Annals of Microbiology, 59 (2) 383-390 (2009) Investigation of stored wheat mycoflora, reporting the Fusarium cf. langsethiae in three provinces of Iran during 2007 Reza KACHUEI1, Mohammad Hossein YADEGARI1*, Sasan REZAIE2, Abdolamir ALLAMEH3, Naser SAFAIE4, Farideh ZAINI2, Fatemeh KHANEZAD YAZDI2 1Department of Medical Mycology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran; 2Department of Medical Parasitology and Mycology, Faculty of Public Health, Tehran University of Medical Sciences, Tehran; 3Department of Biochemistry, Faculty of Medical Sciences, Tarbiat Modares University, Tehran; 4Department of Plant Pathology, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran Received 10 December 2008 / Accepted 16 March 2009 Abstract - Wheat is the most important cereal produced in Iran. A mycological survey was carried out for the first time, on the stored wheat samples in Tehran, East Azarbayejan and Mazandaran provinces in 2007. Exogenous and endogenous fungi, were isolated by the method of flotation with Malachite green agar (MGA 0.25) and Freeze blotter techniques respectively. In this study, 46 species belonging to 23 different genera were isolated. Cladosporium spp. (57.1-89.2%) and Alternaria spp. (82.4-100%) species were the predominant fungal species identified as endogenous mycoflora. The predominant exogenous fungi were Penicillium spp. (78.4-92.8%) and Aspergillus spp. (71.4-85.7%) species. Fusarium proliferatum was the most prevalent species of Fusarium isolates. Aspergillus niger (39.4%) and Aspergillus flavus (36.7%) were the predominant Aspergillus species identified as exogenous mycoflora. Aspergillus flavus (26.6%) was the predominant Aspergillus species identified as endogenous mycoflora. Flotation method with MGA 0.25 recommended for isolating of hyaline fungi from wheat cereals. In this study one isolate from Fusarium species was isolated on the basis of morphology and ribosomal internal transcribed spacer classified as Fusarium langsethiae but on the basis of partial translation elongation factor-1alpha gene grouped with Fusarium sporotrichioides. To our knowledge, this is the first report about F. cf. langsethiae in Iran and Asia. Key words: wheat; Fusarium spp.; Fusarium langsethiae; Aspergillus spp.; flotation method. INTRODUCTION Wheat is an economic and important crop that provides approximately 20% of food calorie in the world (Wiese, 1987). It is one of the main staple foods in developing countries and the aspects of production and consumption is the most important grain in Iran (FAO, 2001). Bread wheat (Triticum aestivum L.) is the main cereal used for human consumption in Iran (Farshadfar et al., 2008). Information about fungi associated with these grains is important in assessing the risk of mycotoxin contamination. A number of fungal species, belonging mainly to the genera Fusarium, Aspergillus and Penicillium, have been reported to produce mycotoxins that cause serious illness and immunorepression in humans and animals (Marasas, 1995). This moulds enter the food chain in the field and during storage after harvest (Petzinger and Weindenbach, 2002). There is more information concerning wheat mycoflora on the field and fresh stored wheat both on the international and national levels (Zamani-Zadeh and Khorsandi, 1995; Zare and * Corresponding Author. Phone: +98 21 82883883; Fax: +98 21 88013030; E-mail: yadegarm@modares.ac.ir Ershad, 1997; Walker et al., 2001; Bottalico and Perrone, 2002; Torp and Nirenberg, 2004; Darvish Nia et al., 2006; Davari et al., 2006; González et al., 2008). There are few reports concerning wheat mycoflora in store compared with field, and