Isolation and Identification of Fungal Species from the Insect Pest Tribolium castaneum in Rice Processing Complexes in Korea.

The red flour beetle, Tribolium castaneum, is one of the most common and economically important pests of stored cereal products worldwide. Furthermore, these beetles can act as vectors for several fungal post-harvest diseases. In this study, we collected T. castaneum from 49 rice processing complexes (RPCs) nationwide during 2016-2017 and identified contaminating fungal species on the surface of the beetles. Five beetles from each region were placed on potato dextrose agar media or Fusarium selection media after wet processing with 100% relative humidity at 27°C for one week. A total of 142 fungal isolates were thus collected. By sequence analysis of the internal transcribed spacer region, 23 fungal genera including one unidentified taxon were found to be associated with T. castaneum. The genus Aspergillus spp. (28.9%) was the most frequently present, followed by Cladosporium spp. (12.0%), Hyphopichia burtonii (9.2%), Penicillium spp. (8.5%), Mucor spp. (6.3%), Rhizopus spp. (5.6%), Cephaliophora spp. (3.5%), Alternaria alternata (2.8%) and Monascus sp. (2.8%). Less commonly identified were genera Fusarium, Nigrospora, Beauveria, Chaetomium, Coprinellus, Irpex, Lichtheimia, Trichoderma, Byssochlamys, Cochliobolus, Cunninghamella, Mortierella, Polyporales, Rhizomucor and Talaromyces. Among the isolates, two known mycotoxin-producing fungi, Aspergillus flavus and Fusarium spp. were also identified. This result is consistent with previous studies that surveyed fungal and mycotoxin contamination in rice from RPCs. Our study indicates that the storage pest, T. castaneum, would play an important role in spreading fungal contaminants and consequently increasing mycotoxin contamination in stored rice.

The control of pests in stored grains is as economically important as increasing the crop yield because, unlike crop damage during the growing season, post-harvest damage of stored grains is not financially compensated. Fungi and animal pests are the major culprits for damage of stored grains, globally estimated to be responsible for 20% of food losses and up to 40–50% in some developing countries.

The red flour beetle (Tribolium castaneum Herbst) is one of the most important pests for stored grains such as rice (Kim and Ryoo, 1982), maize (LeCato and Flaherty, 1973), millet (Roorda et al., 1982), sorghum (Shazali and Smith, 1986), and wheat flour (Birch, 1945; Daniels, 1956) worldwide. Furthermore, T. castaneum beetles cause additional damage by spreading and promoting fungal contamination (Karunakaran et al., 2004; Kim and Ryoo, 1982; Simpanya et al., 2001). Here we chose to investigate the fungal contaminants disseminated by T. castaneum.

The T. castaneum has been reported to increase the moisture and temperature of stored grains to create an environment favorable for fungal proliferation, thereby accelerating grain degradation and decay (Miller, 1995). Degradation of stored grain by fungi results in lower germination rate, weight loss, loss of nutrients, odor and discoloration, which reduce overall grain quality. Fungal contamination of stored grains not only results in enormous economic losses but also has harmful consequences on human health and livestock due to toxic fungal secondary metabolites called mycotoxins (Tipples, 1995).

T. castaneum has also been reported to act as a vector for these toxigenic fungi during storage (Philip and Throne, 2010). When maize flour is co-contaminated with T. castaneum, toxigenic fungi including Aspergillus spp. are approximately 5 times more abundant than in the absence of the beetle vectors (Simpanya et al., 2001). Unlike in other stored grains such as wheat, barley, and corn, insectmediated fungal toxin contamination in stored rice has yet to be reported (Tanaka et al., 2004).

Aspergillus spp., which produces aflatoxin in contaminated rice, has been reported to occur mainly in high temperature and high humidity countries such as India, China, and Iran (Rahmania et al., 2011; Reddy et al., 2008). It has been reported that deoxynivalenol (DON), nivalenol (NIV), zearalenone (ZEA) and fumonisin (FMS), which are mycotoxins of genus Fusarium, were detected in stored rice (Abbas et al., 1998; Lee et al., 2011; Tanaka et al., 2004). Previously, the distribution of toxigenic fungi on rice was investigated in the southern and central regions of Korea and ochratoxin A (OTA), aflatoxin B1 (AFB1), fumonisin B1 (FB1), and zearalenone (ZEN) were detected (Park et al., 2005). According to other reports, which tested the geographic distribution of toxigenic fungi contaminating seven different types of rice samples (paddy, husk, brown, bluetinged, discolored, Broken and polished)from rice processing complexes, Fusarium spp. and Alternaria spp. were common in the southern region, while Aspergillus spp. and Penicillium spp. were common in the central region of Korea (Son et al., 2011).

