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

059

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 + + + ++ + RRL 26112-soil, Georgia, 1992 + ++ ++ + RRL 26113-soil, Georgia, 1992 + +++ +++ + RRL 26114-soil, Georgia, 1992 ++ +++ + ++ RRL 26115-soil, Georgia, 1992 +++ + + RRL 26116--soil, Georgia, 1992 + RRL 26117-soil, Georgia, 1992 ++ + RRL 26118-soil, Georgia, 1992 + ++ + RRL 26119-soil, Georgia, 1992 + ++ +++ RRL 26120-soil, Georgia, 1992 ++ ++ ++ RRL 26121-soil, Georgia, 1992 ++ +++ +++ ++ RRL 26122-soil, Georgia, 1992 + + + RRL 26123-soil, Georgia, 1992 ++ +++ +++ + RRL 26124-soil, Georgia, 1992 ++ +++ +++ + RRL 26125-peanut seed, Georgia, 1992 ++ +++ +++ + NRRL 26126--peanut seed, Georgia, 1992 +++ +++ +++ + NRRL 26127-peanut seed, Georgia, 1992 +++ + + NRRL 26128-peanut seed, Georgia, 1992 +++ +++ ++ RRL 26129-peanut seed, Georgia, 1992 ++ +++ +++ ++ RRL 26130-soil, Georgia, 1992 ++ + ++ RRL 26306--peanut, Texas, 1970 +++ ++ + RRL 26578-soil, japan, 1993 +++ NRRL 26582-soil, japan, 1993 +++ + RRL 26587-soil, japan, 1993 RRL 26592-soil, japan, 1993 +++ RRL 26593-soil, japan, 1993 +++ +

a +++ = abundant, = moderate, + = few. b + + + = >500/plate, + + = 100-500, + = <100, - = not produced. Four replicates/isolate. 940 MYCOLOGlA

l'I:l 5000 NRRL 26119 II NRRL 26119 Ell NRRL 25528 NRRL25528 ~ ~ ell I • Sclerotia II . Synnemata • Sclerotia O• Synnemata "0 en 3 4000 l'I:l E ell c: c: 3000 en>0- ..: ell JJ 2000 E ::J Z c: 1000 l'I:l ell :E a

FIc. 2. Synnema/sclerotium production on different carbon sources and standard deviation of the mean per plate from four replicates by A. caelatus RRL 25528 and TRRL 26119. Cellulose and 0 Carbon did not produce synnemata or sclerotia. M = monosaccharide, D = disaccharide, P = polysaccharide. did not produce sclerotia on CZA also produced no scle pounds (see FIc. 4). CZA without nitrogen served as the rotia on CZA amended with different carbon or nitrogen control. All media were adjusted to pH 7 with 6 HCI or sources. Therefore, only the type species ( RRL 25528), 5 T aOH. which is fairly representative of the black sclerotium-form ing isolates, and an atypical isolate ( RRL 26119), which Carbon and nitrogen concentration.-The effects of carbon produced slightly melanized or tan sclerotia and numerous and nitrogen concentration in CZA on the formation of short synnemata and few sclerotia, were selected for further synnematalsclerotia were determined by varying the investigation because of their contrasting morphological amount of sucrose from 0 to 20% with a fixed 3 giL nitro characteristics. gen, and the amount of aN03 was varied from 0 to 2.0% These media were prepared for synnema and sclerotium with 30 giL of sucrose. production according to Booth (1971) and Atlas (1993) ex C:N ratio.-The amount of a 0 3 in CZA was varied at cept where the commercial brand or source is indicated: 0.0, 0.1, 0.3, 0.6, 0.9, 1.2, 1.5 and 2.0% and paired in all Coon's medium (CM), cornmeal agar (CMA) (Difco, De possible combinations with different sucrose concentrations troit, Michigan), Czapek agar (CZA) , complete medium at 0, 1, 3, 6, 9, 12, 15 and 20% in 200 mL aliquots. This (GYM) (CZA 0.25% yeast extract + 0.75% malt extract), gives g C : g ratios from approximately 1.4 to 500 in which malt-extract agar (MEA), Murashige and Skoog basal me the basal medium (CZA) is approximately 25.7. dium (Sigma Chemical Co., St. Louis, Missouri) plus 3% sucrose and 1.5% agar, oatmeal agar (OA), potato-dextrose Microscopic studies.-Development of sclerotia was observed agar (PDA) , potato-dextrose agar + 0.5% yeast extract at different intervals on CM, CZA and MSA plates under (PDAYE), and V8juice agar (V8). Four replicate 90 mm the light microscope. At various intervals up to 7 mo, sessile diam plastic plates were center-point inoculated with 3 J.LL and stipitate sclerotia were sectioned or crushed and ex of spore suspension (1 X 105 spores/mL of 0.1 % water amined for the presence of ascospores. agar) of A. caelatus RRL 25528 or RRL 26119. Growth, sporulation and synnema/sclerotium production were ob served every 2 or 3 d for 2 wk and weekly thereafter for 6 RES LTS wk in all subsequent experiments. Preliminary experiments on the effects of pH, light Carbon and nitrogen sources.-CZA, consistmg of 3 g and temperature for A. caelatus showed that optimal aN03 , 0.5 g MgS04, 0.5 g KCI, 0.01 g FeS04 ,7H20, 1 g synnema/sclerotium formation occurred between KH2P04 , 30 g sucrose and 15 g agar, was used as the basal pH 6 and 10, that dark conditions were better than medium. The 30 g sucrose with approximately 42% avail light, that 28-30 C promoted synnema and sclero able carbon equivalent to 12.62 g C/L was replaced in the tium production and that 60-80% RH was necessary medium with equivalent amounts of carbon from 22 differ for the formation of conidia on the synnemata (data ent sources: seven monosaccharides, seven disaccharides and eight polysaccharides (see FIc. 2). A control medium not shown). The ability of the 48 A. caelatus isolates with no carbon source was included. Similarly, the 3 g of to produce synnemata and sclerotia was examined

