Abstract
A new monoverticillate Penicillium species (subgenus Aspergilloides Dierckx), Penicillium viticola, was isolated from a grape cultivated in Yamanashi Prefecture, Japan. Morphologically, P. viticola is characterized by the production of slightly roughened conidia, slightly roughened stipes, and rapid growth on 25% glycerol nitrate agar (G25N). This species is phylogenetically close to Penicillium angulare S.W. Peterson, E.M. Bayer & Wicklow, but differs with respect to colonial characteristics and conidia and penicilli morphology.
Avoid common mistakes on your manuscript.
We have isolated numerous fungal strains from soil and plant samples during the course of our screening program to discover antiinfective agents. A novel antimalarial agent was discovered from the secondary metabolites of a fungal strain designated FKI-4410. FKI-4410 was identified as belonging to the genus Penicillium, but it was not one of the known Penicillium species.
The fungus, which was isolated from a grape harvested in Yamanashi Prefecture, Japan, was incubated on potato dextrose agar (PDA) at 25°C.
For determination of the morphological characteristics, the methodology of Pitt (1979) was used.
Digital photographs of colonies were taken with a PowerShot G10 digital camera (Canon, Tokyo, Japan) and adjusted using the PhotoStudio 4 software (Canon).
The Color Harmony Manual, 4th edition (Container Corporation of America, Chicago) was used to determine color names and hue numbers (Jacobson et al. 1958).
For the determination of micromorphological characteristics, microscope slides were examined with a Vanox-S AH-2 microscope (Olympus, Tokyo, Japan), and digital micrographs were taken with a DP25 digital camera (Olympus). For scanning electron microscopy (SEM) of the conidia and conidiophores, agar blocks (3 mm2) were cut from a 7-day-old culture of the strain FKI-4410 growing on malt extract agar (MEA). The agar blocks were fixed with osmium tetroxide (TAAB, Berkshire, UK), air dried, and sputter coated with gold using a JFC-1200 Fine Coater (JEOL, Tokyo, Japan). The samples were observed under a JSM-5600 scanning electron microscope (JEOL).
Genomic DNA of the strain FKI-4410 was isolated using the PrepMan Ultra Sample Preparation Reagent (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instructions. Amplification of the partial calmodulin gene region, the partial beta-tubulin gene region, and internal transcribed spacer (ITS) and large subunit (LSU) rDNA (ID) regions were performed using primers CF1 and CF4 (Peterson et al. 2005), Bt2a and Bt2b (Glass and Donaldson 1995), and ITS1 and NL4 (White et al. 1990; O’Donnell 1993), respectively. Polymerase chain reactions (PCR) were performed with QIAGEN Fast Cycling PCR Kit protocol (Qiagen, Valencia, CA, USA).
Amplifications were performed in a PCR Verity 96-well thermal cycler (Applied Biosystems), programmed for 5 min with denaturation at 95°C, followed by 35 cycles consisting of denaturation at 96°C for 5 s, primer annealing for 5 s at 50°C, extension for 21 s at 68°C, and a final 1 min elongation step at 72°C. After amplification of each gene templates, excess primers and deoxynucleosides (dNTPs) were removed from the reaction mixture using a QIAquick PCR DNA Purification kit protocol (Qiagen). The PCR products were sequenced using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). The cycle sequencing reaction mixture had a total reaction volume of 10 μl, and contained 2.5 μl of template DNA (10–15 ng/μl), 2 μl BigDye terminator premix, 4 μl ultra pure sterile water, and 0.5 μl primer (5 pmol/μl). Reactions were run in a PCR thermal cycler, programmed for 1 min at 96°C, then by 25 cycles of 10 s denaturation at 96°C, followed by primer annealing for 5 s at 50°C, and extension for 4 min at 60°C. Sequencing products were purified by ethanol/ethylenediaminetetraacetic acid (EDTA) precipitation, and samples were analyzed on an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems). Contigs were assembled using the forward and reverse sequences with the SeqMan and SeqBuilder programs from the Lasergene8 package (DNAStar, Madison, WI, USA). The sequences of the strain FKI-4410 were deposited at the DNA Data Bank of Japan (DDBJ) with accession number AB540173 (calmodulin), AB540174 (beta-tubulin), and AB606414 (ID).