Most studies previously conducted in Korea have focused on the regional distribution of contaminating fungal species, and only a few studies have examined the effect of T. castaneum on fungal transmission (Kim and Ryoo, 1982). The purpose of this study was to investigate what type of fungi could be disseminated by T. castaneum collected at rice processing complexes by time periods. This study could be used as a reference for establishing a system to effectively protect stored agricultural products.

Materials and Methods

Study site and insect trapping

T. castaneum beetles were collected in 49 different rice processing complexes (RPCs) nationwide, over three collection dates between April 2016 and August 2017 (Fig. 1A). We installed three or four corrugated traps (300 × 300 × 2 mm) at each RPC. Traps were placed in a variety of positions on the grain surface and collected a week later, placed individually into plastic bags. In the laboratory, the T. castaneum adults from the trap were placed into an insect breeding box (72 × 72 × 100 mm) and stored until just before the experiment.

Fig. 1

Collation map of fungal isolates from the insect pest Tribolium castaneum in rice processing complexes (RPCs) during 2016 to 2017 in Korea. (A) Distribution map of 49 RPCs in Korea. The 49 RPCs are indicated by gray circles. The name of RPCs are noted by two capitalized letter just below the gray circles. The blue-colored letter indicates 8 provinces in Korea and two capitalized letters in parentheses indicates the abbreviation of provinces (See Table 1). The obtained fungal isolates and distribution map from (B) the first round (44 isolates from 17 RPCs), (C) the second round (46 isolates from 22 RPCs), and (D) the third round (52 isolates from 26 RPCs) of collection. The yellow colored circles indicates the location that obtained fungal isolates from the collected T. castaneum. A total number of fungal isolates is noted in the yellow colored circles. The percentage distribution of different fungal isolates from (E) the first round, (F) the second round, and (G) the third round of collection.

Isolation of fungi from T. castaneum adult

The boxes containing T. castaneum were transferred to −15°C for 30 min (Fields, 2012), then five T. castaneum individuals were each placed on a sterilized glass slide inside a 9 cm Petri-dish lined with a single-layer of wet filter paper. The plates were incubated at 27°C for 7 days. After wet processing, the beetles were transferred onto potato dextrose agar (PDA) media containing streptomycin (50 mg/L) or Fusarium selective media (Nash and Snyder, 1962), and incubated at 25°C for 3 to 7 days. The fungal isolates were transferred to PDA medium and identified according to microscopic observations following the taxonomic keys for each genus (Barnett and Hunter, 1972; Samson et al., 1995). All fungal isolates were deposited at the Center for Fungal Genetic Resources (CFGR) at Seoul National University, Seoul, Korea.

Isolation of genomic DNA from fungal cultures

For molecular identification, fungal genomic DNA was extracted from mycelia using DNeasy Plant Mini Kit according to the manufacturer’s protocol (Qiagen, Valencia, CA, USA). Using the purified DNA from the collected isolates, the internal transcribed spacer with 5.8 s rDNA was amplified using ITS5/ITS4 (White et al). For further identification, beta-tubulin, calmodulin, translation elongation factor1 and glyceraldehyde-3-phosphate dehydrogenase sequence data were amplified using primer pairs BT2A/BT2B (Glass and Donaldson, 1995; O’Donnell and Cigelnik, 1997), CL1/CL2A (O’Donnell et al., 2000), 728F/1569R or 728F/EF2 (Carbone and Kohn, 1999; O’Donnell and Cigelnik, 1997), and GDF1/GDR1 (Guerber et al., 2003), respectively.

PCR reactions were performed using AccuPower PCR Premix (Bioneer, Korea) with an initial denaturation for 5 min at 94°C, 30 cycles of 1 min denaturation at 94°C, 1 min annealing at 55°C, 1 min extension at 72°C, followed by a final extension for 5 min at 72°C. PCR products were confirmed by gel electrophoresis, purified with AccuPower PCR purification kit (Bineer, Korea) and bi-directionally sequenced on both strands with the same primers used for PCR amplification. Sequence assembly was performed using SeqMan program of DNA star (Madison, WI). The obtained nucleotide sequences were used for BLASTn search in the GenBank database (http://www.ncbi.nlm.nih.gov/BLAST/).

