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Synnema and Sclerotium Production in Aspergillus Caelatus and the Influence of Substrate Composition on Their Development in Selected Strains

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Synnema and Sclerotium Production in Aspergillus Caelatus and the Influence of Substrate Composition on Their Development in Selected Strains 059 My cologia, 96(5), 2004, pp. 937-947. © 2004 by The Mycological Society of America, Lawrence, KS 66044-8897 Issued 13 October 2004 Synnema and sclerotium production in Aspergillus caelatus and the influence of substrate composition on their development in selected strains Cesaria E. McAlpin12 and synnematal species A. togoensis, which also pro­ Mycotoxin Research Unit, National Center for duces stipitate sclerotia. Agricultural Utilization Research, USDA, Agricultural Key words: amino acids, C:N ratio, carbohydrates, Research Service, 1815 N. University Street, Peoria, development, morphology, Stilbothamnium Illinois 61604 Abstract: The ability of Aspergillus caelatus, a species I TRODUCTIO in Aspergillus section Flavi, to produce synnemata Synnemata and sclerotia are important morphologi­ and sclerotia was investigated. Forty-eight isolates of cal characters for identifying some species of the As­ A. caelatus differed widely in their production of syn­ pergillus, Penicillium and related genera (Christensen nemata and sclerotia; 83% of the isolates produced 1981, Raper and Fennell 1965, Raper and Thorn varying numbers of synnemata and sclerotia, and 1949). Aspergillus togoensis (Henn.) Samson and Sei­ 17% produced neither sclerotia nor synnemata. Most fert is a tropical fungus that forms tall synnemata, strains produced synnemata and mostly sessile and large sessile or stipitate sclerotia/stromata and yellow few stipitate sclerotia on the same Czapek agar (CZA) to greenish yellow radiate conidial heads. Although plate. Two strains of A. caelatus were selected for fur­ Samson and Seifert (1985) placed this species in As­ ther study because of the contrasting morphology of pergillus subgenus Stilbothamnium, it has been sug­ their synnemata and sclerotia. Those strains are gested as an ancestral form of Aspergillus section Fla­ RRL 25528, the type species and a representative vi (Roquebert and icot 1985, Samson and Seifert of the synnema- and black sclerotium-forming iso­ 1985) . lates, and RRL 26119, considered an atypical strain The ability to produce synnemata and stipitate scle­ that produced numerous synnemata and few slightly rotia in Aspergillus section Flavi was not known until melanized or tan sclerotia. The induction and mat­ recently. McAlpin (2001) first described an A. flavus uration of sclerotia in A. caelatus were affected great­ mutant ( RRL 29254) that produced synnemata and ly by the type of media as well as the kind and con­ stipitate sclerotia on different media, on carbon- or centration of the carbon and nitrogen sources. CZA nitrogen-amended Czapek agar (CZA) and on CZA induced synnema and sclerotium production in both with different concentrations of carbon and nitro­ strains, whereas other media did not. Production of gen. The development of these structures was modi­ abundant synnemata and sclerotia also occurred fied by temperature, light and pH. A synnema (pI. when the carbon source in CZA is replaced with dex­ synnemata) is "a conidioma composed of more or trose, xylose, cellobiose, melibiose and trehalose. less compacted groups of erect and sometimes fused CZA amended with serine, threonine, KN03 and conidiophores bearing conidia at the apex only or NaN03 induced the production of numerous scle­ on both apex and sides" (Hawksworth et al 1995). rotia and synnemata. For both strains, the optimal This description fits the synnema-like structures of levels of sucrose and Na 0 3 for maximum produc­ the A. flavus mutant (McAlpin 2001), which pro­ tion of synnemata or sclerotia were 3 and 0.9%, re­ duced white, erect, intricate stipe with conidia borne spectively. The production of synnemata and stipi­ on both apex and sides, similar to the synnemata pro­ tate/sessile sclerotia by several wild-type strains of A. duced by the Aspergillus subgenus Stilbothamnium, al­ caelatus further substantiates previous suggestions for though much smaller. The stipitate sclerotia resem­ an evolutionary link between Aspergillus section Flavi bled the teleomorphic stage of the genus Penicilliop­ sis according to descriptions by Samson and Seifert Accepted for publication March 1, 2004. (1985) . I E-mail: [email protected] Aspergillus caelatus B.W. Horn, a recently described 2 ames are necessary to report factually on available data. How­ species in Aspergillus section Flavi isolated from ag­ ever, the USDA neither guarantees nor warrants the standard of ricultural field soils and insect-damaged peanut seeds the products, and the use of the name by