the most common fungi so far reported that contaminating wheat and flour in Australia are Aspergillus and Penicillium species (Berghofer et al., 2003). But the most common dematiaceous fungi so far reported in Brazil and Germany includes Alternaria spp. and Cladosporium spp. (Furlung et al., 1995; Weidenboner et al., 1997). The relevant study in Iran has been performed in limited range. The report from the first relevant examination on stored wheat performed in location in central province of Iran is indicating that the most abundant species are Cladosporium and Alternaria (Saberi-Riseh et al., 2004). In other studies, Aspergillus has been presented as the most dominant contaminating species concerning stored wheat (Hedayati and Mohammad pour, 2005). There are methods and media for isolation of fungi such as “Blotter” and “Agar plate” methods. The selective media for cultivation of fungi include Malachite green agar (MGA 0.25) and Nash and Snyder medium contain pentachloronitrobenzene (PCNB) (Castella et al., 1997a; Bragulat et al., 2004). The flo- R. KACHUEI et al. 384 tation method has been used for isolation of pathogenic fungi from soil (Smith and Furcolow, 1964; Vanittanakom et al., 1995). Our results based on the flotation method performed on soil samples from Isfahan province indicate that the predominant fungi include Aspergillus, Penicillium and Fusarium species (Kachuei et al., unpublished data). The selective medium Malachite green agar has been presented for the isolation of Fusarium species, specially for the isolation of Fusarium verticilloides. This medium relative to media contain PCNB, which is potentially carcinogen, has lesser risk (Castella et al.,1997a, 1997b; Bragulat et al., 2004). The flotation method accompanied with Malachite green agar so far has not been used for isolation of fungi from grains. Fusarium langsethiae described as a new toxigenic Fusarium species (Torp and Nirenberg, 2004), which has been isolated from oats, wheat and barly in northern, central and east Europe (Norway, Austria, Germany, The Netherlands, Czech Republic, Denmark, England, Italy and Poland) (Torp and Adler, 2004; Torp and Nirenberg, 2004; Infantino et al., 2007; Lukanowski et al., 2008). The basis of morphology is similar to Fusarium poae and identified as a “powdery” form of F. poae. But the profile of trichothecenes produced is very similar to Fusarium sporotrichioides and produced type A trichothecene toxins such as T-2 toxin (Torp and Langseth, 1999; Torp and Nirenberg, 2004). T-2 toxin is highly toxic, inhibit protein synthesis and inducing DNA fragmentation characteristic of apoptosis (Beasley, 1989; Prelusky et al., 1994). The current study has been performed to pursue the following goals: (1) qualitative and quantitative examination of contaminating fungi in stored wheat located in three main wheat producing and storage provinces in Iran including Tehran (north-centre of Iran), East Azarbayejan (region with cold climate in northwest of Iran) and Mazandaran (endemic area of esophageal cancer and region with moderate and humid climate in north of Iran); (2) the presentation of Flotation method (FM) accompanied with Malachite green agar (MGA 0.25) for isolation of toxigenic potential hyaline fungi from grains. FIG. 1 - Map of Iran, showing relative positions of the three provinces of sampling place (1-3) and provinces related to kind of wheat samples (4-15). 