Four replicate 90 mm diam plastic plates were center-point inoculated with 3 µL of spore suspension (1 x 105 spores/mL of 0.1% water agar) of A. caelatus NRRL 25528 or NRRL 26119. Growth, sporulation and synnema/sclerotium production were observed every 2 or 3 d for 2 wk and weekly thereafter for 6 wk in all subsequent experiments.
Carbon and nitrogen sources. –
CZA, consisting of 3 g NaNO3, 0.5 g MgSO4, 0.5 g KCl, 0.01 g FeSO4·7H2O, 1 g KH2PO4, 30 g sucrose and 15 g agar, was used as the basal medium. The 30 g sucrose with approximately 42% available carbon equivalent to 12.62 g C/L was replaced in the medium with equivalent amounts of carbon from 22 different sources: seven monosaccharides, seven disaccharides and eight polysaccharides (see FIG. 2). A control medium with no carbon source was included. Similarly, the 3 g of NaNO3 with approximately 16.5% available nitrogen equivalent to 0.49 g N/L in the basal medium was replaced with 22 amino acids and seven other nitrogen-containing compounds (see FIG. 4). CZA without nitrogen served as the control. All media were adjusted to pH 7 with 6N HCl or 5N NaOH.
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C:N ratio. – The amount of NaNO3 in CZA was varied at 0.0, 0.1, 0.3, 0.6, 0.9, 1.2, 1.5 and 2.0% and paired in all possible combinations with different sucrose concentrations at 0, 1, 3, 6, 9, 12, 15 and 20% in 200 mL aliquots. This gives g C : g N ratios from approximately 1.4 to 500 in which the basal medium (CZA) is approximately 25.7.
Microscopic studies. – Development of sclerotia was observed at different intervals on CM, CZA and MSA plates under the light microscope. At various intervals up to 7 mo, sessile and stipitate sclerotia were sectioned or crushed and examined for the presence of ascospores.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), 39 produced black sclerotia and one isolate (NRRL 26119) produced what appeared at first to be exclusively synnemata on CZA. However, after 21 d of incubation, some were found to produce hard and slightly melanized or tan ovoid to irregularly shaped sclerotia at the apex. On MSA, 34 of the isolates produced only sessile sclerotia but no synnemata, a single isolate (NRRL 26306) produced a few (less than 100 per plate) synnemata with several sessile sclerotia, and the remaining isolates produced neither synnemata nor sclerotia. Two types of conidia were produced: (i) the typical conidia formed on conidiophores on the agar surface and (ii) conidia arising from the apex of synnema stalks. In general, the amount of sporulation on the agar surface was inversely proportional to the number of synnemata/ sclerotia produced, e.g., moderate sporulation supported moderate (approximately 200–500 sclerotia/ plate) to abundant sclerotium production (>500 sclerotia/plate) 14–21 d after inoculation. Beyond this period, the amount of surface sporulation in most isolates continued to increase during incubation, and the synnemata and sclerotia often became covered with surface conidia.
The ability of A. caelatus NRRL 25528 and NRRL 26119 to produce synnemata and sclerotia was tested on different agar media (TABLE II). The best medium for both synnema and sclerotium production by NRRL 25528 was CZA in which 5–10% of the sclerotia from a single plate were stipitate (FIG. 1B). A few synnemata and sessile sclerotia were produced on PDA and PDAYE, whereas only sessile sclerotia were produced on CM, MSA and OA. Abundant spores but no synnemata or sclerotia were produced on CMA, CYM, MEA and V8 by NRRL 25528. NRRL 26119 produced numerous short (1–3 mm) synnemata with conidial heads on CZA; some of these heads later produced hard, slightly pigmented or tan to light brown ovoid sclerotia at the apex, while the remainder withered and desiccated. All tan sclerotia were sessile on CM, CYM, MSA and OA (FIG. 1C). A few synnemata and sclerotia were formed by NRRL 26119 on PDA but none on CMA, MEA, PDAYE and V8.
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). CZA amended with polysaccharides, including apple pectin, citrus pectin or starch, induced only sessile sclerotium formation and completely inhibited synnema formation, except for inulin, which supported formation of synnemata and abundant sclerotia for both isolates. NRRL 26119 produced numerous synnemata on most of the carbon sources tested, including arabinose, dextrose, glycerol, maltose, melibiose, sorbitol, sucrose, trehalose and xylose (FIG. 3A), with some synnemata producing pedicellate sclerotia at maturity. Inulin, mannitol and sorbitol stimulated branching of the synnemata produced by NRRL 26119 (FIG. 3B, C). Cellulose-amended CZA induced sparse sporulation and inhibited synnema/sclerotium formation.