Results and Discussion

In the first round of collection (June 4, 2016), 44 fungal strains were obtained from 17 RPCs (Fig. 1B), 46 were collected in the second round from 22 RPCs (May 18, 2017) (Fig. 1C), and 52 in the third round from 26 RPCs (Aug 1, 2017) (Fig. 1D). Based on the NCBI BLAST search results of the ITS sequences and morphological analysis, Aspergillus spp. including A. flavus were dominant in whole collected periods (Fig. 1E–G).

A total 142 fungal isolates corresponding to 49 species, belonging to 23 genera, were identified from 40 RPCs (Fig. 2A, Table 1 and Table 2). The major fungal species isolated in each sampling period were Aspergillus spp. including A. flavus (40.9%), Cladosporium sp. (15.9%), and Mucor spp. (9.1%) in the first round (June 2016) (Fig. 1E), Aspergillus spp. including A. flavus (28.3%), Cladosporium sp. (17.4%), and Penicillium spp. (10.9%) in the second round (May 2017) (Fig. 1F), and Aspergillus spp. including A. flavus (19.2%), Hyphopichia sp. (15.4%), Mucor spp. (7.7%), and Penicillium spp. (7.7%) for the third round (August 2017).

Fig. 2

Summary of fungal isolates from Tribolium castaneum. (A) Distribution map of 142 fungal isolates were identified from 40 RPCs. (B) The percentage distribution of different fungal isolates among the collected 142 isolates between April 2016 and August 2017. Distribution map of (C) Aspergillus flavus (collected from 12 RPCs), (D) Aspergillus spp. (16 RPCs) (E) Penicillum spp. (9 RPCs), Alternaria alternate and Fusarium spp. were collected from T. castaneum in this study. The number in the circles indicates a total number of identified isolates.

Table 1

Fungal isolates from Tribolium castaneum, GenBank accessions nos. of the ITS region sequences and the Blast search results of the sequences obtained