1: East Azarbayejan, 2: Mazandaran, 3: Tehran, 4: Hamadan, 5: Khoozestan, 6: Kordestan, 7: West Azarbayejan, 8: Zanjan, 9: Ardebil, 10: Fars, 11: Ghazvin, 12: Golestan, 13: Kermanshah, 14: Lorestan, 15: Markazi. MATERIALS AND METHODS Wheat samples. A total of 109 submitted samples (approximately one kg each sample) of wheat grain were collected from silos and store-pits in the three provinces, Tehran, Mazandaran and East Azarbayejan in the main wheat producing and storage areas in Iran. These samples were related to at least 15 wheatproducing provinces in Iran (Fig. 1, Table 1). TABLE 1 - List of locations, number and kind of wheat samples collected silos and store-pits in Iran, during 2007 No. of samples Provinces of related to wheat samples 13 1 2 2 2 1 14 2 2 3 9 6 3 8 3 3 2 7 2 1 East Azarbayejan Hamadan Khoozestan Kordestan West Azarbayejan Zanjan Mazandaran Ardebil Fars Ghazvin Golestan Hamadan Kermanshah Khoozestan Kordestan Lorestan Markazi Tehran West Azarbayejan Zanjan 2 2 3 5 11 Mix*(Mar, Ham, Ker) Mix (Ham, Lor, Kor, W.Az) Mix (Zan, Lor, Kor, Mar) Mix (Zan, Kor, Ham, Ard) Mix No. of samples Provinces of sampling place 21 East Azarbayejan 14 74 Mazandaran Tehran * Mixed of wheat samples in Iran (Mar: Markazi, Ham: Hamadan, Ker: Kermanshah, Lor: Lorestan, Kor: Kordestan, W.Az: West Azarbayejan, Zan: Zanjan, Ard: Ardebil). Ann. Microbiol., 59 (2) 383-390 (2009) Sampling method. The sampling was done according to International Seed Test Association (ISTA) protocol (Mathur and Kongsdal, 2003). Samples were taken from different horizontal and vertical position chosen at random. Samples were packed in paper bags and immediately sent to the laboratory, where they were stored at 5 ± 1 °C. All of the samples were original cultivated and at least stored for 1 year and intended for human consumption and none had visible signs of mould contamination. Isolation of fungi. Flotation method (FM) for isolation of surface mycoflora. Surface contaminating fungal mycoflora from wheat samples without surface disinfection were isolated on the base of FM with MGA 0.25. In this method four hundred kernels (four series, each contains 100 seeds) per subsample were examined. A suspension of 100 kernels in 8 ml sterile distilled water including solutions of Tween 80 (0.005%/ml), penicillin (20000 U/ml) and streptomycin (10 mg/ml), was shaked for 1 min, fixed for 15-30 min and then of the supernatant solution (0.5 ml) was cultured on two plates of MGA 0.25 (Nash-Snyder base containing 0.25 ppm of Malachite Green) by streak method. The plates were incubated in the dark at 28 °C for 4-10 days. Freeze Blotter (FB) method for isolation of internal mycoflora. For isolation of the internal mycoflora in grains, subsamples of wheat kernels from each sample were surface disinfected in a 1% sodium hypochlorite for one min and rinsed twice in sterile distilled water for 30 s. Four hundred kernels (four series, each contains 100 seeds) per subsample were placed in 25 kernels per plate, according to FB method (Singh et al., 1974). The resulting fungal colonies from both methods were recorded and then subcultured in Potato Dextrose agar (PDA). Identification of fungi. Aspergillus species were identified as described by Raper and Fennell (1973), Pitt and Hocking (1997) and Klich (2003). Fusarium isolates were purified by single-spore isolation. Single-spore isolates were cultured onto Carnation Leaf agar (CLA), Spezieller Nahrstoffarmer agar (SNA) and PDA plates and incubated at 25-27 °C for 7-14 days under 12 h near UV (nUV) light/12 h darkness cycle (Leslie and Summerell, 2006). Isolates were identified by conidial morphology and colony characteristic as described by Nelson et al. (1983) and Leslie and Summerell (2006). Identification of Fusarium cf. langsethiae. Morphological observations. Single-spore isolate was transferred onto PDA, CLA and SNA (with a piece of sterile