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). Asparagine, aspartic acid, glutamic acid, glycine and proline supported the production of moderate numbers of sclerotia (NRRL 25528) and synnemata (NRRL 26119), whereas relatively few synnemata or sclerotia were produced on alanine, arginine, glutamine, histidine, hydroxyproline, isoleucine, ammonium tartrate and urea. A slight increase in pedicellate sclerotium production (11–15% of total) and slightly longer synnemata (3–6 mm) were produced on CZA amended with serine, arginine and glutamic acid than on regular CZA. Ammonium sulfate inhibited growth, sporulation and synnema/sclerotium formation by both isolates.
Optimal induction of synnema/sclerotium formation in NRRL 25528 occurred at 3% sucrose; at 6%, approximately equal numbers of synnemata and sclerotia were produced; at 9%, mostly synnemata with few sclerotia formed; at 15%, few synnemata formed; none at 20%; and no sclerotia developed above 9% sucrose (FIG. 5A). Maximum counts of synnemata and sclerotia with NRRL 26119 were obtained at 3% sucrose, gradually decreasing at 6–12%, whereas at 15–20%, only a few synnemata but no sclerotia were formed (FIG. 5B).
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). NRRL 26119 produced numerous synnemata but substantially fewer sclerotia than NRRL 25528 in all the concentrations tested. The highest counts of synnemata and sclerotia were recorded at 0.9% NaNO3, the number declining at higher concentrations (1.2–2.0%) (FIG. 5D). Sclerotia at 0.9% NaNO3 were noticeably more elongated than irregularly shaped. The maximum production for both synnemata (2800/plate) and sclerotia (1680/plate) by the A. caelatus isolates NRRL 25528 and NRRL 26119 occurred when a 3% C concentration and a 0.9% N were used, equivalent to a C:N ratio of 8.6. However, a similar ratio using different amounts of C and N (1% sucrose/0.3% NaNO3 and 6% sucrose/2% NaNO3) induced moderate numbers (<500/plate) of synnemata and sclerotia. The common observations for C:N ratio that confirmed the results obtained above follow. For any fixed concentration of N, the numbers of synnemata/sclerotia increased with the C concentration reaching a maximum at 3% and then decreased as the concentration was increased further. Similarly, for any fixed value of C, the numbers of synnemata/sclerotia increased as the N concentration increased until a maximum value was reached and then declined with further increases in N. No interactive effects between C and N were observed. One major difference between the two isolates was that sclerotium production was much greater than synnema production in NRRL 25528, whereas synnema production was much greater than sclerotium production in NRRL 26119 and that NRRL 26119 produced much fewer sclerotia in all concentrations of C and N.
Sclerotium initiation in NRRL 25528 occurred 4–5 d at 30 C after inoculation with the appearance of highly branched, anastomosing hyphae, often giving a knotted appearance. These initials increased in size and became a globose or somewhat elongated, white hyphal mass 8–10 d after inoculation (FIG. 6A). The white hyphal masses that became sclerotia could be distinguished by their more or less rounded appearance, which at maturity were highly irregular in shape and size. Maturation of the young sclerotia was marked by the melanization and hardening of the structure; it occurred 14–21 d after inoculation (FIG. 6B, C). The same highly anastomosing hyphae that formed a white, elongated mass continued to differentiate into 2–5 mm long stipes with conidial heads at the apex, resulting in the formation of synnemata instead of melanized sclerotia (FIG. 6C). Both structures were intermixed on the agar surface. The synnemata eventually withered after several weeks, whereas the sclerotia remained hard and viable. Thin cross sections of the tan to light brown sclerotia of A. caelatus NRRL 26119 revealed an outer region of slightly melanized, thick-walled cells. Next to the 1–2 layers of rind, cells are more or less closely adhering globose cortical cells and a central medulla comprising loosely interwoven hyphal cells, similar to those produced by darkly pigmented sclerotia. The A. caelatus sclerotia became soft after 5–6 mo of incubation, but no ascospores were found.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) and by A. togoense (Wicklow and McAlpin 1990, McAlpin 2001) (see FIG. 1A, B). The production of only sessile sclerotia on MSA, OA or CZA amended with different kinds of starch by both A. caelatus NRRL 25528 and NRRL 26119 contrasts with A. flavus NRRL 29254, which produced exclusively synnemata on those same media. These results showed striking differences in nutrient requirement between A. flavus NRRL 29254 (McAlpin 2001) and both A. caelatus isolates for the initiation of sclerotium formation.
