Formation of Sclerotia and Production of Indoloterpenes by Aspergillus niger and Other Species in Section Nigri

Several species in Aspergillus section Nigri have been reported to produce sclerotia on well-known growth media, such as Czapek yeast autolysate (CYA) agar, with sclerotia considered to be an important prerequisite for sexual development. However Aspergillus niger sensu stricto has not been reported to produce sclerotia, and is thought to be a purely asexual organism. Here we report, for the first time, the production of sclerotia by certain strains of Aspergillus niger when grown on CYA agar with raisins, or on other fruits or on rice. Up to 11 apolar indoloterpenes of the aflavinine type were detected by liquid chromatography and diode array and mass spectrometric detection where sclerotia were formed, including 10,23-dihydro-24,25-dehydroaflavinine. Sclerotium induction can thus be a way of inducing the production of new secondary metabolites from previously silent gene clusters. Cultivation of other species of the black aspergilli showed that raisins induced sclerotium formation by A. brasiliensis, A. floridensis A. ibericus, A. luchuensis, A. neoniger, A. trinidadensis and A. saccharolyticus for the first time

Ultra high performance liquid chromatography time of flight high resolution mass spectrometry electrospray ionization + base peak (UHPLC-TOF-HRMS ESI+ BP) chromatogram of <i>Aspergillus tubingensis</i> (IBT 16833 = CBS 161.79 = NRRL 4700) extracts.

<p><b>A</b>: Plug extraction from colony area with no sclerotia (CYAR with biotin). <b>B</b>: Plug extraction from colony area with sclerotia (CYAR with biotin). <b>C</b>: Sclerotium extraction (from CYAR with biotin). 1) Nigragillin, 3) Fonsecin, 5) TMC-256A1, 6) Asperazine, 7) Tensidol B, 8) Fonsecin B, 9) Aurasperone C, 10) Aurasperone B, 12) Flavasperone analog, 14) Aflavinine analog, 15) Aflavinine analog, 16) Aflavinine analog, 17) Anominine, 18) 10,23-dihydro-24,25-dehydroaflavinine, 19) Aflavinine analog, 20) Aflavinine analog. From this strain TePaske et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094857#pone.0094857-TePaske1" target="_blank">[20]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094857#pone.0094857-TePaske2" target="_blank">[21]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094857#pone.0094857-TePaske3" target="_blank">[22]</a> isolated 14-epi-14-hydroxy-10,23-dihydro-24,25-dehydroaflavinine, 10,23-dihydro-24,25-dehydro-21-oxo-aflavinine, 10,23-dihydro-24,25-dehydroaflavinine, tubingensin A and B, and dehydrotubingensin A and B.</p

<i>Aspergillus</i> section <i>Nigri</i>: Production of sclerotia on CYA agar or CYA with raisins (most sclerotia were formed around the raisins on the agar surface, and few sclerotia if any on the raisins themselves, see footnotes).

<p>*-: no sclerotia formed, +: Few scattered sclerotia (<10), ++: Several (>10) sclerotia surrounding the raisins, +++: numerous sclerotia formed all over the medium, both with and without raisins.</p><p>**Homothallic sexual state reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094857#pone.0094857-Rajendran1" target="_blank">[2]</a>, this strain readily produced abundant sclerotia, both with and without raisins.</p><p>***Paspa is probably an indoloterpene with a paspalinine chromophore.</p><p>****In this species, sclerotia were only produced on the raisins, not on the medium.</p><p>*****Abundant sclerotia reported by Musallam <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094857#pone.0094857-AlMusallam1" target="_blank">[3]</a>.</p><p>Isolates not producing sclerotia in <i>A. brasiliensis</i>, <i>A. niger</i>, and <i>A. welwitschiae</i> and are listed in Table S2 in File S1. The conidia were frozen at −18°C for three weeks before inoculation.</p

Approximately 41 white sclerotia can be observed on a close-up of <i>Aspergillus niger</i> IBT 29019 growing on Czapek yeast autolysate agar with raisins.

<p>Note the clear or brown exudate droplets associated with most sclerotia.</p

Numerous cream-coloured sclerotia produced on CYAR agar can be seen surrounding the raisins and the usual heavy sporulation caused by black <i>Aspergillus niger</i> IBT 29019 heads.

<p>The ellipses added show the position of the raisins, which are covered with black aspergilla.</p