filter paper placed on the agar surface) to grow at 25 °C with exposure to 12-h alternate cycles of darkness and nUV light (Philips TLD 36W/08 black light, peak 360 nm). All microscopic characters (sporodochial conidia or macroconidia observed under nUV, shape of conidia of aerial mycelium or microconidia globose/napiform, polyphialide and monophialides present, chlamydospore present) and macroscopic characters (powdery appearance on PDA and colony diameter on Potato Sucrose agar (PSA) were according to the Torp and Langseth (1999) and Torp and Nirenberg (2004). DNA extraction. Fungal DNA was extracted using standard protocols, according to Choi et al. (1990) and Rezaie et al. (2000) with slight modifications. Briefly, the harvested mycelial mass was flash-frozen in liquid nitrogen and ground to a fine powder in a porcelain mortar. The mycelial powder was suspended in DNA extraction buffer containing 50 mM Tris-HCl (pH 8.0), 50 mM EDTA, 3% SDS and 50 μl of proteinase-K (20 mg/ml). The 385 suspension was then incubated at 65 °C for 1 h and the cellular debris was removed by centrifugation at 2500 x g for 15 min. After addition of 25 μl RNase H (10 mg/ml), the suspension was incubated at 37 °C for 30 min, extracted once with phenolchloroform-isoamyl alcohol (25:24:1) and once with chloroformisoamyl alcohol (24:1). The DNA was precipitated by addition of an equal volume of isopropanol, followed by centrifugation at 15000 x g for 30 min. Finally, The DNA pellet was rinsed with 70% ethanol and resuspended in distilled water. PCR and DNA sequencing. DNA of the mentioned Fusarium isolate were used in PCR with the F. sporotrichioides-specific primers according to Kulik et al. (2004) and the F. langsethiae-specific primers (Wilson et al., 2004). Internal transcribed spacer (ITS) regions of isolate was amplified using universal primers ITS4 and ITS5 (White et al., 1990). Amplification was performed in a final volume of 50 μl. Reaction contained 5 μl of 10X PCR buffer, each deoxynucleoside triphosphate (dNTP) at 0.2 mM, each forward and reverse primer at 0.2 μM, 1 μl (50-100 ng) of template DNA, and 5 U of Taq DNA polymerase. The PCR condition consisted of the initial denaturation at 94 °C for 2 min, 35 cycles each of 30 s at 94 °C, 30 s at 58 °C, 1 min at 72 °C, and a final extension at 72 °C for 7 min. The obtained PCR product with an approximate size of 568-bp was sequenced. A 618-646 bp fraction of the Translation elongation factor 1 alpha (TEF-1A) gene was amplified using the primers EF1 and EF2 according to O’Donnell et al. (1998) and the protocol described by Carbone et al. (1999) with the exception that we used 50 μl PCR mixtures instead of 20 μl. Two external primers (EF1, EF2) (O’Donnell et al., 1998) and two nested primers (EF15fwd, EF16rev ) (Knutsen et al., 2004) were used for reamplification and cycle sequencing. The sequences were aligned using Mega 3, followed by visual inspection and manual adjustment. The isolation frequency (Fr) and the relative density (RD) of genera and species were calculated according to González et al. (2008): Fr (%) = ( RD (%) = ( ) ) 100 100 where ns = number of samples where a genus or species of fungi occurred; N = total number of samples; ni = number of isolates of a genus or species; Ni = total number of fungal isolates obtained. Statistical analysis. The Fisher exact test was applied to analyse possible differences in the isolation frequencies of fungal genera between provinces and applied methods. The analysis was performed by using the GraphPad InStat software V2.02. RESULTS AND DISCUSSION Fungi associated with wheat grain All together, 109 wheat samples were examined. Out