The induction of sclerotial initials was found in this study to be due to the presence of different carbohydrates as well as to the amounts of these compounds in the agar medium. Dextrose, melibiose, sucrose and xylose were found to stimulate sclerotium development in the A. flavus mutant (McAlpin 2001), as reported here for the two A. caelatus isolates. Inulin had no effect on synnema/sclerotium production by the A. flavus mutant (McAlpin 2001); in contrast, A. caelatus NRRL 26119 and NRRL 25528 produced numerous synnemata and sclerotia on inulin-amended CZA. Inulin had no effect on synnema branching by A. flavus mutant NRRL 29254 (McAlpin 2001) and A. caelatus NRRL 25528, but it induced branching in A. caelatus NRRL 26119 (see FIG. 3C). Mannitol and sorbitol not only suppressed sclerotium production but also induced branching of the synnemata in the A. flavus mutant (McAlpin 2001) and in A. caelatus NRRL 26119 but not in NRRL 25528. Some anamorphic species in the genus Penicilliopsis are also known to have branching synnemata (Samson and Seifert 1985). Lower sucrose concentrations (3–6%) stimulated sclerotium formation, but higher concentrations (15–20%) stimulated synnema formation and suppressed sclerotium initiation in the A. flavus mutant (McAlpin 2001) and both A. caelatus isolates.
The importance of the type and amount of nitrogen source for the formation of sclerotia has been well documented in many Aspergilli (Rudolph 1962, Rai et al 1967, Agnihotri 1968, Paster and Chet 1980). However, the regulatory effects of carbon and nitrogen compounds on sclerotium formation are poorly understood (Chet and Henis 1975, Willets and Bullock 1992). Aspartic acid, glutamic acid, serine and threonine, which were found to stimulate sclerotium production in A. flavus NRRL 29254 (McAlpin 2001) and both A. caelatus strains, also stimulated sclerotium production in S. rolfsii (Chet and Henis 1972), presumably because these amino acids are known to be indirectly associated with the tricarboxylic acid cycle (Wang and Le Torneau 1972). Methionine, cysteine and cystine inhibited sclerotium formation in A. flavus NRRL 29254 (McAlpin 2001), A. caelatus NRRL 26119 and NRRL 25528, and S. rolfsii (Henis et al 1973). The latter authors suggested that the sulfydryl (–SH) group or disulfide (–S–S–) bonds act on the cell walls and cellular enzymes, which may suppress development of sclerotium initials by modifying the metabolic processes involved in the normal development of mycelia.
Sodium nitrate proved to be an excellent source of nitrogen for sclerotium formation, whereas ammonium sulfate inhibited the growth, sporulation, and synnema/sclerotium formation in the A. flavus mutant (McAlpin 2001) and in both A. caelatus strains. Ammonium sulfate has been reported to control S. rolfsii under natural conditions (Punja et al 1982, Fang and Liu 1988). The survival of Macrophomina phaseolina (Tassi) Goidanich sclerotia in sandy loam and sandy clay loam soils amended with ammonium sulfate declined more rapidly and drastically than in soils amended with sodium nitrate within 30–40 d of treatment (Filho and Dhingra 1980). Similar reduction in A. flavus propagules may be achieved with application of ammonium sulfate in field soils instead of sodium nitrate.
The amount of C and N affected both sessile and stipitate sclerotium formation in A. caelatus, but the C:N ratio had no significant effect, although the synnema/sclerotium production peaked at a ratio of 8.6. The concentrations of both C and N were important, but both nutrients acted independently. Nitrogen (NaNO3) induced sclerotium production more gradually at higher concentrations (1.5 and 2.0%), whereas sucrose suppressed it abruptly at 12%, most likely due to an osmotic effect. The same conclusions were obtained with the A. flavus mutant NRRL 29254 (McAlpin 2001). However, an increase in C:N ratio stimulated sclerotium formation in other Aspergilli (Rudolph 1962), in S. rolfsii (Wheeler and Sharan 1965) and Verticillium (Wyllie and de Vay 1970).
