Re-introduction of <italic>A. luchuensis</italic>
Inui [1], [2] described A. luchuensis as a major fermentation agent in awamori-koji in Okinawa. He reported that the species had blackish brown, uniseriate conidial heads and finely roughened conidia. Nakazawa [3], [4] also isolated molds from awamori koji and he found black and golden yellow cultures and named them A. awamori and A. aureus, respectively. He examined the A. luchuensis strain obtained from T. Inui, and reported that the strain had biseriate conidial heads and had similar morphological features as A. awamori. He rejected A. luchuensis because it was described as a uniseriate species [3], [4], [8]. However, Thom and Raper [21] accepted A. luchuensis and accommodated the species in the Aspergillus luchuensis series. They described it as an Aspergillus with uniseriate heads with occasionally biseriate sterigmata. In contrast, Nehira [22] considered A. luchuensis a synonym of A. awamori. Raper and Fennell [7] reported that A. luchuensis was accepted by Japanese workers as a biseriate species but based on the original description of A. luchuensis with uniseriate conidial heads they rejected this taxon and accepted A. awamori with NRRL 4948 ( = CBS 557.65) as representative culture. Nevertheless, A. luchuensis has been used by Japanese mycologists, and Murakami [8] suggested that if A. luchuensis was corrected as a biseriate species, it was reasonable that A. luchuensis was used as the main awamori koji mold. Recently, Yamada et al. [11] proposed that industrial black-koji molds should be classified as A. luchuensis.
There has been confusion about the existence of the ex-type culture of A. luchuensis, but a study of the data of strain deposition in NBRC showed that Inui [1], [2] sent his strain to K. Sakaguchi at the ACTU (now ATU) collection. Our observations of this ex-type of A. luchuensis (NBRC 4281T) grown on steamed rice confirm the original description of Inui (Fig. 1). In our opinion, Inui [1], [2] described the species at a young stage. This can be seen from his illustrations, where young, not fully developed conidiophores were depicted. Secondly, Inui [1], [2], in the description in the Japanese article, mentioned the single seriation of the conidial head, but he did not place importance to this feature. In his German article he did not mention the seriate structure of the conidial head either. An indication that the conidial heads could be biseriate was, that Inui [1], [2] described that the phialides (sterigmata) were longer than other Aspergillus species. Based on the data mentioned above, A. luchuensis should be accepted as a valid species.
Aspergillus kawachii, a white-koji mold that has been used widely in shochu making on the Kyusyu in Japan, which was probably formed by the mutation of a certain black Aspergillus species, is considered similar with A. luchuensis [5], [23]. Brewers have used the name A. kawachii, but Japanese mycologists have used the name, A. luchuensis mut. kawachii [23], [24], [25], because A. kawachii was not validly published. Yamada et al. [11] proposed that A. kawachii should be classified as A. luchuensis based on their multilocus sequence typing of ITS, D1D2 of LSU, histon3, β-tubulin and cytochrome b sequence data. Our results of RAPDs and MLST of ITS, β-tubulin and calmodulin also support the findings of Kitahara and Kurushima [23] and Yamada et al. [11]. Like A. luchuensis, also A. kawachii was described with uniseriate conidial heads [23]. In our opinion, the same situation and observations occurred here as with the description of A. luchuensis. In conclusion, we propose to place A. kawachii in synonymy with A. luchuensis.
Nakazawa et al. [26] isolated black-koji molds from various awamori fermentations and proposed A. awamori with five varieties, A. aureus with five varieties and A. miyakoensis. One of the five varieties of A. aureus is variety acidus, which was renamed as A. foetidus var. acidus by Thom and Raper [21 Later the variety was raised to species level by Kozakiewicz [27] based on morphological features including the conidial ornamentation observed by scanning electron microscopy. Varga et al. [9] re-validated the species. Our RAPD data and multigene sequence data show that A. acidus shows no difference with A. luchuensis and A. kawachii, and based on priority, the name A. luchuensis is proposed as the correct name for these strains.
From our study we can conclude that A. luchuensis is the correct name for the fungus used in awamori fermentations and that A. acidus and A. kawachii are synonyms. Since there has been confusion about the uni- or biseriate feature of the conidial heads of this species, A. luchuensis is re-described below.
Aspergillus luchuensis Inui [1], [2].
= Aspergillus perniciosis Inui [1], [2]
= Aspergillus awamori Nakazawa pro parte [4]
= Aspergillus aureus var. acidus Nakaz., Simo & A. Watan. [26]
= Aspergillus foetidus var. acidus (Nakaz., Simo & A. Watan.) Thom & Raper [21]
= Aspergillus kawachii Kitahara & Yoshida [5]
= Aspergillus acidus Kozak. [27]
= Aspergillus coreanus Yu et al. [6]
Colony diameters at 7 days, in mm: CYA, 37; DG18, 33; CREA, 29; MEA, 53; MEA37°C, 50.