of which 681 fungal isolates were recovered on the basis of FM method and 485 fungal isolates were isolated on the basis of FB method. The isolates were belonged to 46 species of 23 fungal genera. Based on Fr, the Penicillium spp. (78.4-92.8%), Aspergillus spp. (71.4-85.7%) and Fusarium spp. (28.6-55.4%) were predominant using FM method. However, dematiaceous fungi such as Alternaria spp. (82.4-100%) and Cladosporium spp. R. KACHUEI et al. 386 (57.1-89.2%) were defected from FB method. The difference was apparent in all three provinces (P < 0.01 to P < 0.0001). Obviously, the isolating method for identification of Aspergillus spp. in east Azarbayejan province and identification of Fusarium spp. in Mazandaran province was not significant (Table 2). The exogenous mycoflora of the examined grains has been consisted of Penicillium spp., Aspergillus spp. and Fusarium spp. and the endogenous mycoflora was consisted of Alternaria spp. and Cladosporium spp. The result of this study is in accordance with other studies (Furlung et al., 1995; Weidenboner et al., 1997; Krysinska-Traczyk et al., 2003; Saberi-Riseh et al., 2004). The grains obtained from Mazandaran province were contaminated more by Aspergillus spp., Penicillium spp. and Alternaria spp., but the grains obtained from Tehran and East Azarbayejan were contaminated more with Fusarium spp. and Cladosporium spp. (P < 0.05 to P < 0.0001) (Table 2). According to FM method, among the isolated Aspergillus species, the Fr and RD of the A. niger (39.4%, 6.3%) and A. flavus (36.7%, 5.9%) were more than other species. The significant difference between these two species was not observed (P > 0.05). In other words, these two mentioned species were dominant exogenous contaminants of the examined grains. Among these three examined provinces, the abundance of these two species was more in Tehran and East Azarbayejan provinces compared with Mazandaran province (P < 0.05 to P < 0.0001). According to FB method, the abundance of A. flavus (26.6%) and A. fumigatus (18.3%) was more than A. niger (10.1%). The Fisher exact test method indicated a significant difference between A. flavus and A. niger (P < 0.01). But there was no significant difference between A. fumigatus and A. niger. Aspergillus flavus was the most dominant endogenous Aspergillus species contaminating grains. The reports from other countries indicate that the genus Aspergillus is the most dominant contaminant of stored wheat and flour and the A. flavus is the most abundant Aspergillus contaminating wheat and flour (Halt, 1994; Abdullah, 1998; Berghofer et al., 2003; Hedayati and Mohammad pour, 2005; Riba et al., 2008). The potential risk of A. flavus in foodstuff due to aflatoxin production is considerable. Aflatoxin B1 is the most toxic form for mammals and presents mutagenic, carcinogenic and teratogenic properties and has been implicated as causative agent in human hepatic and extrahepatic carcinomas (IARC, 1993). Other Aspergillus species that were present at low frequency levels were A. nidulans, A. terreus, A. restrictus, A. ochraceus, A. parasiticus, A. candidus and A. amstelodami. Surveys carried out in Iran have shown that natural occurrence of aflatoxins in bread (Hormozdiari et al., 1975) as well as ochratoxin A in red wheat (Lacey, 1988). The Fr and RD of the Acremonium species on the basis of FM (58.7%, 9.4%) compared with FB method (1.8%, 0.4%) was highly significant (P < 0.0001) (Table 3). Therefore one can present it as exogenous contaminant of grains. Among the isolated Fusarium species (FM method), the Fr and RD of F. proliferatum (34%) (5.4%) and F. verticilloides (12.8%) (2.3%) was more