a 0 3 with approximately 16.5% available nitrogen equiv based on the conditions obtained from this prelimi alent to 0.49 g IL in the basal medium was replaced with nary work. Of the 48 isolates, 40 (83%) produced 22 amino acids and seven other nitrogen-containing com- synnemata and sclerotia on the same CZA plate (TA- McALPIN: SCLEROTIUM PRODUCTIO IN ASPERGILLUS CAELATUS 941

TABLE II. Formation of sclerotia and synnemata on different microbiological media by A. caelatus RRL 25528 and 26119

RRL 25528 Agar media Synnemata Sclerotia Synnemata Sclerotia Coon's medium (CM) 0.0 553.3 ± 46.0a 103.0 ± 16.0 2537.2 ± 247.7 Cornmeal agar (CMA) 0.0 0.0 0.0 0.0 Complete medium (CYM) 0.0 0.0 0.0 260.3 ± 9.6 Czapek agar (CZA) 440.3 ± 77.3 1884.8 ± 260.0 2355.0 ± 183.7 1068.3 ± 124.0 Malt-extract agar (MEA) 0.0 0.0 0.0 0.0 Murashige-Skoogs agar (MSA) 0.0 208.0 ± 26.1 0.0 241.3 ± 12.8 Oatmeal agar (OA) 0.0 144.3 ± 45.1 0.0 269.5 ± 36.5 Potato-dextrose agar (PDA) 14.0 ± 3.4 0.0 291.5 ± 46.9 5.3 ± 3.4 PDA yeast extract (PDAYE) 27.5 ± 12.7 0.0 92.3 ± 13.0 0.0 V8 Juice agar (V8) 0.0 0.0 0.0 0.0

a Mean values per plate from four replicates and standard deviation of the mean.