USDA implies no ap­ proval of the product to the exclusion of others that may also be in the Southern United States (Horn 1997, Horn and suitable. Dorner 1998) as well as from tea field soils in Japan 937 938 MYCOLOCIA ducing mutant strain of A. flavus NRRL 29254 (McAlpin 2001), the A. caelatus strains described in this study are wild types. Sclerotia are important survival structures in the life cycle of many fungi. Studies on the conditions responsible for sclerotium initiation might be impor­ tant in developing methods for suppressing the for­ mation of sclerotia, resulting in reduced survival of the fungus and better disease management. Willets and Bullock (1992) reviewed the development of sclerotia, mainly in Sclerotinia sclerotiorum (Libert) de Bary and Sclerotium rolfsii Saccardo, and observed that relatively few studies described the development of sclerotia in the genera Aspergillus and Penicillium. In this study, the nutritional factors critical to syn­ nema/sclerotium initiation and maturation of some strains of A. caelatus were compared with those for the mutant A. flavus RRL 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 scle­ rotium production as morphological bases for eval­ uating the relationship among A. caelatus, A. flavus and A. togoensis. MATERIALS AND METHODS Fungal strains.-Forty-three strains of A. caelatus obtained from the RRL Culture Collection at the USDA, ARS, a­ tional Center for Agricultural Utilization Research in Pe­ oria, Illinois, are listed in TABLE 1. Media and cultural conditions.-Preliminary experiments were undertaken to determine the best agar medium, pH, temperature and light versus dark conditions for synnema and sclerotium production in A. caelatus based on proce­ dures previously described for A. flavus (McAlpin 2001). The relative humidity inside the incubation boxes was checked (Traceable Memory Hygrometer, Control Co., Friendswood, Texas) and maintained within 60-80%. Mter FIc. 1. A. Sclerotia of A. flavus mutant RRL 29254 these conditions were determined, each of the 48 A. cae­ (small stipitate) and A. togoense RRL 13550 (large stipitate latus strains was inoculated centrally on four replicate CZA and sessile) sclerotia from Czapek agar (CZA) and oatmeal plates with 3 f..LL of spore suspension (1 X 105 spores/mL agar (OA), respectively. B. A. caelatus RRL 25528 with ir­ in 0.1 % agar) obtained from 5 d old CZA slants, then regularly shaped stipitate and sessile sclerotia from CZA at placed in plastic boxes, covered with aluminum foil and 30 C. C. A. caelatus RRL 26119 with sessile sclerotia pro­ incubated at 30 C. All 48 isolates also were grown on Mu­ duced on Murashige-Skoog agar (MSA). Bars: A, B, C = rashige-Skoog agar (MSA) for comparison because this me­ 500 f..Lm. dium induced only synnema production in the A. flavus mutant RRL 29254 (McAlpin 2001). (Peterson et al 2000), was found to produce both Another preliminary study was conducted on the nutri­ tional requirements of four representative A. caelatus synnemata and sessile and stipitate sclerotia on the strains with black sclerotia ( RRL 26114, 25528, 26105, same CZA plate (personal observation). As in the A. 25577), using two replicate plates for each isolate. Their flavus mutant (McAlpin 2001), the synnemata and response to different media and different concentrations of stipitate sclerotia produced by A. caelatus, resembled carbon and nitrogen on CZA were similar regardless of the those of A. togoensis in miniature form (see FIc. lA, number of synnemata/sclerotia produced by each strain B). Unlike the synnema- and stipitate sclerotium-pro- (data not shown). Two strains ( RRL 26108, 25568) that McALPI : SCLEROTIUM PROD CTIO 1 I ASPERGILLUS CAELATUS 939 TABLE I. Sporulation and production of sclerotia and synnemata by Aspergillus caelatus isolates on Czapek agar (CZA) and Murashige-Skoog Agar (MSA) 14-21 d after inoculation CZA MSA Isolate and source Sporulationa Sclerotiab Synnematab Sclerotiab RRL 25404-soil, japan, 1994 ++ + ++ + RRL 25528-soil, Georgia, 1992 ++ +++ +++ + RRL 25566--soil, japan, 1995 ++ ++ +++ + RRL 25567-soil, japan, 1995 ++ ++ ++ RRL 25568-soil, japan, 1995 +++ RRL 25569-soil, japan, 1995 +++ RRL 25571-soil, japan, 1995 + ++ ++ + RRL 25576--soil,japan, 1996 ++ +++ + + RRL 25577-soil, japan, 1996 ++ + ++ + RRL 26015-soil, Louisiana, 1993 +++ +++ RRL 26017-soil, Mississippi, 1993 ++ RRL 26100-soil, Georgia, 1992 ++ ++ ++ ++ RRL 26101-soil, Georgia, 1992 + + + RRL 26102-soil, Georgia, 1992 +++ + RRL 26103-soil, Georgia, 1992 + NRRL 26104-soil, Georgia, 1992 ++ ++ + ++ RRL 26105-soil, Georgia, 1992 ++ +++ +++ + RRL 26106--soil, Georgia, 1992 ++ +++ +++ ++ RRL 26107-soil, Georgia, 1992 +++ + + ++ RRL 26108-soil, Georgia, 1992 +++ RRL 26109-soil, Georgia, 1992 ++ +++ +++ ++ RRL 26110-soil, Georgia, 1992 + +++ +++ ++ RRL 26111-soil, Georgia, 1992 + + + ++ +
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