than other species. The wheat obtained from Tehran province was more contaminated by F. proliferatum compared with other provinces (P < 0.01) (Table 3). The distribution of this species has implications for human and animal health due to its ability to produce fumonisins, a group of toxic and carcinogenic metabolites that designated as Group 2B carcinogens, that is ‘possibly carcinogenic to humans’ (Gelderblom et al., 1988; IARC, 1993; Jackson et al., 1996; Ghiasian et al., 2004). The results of the current study are in accordance with the performed study on the field in 23 provinces of Iran (Darvish Nia et al., 2006), Golestan province (Zare and Ershad, 1997) and Khozestan province (Moosawi-Jorf et al., 2007) and other studies (Lacey, 1988; Saremi and Okhovvat, 2006). However, our results was not in accordance with two other field studies (Zamani-Zadeh and Khorsandi, 1995; Davari et al., 2006). In the USA, Canada and Argentina the predominant Fusarium species isolated in the field were F. graminearum (Walker et al., 2001; González et al., 2008), while in Europe the most common isolates were F. graminearum, F. culmorum, F. avenaceum, F. poae and F. langsethiae (Bottalico and Perrone, 2002; Torp and Nirenberg, 2004). Other Fusarium species that were present at low frequency levels were F. subglutinans, F. oxysporum, F. nygamai, F. culmorum, F. acuminatum, F. equiseti, F. pseudonygamai, F. tricinctum, F. lateritium, F. chlamydosporum and F. babinda. Studies on fungal species isolated from Iranian cereals have demonstrated their ability to produce trichothecenes (Rezayat et al., 1996; Haratian et al., 2008), moniliformin (Zamani-zadeh, 1996), fumonisins (Ghiasian et al., 2005) and zearalenone (Zamani-Zadeh and Khorsandi, 1995; Saremi and Okhovvat, 2006). In this study, we have also isolated a rare species known as F. xylarioides in Iran. The latter case has been recently reported as Fusarium head blight by Moosawi-Jorf et al. (2007).This species is rarely isolated from wheat and may cause coffee wilt disease (Nelson et al., 1983). TABLE 2 - Major fungal genera (hyaline and dematiaceae) recovered from wheat kernels collected of silos and storepits in three provinces in Iran, during 2007 Species East Azarbayejan FB FM RD Fr RD Fr (%) (%) (%) (%) Mazandaran FB FM RD Fr RD Fr (%) (%) (%) (%) Aspergillus spp. 21.3 66.6 71.4 21.7 57.1 30.7 85.7 Penicillium spp. 12.3 52.4 14.7 80.9 6.7 28.5 32.3 92.8 Fusarium spp. 4.5 10 28.6 9.2 28.6 19 25 9.5 42.8 Tehran FB RD (%) FM Fr (%) RD (%) 16.7 50 24.1 12.5 54 18 4.7 14.8 12.3 Fr (%) 73 Overall mean FB FM RD Fr RD Fr (%) (%) (%) (%) 18.1 54.1 25 74.3 78.4 11.7 50.4 18.6 80.7 55.4 17.4 11.4 49.5 Cladosporium spp. 20.2 85.7 8 52.4 13.3 57.1 3 14.3 25 89.2 11.2 22.7 84.4 9.8 58.7 Alternaria spp. 20.2 85.7 5.1 33.3 31.6 100 nd nd 19 82.4 2.9 18.9 20.8 85.3 3.1 19.2 480 485 681 Total isolates Total samples 89 136 21 60 65 14 336 74 69 5.4 109 FB: Freeze Blotter, FM: Flotation method with MGA 0.25, RD: relative density, Fr: frequency of isolation, nd: not detected. Ann. Microbiol., 59 (2) 383-390 (2009) 387 TABLE 3 - Fungal species recovered from wheat kernels collected of silos and store-pits in three provinces in Iran, during 2007 Species East Azarbayejan FB RD (%) Absidia spp. 2.2 Acremonium spp. 2.2 Alternaria spp. 20.2 Aspergillus amstelodami nd Aspergillus candidus nd Aspergillus flavus 7.8 Aspergillus fumigatus 5.6 Aspergillus nidulans nd Aspergillus niger 4.5 Aspergillus ochraceus nd Aspergillus parasiticus nd Aspergillus restrictus nd Aspergillus terreus nd Aspergillus spp. 3.4 Aureobasidium pullulans 4.5 Bipolaris spp. 1.1 Cladosporium spp. 20.2 Curvularia spp. nd Epicoccum