Isolates	Collection Date	Region, Province	GenBank Accession No.	Most closely related fungi (GenBank Accession No.)	Similarity (%)
CB-BE-3-1	June 4, 2016	Boeun, Chungbuk	MG554270	Aspergillus sp. (KX148624.1)	100
CB-JC-3-2	June 4, 2016	Jincheon, Chungbuk	MG554234	Aspergillus flavus (AM745114.1)	100
CB-JC-3-3	June 4, 2016	Jincheon, Chungbuk	MG554235	Mucor circinelloides (KT336541.1)	99
CB-JC-3-1	June 4, 2016	Jincheon, Chungbuk	MG554233	Mucor racemosus (LN809049.1)	99
CN-GJ-3-2	June 4, 2016	Gongju, Chungnam	MG554260	Alternaria alternata (HQ380767.1)	100
CN-GJ-3-1	June 4, 2016	Gongju, Chungnam	MG554259	Alternaria alternata (HQ380767.1)	100
GB-GR-3-1	June 4, 2016	Goryeong, Gyeongbuk	MG554250	Irpex lacteus (KU761586.1)	99
GB-GR-3-2	June 4, 2016	Goryeong, Gyeongbuk	MG554251	Mucor racemosus (HM641690.1)	99
GB-MG-3-3	June 4, 2016	Mungyeong, Gyeongbuk	MG554264	Aspergillus sp. (KC178674.1)	100
GB-MG-3-4	June 4, 2016	Mungyeong, Gyeongbuk	MG554265	Aspergillus sp. (KC178674.1)	100
GB-MG-3-2	June 4, 2016	Mungyeong, Gyeongbuk	MG554263	Monascus sp. (HQ214967.1)	100
GB-MG-3-1	June 4, 2016	Mungyeong, Gyeongbuk	MG554262	Polyporales sp. (JQ312163.1)	98
GB-SJ-3-3	June 4, 2016	Sangju, Gyeongbuk	MG554242	Cladosporium sp. (KJ957785.1)	100
GB-SJ-3-1	June 4, 2016	Sangju, Gyeongbuk	MG554240	Rhizopus oryzae (JQ745257.1)	100
GB-SJ-3-2	June 4, 2016	Sangju, Gyeongbuk	MG554241	Rhizopus oryzae (AB109755.1)	100
GB-YC-3-2	June 4, 2016	Yecheon, Gyeongbuk	MG554237	Aspergillus flavus (KR611590.1)	99
GB-YC-3-1	June 4, 2016	Yecheon, Gyeongbuk	MG554236	Aspergillus sp. (KJ863514.1)	99
GG-YJ-3-1	June 4, 2016	Yeoju, Gyeonggi	MG554253	Aspergillus sp. (HQ832844.1)	99
GG-YJ-3-4	June 4, 2016	Yeoju, Gyeonggi	MG554256	Aspergillus sp. (KY086232.1)	100
GG-YJ-3-3	June 4, 2016	Yeoju, Gyeonggi	MG554255	Lichtheimia ramosa (KP132378.1)	100
GG-YJ-3-2	June 4, 2016	Yeoju, Gyeonggi	MG554254	Mucor racemosus (JF723566.1)	100
GN-HY-3-1	June 4, 2016	Hamyang, Gyeongnam	MG554257	Cladosporium sp. (KT826674.1)	100
GN-HY-3-2	June 4, 2016	Hamyang, Gyeongnam	MG554258	Rhizopus oryzae (JQ745257.1)	99
JJ-JJ-3-2	June 4, 2016	Jeju, Jeju	MG554239	Chaetomium globosum (KM268670.1)	99
JJ-JJ-3-1	June 4, 2016	Jeju, Jeju	MG554238	Irpex lacteus (JX290570.1)	99
JB-GC-3-3	June 4, 2016	Gochang, Jeonbuk	MG554245	Aspergillus flavus (EF409804.1)	99
JB-GC-3-2	June 4, 2016	Gochang, Jeonbuk	MG554244	Aspergillus oryzae (KX462757.1)	100
JB-GC-3-1	June 4, 2016	Gochang, Jeonbuk	MG554243	Cladosporium sp. (KX757230.1)	100
JB-GC-3-5	June 4, 2016	Gochang, Jeonbuk	MG554247	Cladosporium sp. (KJ957785.1)	100
JB-GC-3-4	June 4, 2016	Gochang, Jeonbuk	MG554246	Penicillium citrinum (KY921947.1)	100
JB-IK-3-2	June 4, 2016	Iksan, Jeonbuk	MG554267	Aspergillus flavus (GQ370372.1)	99
JB-IK-3-1	June 4, 2016	Iksan, Jeonbuk	MG554266	Cladosporium cycadicola (KJ869122.1)	99
JB-IM-3-1	June 4, 2016	Imsil, Jeonbuk	MG554261	Cladosporium sp. (KT329207.1)	100
JN-GJ-3-1	June 4, 2016	Gangjin, Jeonnam	MG554248	Hyphopichia burtonii (DQ683006.1)	99
JN-GJ-3-2	June 4, 2016	Gangjin, Jeonnam	MG554249	Penicillium sp. (HE608805.1)	99
