High prevalence of B and G aflatoxin-producing fungi in sugarcane field soil in Japan: heteroduplex panel analysis identifies a new genotype within Aspergillus Section Flavi and Aspergillus nomius

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High prevalence of B and G a£atoxin-producing fungi in sugarcane ¢eld soil in Japan: heteroduplex panel analysis identi¢es a new genotype within Aspergillus Section Flavi and Aspergillus nomius a;

Yuko Kumeda a b

, Tsutomu Asao a , Haruo Takahashi b , Masakazu Ichinoe

c

Osaka Prefectural Institute of Public Health, Nakamichi, Osaka 537-0025, Japan Public Health Laboratory of Chiba Prefecture, Nitona-cho, Chiba 260-8715, Japan c Tokyo Kasei University, Kaga, Tokyo 173-8602, Japan

Received 3 February 2003; received in revised form 2 May 2003 ; accepted 7 May 2003 First published online 26 June 2003

Abstract Heteroduplex panel analysis (HPA) was previously developed for genetic identification of Aspergillus Section Flavi strains, utilizing polymerase chain reaction-amplified fragments of the internal transcribed spacer (ITS) regions of the rRNA gene. Application of HPA to a field study demonstrated that a new type of FP-1 strains belonging to Section Flavi is predominantly distributed throughout sugarcane field soil in the southernmost islands of Japan, and such a trend may also be the case in Vietnam. All of the 71 tested isolates of type FP-1 were able to produce aflatoxins B and G. The morphological observations of the type FP-1 isolates showed that a major part of them had broad interfaces with Aspergillus parasiticus and the remainder with Aspergillus flavus. Phylogenetic analysis based on the ITS sequences indicated that type FP-1 formed an independent clade positioned between A. parasiticus and A. flavus, and was more closely related to the former species. This is also the first report on the distribution of Aspergillus nomius in sugarcane field soil and/or sugarcane stems in Japan and Vietnam. ; 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords : Aspergillus parasiticus ; Aspergillus nomius; Aspergillus £avus; A£atoxin; Sugarcane ¢eld soil; Heteroduplex panel analysis ; Internal transcribed spacer region

1. Introduction Aspergillus Section Flavi is one of the most widely investigated fungi because of the ability of some isolates to produce potent carcinogenic a£atoxins [1]. Three known a£atoxigenic species, Aspergillus £avus, Aspergillus parasiticus and Aspergillus nomius, often invade susceptible crops such as corn, cotton, peanuts and tree nuts before or after harvest causing a£atoxin contamination of food and feed [1]. Agricultural soil serves as the main reservoir of inocula for these fungi [2]. When unusual weather associated with drought occurs in agricultural ¢elds, a£atoxigenic fungi become highly competitive and may become the dominant fungal species in soil [3]. This may be due to the ability of

* Corresponding author. Tel. : +81 (6) 6972-1321; Fax : +81 (6) 6972-1329. E-mail address : [email protected] (Y. Kumeda).

these fungi to grow at very high temperatures (43^48‡C) and at low water activities (0.8 at 37‡C) [4]. Sugarcane is a major crop in the southernmost islands of Japan. The sugarcane product of muscovado (crude sugar) is well known as a traditional natural food, and is sometimes used for confectionery or other dishes. Since 1993, a£atoxin B1 has been detected in muscovado that is produced in the southernmost islands of Japan [5]. Our previous survey revealed that Aspergillus Section Flavi isolates, producing a£atoxins B and G, are predominantly distributed in sugarcane ¢eld soil [6]. These fungi were classi¢ed as atypical and typical A. parasiticus or A. £avus according to their morphological characteristics [7]. Recently, some new species belonging to Aspergillus Section Flavi have been described as follows. Aspergillus caelatus was found in agricultural soil and peanut seeds in the USA [2,8]. Aspergillus pseudotamarii and Aspergillus bombycis were found in tea ¢eld soil and silkworm rearing houses in Japan, respectively [9^11]. This diversi¢cation of

0168-6496 / 03 / $22.00 ; 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/S0168-6496(03)00154-5

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Section Flavi species makes it more di⁄cult to identify species with only conventional morphological and physiological methods because of their overlapping characteristics [11,12]. For genetic identi¢cation of Section Flavi isolates, we have previously developed single-strand conformation polymorphism analysis [13] and heteroduplex panel analysis (HPA) [14] utilizing polymerase chain reaction (PCR)-ampli¢ed fragments of the internal transcribed spacer regions (ITS1^5.8S^ITS2) of the rRNA gene. The HPA method had the highest sensitivity at detecting genetic variants within a species, and allowed distinct identi¢cation of the species in accordance with the taxonomy. The aim of this study was to identify and characterize the fungi of Aspergillus Section Flavi that inhabit sugarcane ¢eld soil in the southernmost islands of Japan using the HPA method.