To determine the most closely related Penicillium species, the DNA sequences in the calmodulin gene region, the beta-tubulin gene region, and the ID region of FKI-4410 were compared to other sequences in the GenBank database by BLASTN 2.2.21 analysis. The calmodulin gene sequences of phylogenetically related Penicillium species were obtained from GenBank (see Fig. 1 for the GenBank accession numbers) and aligned using Clustal X 2.0.11 (Tompson et al. 1997) with the pairwise and multiple alignment parameters set at 15.0 for gap opening and 0.2 for gap extension. Alignment was refined using SeaView (Galtier et al. 1996), and the alignment was deposited in TreeBASE (http://www.treebase.org/) with accession number S11170. Phylogenetic analyses were based on the neighbor-joining (NJ) method (Saitou and Nei 1987) using Clustal X 2.0.11. Bootstrap analyses were performed on NJ trees with 1,000 bootstrap replicates. The trees were rooted with Aspergillus spp. and viewed with NJplot (Perrière and Gouy 1996).
Morphologically, FKI-4410 is classified (Pitt 1979) in the subgenus Aspergilloides sect. Exilicaulis Pitt based on monoverticillate penicilli that are nonvesiculate. However, FKI-4410 is different from all species of the section in having slightly roughened stipes and rapid growth on 25% glycerol nitrate agar (G25N) (>20 mm) (Pitt 1979; Peterson and Sigler 2002; Peterson et al. 2004; Peterson and Horn 2009). In contrast, the other newly described three species (P. dravuni Janso, P. coffeae S.W. Peterson, F.E. Vega, Posada & Nagai, and P. macrosclerotiorum L. Wang, X.M. Zhang & W.Y. Zhuang) belong to the section Aspergilloides (Janso et al. 2005; Peterson et al. 2005; Wang et al. 2007).
In a BLAST search using the blastn from the National Center for Biotechnology Information (NCBI) (Altschul et al. 1990), FKI-4410 had 91.5% similarity to the partial calmodulin gene sequences of P. angulare (EF198582). In the NJ analysis, FKI-4410 is most closely related to P. angulare (subgenus Aspergilloides sect. Exilicaulis), and the group was supported by a high bootstrap value (see Fig. 1). Nevertheless, FKI-4410 differs from P. angulare in colonial texture, growth rates, and length of stipes (Peterson et al. 2004). Conversely, in a BLAST search, FKI-4410 had 88.3% similarity to the partial beta-tubulin gene sequences of P. multicolor Grig.-Man. & Porad. (EU427265) and 98.4% similarity to the ID sequences of P. sclerotiorum J.F.H. Beyma (AF033404). However, the stipe of FKI-4410 (nonvesiculate) is different from those of P. multicolor and P. sclerotiorum (vesiculate). Therefore, FKI-4410 represents a novel species of the genus Penicillium, for which the name Penicillium viticola sp. nov. is proposed.
We discovered that the novel P. viticola produces the tropolone compounds puberulic acid and stipitatic acid and their novel analogues, viticolins A–C. In previous reports, puberulic acid has only been isolated from Penicillium puberulum Bainier belonging to the subgenus Penicillium (Birkinshaw and Raistrick 1932). We have observed that the compounds produced by P. viticola, notably puberulic acid, had antimalarial properties, and we reported these findings elsewhere (Iwatsuki et al. 2010).
MycoBank no.: MB 516048
Coloniae in agaro CYA post 7 dies 25°C 34–35 mm diametro. Coloniae in agaro MEA post 7 dies 25°C 26–28 mm diametro. Coloniae in agaro G25N post 7 dies 25°C 20–21 mm diametro. Conidiophora plerumque ex hyphis basalibus. Stipes scabridis, 20–85 × 3.0–3.5 μm. Penicilli monoverticillati, phialides ampulliformes, 8.0–12.5 × (2.0–)3.0–4.2 μm. Conidia subglobosa ad ellipticus, leviter scabrida, 2.7–4.0(–4.3) × 2.2–3.7 μm.