spp. nd Fusarium acuminatum 1.1 Fusarium babinda nd Fusarium chlamydosporum nd Fusarium culmorum nd Fusarium equiseti nd Fusarium cf. langsethiae nd Fusarium lateritium nd Fusarium nygamai nd Fusarium oxysporum 1.1 Fusarium proliferatum nd Fusarium pseudonygamai nd Fusarium subglutinans nd Fusarium tricinctum nd 2.2 Fusarium verticillioides Fusarium xylarioides nd Fusarium spp. nd Mucor spp. 3.4 Nigrospora spp. nd Paecilomyces spp. nd Penicillium spp. 12.3 Rhizomucor spp. nd Rhizopus spp. 3.4 Scopulariopsis spp. nd Stemphillium spp. nd Syncephalastrum spp. nd Torula spp. nd Trichoderma spp. 1.1 Trichothecium spp. 1.1 Ulocladium spp. 1.1 Yeast spp. 1.1 Un known spp. nd Total isolates Total samples Mazandaran FM Fr RD (%) (%) 9.5 2.2 9.5 8.8 85.7 5.1 nd nd nd nd 33.3 6.6 23.8 6.6 nd 0.7 19 6.6 nd nd nd nd nd nd nd nd 14.3 4.4 19 0.7 4.7 nd 85.7 8 nd nd nd nd 4.7 0.7 nd nd nd nd nd nd nd nd nd nd nd 0.7 nd nd 4.7 0.7 nd 2.9 nd nd nd 0.7 nd nd 9.5 2.9 nd 0.7 nd nd 14.3 0.7 nd nd nd nd 52.4 14.7 nd nd 14.3 3.7 nd 0.7 nd nd nd nd nd nd 4.7 nd 4.7 0.7 4.7 0.7 4.7 11 nd 8 89 136 21 FB Fr RD (%) (%) 14.3 nd 47.6 nd 33.3 31.6 nd nd nd nd 42.8 10 42.8 1.6 4.7 nd 42.8 nd nd nd nd nd nd nd nd nd 23.8 10 4.7 nd nd 1.6 52.4 13.3 nd 1.6 nd nd 4.7 nd nd nd nd nd nd 1.6 nd nd nd nd 4.7 nd nd nd 4.7 nd 19 3.3 nd nd 4.7 nd nd nd 19 1.6 4.7 nd nd 3.3 4.7 nd nd nd nd nd 80.9 6.7 nd nd 23.8 nd 4.7 1.6 nd nd nd nd nd nd nd nd 4.7 nd 4.7 10 61.9 nd 33.3 1.6 Tehran FM FB Fr RD Fr RD Fr RD (%) (%) (%) (%) (%) (%) nd 1.5 7.1 nd nd 1 nd 6.1 28.5 2 9.4 10 100 nd nd 19 82.4 2.9 nd nd nd 0.3 1.3 0.2 nd nd nd 0.3 1.3 0.2 42.8 4.6 21.4 4.7 21.6 5.8 7.1 4.6 21.4 4.2 18.9 3.7 nd 3 14.3 nd nd 1.9 nd 3 14.3 2 9.4 6.7 nd nd nd nd nd 0.2 nd nd nd 0.3 1.3 0.2 nd nd nd 0.6 2.7 nd nd 1.5 7.1 1.2 5.4 1.6 35.7 13.8 35.7 3 13.5 3.5 nd nd nd nd nd nd 7.1 nd nd 1.2 5.4 0.2 57.1 3 14.3 25 89.2 11.2 7.1 nd nd 0.3 1.3 nd nd 1.5 7.1 nd nd 0.2 nd nd nd nd nd nd nd nd nd 0.3 1.3 0.2 nd nd nd nd nd 0.2 7.1 nd nd 0.3 1.3 nd nd nd nd nd nd 0.2 nd nd nd 0.3 1.3 nd nd nd nd nd nd nd nd nd nd nd nd 1.2 nd nd nd 0.3 1.3 0.6 14.3 4.6 21.4 1.8 8.1 6.2 nd nd nd nd nd 0.2 nd 1.5 7.1 0.9 4 1.6 nd nd nd 0.3 1.3 nd 7.1 3 14.3 0.3 1.3 1.6 nd nd nd nd nd nd 14.3 nd nd 0.3 1.3 0.2 nd 1.5 7.1 1.5 6.7 2.1 nd nd nd 0.3 1.3 nd nd nd nd nd nd 0.2 28.5 32.3 92.8 12.5 54 18 nd nd nd nd nd 0.2 nd 7.7 35.7 2.7 12.1 6.4 7.1 nd nd nd nd 1.2 nd nd nd 0.3 1.3 0.2 nd nd nd nd nd 0.4 nd nd nd 0.3 1.3 nd nd 3 14.3 nd nd nd nd nd nd nd nd nd 42.8 nd nd 6.5 29.7 0.2 nd nd nd 2.4 10.8 5.8 7.1 3 14.3 4.5 20.3 2.9 60 65 14 Overall mean FM 336 480 74 Fr (%) 6.7 64.8 18.9 1.3 1.3 37.8 24.3 12.1 43.2 1.3 1.3 nd 10.8 22.9 nd 1.3 69 nd 1.3 nd 1.3 1.3 nd 1.3 nd nd 8.1 4 40.5 1.3 10.8 nd 10.8 nd 1.3 13.5 nd 1.3 78.4 1.3 41.8 8.1 1.3 2.7 nd nd nd 1.3 37.8 18.9 FB RD (%) 0.4 1.8 20.8 0.2 0.2 6 4.1 nd 2.3 nd 0.2 0.4 0.8 3.9 0.8 1.2 22.7 0.4 nd 0.2 0.2 nd 0.4 nd 0.2 nd nd 0.4 1.6 nd 0.6 0.2 0.8 nd 0.6 1.2 0.2 nd 11.7 nd 2.5 0.2 0.2 nd 0.2 0.2 0.2 6 1.8 3.3 FM Fr RD Fr (%) (%) (%) 1.8 1.3 8.2 8.2 9.4 58.7 85.3 3.1 19.2 0.9 0.1 0.9 0.9 0.1 0.9 26.6 5.9 36.7 18.3 4.4 27.5 nd 1.8 11 10.1 6.3 39.4 nd 0.1 0.9 0.9 0.1 0.9 1.8 nd nd 3.7 1.3 8.2 17.4 4.7 31.2 3.7 0.1 0.9 5.5 0.1 0.9 84.4 9.8 58.7 1.8 nd nd nd 0.3 1.8 0.9 0.1 0.9 0.9 nd nd nd 0.1 0.9 1.8 nd nd nd 0.1 0.9 0.9 nd nd nd 0.1 0.9 nd 0.9 5.5 1.8 0.6 3.7 7.3 5.4 34 nd 0.1 0.9 2.7 1.5 9.2 0.9 nd nd 3.7 2.3 12.8 nd 0.1 0.9 2.7 0.1 0.9 7.3 1.8 11 0.9 nd nd nd 0.1 0.9 50.4 18.6 80.7 nd 0.1 0.9 11 6 37.6 0.9 1 6.4 0.9 0.1 0.9 nd 0.3 1.8 0.9 nd nd 0.9 0.3 1.8 0.9 0.1 0.9 26.6 0.3 1.8 8.2 6.3 37.6 14.7 4 21.1 485 681 109 FB: Freeze Blotter, FM: Flotation method