2. Materials and methods 2.1. Culture collections used in this study In this study we used 95 Aspergillus Section Flavi isolates from our laboratory collections. Eight Thai isolates, 12 Vietnamese isolates, and A. bombycis (NRRL 28900 and NRRL 28901) have been previously isolated by us. A. caelatus 94K02 and A. pseudotamarii NRRL 25517 were a gift from T. Goto. Zambian isolates, four A. parasiticus, two Aspergillus tamarii and 61 A. £avus isolates, were a gift from H. Sunagawa. Four Benin S strain isolates of A. £avus (BN008R, BN038G, BN026G and BN040B) were a gift from P.J. Cotty [15]. 2.2. Heteroduplex panel analysis DNA extraction, PCR ampli¢cation and subsequent HPA were carried out as described by Kumeda and Asao [14]. Brie£y, reference fragments for HPA were prepared by PCR ampli¢cation of the ITS regions of the representative A. £avus, A. parasiticus, A. tamarii and A. nomius isolates. To form the heteroduplex molecules, tested fragments ampli¢ed from the isolates and each reference fragment were mixed and then denatured at 94‡C for 5 min, followed by slow cooling to 20‡C for 10 min. After electrophoresis, the resulting heteroduplexes appeared on a hydrolink-mutation detection enhancement gel (BMA, Rockland, ME, USA) as characteristic banding patterns. 2.3. Sugarcane ¢eld soil A total of 56 soil samples, comprising ¢ve from Hateruma Island, 13 from Ishigaki, ¢ve from Iriomote, 11 from Okinawa, 17 from Tokunoshima and ¢ve from Amamioshima Island, were collected from 1991 to 2002. These

southernmost islands of Japan are located at 24^28‡N and 124^130‡E. Three subsamples of each ¢eld soil (approximately 100 cm3 ) were taken with a sterile trowel at 8^10-m intervals after removing the surface soil. The subsamples were combined and well mixed in a polyethylene bag, and then allowed to dry on a paper dish covered with a sheet of paper at 5‡C for 7 days in a refrigerator. In Vietnam, eight sugarcane ¢eld soil samples, 24 sugarcane stem samples, and 12 peanut samples were taken in 1999. 2.4. Isolation of fungi from soil and sugarcane The dried soil samples were passed through testing sieves (0.83 mm mesh), and a 25-g portion of sieved soil was then blended with 225 ml of distilled water containing 0.05% agar in a 500-ml £ask. After serial dilutions of 1031 to 1035 with distilled water containing 0.05% agar, 0.5-ml aliquots of each dilution were spread onto two plates of Aspergillus £avus/parasiticus agar (Oxoid, Ogdensburg, NY, USA) and the plates were incubated for 3 days at 30‡C. Sugarcane stems were cut into 10^15-cm-long pieces and cultured in a polyethylene bag for 1^2 days at 30‡C, and fungi that had grown on the cut surfaces of the stems were isolated. Potential Aspergillus Section Flavi isolates were transferred onto potato dextrose agar (PDA) slants and then puri¢ed by the single-spore technique. 2.5. Morphological observation Observation of the macroscopic characteristics of the isolates was made from inoculations onto Blakeslee’s malt extract agar and PDA plates following incubation in the dark for 7 and 14 days at 25‡C. For scanning electron microscopic (SEM) observation, 14-day-old conidia were washed with 0.1% Tween 80 and ¢xed in modi¢ed Karnovsky solution (2% glutaraldehyde, 2% paraformaldehyde and 0.1 M phosphate bu¡er, pH 7.2). Fixed conidia were dehydrated in a graded ethanol series and dried with a t-butyl alcohol freeze-dryer (RMC-Eiko Corp ID2, Tokyo, Japan). Specimens were coated with gold to a thickness of 25 nm by an ion sputter coater (JFC-1100, Jeol, Tokyo, Japan) and observed with a scanning electron microscope (JSM-T100, Jeol, Tokyo, Japan). 2.6. Mycotoxin analysis Isolates were inoculated on unpolished rice (5 g) moistened with 2.5 ml water in a 50-ml £ask, and cultured for 7 days at 27‡C in the dark. A£atoxins extracted with 10 ml of chloroform were concentrated by a rotary evaporator and dried under a stream of nitrogen gas. The residue was dissolved in 0.4 ml of benzene^acetonitrile (95:5, v/v) and then spotted on a pre-coated Kieselgel plate (Merck, Darmstadt, Germany). After developing with chloroform^acetone (9:1, v/v), a£atoxins were detected under UV light at 365 nm wavelength. High-performance liquid