Holotypus: TNS-F-38702 colonia exsiccata in cultura ex uva sativo (Vitis sp.), Yamanashi Pref., Japonia, 11.8.2006, a K. Nonaka isolata et in herbario fungorum TSN conservata; cultura viva ex holotypo in JCM ut 17636.
Etymology: Latin, viticola, referring to dweller on the vine (Vitis sp.)
Colonies on Czapek yeast extract agar (CYA) 34–35 mm in diameter after 7 days at 25°C (Fig. 2a), radially sulcate, velutinous, with white (a) mycelium at the margins, covered with olive gray (1 ig) conidia, exudate lacking, the reverse luggage tan (4 ne), the margin entire, soluble pigment not produced.
Colonies on malt extract agar (MEA) 26–28 mm in diameter after 7 days at 25°C (Fig. 2b), plane, with bamboo (2 gc) floccose aerial mycelium, covered with moss green (24 pi) conidia, exudate sparse clear drops, the reverse pale olive (1 ie), the margin entire, soluble pigment not produced.
Colonies on G25N 20–21 mm in diameter after 7 days at 25°C (Fig. 2c), zonate, velutinous, with white (a) mycelium at the margins, covered with olive gray (1 ig) conidia, exudate lacking, the reverse luggage tan (4 ne), the margin entire, soluble pigment not produced.
Colonies on CYA at 5°C, 37°C: no growth.
Conidiophores on MEA borne on a basal felted hyphae or directly from the agar, stipes were simple, slightly roughened, nonvesiculate, 20–85 × 3.0–3.5 μm, with a heavy wall. Penicilli typically monoverticillate (Figs. 3, 4, 7).
Phialides ampulliform, 8.0–12.5 × (2.0–)3.0–4.2 μm, collula 0.5–1.5 μm wide. Conidia borne in chains, subglobose to ellipsoidal, less commonly globose, slightly roughened, 2.7–4.0(–4.3) × 2.2–3.7 μm in size (Figs. 5, 6, 8). Sclerotia not produced.
Specimen, culture examined: TNS-F-38702 (holotype), a dried culture derived from the isolate (FKI-4410) from a grape, Yamanashi, Japan, 11 July 2006, isolated by K. Nonaka. The holotype deposited in the herbarium at The National Science Museum, Ibaraki, Japan (TNS). JCM 17636 (ex-type) deposited at the Japan Collection of Microorganisms (JCM), Saitama, Japan.
Note. Penicillium viticola is classified (Pitt 1979) in the subgenus Aspergilloides sect. Exilicaulis ser. Citreonigra Pitt based on typically monoverticillate penicilli, stipes nonvesiculate, and moderate growth on CYA and MEA for 7 days at 25°C. Morphologically, P. viticola resembles P. decumbens Thom and P. adametzii K.M. Zalessky: these all have a similar growth rate on CYA and MEA and short stipes (Pitt 1979). However, the colonial texture of P. viticola (velutinous) differs from that of P. adametzii (funiculose) and the conidial shape of P. viticola (subglobose to ellipsoidal) is different from that of P. adametzii (spheroidal) (Table 1). The growth rate of P. viticola on CYA at 37°C (no growth) is different from that of P. decumbens (5–20 mm) and the conidial texture of P. viticola (slightly roughened) differs from that of P. decumbens (smooth walled) (Pitt 1979). Moreover, P. viticola showed greater distance from P. decumbens in the phylogenetic tree (Fig. 1).
Penicillium viticola is phylogenetically close to P. angulare, P. brocae S.W. Peterson, Jeann. Pérez, F.E. Vega & Infante, and P. adametzii (Fig. 1). The growth rate and colonial margin on CYA of P. viticola (more than 30 mm; entire) are different from those of P. angulare (less than 20 mm; irregular), and the stipes of P. viticola (20–85 μm) are shorter than those of P. angulare (100–150 μm) (Table 1). The stipe of P. viticola (nonvesiculate) is different from that of P. brocae (vesiculate), and the conidial shape of P. viticola (subglobose to ellipsoidal) is different from that of P. brocae (spheroidal) (Peterson et al. 2003). Moreover, FKI-4410 produced notably puberulic acid. The results of our studies showed that P. viticola was a distinct species, and it is one example of the production of novel compounds by novel species.
