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National Peanut Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Dawson, Georgia 39842
Stephen W. Peterson
National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, Peoria, Illinois 61604
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The genus Penicillium comprises species that mostly colonize plant matter. However early reports suggest that several species are capable of parasitizing Aspergillus and sporulating on the conidial heads of the host. More recently Eupenicillium ochrosalmoneum and E. cinnamopurpureum, both with Penicillium anamorphs, have been observed sporulating on the heads of Aspergillus species belonging to section Flavi during the colonization of peanut seeds. Little is known about the host specificity underlying these Aspergillus-Penicillium associations. In this study Aspergillus species representing nine taxonomic sections were paired in culture with E. ochrosalmoneum, E. cinnamopurpureum and two unnamed Penicillium species. Eupenicillium ochrosalmoneum, E. cinnamopurpureum and Penicillium sp. 1 sporulated predominantly on the heads of section Flavi species. In contrast Penicillium sp. 2 was restricted to the heads of section Nigri species. All species spread across Aspergillus colonies by means of aerial hyphae that grew from head to head. Additional studies are required to clarify whether Eupenicillium and Penicillium species are parasitic or simply epibiotic on their hosts.
Key words: Aspergillus flavus, Aspergillus niger, epibiosis, mycoparasitism, Trichocomaceae
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Aspergillus niger was parasitized by P. rugulosum during the fermentation process, resulting in host death in the infected areas and reduced citric acid yield. Infection involved both external and intracellular hyphal growth of P. rugulosum up the host conidiophore to the upper stipe and head of A. niger, where sporulation by the parasite occurred in a radiate manner (Thom and Raper 1945).
Host specificity in Aspergillus-Penicillium associations has not been examined in a systematic manner. Species of Aspergillus from section Flavi (A. flavus, A. oryzae and A. tamarii) and section Nigri (A. niger) are reported to be parasitized by P. rugulosum and P. purpurogenum from the subgenus Biverticillium (Raper and Thom 1949, Tatarenko 1959, Pitt 1979). Thom and Raper (1945) stated without elaboration that parasitism of Aspergillus by P. rugulosum depends on the host species and even the strain within a single species. Pitt (1979) in his treatment of P. purpurogenum listed two parasitic strains, one from A. oryzae and the other from A. niger, but it is not known whether the strains have different host specificities.
Eupenicillium ochrosalmoneum (anamorph = P. ochrosalmoneum) and E. cinnamopurpureum (anamorph = P. cinnamopurpureum) recently were described as sporulating on the conidial heads of section Flavi species (Horn 2005, 2006). Peanut seeds were wounded and the wounds were inoculated with soil directly from the field. In those experiments Aspergillus and Eupenicillium species both originated from soil and became associated during seed colonization. Some degree of host specificity was observed on the seeds based on observations that the two Eupenicillium species did not appear to sporulate on the heads of Aspergillus species outside of section Flavi.
For this study host specificity in E. ochrosalmoneum, E. cinnamopurpureum, and two unnamed Penicillium species was examined in culture using Aspergillus species belonging to different taxonomic sections.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) and are deposited in the Agricultural Research Service Culture Collection (NRRL), Peoria, Illinois, USA. Eupenicillium ochrosalmoneum, E. cinnamopurpureum, Penicillium sp. 1 and Penicillium sp. 2 were isolated from the conidial heads of Aspergillus species during the course of experiments involving soil dilution plating, plating of surface-sterilized peanut seeds and corn kernels, and inoculation of wounded peanut seeds with field soils (Horn et al 1994, 1995; Horn and Dorner 1998; Horn 2005, 2006). For Aspergillus species tested as possible hosts, strains showing good sporulation, including the type strains, were chosen from Peterson (2000) in which the rDNA gene was sequenced for establishing phylogenetic relationships within the genus. Strains of Aspergillus species from Peterson (2000) that sporulated poorly were substituted with representative strains for the species.