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Fig. 1. HPA panels of Aspergillus Section Flavi strains obtained by using ITS region DNAs ampli¢ed by PCR. Panels: F-1, A. £avus P-39-1; F-2, A. £avus IFO 5375; F-3, A. £avus OPS 397; F-4, A. £avus OPS 409; F-5, A. £avus OPS 249; F-6, A. £avus BN026-G; FP-1, OPS 376; P-1, A. parasiticus IFO 4082 ; P-2, A. parasiticus OPS 371; T-1, A. tamarii A 0754 ; T-2, A. pseudotamarii NRRL 25517; T-3, A. caelatus 94K02; TN-1, A. nomius IMI 358749; TN-2, A. bombycis NRRL 28901; N-1, A. nomius ATCC 15546; N-2, A. nomius NRRL 6552; N-3, A. nomius IMI 358751; N-4, A. nomius OPS 367; N-5, A. nomius OPS 418. Heteroduplex bands in lanes f, p, t and n were formed with reference strains of A. £avus P-39-1, A. parasiticus IFO 4082, A. tamarii A0754 and A. nomius ATCC 15546, respectively. F-3 to F-6, FP-1, P-2, T-2, T-3, TN-2, N-4 and N-5 are additional new panels of HPA types obtained in this study.

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Table 1 HPA types of Aspergillus Section Flavi identi¢ed in our studies HPA type

Number of isolates

A£atoxin production ND

a

B

A. £avus/A. oryzae F-1

76 (11)c

39 (11)

24

F-2

50 (2)c

28 (2)

19

F-3 6 F-4 3 F-5 1 F-6 1 A. parasiticus/A. sojae P-1 34 (14)d

P-2e A new genotype FP-1e A. tamarii T-1

A. pseudotamarii T-2 A. caelatus T-3 A. bombyciss TN-1 TN-2 A. nomius N-1 N-2 N-3 N-4 N-5

2 1

B and G 4

4 1 1

Source

9

Japan, USA, Turkey, The Philippines, South Paci¢c, New Guinea, Vietnam, Thailand, Benin, Zambia

3

Japan, Vietnam, Thailand, Zambia

Peanut, soil, hazelnut, soybean lees, shoe sole, bran, groundnut, beef, sake koji, miso, milk, amazake, cereal, sugarcane ¢eld soil, rice, peanut, co¡ee bean, bean, chick pea, wheat, mill meal, agricultural ¢eld Buckwheat, beef top silage, tane-koji, butter, sugarcane, sugarcane ¢eld soil, rice, peanut, bean, chick pea, wheat, mill meal, sorghum Chick pea, sugarcane ¢eld soil Peanut, mill meal Chick pea Agricultural ¢eld

NT

Zambia, Japan Vietnam, Zambia Zambia Benin

1 1

14 (14)

20

1

1

71

71

16

Geographical origin

8

1

8

1

1

1

1 2

1

1 1

24

23

2 2 14 2

2 2 14 2

b

1

USA, Argentina, Sudan, Uganda, Vietnam, Japan, Zambia Vietnam

Mealy bug, peanut, macadamia nut, koji, soy sauce, forest soil, bean Sugarcane

Japan, Vietnam, Egypt

Sugarcane, sugarcane ¢eld soil

Cuba, Japan, The Coconut, co¡ee bean, soil, feed, chili Philippines, Thailand, USA, powder, soy sauce, chick pea, China, Zambia sugarcane ¢eld soil Japan

Tea ¢eld soil

Japan

Soil

South America Japan

Brazil nut Silkworm

USA, Vietnam, Japan

Moldy wheat, sugarcane, sugarcane ¢eld soil, rice Pine saw£y, cottonseed Soil Spice, sugarcane, sugarcane ¢eld soil Sugarcane ¢eld soil, unknown