Colony colors and textures
On CYA, sterile mycelium white, conidial areas gray to black, radially sulcate, floccose; reverse cream to brown, radially sulcate. On MEA, obverse, similar to CYA. On CREA, poor growth but with strong acid production.
Microscopic characteristics
Conidial heads, radiate; stipes length up to 1.5 mm, width 10–13 µm, walls thick, smooth and hyaline; Vesicles, 20–40 µm wide, nearly spherical; when young uniseriate with irregular formation of metulae, later predominantly biseriate, metulae covering almost entire surface of the vesicle 17.0–26.1 × 4.5–8.1 µm, phialides ampulliform 5.6–8.4 × 3.5–4.9 µm; Conidia, globose, smooth, 3.5–4.5 µm, dark brown.
Type strain: NBRC 4281 = KACC 46772 = CBS 205.80 = IFM 47726 = RIB 2642, isolated from awamori-koji in Okinawa, Japan, sent by T. Inui to K. Sakaguchi (University of Tokyo).
Note: The description above is based on the ex-type strain. As shown in Table S1 and Figure S1 A. luchuensis strains show variable morphological characteristic. The colony color is white to gray, or brown to black; stipes have a width 8–30 µm and length up to 1.5 mm; conidial heads are predominantly biseriate, but aberrant uniseriate heads, vesicles are 15–90 µm; metulae 5.0–26.1 µm; phialides are 5.4–12.5 µm; conidia are 3.0–4.5 µm, smooth, finely rough, or rarely rough.
The morphological characteristics are similar with those produced by A. niger and A. tubingensis in Aspergillus section Nigri, and it is difficult to differentiate the species based on only morphology. DNA sequences of β-tubulin and calmodulin are useful to differentiate the species from the others, and GenBank JX500062 for β-tubulin and JX500071 for calmodulin could be good molecular markers for the species. Chemically, the extrolites antafumicins [28] and/or “luchuensin” sets A. luchuensis apart from all other black Aspergillus strains. Antafumicins were first reported from A. niger NH-401. We were not able to study this strain, but it is probably A. luchuensis.
Inui [1], [2] also described A. perniciosis as a contaminant found during awamori-koji fermentation. He distinguished this species by yellow green mycelium with larger conidial heads similar to A. niger. No type culture is available, but the description of this taxon resembles A. coreanus [6] on the basis of yellow green mycelium. Our molecular analysis shows that A. coreanus is a synonym of A. luchuensis(Fig. 3). Therefore we consider A. perniciosis a further synonym of A. luchuensis.
Most isolates of Aspergillus luchuensis specifically produce antafumicin and/or a partially characterized metabolite provisionally called “luchuensin”, in addition to asperazine (shared with A. tubingensis and A. vadensis), they often produce atromentin, funalenone, pyranonigrin A, and occasionally tensidol B [13, 29, 30, Table S2]. None of these extrolites are considered toxic to vertebrates [31]. Furthermore none of these strains produced fumonisin B2, B4 or B6 or any ochratoxins. A total of 52 strains from tea, coffee, and other substrates in addition to industrial strains were analyzed chemically, and none of them produced fumonisins or ochratoxins [13, 32, see also Table S2).
Aspergillus luchuensis is the representative fungus of industrial black- and white-koji mold in Japan [11], and it is also the dominant microorganism of Puerh tea in China [13]. The species is also known from nuruk (KACC 41731, 46420), Korean traditional fermentation starter for makgeolli, and meju (KACC 46490), a starter for soybean paste and soy sauce. In the case of meju, out of 54 strains of black Aspergillus, 15 strains were A. luchuensis (unpublished data). These data suggest that A. luchuensis is common in the food fermentation environment and the species play an important role in industry of East Asia.
Taxonomic Position of <italic>Aspergillus awamori</italic>
The taxonomic position of A. awamori Nakazawa proved to be complex. This is mainly caused by the fact that no type of A. awamori exists. CBS 557.65 ( = NRRL 4948), which does not originate from awamori koji and received from Instituto Ozwaldo Cruz, Brazil, was designated as neotype of A. niger var. awamori by Al-Musallam [14]. The selection of this neotype was based on the fact that Raper and Fennell [7] used this isolate for their description of A. awamori. In this study, the neotype of A. awamori (CBS 557.65) is located near to A. niger (CBS 554.65T), but distant from Inui’s A. luchuensis (NBRC 4281) (Fig. 3).
Based on the neotype (CBS 557.65), Perrone et al. [15] regarded A. awamori as a cryptic phylogenetic species in section Nigri. In our opinion, the taxonomic position of A. awamori is not clear, because no ex-type culture is known while the majority of strains found in culture collections under this name belong to A. niger (n = 24), A. luchuensis (n = 10), A. tubingensis (n = 2) or A. awamori sensu Perrone et al. (n = 2) (Table 2). According to Yamada et al. [11], 14 of 20 strains of A. awamori were A. niger and six strains were A. luchuensis. Out of six of Nakazawa’s strains of A. awamori, three strains were the A. niger and the other three were A. luchuensis. This indicates that Nakazawa’s species concept is not clear and that he dealt with both A. luchuensis and A. niger. A. awamori as described by Nakazawa can be best treated as a doubtful synonym of A. niger or A. luchuensis.