BLE I), 39 produced black sclerotia and one isolate to light brown ovoid sclerotia at the apex, while the ( RRL 26119) produced what appeared at first to be remainder withered and desiccated. All tan sclerotia exclusively synnemata on CZA. However, after 21 d were sessile on CM, CYM, MSA and OA (FIc. 1C). A of incubation, some were found to produce hard and few synnemata and sclerotia were formed by RRL slightly melanized or tan ovoid to irregularly shaped 26119 on PDA but none on CMA, MEA, PDAYE and sclerotia at the apex. On MSA, 34 of the isolates pro V8. duced only sessile sclerotia but no synnemata, a sin The monosaccharides arabinose, dextrose and xy gle isolate ( RRL 26306) produced a few (less than lose and the disaccharides cellobiose, melibiose, su 100 per plate) synnemata with several sessile sclero crose and trehalose stimulated the most synnema/ tia, and the remaining isolates produced neither syn sclerotium formation in RRL 25528 (FIc. 2). CZA nemata nor sclerotia. Two types of conidia were pro amended with polysaccharides, including apple pec duced: (i) the typical conidia formed on conidio tin, citrus pectin or starch, induced only sessile scle phores on the agar surface and (ii) conidia arising rotium formation and completely inhibited synnema from the apex of synnema stalks. In general, the formation, except for inulin, which supported for amount of sporulation on the agar surface was in mation of synnemata and abundant sclerotia for both versely proportional to the number of synnemata/ isolates. RRL 26119 produced numerous synnemata sclerotia produced, e.g., moderate sporulation sup on most of the carbon sources tested, including arab ported moderate (approximately 200-500 sclerotia/ inose, dextrose, glycerol, maltose, melibiose, sorbitol, plate) to abundant sclerotium production (>500 sucrose, trehalose and xylose (FIc. 3A), with some sclerotia/plate) 14-21 d after inoculation. Beyond synnemata producing pedicellate sclerotia at maturi this period, the amount of surface sporulation in ty. Inulin, mannitol and sorbitol stimulated branch most isolates continued to increase during incuba ing of the synnemata produced by RRL 26119 (FIc. tion, and the synnemata and sclerotia often became 3B, C). Cellulose-amended CZA induced sparse spor covered with surface conidia. ulation and inhibited synnema/sclerotium forma The ability of A. caelatus RRL 25528 and RRL tion. 26119 to produce synnemata and sclerotia was tested The development of synnemata and sclerotia also on different agar media (TABLE II). The best medium was affected by different kinds of nitrogen sources. for both synnema and sclerotium production by Serine, threonine, potassium nitrate and sodium ni NRRL 25528 was CZA in which 5-10% of the scle trate induced the most sclerotium formation in A. rotia from a single plate were stipitate (FIc. 1B). A caelatus RRL 25528 and synnema formation in few synnemata and sessile sclerotia were produced on RRL 26119, whereas cysteine, cystine, leucine, ly PDA and PDAYE, whereas only sessile sclerotia were sine methionine, phenylalanine tryptophan, tyrosine produced on CM, MSA and OA. Abundant spores and valine were inhibitory to both isolates (FIc. 4). but no synnemata or sclerotia were produced on Asparagine, aspartic acid, glutamic acid, glycine and CMA, CYM, MEA and V8 by RRL 25528. RRL proline supported the production of moderate num 26119 produced numerous short (1-3 mm) synne bers of sclerotia ( RRL 25528) and synnemata mata with conidial heads on CZA; some of these (NRRL 26119), whereas relatively few synnemata or heads later produced hard, slightly pigmented or tan sclerotia were produced on alanine, arginine, gluta- 942 MYCOLOCIA

Optimal induction of synnema/sclerotium forma tion in RRL 25528 occurred at 3% sucrose; at 6%, approximately equal numbers of synnemata and scle rotia 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 (FIc. 5A). Maximum counts of synnemata and sclerotia with RRL 26119 were obtained at 3% sucrose, gradually decreasing at 6-12%, whereas at 15-20%, only a few synnemata but no sclerotia were formed (FIc. 5B). The optimal nitrogen concentration for sclerotium

production in RRL 25528 was 0.9% 0 3 ; the least favorable was 0.1 % and the numbers of synne mata and sclerotia were reduced greatly above 0.9%

aN03 (FIc. 5C). RRL 26119 produced numerous synnemata but substantially fewer sclerotia than RRL 25528 in all the concentrations tested. The highest counts of synnemata and sclerotia were re

corded at 0.9% 0 3, the number declining at higher concentrations (1.2-2.0%) (FIc. 5D). Sclero

tia at 0.9% 0 3 were noticeably more elongated than irregularly shaped. The maximum production for both synnemata (2800/plate) and sclerotia (1680/plate) by the A. caelatus isolates RRL 25528 and RRL 26119 oc curred when a 3% C concentration and a 0.9% were used, equivalent to a C: ratio of 8.6. However, a similar ratio using different amounts of C and