USA USA Unknown, Japan Vietnam, Unknown

a

ND, not detected. NT, not tested. c The numbers within parentheses indicate the numbers of A. oryzae. d The numbers within parentheses indicate the numbers of A. sojae. e The details of types are described in the text. b

chromatography (HPLC) was used for the quantitative analysis of a£atoxins [16]. Cyclopiazonic acid production was assayed from isolates grown on modi¢ed Czapek’s broth using HPLC [17]. 2.7. Nucleotide sequencing and phylogenetic analysis PCR products were sequenced in both directions using primers ITS1 and ITS4 [18] with a BigDye terminator cycle sequencing FS Ready Reaction kit (PE Biochem-

icals, Foster City, CA, USA). The sequence of the labeled DNA sample was read by an ABI Prism 310 genetic analyzer (PE Biochemicals) and analyzed with Factura software (PE Biochemicals). The DNA sequence data were aligned, and neighbor-joining trees were constructed using ClustalX version 1.8 (http://inn-prot.weizmann.ac.il/ software/ClustalX.html) [19]. The phylogenetic trees were displayed using the Njplot program. Bootstrap analyses were performed by 1000 resamplings of the data sets [20].

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morphological diversity as described below, and 34 (18%) were identi¢ed as A. £avus with the ability to produce a£atoxins B and G. A. parasiticus was isolated from all islands with the highest incidence (2/5 to 10/11) except from Hateruma Island, where A. £avus (5/5) was isolated with a higher incidence (3/5). On Okinawa Island, the incidence of A. £avus, with the ability to produce a£atoxins B and G (10/11), was apparently higher than that of the other A. £avus isolates (1/11). Conversely, the incidence of the former A. £avus (3/45) was lower than that of the latter one (18/45) on the other islands. A. tamarii was a rare species in the tested sugarcane ¢elds. The colony counts of Section Flavi ranged from 1.3U102 to 1.2U105 CFU g31 .

3. Results 3.1. HPA panels and types Eight HPA panels, F-1 and F-2 (A. £avus/A. oryzae), P-1 (A. parasiticus/A. sojae), T-1 (A. tamarii), TN-1 (atypical A. nomius) and N-1 to N-3 (A. nomius), have been previously reported [14]. In the present study, nine additional panels, F-3 to F-6 (A. £avus), T-2 (A. pseudotamarii), T-3 (A. caelatus), TN-2 (A. bombycis), N-4 and N-5 (A. nomius), were generated from 95 examined isolates (Fig. 1). FP-1 (a new genotype) and P-2 (A. parasiticus) panels were generated from the sugarcane ¢eld isolates described below. Although A. bombycis did not form any single band with the reference strains, the banding pattern was similar to that of the TN-1 panel. In addition, A. bombycis formed a single band with TN-1 isolate (data not shown), thus the panel was designated TN-2. A. pseudotamarii and A. caelatus formed heteroduplex bands with the A. tamarii reference strain, which migrated at nearly the same position as each homoduplex band, thus we designated the HPA panels T-2 and T-3, respectively. Consequently, 19 HPA types, including type P-2 and type FP-1, were identi¢ed from a total of 308 examined Aspergillus Section Flavi isolates. The number of isolates, the range of origins and sources, and a£atoxin production for each type are shown in Table 1. F-1 and F-2 were dominant types in A. £avus, and there was no relationship between HPA types and a£atoxin production. Six A. £avus isolates producing a£atoxin B and G, all of which derived from Africa (Zambia and Benin), were identi¢ed as type F-1, F-4 and F-6.

3.3. HPA typing of isolates from sugarcane ¢elds in Japan Of the 186 isolates, 110 were maintained as stock cultures and used for the HPA typing (Table 3). Surprisingly, 66 isolates displayed a new HPA panel, as two retarded but clear heteroduplex bands were shown in all lanes (Fig. 1). These unique and identical HPA panels were designated FP-1 based on the DNA sequence data and phylogenetic analysis (Fig. 4). All type FP-1 isolates were initially morphologically identi¢ed as atypical or typical A. parasiticus based on whether they formed metulae or not [7]. Of the 110 stored isolates, 30 that were initially identi¢ed as A. £avus with the ability to produce a£atoxins B and G (Table 2) were re-identi¢ed as A. nomius. Of these 30 strains, 18 belonged to type N-1 and 12 to type N-4 (Table 3). From these HPA results, 121 A. parasiticus and 34 A. £avus isolates, both having the ability to produce a£atoxins B and G, were considered to be type FP-1 and A. nomius, respectively. Of the 110 stored isolates, 10 were identi¢ed as A. £avus. Of these 10 isolates, eight belonged to type F-1, one belonged to type F-2 and one to type F-3. The remaining four isolates were identi¢ed as type T-1 of A. tamarii. From two samples of the sugarcane stems harvested on Okinawa island, types N-1 and N-4 of A. nomius were isolated.