JN-GR-3-1	June 4, 2016	Gurye, Jeonnam	MG554252	Irpex lacteus (KU761586.1)	99
JN-NJ-3-2	June 4, 2016	Naju, Jeonnam	MG554269	Aspergillus flavus (MF377553.1)	100
JN-NJ-3-1	June 4, 2016	Naju, Jeonnam	MG554268	Aspergillus sp. (KC834792.1)	100
JN-YG-3-2	June 4, 2016	Yeonggwang, Jeonnam	MG554228	Aspergillus candidus (JQ781823.1)	99
JN-YG-3-5	June 4, 2016	Yeonggwang, Jeonnam	MG554231	Aspergillus flavus (KR611590.1)	100
JN-YG-3-3	June 4, 2016	Yeonggwang, Jeonnam	MG554229	Aspergillus sp. (KC178662.1)	100
JN-YG-3-1	June 4, 2016	Yeonggwang, Jeonnam	MG554227	Aspergillus sydowii (KP131616.1)	100
JN-YG-3-6	June 4, 2016	Yeonggwang, Jeonnam	MG554232	Cladosporium sp. (HQ166315.1)	100
JN-YG-3-4	June 4, 2016	Yeonggwang, Jeonnam	MG554230	Penicillium citrinum (KY921947.1)	99
CB-CJ-1-2	May 18, 2017	Chungju, Chungbuk	MG554302	Aspergillus flavus (LC133097.1)	99
CB-JC-1-5	May 18, 2017	Jincheon, Chungbuk	MG554303	Mucor sp. (KX099678.1)	100
CN-NS-1-1	May 18, 2017	Nonsan, Chungnam	MG554309	Aspergillus sydowii (KX958061.1)	100
CN-NS-1-2	May 18, 2017	Nonsan, Chungnam	MG554310	Lichtheimia corymbifera (KU147463.1)	100
CN-SC-1-1	May 18, 2017	Seocheon, Chungnam	MG554306	Hyphopichia burtonii (KY103598.1)	100
GW-GS-1-5	May 18, 2017	Goseong, Gangwon	MG554277	Aspergillus versicolor (AJ937749.1)	100
GW-GS-1-1	May 18, 2017	Goseong, Gangwon	MG554275	Beauveria bassiana (KM249032.1)	100
GW-GS-1-4	May 18, 2017	Goseong, Gangwon	MG554276	Penicillium neoechinulatum (AJ005481.1)	100
GB-GJ-1-1	May 18, 2017	Gyeongju, Gyeongbuk	MG554304	Aspergillus flavus (KY593504.1)	100
GB-GJ-1-2	May 18, 2017	Gyeongju, Gyeongbuk	MG554305	Aspergillus sp. (KX450911.1)	100
GB-MG-1-3	May 18, 2017	Mungyeong, Gyeongbuk	MG554279	Cladosporium velox (KX788192.1)	100
GB-MG-1-2	May 18, 2017	Mungyeong, Gyeongbuk	MG554278	Hyphopichia burtonii (EU714323.1)	100
GB-MG-1-4	May 18, 2017	Mungyeong, Gyeongbuk	MG554280	Hyphopichia burtonii (EU714323.1)	100
GB-MG-1-5	May 18, 2017	Mungyeong, Gyeongbuk	MG554281	Hyphopichia burtonii (KY103602.1)	100
GB-SJ-1-4	May 18, 2017	Sangju, Gyeongbuk	MG554285	Cladosporium halotolerans (KP701942.1)	100
GB-SJ-1-2	May 18, 2017	Sangju, Gyeongbuk	MG554283	Cladosporium sp. (KR081401.1)	100
GB-SJ-1-3	May 18, 2017	Sangju, Gyeongbuk	MG554284	Cladosporium sphaerospermum (KY987535.1)	100
GB-SJ-1-1	May 18, 2017	Sangju, Gyeongbuk	MG554282	Cladosporium velox (KX788192.1)	99
GB-US-1-3	May 18, 2017	Uisung, Gyeongbuk	MG554273	Cladosporium sp. (KX148680.1)	99
GB-US-1-4	May 18, 2017	Uisung, Gyeongbuk	MG554274	Nigrospora oryzae (KX986075.1)	100
GG-PJ-1-1	May 18, 2017	Paju, Gyeonggi	MG554314	Aspergillus creber (LN898694.1)	100
GN-GS-1-5	May 18, 2017	Goseong, Gyeongnam	MG554272	Cladosporium sphaerospermum (KP701988.1)	100
GN-GS-1-1	May 18, 2017	Goseong, Gyeongnam	MG554271	Mortierella oligospora (KM265101.1)	100
GN-HA-1-3	May 18, 2017	Haman, Gyeongnam	MG554311	Cephaliophora tropica (FJ792583.1)	99
GN-HY-1-6	May 18, 2017	Hamyang, Gyeongnam	MG554298	Aspergillus sp. (KX928745.1)	100
GN-HY-1-5	May 18, 2017	Hamyang, Gyeongnam	MG554297	Beauveria bassiana (KY682175.1)	100
GN-HY-1-1	May 18, 2017	Hamyang, Gyeongnam	MG554296	Penicillium crustosum (MF188258.1)	100