References
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410
Birkinshaw JH, Raistrick H (1932) LIII. Studies in the biochemistry of micro-organisms. XXIII. Puberulic acid C8H6O6 and an acid C8H4O6, new products of the metabolism of glucose by Penicillium puberulum Bainier and Penicillium aurantio-virens Biourge. With an appendix on certain dihydroxy-benzenedicarboxylic acids. Biochem J 26:441–453
Galtier N, Gouy M, Gautier C (1996) SeaView and Phylo_win, two graphic tools for sequence alignment and molecular phylogeny. Comput Appl Biosci 12:543–548
Glass NL, Donaldson G (1995) Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Appl Environ Microbiol 61:1323–1330
Iwatsuki M, Takada S, Ishiyama A, Namatame M, Tukashima-Nishihara A, Nonaka K, Masuma R, Mori M, Shiomi K, Otoguro K, Ōmura S (2010) In vitro and in vivo antimalarial activities of puberulic acid and its new analogs, viticolins A-C, produced by Penicillium sp. FKI-4410. J Antibiot (Tokyo) 64:183–188
Jacobson E, Granville WC, Foss CE (1958) Color harmony manual, 4th edn. Container Corporation of America, Chicago
Janso JE, Bernan VS, Greenstein M, Bugni TS, Ireland CM (2005) Penicillium dravuni, a new marine-derived species from an alga in Fiji. Mycologia 97:444–453
O’Donnell K (1993) Fusarium and its near relatives. In: Reynolds DR, Taylor JW (eds) The fungal holomorph: miotic meiotic and pleomorphic speciation in fungal systematics. CAB International, Wallingford, pp 225–233
Perrière G, Gouy M (1996) WWW-Query: An on-line retrieval system for biological sequence banks. Biochimie 78:364–369
Peterson SW, Horn BW (2009) Penicillium parvulum and Penicillium georgiense, sp. nov., isolated from the conidial heads of Aspergillus species. Mycologia 101:71–83
Peterson SW, Sigler L (2002) Four new Penicillium species having Thysanophora-like melanized conidiophores. Mycol Res 106:1109–1118
Peterson SW, Pèrez J, Vega FE, Infante F (2003) Penicillium brocae, a new species associated with the coffee berry borer in Chiapas, Mexico. Mycologia 95:141–147
Peterson SW, Bayer EM, Wicklow DT (2004) Penicillium thiersii, Penicillium angulare and Penicillium decaturense, new species isolated from wood-decay fungi in North America and their phylogenetic placement from multilocus DNA sequence analysis. Mycologia 96:1280–1293
Peterson SW, Vega FE, Posada F, Nagai C (2005) Penicillium coffeae, a new endophytic species isolated from a coffee plant and its phylogenetic relationship to P. fellutanum, P. thiersii and P. brocae based on parsimony analysis of multilocus DNA sequences. Mycologia 97:659–666
Pitt JI (1979) The genus Penicillium, and its teleomorphic states Eupenicillium and Talaromyces. Academic Press, London
Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425
Tompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The Clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882
Wang L, Zhang XM, Zhuang WY (2007) Penicillium macrosclerotiorum, a new species producing large sclerotia discovered in south China. Mycol Res 111:1242–1248
White TJ, Bruns T, Lee S, Taylor JW (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand RH, Sninsky JJ, White TJ (eds) PCR protocols: a guide to methods and applications. Academic Press, San Diego, pp 315–332
Acknowledgments
This study was supported, in part, by funds from the Quality Assurance Framework of Higher Education from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) in Japan.
Open Access
This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
This article is published under an open access license. Please check the 'Copyright Information' section either on this page or in the PDF for details of this license and what re-use is permitted. If your intended use exceeds what is permitted by the license or if you are unable to locate the licence and re-use information, please contact the Rights and Permissions team.
About this article
Cite this article
Nonaka, K., Masuma, R., Iwatsuki, M. et al. Penicillium viticola, a new species isolated from a grape in Japan. Mycoscience 52, 338–343 (2011). https://doi.org/10.1007/s10267-011-0114-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10267-011-0114-7