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). After inocula had dried on the agar medium surface (<3 h), plates were re-inoculated with a loopful of Eupencillium or Penicillium species (106/mL) directly over each Aspergillus inoculation point. Eupenicillium ochrosalmoneum and Penicillium sp. 2 overgrew both A. versicolor and A. sydowii; therefore plates inoculated with those Aspergillus species were incubated 15–25 h (30 C) before inoculation with E. ochrosalmoneum and Penicillium sp. 2. Plates were incubated in the dark at 30 C in trays enclosed by plastic bags to prevent drying. Colonies were examined under the stereomicroscope after 7, 14 and 21 d. All host specificity experiments were performed twice. Digital images of Aspergillus heads with sporulating Eupenicillium and Penicillium species were obtained with a Leica MZ16FA stereomicroscope equipped with a DFC-300FX digital camera. Stacked images were combined using Image Pro Express v.5.0 software module.
The ability of conidia of Eupenicillium and Penicillium species to germinate on the heads of susceptible Aspergillus hosts also was examined. Conidial suspensions (5 x 105/mL) of A. flavus NRRL 1957, A. parasiticus NRRL 29555, and A. caelatus NRRL 25528 were spread onto plates of malt extract agar (Raper and Fennell 1965) (0.2 mL/plate), which resulted in a dense lawn of conidial heads. After 8 d of incubation (30 C), dry conidia of E. ochrosalmoneum NRRL 35496, E. cinnamopurpureum NRRL 35500 and Penicillium sp. 1 NRRL 35504 from CZ slants were removed with a transfer needle and sprinkled onto the Aspergillus colonies. Plates of A. niger NRRL 326, A. tubingensis NRRL 35599 and A. aculeatus NRRL 5094 were inoculated with Penicillium sp. 2 NRRL 35507 in a similar manner. Aspergillus heads were examined with a stereomicroscope under high magnification after 3, 6 and 10 d of incubation at 30 C.
Light microscopy.— – Aspergillus stipes from heads showing sporulation with Eupenicillium and Penicillium species also were examined under phase-contrast microscopy (400x) for evidence of parasitism after 5, 7 and 10 d of incubation on mCMA at 30 C. Fungal strain combinations and numbers of stipes examined (in parentheses) were: E. ochrosalmoneum NRRL 35496 + A. flavus NRRL 29525 (89) or A. parasiticus NRRL 29555 (74) or A. parasiticus NRRL 29568 (62); E. cinnamopurpureum NRRL 35500 + A. flavus NRRL 29525 (80) or A. parasiticus NRRL 29555 (95) or A. parasiticus NRRL 29568 (74); Penicillium sp. 1 NRRL 35506 + A. flavus NRRL 29525 (91) or A. parasiticus NRRL 29555 (89) or A. parasiticus NRRL 29568 (73); and Penicillium sp. 2 NRRL 35509 + A. tubingensis NRRL 35599 (81). Stipes of A. flavus NRRL 29525 (180), A. parasiticus NRRL 29555 (154), and A. parasiticus NRRL 29568 (165) also were examined when grown in the absence of Eupenicillium and Penicillium species.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), A. tamarii, A. nomius, A. alliaceus and A. leporis (TABLE II). These fungi also sporulated sporadically on the heads of several other Aspergillus species outside of section Flavi, most notably the sporulation of E. cinnamopurpureum and Penicillium sp. 1 on A. sclerotiorum (section Circumdati) and A. terreus (section Terrei) and the sporulation of E. cinnamopurpureum on A. sydowii (section Nidulantes). Penicillium sp. 1 NRRL 35505 was isolated from the heads of A. wentii (section Cremei; Peterson 1995, 2000) (TABLE I
), but this host species was not included in the study. In contrast Penicillium sp. 2 was restricted to hosts within section Nigri (A. niger, A. tubingensis and A. aculeatus), with the exception of light sporulation on the heads of one A. tamarii strain (NRRL 20818) (TABLE II).
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), E. cinnamopurpureum (FIGS. 5–8), Penicillium sp. 1 (FIGS. 9, 10), and Penicillium sp. 2 (FIGS. 11, 12). Immature ascostromata of E. cinnamopurpureum sometimes were present on Aspergillus heads along with penicilli. Eupenicillium ochrosalmoneum and Penicillium sp. 2 sporulated on the agar medium in addition to the heads of their respective section Flavi and section Nigri hosts. In contrast sporulation by E. cinnamopurpureum and Penicillium sp. 1 was confined mostly to section Flavi heads and was observed uncommonly on the agar medium; in those instances sporulation on the medium was restricted to the point of inoculation. Dry conidia of Eupencillium and Penicillium species when sprinkled on mature colonies of susceptible Aspergillus species did not germinate and grow on the host heads.