The strains named as A. awamori by Perrone et al. [15] based on the incorrect selected neotype (CBS 557.65NT) are identical with CBS 139.54, a strain deposited at CBS from Welwitschia in Namibia. Figure S2 shows two phylogenetic trees produced from the combined sequence data of two loci (CaM, benA) of 30 taxa belonging to A. niger “aggregate” group. The epitype of A. welwitschiae is accommodated in the group of A. awamori sensu Perrone et al. [15].
Isolates of A. welwitschiae have been reported on various substrates as mycotoxin producers or plant pathogens including grapes [15], onions as causative agents of black mold rot and fumonisin contamination [33] and Welwitschia mirabilis seeds causing seedling rot (van Diepeningen et al. unpublished data). In addition, this species was also identified in various otomycosis cases both in Iran and Hungary [34], [35]. Although the name A. awamori was used taxonomically correctly by Perrone et al. [15], this currently used name is misleading, as this species is rarely identified in awamori fermentation processes, where mostly A. niger and A. luchuensis could be isolated [11]. It should also be mentioned that isolates of A. awamori sensu Perrone are able to produce ochratoxins and/or fumonisins [15]; [33], making the application of the name of an oriental fermentation process as the basis of the name of this fungus is inadequate.
Aspergillus welwitschiae (Bresadola) Hennings apud Wehmer.
= Ustilago welwitschiae Bres. in Saccardo, Bolm Soc. Broteriana 11∶9–90. 1893 = Sterigmatomyces welwitschiae (Bres.) Henn. in H. Baum Kunene-Zambesi Expedit., p. 168, 1903 = Aspergillus welwitschiae (Bres.) Henn. apud Wehmer in Centrbl. Bakteriol. ParasitK. II, 18∶294, 1907
= Aspergillus awamori sensu Perrone et al. [15].
Epitype: CBS 139.54, isolated by H.J. Swart from female inflorescence of Welwitschia mirabilis collected in Namibia, stored for 2 years (sample no. 236).
Colonies on CYA, MEA, YES grow similar as A. niger. Vesicles 45–85 µm in diameter. Conidia globose, finely to distinctly roughened, brown to dark brown, 3.5–5.5 µm.
Isolates of Aspergillus niger and A. welwitschiae have overlapping features concerning conidium size, ornamentation, stipe ornamentation, stipe length, and conidium colour. Both species contain mutant isolates that have more brownish conidium colours. A. welwitschiae produced conidia which were mostly globose and finely to distinct roughened. The conidial dimensions vary from 3.5–5.5 µm. Vesicles were 45–85 µm in diameter. These morphological characters are identical with the morphological structures in typical A. niger strains [30], A. welwitschiae has the same ranges of growth rates of A. niger on the media CYA at 5, 25 and 37°C, on G25N, CZA, MEA at 25°C in the dark, and also at reduced water activity on CZ20, M40Y at 25°C. It had also a strong acid production on CREA substrate.
Notes: The name Aspergillus welwitschiae was first mentioned by Wehmer [36] who reported that this name was proposed by P. Hennings in a written communication. This species has been reported from Welwitschia mirabilis by several authors and is mostly called A. niger or A. niger var. phoenicis [37]–[39]. It frequently infects and destroys germinating seeds which has an impact on a Welwitschia colony which can sometimes go many years without successfully reproducing.
Aspergillus welwitschiae is morphologically indistinguishable from A. niger but some fixed nucleotide differences between these two species could be useful for their identification. Sequence positions and differences of A. welwitschiae compared to A. niger in the four regions sequenced are: calmodulin: 442 (T), 465 (C), 486–87 (CT), 493–494 (TT), 518 (-) 801 (T); β-tubulin: 173339 (T), 173346 (A); - translation elongation factor-1∶665 (G), 669 (A); RNA polymerase II subunit RPB2∶1281 (C), 1449 (C), 1689 (A), 1692 (C), 1719 (C), 1770 (T), 1947 (C), 2103 (T), 2220 (T).
Besides its specific occurrence on Welwitschia plants strains of A. welwitschiae have been found on grapes, dried fruits, coffee, cocoa, and other sources and it has also a worldwide distribution from all the continents. In particular A. welwitschiae resulted to grow very poorly or no growth in the majority of the strains tested on to 2-deoxy-D-glucose substrate, respect to A. niger which grows well on this substrate [9].
Most isolates of A. welwitschiae produce large amounts of fumonisins and occasionally ochratoxins [32]. The best producer of fumonisin B2, 4 & 6 found yet is NRRL 567, a strain identified by us as A. welwitschiae and used for citric acid production [32].