The developmental differences between the two isolates of A. caelatus (NRRL 25528 and NRRL 26119) and the A. flavus mutant NRRL 29254 (McAlpin 2001) indicated that interaction between the genome and physiological conditions affects synnema and sclerotium formation. The expression of genes for synnema and stipitate sclerotium production depended on the strain and the type and amount of nutrients in the medium. In most strains of A. alliaceus, a close relative of A. flavus (Peterson 1995, 2000; Rigo et al 2002), the sclerotial initials that developed into sclerotia became dark and hard, whereas those that did not differentiate into sclerotia formed a white cottony background on the plate instead of erect structures with conidial heads (synnemata), although some isolates are also capable of making synnemata or synnema-like structures on CZA and other media (unpublished results).
The production of synnemata and stipitate sclerotia in Aspergillus section Flavi are considered to be primitive characteristics (Samson and Seifert 1985). The ability to produce synnemata and stipitate sclerotia by A. flavus was discovered by a chance encounter of a mutant, which indicated that this characteristic might have been a genetic component of the Aspergillus section Flavi (McAlpin 2001). The presence of wild-type strains of Aspergillus species exhibiting the ability to form synnemata and stipitate/ sessile sclerotia demonstrates that these phenotypes also might exist in nature under favorable conditions. The existence of an A. flavus mutant and wild-type strains of A. caelatus, capable of producing morphologically similar but much smaller synnemata and stipitate sclerotia, provides a link between the primitive and more advanced form of Aspergillus section Flavi and further strengthens previous suggestions that Stilbothamnium become a synonym for Aspergillus (Samson and Seifert 1985) and substantiates their placement in the section Flavi (Roquebert and Nicot 1985, Samson and Seifert 1985). The evolution of the synnemata in the Trichocomaceae has been suggested to be toward more compact or reduced forms with the more advanced types possessing enlarged vesicles bearing phialides and conidia, while the more primitive types have more complex and highly branched conidiophores (Malloch and Cain 1972). Presumably, some putative selection pressure against large, showy synnemata occurred as the ancestors (Stilbothamnium) moved from humid habitats in tropical rain-forests to drier environments in agricultural soils and grains (Samson and Seifert 1985). Evaluations of several synnematous and nonsynnematous Aspergillus isolates for genetic divergence by rRNA sequencing analyses of the 28S RNA showed that A. flavus Link, A. flavus var. columnari Raper and Fennell, A. parasiticus Speare and A. coremiformis Bartoli & Maggi were similar and found to be related closely to A. togoensis (Dupont et al 1990). DNA fingerprinting showed distinct hybridization bands in A. togoensis NRRL 13550 and 13551 when tested with pAF28, a repetitive DNA probe derived from A. flavus, indicating some degree of homology between these two fungi (McAlpin 2001). The DNA probe also hybridized with A. caelatus, suggesting a close relationship between this species and A. togoensis (McAlpin 2002).
As was observed in the A. flavus mutant NRRL 29254 (McAlpin 2001), the synnemata and sessile/ stipitate sclerotia in A. caelatus originated from sclerotial initials, which continued to differentiate into either spore-bearing structures or hard sclerotia at maturity (see FIG. 6A–C). This substantiates assertions that certain reproductive structures, such as coremia (synnemata) and sporodochia, have the same developmental characteristics that are shared with sclerotia (Cooke 1983) and further illustrates the similarities of sclerotial development to that of other vegetative structures such as synnemata (Cooke 1983, McAlpin 2001). This study further provides evidence that the synnemata and the hard, melanized or slightly melanized pedicellate sclerotia share the same origin in some Aspergillus species. The mechanism or mechanisms by which sclerotial initials differentiate into either synnemata or sclerotia may be elucidated only at the molecular level. The presence of developmentally regulated proteins in the mycelia, sclerotial initials, sclerotia and conidia have been reported in five Aspergillus species, but their function has not been defined (Novak and Kohn 1990). The A. flavus mutant NRRL 29254 (McAlpin 2001) and two strains of A. caelatus (NRRL 25528 and 26119) could be used as model systems for better understanding of sclerotium morphogenesis and evolution in Aspergilli.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 E-mail: mcalpice{at}mail.ncaur.usda.gov 
2 Names are necessary to report factually on available data. However, the USDA neither guarantees nor warrants the standard of the products, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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