GN-JJ-1-4	May 18, 2017	Jinju, Gyeongnam	MG554308	Rhizopus oryzae (KY244030.1)	100
GN-US-1-1	May 18, 2017	Ulsan, Gyeongnam	MG554291	Aspergillus sp. (KX008655.1)	99
GN-US-1-3	May 18, 2017	Ulsan, Gyeongnam	MG554293	Cochliobolus miyabeanus (KC315937.1)	100
GN-US-1-2	May 18, 2017	Ulsan, Gyeongnam	MG554292	Fusarium proliferatum (MG625088.1)	100
JB-GC-1-3	May 18, 2017	Gochang, Jeonbuk	MG554316	Aspergillus sclerotiorum (AY373866.1)	100
JB-GC-1-1	May 18, 2017	Gochang, Jeonbuk	MG554315	Penicillium polonicum (KX674637.1)	100
JB-IS-1-1	May 18, 2017	Iksan, Jeonbuk	MG554294	Fusarium oxysporum (KY508368.1)	100
JB-IS-1-3	May 18, 2017	Iksan, Jeonbuk	MG554295	Penicillium sp. (KY401140.1)	100
JB-NW-1-3	May 18, 2017	Namwon, Jeonbuk	MG554300	Aspergillus flavus (KX912161.1)	100
JB-NW-1-1	May 18, 2017	Namwon, Jeonbuk	MG554299	Cephaliophora tropica (KR809561.1)	100
JB-NW-1-4	May 18, 2017	Namwon, Jeonbuk	MG554301	Rhizopus microsporus (KY606252.1)	99
JN-HN-1-2	May 18, 2017	Haenam, Jeonnam	MG554307	Monascus sp. (KY511749.1)	100
JN-NJ-1-1	May 18, 2017	Naju, Jeonnam	MG554312	Aspergillus versicolor (JN638793.1)	100
JN-NJ-1-2	May 18, 2017	Naju, Jeonnam	MG554313	Aspergillus versicolor (GU232767.1)	100
JN-YG-1-5	May 18, 2017	Yeonggwang, Jeonnam	MG554289	Aspergillus flavus (JQ316530.1)	100
JN-YG-1-1	May 18, 2017	Yeonggwang, Jeonnam	MG554286	Beauveria bassiana (KY682175.1)	100
JN-YG-1-6	May 18, 2017	Yeonggwang, Jeonnam	MG554290	Cephaliophora tropica (KR809561.1)	100
JN-YG-1-3	May 18, 2017	Yeonggwang, Jeonnam	MG554288	Cladosporium sp. (KY643766.1)	100
JN-YG-1-2	May 18, 2017	Yeonggwang, Jeonnam	MG554287	Penicillium crustosum (KY906188.1)	100
CB-CJ-2-1	Aug 1, 2017	Chungju, Chungbuk	MG554336	Mucor circinelloides (LN809028.1)	100
CB-CJ-2-2	Aug 1, 2017	Chungju, Chungbuk	MG554337	Mucor circinelloides (LN809028.1)	100
CB-CJ-2-3	Aug 1, 2017	Chungju, Chungbuk	MG554338	Mucor circinelloides (LN809028.1)	100
CN-HS-2-1	Aug 1, 2017	Hongseong, Chungnam	MG554358	Hyphopichia burtonii (KY103598.1)	100
CN-NS-2-1	Aug 1, 2017	Nonsan, Chungnam	MG554344	Aspergillus sclerotiorum (AY373866.1)	100
CN-NS-2-3	Aug 1, 2017	Nonsan, Chungnam	MG554346	Aspergillus sclerotiorum (AY373866.1)	100
CN-NS-2-2	Aug 1, 2017	Nonsan, Chungnam	MG554345	Penicillium sp. (KX148628.1)	100
CN-TA-2-1	Aug 1, 2017	Taean, Chungnam	MG554349	Cephaliophora tropica (KR809561.1)	100
CN-TA-2-2	Aug 1, 2017	Taean, Chungnam	MG554350	Cephaliophora tropica (KR809561.1)	100
CN-TA-2-3	Aug 1, 2017	Taean, Chungnam	MG554351	Rhizopus oryzae (AB109754.1)	100
CN-TA-2-4	Aug 1, 2017	Taean, Chungnam	MG554352	Rhizopus oryzae (AB109754.1)	100
GW-GN-2-1	Aug 1, 2017	Gangneung, Gangwon	MG554353	Aspergillus flavus (KX462773.1)	100
GW-GN-2-2	Aug 1, 2017	Gangneung, Gangwon	MG554354	Aspergillus flavus (MF120213.1)	100
GW-GN-2-4	Aug 1, 2017	Gangneung, Gangwon	MG554356	Aspergillus flavus (MF120213.1)	100
GW-GN-2-3	Aug 1, 2017	Gangneung, Gangwon	MG554355	Fusarium equiseti (KY963137.1)	100
GB-GR-2-1	Aug 1, 2017	Goryeong, Gyeongbuk	MG554330	Coprinellus sp. (MF136551.1)	100