with MGA 0.25, RD: relative density, Fr: frequency of isolation, nd: not detected. Description of Fusarium cf. langsethiae Macroscopic observation In primary culturing on PDA medium, they appeared as pink colour, had slow growth and possessed small air mycelium. On repeated passage on PDA medium they appeared as white coloured with powdered characteristic. On PSA medium they demonstrated a rapid growth and possessed more air mycelium. Radial growth rate on PSA and PDA at 25 °C in complete darkness was 8.5 and 7.5 mm/day, respectively, at 20 ± 0.5 °C on PDA, 6.4 mm/day. Microscopic observation Conidia napiform or globose, nonseptate, measuring: 5.1-9.4 x 3.8-6.4 μm with average and S.D. of 6.7 ± 1 x 5 ± 0.6 μm. Chlamydospores not formed, no sporodochial and macroconidia under nUV, conidiophores short and branched, branched monophialides, polyphialide, and ampuliform phialides (Fig. 2). Molecular properties The sequencing result of PCR product that was based on universal primers ITS4 and ITS5 (568 bp) with two registered records R. KACHUEI et al. 388 in GenBank/EMBL database belonged to F. langsethiae with accession number EF526078 and AY680864. This result indicated 100% identity. The isolated species did not respond to specific primers belonging to F. sporotrichioides as Kulik et al. (2004) but did respond to specific primers belonging to F. sporotrichioides that designed by Wilson et al. (2004). The specific primers belonging to F. langsethiae that designed by these authors did not any positive PCR. The sequencing result of PCR product that was based on external primers (EF1, EF2) and nested primers (EF15, EF16) for TEF-1A gene with registered sequences recorded in GenBank/EMBL database related to F. langsethiae (accession Nr. AJ427272), revealed high homology with the same genes from F. langsethiae (99%). However, the same results have been detected by homology search with F. sporotrichioides spe- cies (accession Nr. EF521146, AY337442, AJ420840, AJ420820) as well as 11 other F. sporotrichioides species recorded. With regard to the morphological results, ITS1, ITS2 and TEF-1A gene sequences, this species is similar to F. langsethiae (IBT 9959). On the basis of morphology, Kristensen et al. (2005) has classified the IBT 9959 species as F. cf. langsethiae, however on the basis of TEF-1A gene it is classified as F. sporotrichioides. Thus this isolate could be considered as the intermediat species of F. langsethiae and F. sporotrichioides. This isolated species has been discovered for first time in Iran. According our knowledge it has not been reported so far such case in Asia. This is the second report in the literature. With regard to our experience in using the flotation method on soil for isolation of the potentially toxigenic species including Penicillium spp., Aspergillus spp., and Fusarium spp. as dominant species, we applied this method by some modification for isolation of fungal exogenous species from grains. In this method instead of using MGA 2.5 that introduced by Castella et al. (1997a, 1997b) and Bragulat et al. (2004) for isolation of Fusarium species specially F. verticilloides, we used of MGA 0.25 ppm for isolation of other fungi specially Fusarium spp. and Aspergillus spp. that also mucoral fungi growth restricted. CONCLUSIONS With regard to our results by application of FM method, we can recommend this method for isolation of toxigenic potential hyaline fungi such as Penicillium spp., Aspergillus spp., Fusarium spp. and Acremonium spp. from grains. In most cases, moreover, this method can observe only one colony on MGA 0.25 ppm medium. 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