3.2. Incidence of Aspergillus Section Flavi in sugarcane ¢elds A total of 186 Aspergillus Section Flavi isolates were identi¢ed from 38 of the 56 soil samples of sugarcane ¢elds in all six southernmost islands (Table 2). Of the 186 isolates, 121 (65%) were identi¢ed as A. parasiticus showing

Table 2 Incidence of and a£atoxin production by Aspergillus Section Flavi in sugarcane ¢eld soil in the southernmost islands of Japan Island

Number of positive samples/number tested

ND Okinawa Ishigaki Tokunoshima Hateruma Amamioshima Iriomote Total

10/11 7/13 9/17 5/5 3/5 4/5 38/56

A. parasiticusa

A. £avus b

B

B and G

1 (1)d

10 (31)

4 (6) 1 (1) 2 (4) 7 (11)

4 (4) 5 (8) 2 (2) 12 (15)

a

Re-identi¢ed as type FP-1 by HPA (Table 3). Not detected. c Re-identi¢ed as A. nomius by HPA (Table 3). d The numbers within parentheses indicate the numbers of strains isolated. b

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1 (1) 1 (1) 1 (1) 13 (34)

c

A. tamarii

B and G

ND

10 (67) 6 (14) 7 (17) 3 (4) 2 (15) 2 (4) 30 (121)

1 (1) 1 (2)

1 (2) 3 (5)

234

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Fig. 2. Diversity of conidial wall texture among type FP-1 strains observed by SEM. Roughness of the conidial wall is gradually decreased in order of photograph number. Numbers 1 and 6 are A. parasiticus NRRL 1731 and A. £avus NRRL 1957, respectively. Numbers 2^5 are the type FP-1 strains of OPS 458, CBS 108.30, OPS 473 and OPS 474, respectively. The scale bars indicate 5 Wm.

Type N-1 and N-4 isolates of A. nomius could clearly be distinguished from each other in their appearance on PDA agar slants incubated for 14 days. Type N-1 strains generated yellowish-green colonies with heavy sporulation and little or no sclerotia formation, while type N-4 isolates generated orange colonies with light sporulation and abundant sclerotia formation. Three HPA types (F-1 to F-3) of A. £avus isolates were morphologically indistinguishable from each other.

and N-5 of A. nomius were isolated from sugarcane stems and sugarcane ¢eld soil samples, respectively. Type P-1 of A. parasiticus was isolated from one out of 12 peanut samples, but not from any sugarcane stems (24 samples) or their ¢eld soil (¢ve samples). The CBS 108.30 strain, deposited as A. £avus var. £avus, which was recorded to be isolated from Pseudococcus sacchari on sugarcane in Egypt in 1930, was identi¢ed as type FP-1. 3.5. Morphological diversity of type FP-1 strains

3.4. HPA typing of isolates from sugarcane ¢eld soil and peanuts in Vietnam Type FP-1 of a new genetic variant within A. parasiticus was isolated from four samples (out of 24) of sugarcane stems, and type P-2 from one sample. The type P-2 isolate produced round sclerotia and a few conidia. Types N-1 Table 3 HPA types of Aspergillus Section Flavi strains isolated from sugarcane ¢eld soil in the southernmost islands of Japan Island

HPA types of: A. nomiusa

A. £avus F-1 Okinawa Ishigaki Tokunoshima Hateruma Iriomote Total a b

F-2

F-3

1 6 1

N-1

N-4

18

11

18

1 12

1 8

1

1 1

FP-1b A. tamarii T-1 48 12

1 2

3 3 66

1 4

All tested type FP-1 isolates sporulated heavily without sclerotia production. Approximately half of type FP-1 isolates produced more than 30% of metulated heads, while the remainder was uniseriate. Based mainly on colony color and conidial roughness, the majority of the type FP-1 strains were more similar to A. parasiticus than to A. £avus. The colony colors of the majority were dark green [21], slightly lighter than that of A. parasiticus, while the minority was herbage green [21], slightly deeper than that of A. £avus. The conidial features of the majority showed globose and conspicuously echinulate walls, while the minority showed subglobose and moderately echinulate walls. However, the two groups could not be clearly di¡erentiated from each other due to the morphologically intergrading characteristics. Indeed, SEM observation showed that the echinulate walls of the type FP-1 strains had broad diversity (Fig. 2). 3.6. Mycotoxin production of type FP-1 strains

Initially identi¢ed as A. £avus (Table 2). Initially identi¢ed as typical and atypical A. parasiticus (Table 2).