(1 % sucrose/0.3% a 0 3 and 6% sucrose/2%

a 0 3 ) induced moderate numbers «500/plate) of synnemata and sclerotia. The common observa tions for C: ratio that confirmed the results ob tained above follow. For any fixed concentration of , the numbers of synnemata/sclerotia increased with the C concentration reaching a maximum at 3% and then decreased as the concentration was in creased further. Similarly, for any fixed value of C, the numbers of synnemata/sclerotia increased as the FIc. 3. Synnemata of A. caelatus RRL 26119 from dif concentration increased until a maximum value ferent media. A. Typical synnemata produced on Czapek was reached and then declined with further increases agar (CZA) 15 d after inoculation B. Branching synnemata in 0 interactive effects between C and were on CZA amended with sorbitol 12 d after inoculation. C. observed. One major difference between the two iso Branching synnemata on CZA amended with inulin at 15 lates was that sclerotium production was much great d, showing some tips turning into slightly melanized scle er than synnema production in RRL 25528, where rotia. Bars: A, B, C = 500 J.Lm. as synnema production was much greater than scle rotium production in NRRL 26119 and that RRL mine, histidine, hydroxyproline, isoleucine, ammo 26119 produced much fewer sclerotia in all concen nium tartrate and urea. A slight increase in pedicel trations of C and late sclerotium production (11-15% of total) and Sclerotium initiation in RRL 25528 occurred 4 slightly longer synnemata (3-6 mm) were produced 5 d at 30 C after inoculation with the appearance of on CZA amended with serine, arginine and glutamic highly branched, anastomosing hyphae, often giving acid than on regular CZA. Ammonium sulfate inhib a knotted appearance. These initials increased in size ited growth, sporulation and synnema/sclerotium and became a globose or somewhat elongated, white formation by both isolates. hyphal mass 8-10 d after inoculation (FIc. 6A). The McALPI T: SCLEROTI M PRODUCTIO T I ASPERGILLUS CAELATUS 943 eco CIl 4000 NRRL 26119 IllII NRRL 26119 ~ NRRL 25528 NRRL25528 13 ~ II) I • Sclerotia IllIl . Synnemata • Sclerotia O• Synnemata ] co 3000 E CIl c: c: >- 2000 II)...- CIl .c E 1000 ::l Z c: co CIl 0 :i: $·~~~t¢-~0'f1 -!!I -!!I.' -!!I;!.' ~ ~ I '" ~ ....o0 § § ~ S § S ~ ~ :Q. ~ ~ ..; ".)0 .; :f' § 0 o s ~f

FIG. 4. Synnema and sclerotium production on different nitrogen sources and standard deviation of the mean per plate from four replicates by A. caelatus NRRL 25528 and RRL 26119. Cysteine, Cystine, Leucine, Lysine, Methionine, Phenyl alanine, Tryptophan, Tyrosine, Valine, and No Nitrogen did not produce synnemata/sclerotia or produced negligible num bers.

4000 4000 ....------, A. NRRL 25528 0 B. NRRL 26119 o Synnemata Synnemata C'Il C'Il :;:; ...... ~ 3000 m ... e 3000 m C1l C1l Sclerotia C1l C1l Sclerotia .0- ,D- E~ E~ ::::l- z!!l 2000 ~~ 2000 c: C'Il c: C'Il C'Il E C'Il E C1l C1l C1l C1l :E~ :EC:c: >- >- CJ) 1000 CJ) 1000

0 1.0 2.0 3.0 6.0 9.0 12.0 15.0 20.0 1.0 2.0 3.0 6.0 9.0 12.0 15.0 20.0 % Sucrose % Sucrose

4000 4000 C. NRRL 25528 0 D. NRRL 26119 0 Synnemata Synnemata C'Il C'Il ...... ~... 3000 m ...... :g 3000 m C1l C1l Sclerotia C1l C1l Sclerotia .0- ,D- E~ E~ ::::l- ::::l- z!!l 2000 z!!l 2000 ... c: C'Il c: C'Il C'Il E C'Il E C1l C1l C1l C1l :EC:c: :E~ >- >- f0- CJ) 1000 CJ) 1000 ~~ 0 o nDa I~ II I 0.1 0.2 0.3 0.6 0.9 1.2 1.5 2.0 0.1 0.2 0.3 0.6 0.9 1.2 1.5 2.0

% Nitrogen (NaN03) % Nitrogen (NaN03)

FIG. 5. Synnema and sclerotium formation by A. caelatus at different sucrose or nitrogen ( 03) concentrations and standard deviation of the mean per plate from four replicates. A. RRL 25528 B. RRL 26119 C. NRRL 25528 D. RRL 26119. 944 MYCOLOCIA

formed a white, elongated mass continued to differ entiate into 2-5 mm long stipes with conidial heads at the apex, resulting in the formation of synnemata instead of melanized sclerotia (FIc. 6C). Both struc tures were intermixed on the agar surface. The syn nemata 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 RRL 26119 revealed an outer region of slightly melanized, thick-walled cells. ext to the 1 2 layers of rind, cells are more or less closely adher ing globose cortical cells and a central medulla com prising loosely interwoven hyphal cells, similar to those produced by darkly pigmented sclerotia. The A. caelatus sclerotia became soft after 5-6 mo of in cubation, but no ascospores were found.