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). The stolons did not appear to be morphologically distinct from vegetative hyphae, and they sometimes supported scattered penicilli (FIGS. 4, 10). Penicilli were not present on immature Aspergillus heads but only on those with conidial chains, where they occasionally were observed arising between columns of adhering conidial chains on the Aspergillus heads. Mostly unbranched hyphae of approximately 1.5–2.5 µ m diam were detected internally within Aspergillus stipes of these strain combinations (incidences in parentheses): E. ochrosalmoneum NRRL 35496 + A. flavus NRRL 29525 (7%) or A. parasiticus NRRL 29555 (3%); E. cinnamopurpureum NRRL 35500 + A. flavus NRRL 29525 (5%); and Penicillium sp. 1 NRRL 35506 + A. flavus NRRL 29525 (2%). Stipes from the other six strain combinations (including Penicillium sp. 2 NRRL 35509 + A. tubingensis NRRL 35599) as well as stipes from Aspergillus hosts grown alone did not show internal hyphae. Hyphae were not observed in close association with the outer walls of the stipes in any of the pairings. The presence of numerous conidia adhering to Aspergillus heads prevented the examination of host vesicles for internal hyphae.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The mechanism underlying these host specificities necessitates a clear understanding of the nutritional basis of the associations. The present research did not unequivocally determine source of nutrition for the Eupenicillium and Penicillium species. Sporulation on the agar medium by E. ochrosalmoneum and Penicillium sp. 2, as well as the inability of conidia to germinate and grow on mature Aspergillus heads, suggest that the two species are obtaining nutrients directly from the agar medium. The nutritional basis for E. cinnamopurpureum and Penicillium sp. 1 is more problematic however because sporulation by these species was restricted primarily to the Aspergillus heads. Death of Aspergillus colonies as reported for other Aspergillus-Penicillium associations (Thom and Raper 1945, Raper and Thom 1949) was not observed with any of the four species in this study. Furthermore the incidences of internal hyphae within the Aspergillus stipes were low (0–7%) and a close association of hyphae with the external walls of stipes was not observed. The absence of internal hyphae in the stipes of Aspergillus hosts grown alone suggests that the observed internal hyphae were from Eupenicillium and Penicillium species and were not due to self-parasitism in Aspergillus species as reported by Raper and Fennell (1965) and Boller and Schroeder (1972). Additional examination of the fungal association on Aspergillus heads with transmission electron microscopy, particularly in the region of the vesicle and associated metulae and phialides, might provide clues as to whether mycoparasitism is involved or whether the fungi are simply epibiotic on the host heads.
The spread of Eupenicillium and Penicillium species across Aspergillus colonies is due in part to their ability to bridge Aspergillus heads with aerial hyphae. Such hyphae are referred to in this paper as stolons, defined as horizontal hyphae that connect groups of hyphae or sporulating structures. The term is used in a functional sense because the stolons are not morphologically different from vegetative hyphae and often support sporulating penicilli. There is precedence in the use of this term for the genus Penicillium. Raper and Thom (1949) in their treatment of the P. brevicompactum series use the term stolon to describe the aerial hyphae that extend from the growing margin of the colony and re-enter the agar medium beyond the limits of the submerged mycelium.
Eupenicillium species are predominantly isolated from soil, although a few species such as E. ochrosalmoneum and E. cinnamopurpureum also occur on agricultural commodities (Pitt 1979, Stolk and Samson 1983). Eupenicillium ochrosalmoneum colonizes corn and peanuts and contaminates them with yellow-pigmented citreoviridin (Wicklow et al 1988, Horn 2005), a neurotoxin implicated in human cases of cardiac beriberi in Japan and Asia from ingestion of contaminated rice (Ueno and Ueno 1972). In addition immature ascostromata of E. ochrosalmoneum are produced during preharvest colonization of corn and peanuts (Wicklow et al 1984, Horn 2005). During combine harvesting of corn in the southern United States, ascostromata are dispersed onto the soil surface (Wicklow et al 1984) where they eventually ripen to form ascospores (Horn and Wicklow 1986). Eupenicillium cinnamopurpureum has been reported from stored corn and other grains and seeds (Pitt 1979, Wicklow et al 1998).