All type FP-1 isolates assayed by thin-layer chromatog-

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Fig. 3. Partial nucleotide sequence alignments for ITS1 and ITS2 of rRNA genes from A. £avus, A. parasiticus and FP-1 strains. Codes correspond to those presented in Fig. 1. Dots indicate identity to the sequence of F-1. Dashes indicate alignment gaps (insertion or deletion di¡erences).

by one nucleotide (T) insertion, one nucleotide (T/C) substitution and four nucleotides (CCAT) deletions, at positions 108, 512 and 507^510, respectively. This conspicuous deletion (4 bp) was located at nearly the same positions as those common to A. £avus. Overall, the sequence alignment of type FP-1 was more similar to that of A. parasiticus than to A. £avus. Type FP-1 had 6- and 7-bp di¡erences from types P-1 and F-1, respectively (Table 4). A. caelatus (type T-2) and A. pseudotamarii (type T-3) each di¡ered from A. tamarii (type T-1) by 3 bp. A. bombycis (type TN-2) had only 1-bp di¡erence from IMI 358749 (type TN-1), originally designated A. nomius [1]. Type P-2 of OPS 371 isolated from sugarcane stem in Vietnam had 1-bp intraspeci¢c diversity from type P-1. The highest intraspeci¢c diversity was shown in A. nomius (5 bp), followed by A. £avus (3 bp). Consequently, the HPA method generated 19 types within

raphy produced a£atoxins B and G. The production of a£atoxins determined by HPLC was variable among the seven tested isolates (B1 , 0.94^58.0 ng g31 ; B2 , 0.08^2.4 ng g31 ; G1 , 0.06^26.0 ng g31 ; G2 , 0.32^0.82 ng g31 ). Two of the isolates did not produce a£atoxin G2 . None of the type FP-1 isolates produced detectable cyclopiazonic acid. 3.7. Nucleotide sequence analysis and HPA types The type FP-1 isolates from the six southernmost islands of Japan, as well as the isolates from Vietnam and Egypt (CBS 108.30), all had identical nucleotide sequences of the ITS regions when ampli¢ed by PCR. Excluding the primer regions, the sequences of types F-1 to F-6, FP-1, P-1 and P-2 were aligned for comparison with type F-1. Sequence di¡erences were all located together in the ITS1 and ITS2 genes (Fig. 3). Type FP-1 di¡ered from type P-1

Table 4 Nucleotide diversity of ITS regions among HPA types of Aspergillus Section Flavi strains Typea

Nucleotide diversity (number of bases) from : F-1 F-2 F-3 F-4 F-5 F-6 FP-1 P-1 P-2 T-1 T-2 T-3 TN-1 (595) (594) (595) (594) (594) (594) (592) (595) (595) (598) (599) (599) (597)

F-1 F-2 F-3 F-4 F-5 F-6 FP-1 P-1 P-2 T-1 T-2 T-3 TN-1 TN-2 N-1 N-2 N-3 N-4 N-5

0 1 1 2 2 2 7 8 9 13 13 12 15 13 20 19 22 20 20

0 2 1 1 1 6 9 9 14 14 13 17 15 21 20 23 21 21

0 1 3 3 8 9 9 14 14 13 16 14 22 21 24 22 21

0 2 2 7 10 11 15 15 14 18 16 24 21 24 21 21

0 2 7 10 11 15 15 14 17 16 23 22 23 21 21

0 7 10 11 14 14 14 17 15 23 21 22 22 22

0 6 5 13 14 12 12 11 18 18 17 17 17

0 1 9 8 8 8 7 14 13 13 13 13

0 8 9 7 7 6 14 12 12 12 12

0 3 3 5 4 13 12 12 12 12

0 2 7 6 15 13 14 14 14

a

0 7 6 13 12 12 12 12

0 1 8 8 7 7 7

TN-2 (597)

N-1 (598)

N-2 (598)

N-3 (598)

N-4 (597)

N-5 (598)

0 9 8 8 8 8

0 2 1 3 1

0 3 5 1

0 2 2

0 4

0

F-1: A. £avus P-39-1; F-2: A. oryzae IFO 5375; F-3: A. £avus OPS 247; F-4: A. £avus OPS 255; F-5: A. £avus OPS 249; F-6: A. £avus BN026-G ; FP-1: A. parasiticus OPS 376; P-1: A. parasiticus IFO 4082; P-2: A. parasiticus OPS 371; T-1: A. tamarii A0754; T-2: A. pseudotamarii NRRL 25517; T-3: A. caelatus 94K02; TN-1: A. nomius IMI 358749; TN-2: A. bombycis NRRL 28901 ; N-1: A. nomius ATCC 15546 ; N-2: A. nomius NRRL 6552; N-3: A. nomius IMI 358751; N-4: A. nomius OPS 367; N-5: A. nomius OPS 418.