DISCUSSIO Many A. caelatus strains differed widely in their ca pabilities for synnema and sclerotium formation, and results indicate that the initiation of sclerotium de velopment depended on various nutrients in the agar medium. A. caelatus strains produced synnemata and stipitate/sessile sclerotia on the same CZA plate, which resembled the structures produced by the A. flavus mutant (McAlpin 2001) and by A. togoense (Wicklow and McAlpin 1990, McAlpin 2001) (see FIc. lA, 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 RRL 26119 contrasts with A. flavus RRL 29254, which produced exclusively synnemata on those same me dia. These results showed striking differences in nu trient 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 carbo FIG. 6. Sclerotium development in A. caelatus RRL hydrates as well as to the amounts of these com 25528 A. White hyphal mass of globose or slightly elongated pounds in the agar medium. Dextrose, melibiose, su sclerotial/synnematal structures 9 d after inoculation on crose and xylose were found to stimulate sclerotium Czapek agar (CZA). B. More elongated young synnemata development in the A. flavus mutant (McAlpin and darkening sclerotia (with water droplets) at 14 d. C. 2001), as reported here for the two A. caelatus iso Mature black sclerotia and synnemata bearing mature co lates. Inulin had no effect on synnema/sclerotium nidia at 21 d. syn = synnemata, scl = sclerotia. Bars: A, B, production by the A. flavus mutant (McAlpin 2001); C = 500 ~m. in contrast, A. caelatus NRRL 26119 and RRL 25528 produced numerous synnemata and sclerotia on in white hyphal masses that became sclerotia could be ulin-amended CZA. Inulin had no effect on synnema distinguished by their more or less rounded appear branching by A. flavus mutant TRRL 29254 (Mc ance, which at maturity were highly irregular in Alpin 2001) and A. caelatus RRL 25528, but it in shape and size. Maturation of the young sclerotia was duced branching in A. caelatus RRL 26119 (see FIc. marked by the melanization and hardening of the 3C). Mannitol and sorbitol not only suppressed scle structure; it occurred 14-21 d after inoculation (FIc. rotium production but also induced branching of the 6B, C). The same highly anastomosing hyphae that synnemata in the A. flavus mutant (McAlpin 2001) McALPI : SCLEROTIUM PRODUCTION IN ASPERGILLUS CACLA TUS 945 and in A. caelatus RRL 26119 but not in NRRL (Na 03) induced sclerotium production more grad 25528. Some anamorphic species in the genus Peni ually at higher concentrations (1.5 and 2.0%), where cilliopsis are also known to have branching synnemata as sucrose suppressed it abruptly at 12%, most likely (Samson and Seifert 1985). Lower sucrose concen due to an osmotic effect. The same conclusions were trations (3-6%) stimulated sclerotium formation, but obtained with the A. flavus mutant RRL 29254 higher concentrations (15-20%) stimulated synnema (McAlpin 2001). However, an increase in C:N ratio formation and suppressed sclerotium initiation in the stimulated sclerotium formation in other Aspergilli A. flavus mutant (McAlpin 2001) and both A. caela (Rudolph 1962), in S. rolfsii (Wheeler and Sharan tus isolates. 1965) and Verticillium (Wyllie and de Yay 1970). The importance of the type and amount of nitro The developmental differences between the two gen source for the formation of sclerotia has been isolates of A. caelatus (NRRL 25528 and RRL well documented in many Aspergilli (Rudolph 1962, 26119) and the A. flavus mutant RRL 29254 Rai et al 1967, Agnihotri 1968, Paster and Chet (McAlpin 2001) indicated that interaction between 1980). However, the regulatory effects of carbon and the genome and physiological conditions affects syn nitrogen compounds on sclerotium formation are nema and sclerotium formation. The expression of poorly understood (Chet and Henis 1975, Willets genes for synnema and stipitate sclerotium produc and Bullock 1992). Aspartic acid, glutamic acid, ser tion depended on the strain and the type and ine and threonine, which were found to stimulate amount of nutrients in the medium. In most strains sclerotium production in A. flavus RRL 29254 of A. alliaceus, a close relative of A. flavus (Peterson (McAlpin 2001) and both A. caelatus strains, also 1995, 2000; Rigo et al 2002), the sclerotial initials that stimulated sclerotium production in S. rolfsii (Chet developed into sclerotia became dark and hard, and Henis 1972), presumably because these