Horn (2005) postulated that E. ochrosalmoneum and E. cinnamopurpureum have had a long evolutionary history of coexistence with section Flavi on seeds and grain and that sporulation on Aspergillus heads is an adaptation for efficient conidium dispersal by Eupenicillium species. Therefore Penicillium sp. 1 and Penicillium sp. 2 would be expected to share similar, but currently undefined, ecological niches with their respective section Flavi and section Nigri hosts.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. E-mail: bruce.horn{at}ars.usda.gov
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Horn BW. 1997. Aspergillus caelatus, a new species in section Flavi. Mycotaxon 61:185–191.
———. 2005. Colonization of wounded peanut seeds by soil fungi: selectivity for species from Aspergillus section Flavi. Mycologia 97:202–217.
———. 2006. Relationship between soil densities of Aspergillus species and colonization of wounded peanut seeds. Can J Microbiol 52:951–960.[CrossRef][Medline]
———, Dorner JW. 1998. Soil populations of Aspergillus species from section Flavi along a transect through peanut-growing regions of the United States. Mycologia 90:767–776.[CrossRef]
———, ———, Greene RL, Blankenship PD, Cole RJ. 1994. Effect of Aspergillus parasiticus soil inoculum on invasion of peanut seeds. Mycopathologia 125:179–191.[CrossRef][Medline]
———, Greene RL, Dorner JW. 1995. Effect of corn and peanut cultivation on soil populations of Aspergillus flavus and A. parasiticus in southwestern Georgia. Appl Environ Microbiol 61:2472–2475.
———, Wicklow DT. 1986. Ripening of Eupenicillium ochrosalmoneum ascostromata on soil. Mycologia 78: 248–252.[CrossRef]
Peterson SW. 1995. Phylogenetic analysis of Aspergillus sections Cremei and Wentii, based on ribosomal DNA sequences. Mycol Res 99:1349–1355.[CrossRef]
———. 2000. Phylogenetic relationships in Aspergillus based on rDNA sequence analysis. In: Samson RA, Pitt JI, eds. Integration of modern taxonomic methods for Penicillium and Aspergillus classification. Amsterdam: Harwood Academic Publishers. p 323–355.
Pitt JI. 1979. The genus Penicillium and its teleomorphic states Eupenicillium and Talaromyces. London: Academic Press. 634 p.
Raper KB, Fennell DI. 1965. The genus Aspergillus. Baltimore: Williams & Wilkins. 686 p.
———, Thom C. 1949. A manual of the penicillia. Baltimore: Williams & Wilkins. 875 p.
Stolk AC, Samson RA. 1983. The ascomycete genus Eupenicillium and related Penicillium anamorphs. Stud Mycol 23:1–149.
Tatarenko ES. 1959. Parasitism of mold fungi. Mikrobiologiya 28:887–893.[Medline]
Thom C, Raper KB. 1945. A manual of the aspergilli. Baltimore: Williams & Wilkins. 373 p.
Ueno Y, Ueno I. 1972. Isolation and acute toxicity of citreoviridin, a neurotoxic mycotoxin of Penicillium citreoviride Biourge. Jpn J Exp Med 42:91–105.[Medline]
Wicklow DT, Horn BW, Burg WR, Cole RJ. 1984. Sclerotium dispersal of Aspergillus flavus and Eupenicillium ochrosalmoneum from maize during harvest. Trans Br Mycol. Soc 83:299–303.
———, Stubblefield RD, Horn BW, Shotwell OL. 1988. Citreoviridin levels in Eupenicillium ochrosalmoneum-infested maize kernels at harvest. Appl Environ Microbiol 54:1096–1098.
———, Weaver DK, Throne JE. 1998. Fungal colonists of maize grain conditioned at constant temperatures and humidities. J Stored Prod Res 34:355–361.[CrossRef]
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