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Fig. 4. Neighbor-joining phylogenetic analysis of 19 HPA types of Aspergillus Section Flavi based on nucleotide sequence comparison of ITS regions. Codes correspond to those presented in Fig. 1 and Table 4. Numbers indicate bootstrap values (percent). The scale indicates 1.0% divergence.

Aspergillus Section Flavi species, and the ITS regions of these types had nucleotide diversity within 24 bp.

were the same as those of A. caelatus NRRL 25528 (AF004930).

3.8. Phylogenetic analysis 4. Discussion On the basis of the ITS1 and ITS2 sequence data from 19 HPA types, a phylogenetic tree was constructed using the neighbor-joining method to show the genetic relationships among Aspergillus Section Flavi species (Fig. 4). The sequence data of ITS1 and ITS2 from Aspergillus wentii (EMBL database accession numbers U03522 and U03523, respectively) served as the outgroup. Type FP-1 formed an independent clade from those of A. parasiticus and A. £avus and was positioned between the two groups. Type FP-1 was more closely related to A. parasiticus than to A. £avus. 3.9. Nucleotide sequence accession numbers The ITS1^5.8S^ITS2 sequence data from the strains obtained in this study have been deposited in the DDJB database. The accession numbers are as follows : A. £avus OPS 247 (type F-3), AB074991; A. £avus OPS255 (type F-4), AB074992; A. £avus OPS 249 (type F-5), AB074993; A. £avus BN026-G (type F-6), AB074994; A. parasiticus OPS 371 (type P-2), AB074995; A. parasiticus OPS 376 (type FP-1), AB074996; A. nomius OPS 367 (type N-4), AB074997; A. nomius OPS 418 (type N-5), AB074998. A. pseudotamarii NRRL 25517 (type T-2) and A. bombycis NRRL 28901 (type TN-2) were previously deposited with accession numbers AF272574 and AF338631. The ITS1^5.8S^ITS2 sequence data of A. caelatus 94K02

Application of HPA to a ¢eld study demonstrated that a new type of FP-1 belonging to Aspergillus Section Flavi is predominantly distributed throughout sugarcane ¢eld soil in the southernmost islands of Japan. During the study on the incidence of a£atoxigenic fungi isolated from soil samples, we encountered unusual isolates that resembled A. parasiticus but di¡ered in the metulation of conidial heads and the lesser roughness of the conidial wall. Hence, these isolates were initially identi¢ed as atypical A. parasiticus having the ability to produce a£atoxins B and G [7]. Unexpectedly, atypical as well as typical A. parasiticus isolates generated a new identical HPA panel designated type FP-1. Re-examination of the morphological characteristics of the type FP-1 isolates showed that a major part of them had broad interfaces with A. parasiticus and the remainder did with A. £avus. These variable characteristics made it more di⁄cult or impossible to accurately identify the type FP-1 isolates without using HPA. Type FP-1 isolates might resemble Aspergillus toxicarius [22] in the fact that they di¡ered from A. parasiticus in producing some metulate conidial heads. The type strain of A. toxicarius RIB 4002 was, however, identi¢ed as the typical type P-1 A. parasiticus. These results were in accordance with those of Klich and Pitt [23] and Christensen [24], who reported that metulation is less important be-