GB-GR-2-4	Aug 1, 2017	Goryeong, Gyeongbuk	MG554331	Coprinellus sp. (MF136551.1)	100
GB-GJ-2-2	Aug 1, 2017	Gyeongju, Gyeongbuk	MG554319	Alternaria alternata (KY814634.1)	100
GB-GJ-2-3	Aug 1, 2017	Gyeongju, Gyeongbuk	MG554320	Alternaria alternata (KY814634.1)	100
GB-GJ-2-1	Aug 1, 2017	Gyeongju, Gyeongbuk	MG554318	Alternaria alternata (MF575850.1)	100
GB-MG-2-1	Aug 1, 2017	Mungyeong, Gyeongbuk	MG554334	Hyphopichia burtonii (KY103598.1)	99
GB-MG-2-3	Aug 1, 2017	Mungyeong, Gyeongbuk	MG554335	Hyphopichia burtonii (KY103602.1)	99
GB-SJ-2-4	Aug 1, 2017	Sangju, Gyeongbuk	MG554329	Cladosporium sp. (HQ832966.1)	100
GB-SJ-2-1	Aug 1, 2017	Sangju, Gyeongbuk	MG554328	Cladosporium sphaerospermum (KY859397.1)	100
GB-US-2-1	Aug 1, 2017	Uisung, Gyeongbuk	MG554317	Trichoderma asperellum (KY623504.1)	100
GB-YD-2-4	Aug 1, 2017	Yeongdeok, Gyeongbuk	MG554327	Aspergillus flavus (KX912161.1)	100
GG-GP-2-4	Aug 1, 2017	Gimpo, Gyeonggi	MG554360	Rhizomucor pusillus (KJ527032.1)	100
GG-HS-2-4	Aug 1, 2017	Hwaseong, Gyeonggi	MG554357	Cunninghamella echinulata (KX179502.1)	100
GG-YP-2-1	Aug 1, 2017	Yangpyeong, Gyeonggi	MG554365	Trichoderma atroviride (KY305043.1)	99
GG-YJ-2-1	Aug 1, 2017	Yeoju, Gyeonggi	MG554359	Uncultured fungus (GU054203.1)	99
GN-GC-2-3	Aug 1, 2017	Geochang, Gyeongnam	MG554333	Chaetomium globosum (MF663683.1)	100
GN-GC-2-2	Aug 1, 2017	Geochang, Gyeongnam	MG554332	Coprinellus sp. (MF136551.1)	100
GN-GS-2-5	Aug 1, 2017	Goseong, Gyeongnam	MG554322	Chaetomium globosum (KX013209.1)	100
GN-GS-2-4	Aug 1, 2017	Goseong, Gyeongnam	MG554321	Hyphopichia burtonii (KY103598.1)	100
GN-GS-2-7	Aug 1, 2017	Goseong, Gyeongnam	MG554323	Monascus sp. (KY511749.1)	100
GN-HA-2-2	Aug 1, 2017	Haman, Gyeongnam	MG554324	Hyphopichia burtonii (KX965648.1)	100
GN-HA-2-3	Aug 1, 2017	Haman, Gyeongnam	MG554325	Hyphopichia burtonii (KX965648.1)	100
GN-HA-2-4	Aug 1, 2017	Haman, Gyeongnam	MG554326	Monascus sp. (KY511749.1)	100
GN-SC-2-1	Aug 1, 2017	Sancheong, Gyeongnam	MG554363	Byssochlamys spectabilis (KC009788.1)	100
GN-US-2-3	Aug 1, 2017	Ulsan, Gyeongnam	MG554343	Aspergillus flavus (KX462773.1)	100
GN-US-2-2	Aug 1, 2017	Ulsan, Gyeongnam	MG554342	Aspergillus sp. (KX928745.1)	100
GN-US-2-1	Aug 1, 2017	Ulsan, Gyeongnam	MG554341	Talaromyces islandicus (JN899318.1)	100
JB-GC-2-1	Aug 1, 2017	Gochang, Jeonbuk	MG554361	Rhizopus microsporus (AB381937.1)	100
JB-IM-2-1	Aug 1, 2017	Imsil, Jeonbuk	MG554362	Hyphopichia burtonii (KY103598.1)	99
JB-NW-2-1	Aug 1, 2017	Namwon, Jeonbuk	MG554368	Penicillium steckii (KX674639.1)	100
JN-BS-2-2	Aug 1, 2017	Boseong, Jeonnam	MG554366	Mucor circinelloides (KX620480.1)	99
JN-GR-2-1	Aug 1, 2017	Gurye, Jeonnam	MG554367	Aspergillus protuberus (LN898712.1)	100
JN-HN-2-1	Aug 1, 2017	Haenam, Jeonnam	MG554347	Lichtheimia hyalospora (GQ342894.1)	100
JN-HN-2-3	Aug 1, 2017	Haenam, Jeonnam	MG554348	Penicillium citrinum (MF663545.1)	100
JN-NJ-2-1	Aug 1, 2017	Naju, Jeonnam	MG554364	Penicillium steckii (KX674639.1)	100
JN-YA-2-2	Aug 1, 2017	Yeongam, Jeonnam	MG554340	Aspergillus terreus (KT778597.1)	100
JN-YA-2-1	Aug 1, 2017	Yeongam, Jeonnam	MG554339	Hyphopichia burtonii (KY103598.1)	100