amino whereas those that did not differentiate into sclerotia acids are known to be indirectly associated with the formed a white cottony background on the plate in tricarboxylic acid cycle (Wang and Le Torneau 1972). stead of erect structures with conidial heads (synne Methionine, cysteine and cystine inhibited sclerotium mata) , although some isolates are also capable of formation in A. flavus RRL 29254 (McAlpin 2001), making synnemata or synnema-like structures on A. caelatus RRL 26119 and RRL 25528, and S. CZA and other media (unpublished results). rolfsii (Henis et al 1973). The latter authors suggested The production of synnemata and stipitate sclero that the sulfydryl (-SH) group or disulfide (-S-S-) tia in Aspergillus section Flavi are considered to be bonds act on the cell walls and cellular enzymes, primitive characteristics (Samson and Seifert 1985). which may suppress development of sclerotium ini The ability to produce synnemata and stipitate scle tials by modifying the metabolic processes involved rotia by A. flavus was discovered by a chance en in the normal development of mycelia. counter of a mutant, which indicated that this char Sodium nitrate proved to be an excellent source acteristic might have been a genetic component of of nitrogen for sclerotium formation, whereas am the Aspergillus section Flavi (McAlpin 2001). The monium sulfate inhibited the growth, sporulation, presence of wild-type strains of Aspergillus species ex and synnema/sclerotium formation in the A. flavus hibiting the ability to form synnemata and stipitate/ mutant (McAlpin 2001) and in both A. caelatus sessile sclerotia demonstrates that these phenotypes strains. Ammonium sulfate has been reported to con also might exist in nature under favorable conditions. trol S. rolfsii under natural conditions (Punja et al The existence of an A. flavus mutant and wild-type 1982, Fang and Liu 1988). The survival of Macro strains of A. caelatus, capable of producing morpho phomina phaseolina (Tassi) Goidanich sclerotia in logically similar but much smaller synnemata and sandy loam and sandy clay loam soils amended with stipitate sclerotia, provides a link between the prim ammonium sulfate declined more rapidly and dras itive and more advanced form of Aspergillus section tically than in soils amended with sodium nitrate Flavi and further strengthens previous suggestions within 30-40 d of treatment (Filho and Dhingra that Stilbothamnium become a synonym for Aspergil 1980). Similar reduction in A. flavus propagules may lus (Samson and Seifert 1985) and substantiates their be achieved with application of ammonium sulfate in placement in the section Flavi (Roquebert and icot field soils instead of sodium nitrate. 1985, Samson and Seifert 1985). The evolution of the The amount of C and affected both sessile and synnemata in the Trichocomaceae has been suggest stipitate sclerotium formation in A. caelatus, but the ed to be toward more compact or reduced forms with C: ratio had no significant effect, although the syn the more advanced types possessing enlarged vesicles nema/sclerotium production peaked at a ratio of8.6. bearing phialides and conidia, while the more prim The concentrations of both C and were important, itive types have more complex and highly branched but both nutrients acted independently. Nitrogen conidiophores (Malloch and Cain 1972). Presumably, 946 MYCOLOCIA

some putative selection pressure against large, showy LITERAT RE CITED synnemata occurred as the ancestors (Stilbotham nium) moved from humid habitats in tropical rain Agnihotri VP. 1968. Effects of nitrogenous compounds on sclerotium formation in Aspergillus niger. Can J Micro forests to drier environments in agricultural soils and bioI 14:1253-1258. grains (Samson and Seifert 1985). Evaluations of sev Atlas R.M. 1993. Handbook of microbiological media. Boca eral synnematous and nonsynnematous Aspergillus Raton, Florida: CRC Press. 1079 p. isolates for genetic divergence by r A sequencing Booth C. 1971. Fungal culture media. In: Booth C, ed. analyses of the 28S RNA showed that A. flavus Link, Methods in microbiology. Vol. IV. London: Academic A. flavus var. columnan Raper and Fennell, A. par Press. p 49-94. asiticus Speare and A. coremiformis Bartoli & Maggi Chet I, Henis Y 1972. 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S pp ied 'ne .5. Department of Agriculture, National Center for Agricultural Utilization Research, Peoria, Illinois