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cause it is a variable characteristic in most members of Aspergillus Section Flavi. Klich and Pitt [23] also concluded that the conidial wall texture is the most useful key to di¡erentiate A. parasiticus from A. £avus, however, this may become less useful due to the emergence of the type FP-1 isolates. There is a good correlation between the ITS-based phylogenetic relationship and the traditional taxonomic arrangements in all Aspergillus Section Flavi species reported. Type FP-1 formed an independent clade positioned between A. parasiticus and A. £avus and was more closely related to the former species (Fig. 4). These ¢ndings are in agreement with those of Wong et al. [25], who analyzed the mitochondrial cytochrome b genes of Section Flavi isolates, including A. £avus var. £avus CBS 108.30 identi¢ed as type FP-1 in the present study. Phylogenetic analyses of multiple independent loci are essential to establish a new species [11,26,27]. Further study is therefore required to clarify whether type FP-1 represents a new species or a genetic variant within A. parasiticus. The high prevalence of type FP-1 in the sugarcane ¢elds of Japan may re£ect its adaptation to the soil environment. Sugarcane is a perennial crop and has been cultivated for more than several decades throughout the southernmost islands of Japan. Such long-term cultivation of a single crop might homogenize the soil resources and help to maintain type FP-1 as a dominant species. We were able to ¢nd type FP-1 isolates in the sugarcane ¢elds in Vietnam, although we failed to ¢nd them in culture collections derived from other sources. Interestingly, CBS 108.30 also originated from sugarcane in Egypt. These results may imply that type FP-1 is associated with sugarcane ¢elds in Asia as A. parasiticus is associated with peanut ¢elds in the USA [1^3]. Three new species belonging to Aspergillus Section Flavi have recently been reported based on their unique morphological characteristics, speci¢c mycotoxin pro¢les and growth rates at 42‡C [8,9,11]. However, these features did not always permit reliable identi¢cation of the new species [11,12]. Indeed, A. caelatus isolates, which share some characteristics with A. tamarii, were initially identi¢ed as A. £avus, A. parasiticus or A. tamarii [12]. Many morphological characteristics of A. bombycis overlap those of A. nomius, thus DNA sequence analysis has become the most useful tool for distinguishing them from each other [11]. HPA produced banding patterns unique for each of the recently described A. caelatus (type T-2), A. bombycis (type TN-2) and A. pseudotamarii (type T-3) isolates; therefore, it is also applicable for identi¢cation of these new species. IMI 358749 from Brazil nuts di¡ered greatly from the other A. nomius isolates in the polymorphism of the Takaamylase gene [28]. We also reported that the isolate was intermediate between A. tamarii and A. nomius, and was identi¢ed as type TN-1 by HPA [14]. In the present study, the phylogenetic analysis indicates that IMI 358749 is one

237

of the genetic variants within A. bombycis designated type TN-2. These phylogenetic data were supported by a previous report [29] of IMI 358749 being closely related to A. bombycis. Peterson et al. [11] demonstrated that A. nomius was able to grow at 42‡C, while A. bombycis was not. IMI 358749 failed to grow at 42‡C after a 7-day incubation (data not shown). These results imply that IMI 358749 is A. bombycis. This is the ¢rst report on the distribution of A. nomius in sugarcane ¢eld soil in the southernmost islands of Japan. Extensive regional di¡erences between Okinawa Island and other southernmost islands were shown in the soil population of A. nomius and A. £avus (Table 2). On Okinawa Island, where the incidence of A. nomius was very high, the history of sugarcane cultivation is longer and the soil management practice has been more active than on other islands. Such soil conditions might in£uence the fungal population in the sugarcane ¢elds [1]. A few previous studies have reported on the distribution of A. nomius in Japan [30], USA [2] and Thailand [31], where it has been isolated from agricultural soil, insects and some foods including crops. Distinguishing A. nomius from A. £avus has always been di⁄cult because of their morphological diversity and overlapping characteristics [32]. This may lead to the underestimation of A. nomius prevalence. Using HPA we identi¢ed A. nomius isolates for the ¢rst time from rice and sugarcane, harvested in Vietnam. From these observations, A. nomius is likely to be more widely distributed in agricultural ¢elds in Southeast Asian countries or warmer countries. None of the single heteroduplex bands was displayed in any lanes in the FP-1 panel as shown in Fig. 1. A large bulge generated in the heteroduplex, when formed between type FP-1 and each reference strain, might cause a drastic reduction in the electric mobility [14,33,34]. By rich formation of the heteroduplexes, these unusual bands could easily be distinguished from those of fungi other than Aspergillus Section Flavi, such as Penicillium and non-Section Flavi Aspergillus isolates [14]. In conclusion, application of HPA to a ¢eld study demonstrated that a new type of FP-1 strains is predominantly distributed throughout sugarcane ¢eld soil in the southernmost islands of Japan. A. nomius as well as type FP-1 isolates were more frequently identi¢ed in Okinawa Island than other islands, possibly due to greater human activities. Type FP-1 consists of isolates that are morphologically intergrading between A. parasiticus and A. £avus, whereas their HPA panels have a uniform electrophoretic mobility pattern. Consequently, the HPA method identi¢ed 19 types within Aspergillus Section Flavi.

Acknowledgements We thank Peter J. Cotty (Southern Regional Research Center, Agricultural Research Service, U.S. Department of

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Agriculture), Koichi Sunagawa (Hokkaido Institute of Public Health) and Tetsuhisa Goto (National Food Research Institute) for providing Aspergillus Section Flavi isolates. We also thank Takashi Nishimura (Osaka Prefectural Institute of Public Health) for his assistance with SEM.

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