Table 2

Identification of four geni including Aspergillus spp., Penicillium spp., Alternaria sp. and Fusarium spp. using partial beta-tubulin, calmodulin, tef1 and glyceraldehyde-3-phosphate gene sequences

Isolates	GenBank Accessions				Identified species
	b-tubulin	Calmodulin	tef1	GAPDH
Aspergillus spp.
CB-BE-3-1	MH424078	MH424038	-	-	Aspergillus flavus
CB-JC-3-2	MH424051	MH424011	-	-	Aspergillus flavus
GB-MG-3-3	MH424073	MH424033	-	-	Aspergillus flavus
GB-MG-3-4	MH424074	MH424034	-	-	Aspergillus flavus
GB-YC-3-2	MH424055	MH424015	-	-	Aspergillus flavus
GB-YC-3-1	MH424076	MH424036	-	-	Aspergillus flavus
GG-YJ-3-1	MH424071	MH424031	-	-	Aspergillus flavus
JB-GC-3-3	MH424052	MH424012	-	-	Aspergillus flavus
JB-IK-3-2	MH424053	MH424013	-	-	Aspergillus flavus
JN-NJ-3-2	MH424065	MH424025	-	-	Aspergillus flavus
JN-NJ-3-1	MH424075	MH424035	-	-	Aspergillus flavus
JN-YG-3-5	MH424056	MH424016	-	-	Aspergillus flavus
JN-YG-3-3	MH424072	MH424032	-	-	Aspergillus flavus
CB-CJ-1-2	MH424062	MH424022	-	-	Aspergillus flavus
GB-GJ-1-1	MH424061	MH424021	-	-	Aspergillus flavus
GB-GJ-1-2	MH424079	MH424039	-	-	Aspergillus flavus
GN-HY-1-6	MH424080	MH424040	-	-	Aspergillus flavus
GN-US-1-1	MH424077	MH424037	-	-	Aspergillus flavus
JB-NW-1-3	MH424059	MH424019	-	-	Aspergillus flavus
JN-YG-1-5	MH424054	MH424014	-	-	Aspergillus flavus
GW-GN-2-1	MH424057	MH424017	-	-	Aspergillus flavus
GW-GN-2-2	MH424063	MH424023	-	-	Aspergillus flavus
GW-GN-2-4	MH424064	MH424024	-	-	Aspergillus flavus
GB-YD-2-4	MH424060	MH424020	-	-	Aspergillus flavus
GN-US-2-3	MH424058	MH424018	-	-	Aspergillus flavus
GN-US-2-2	MH424081	MH424041	-	-	Aspergillus flavus
JB-GC-1-3	MH424068	MH424028	-	-	Aspergillus sclerotiorum
CN-NS-2-1	MH424069	MH424029	-	-	Aspergillus sclerotiorum
CN-NS-2-3	MH424070	MH424030	-	-	Aspergillus sclerotiorum
JN-YG-3-1	MH424083	MH424043	-	-	Aspergillus sydowii
GG-YJ-3-4	MH424082	MH424042	-	-	Aspergillus sydowii
CN-NS-1-1	MH424084	MH424044	-	-	Aspergillus sydowii
GW-GS-1-5	MH424086	MH424046	-	-	Aspergillus versicolor
JN-NJ-1-1	MH424088	MH424048	-	-	Aspergillus versicolor
JN-NJ-1-2	MH424087	MH424047	-	-	Aspergillus versicolor
JN-YG-3-2	MH424049	MH424009	-	-	Aspergillus candidus
GG-PJ-1-1	MH424050	MH424010	-	-	Aspergillus creber
JB-GC-3-2	MH424066	MH424026	-	-	Aspergillus oryzae
JN-GR-2-1	MH424067	MH424027	-	-	Aspergillus protuberus
JN-YA-2-2	MH424085	MH424045	-	-	Aspergillus terreus
Penicillium spp.
JB-GC-3-4	MH423997	MH423985	-	-	Penicillium citrinum
JN-YG-3-4	MH423998	MH423986	-	-	Penicillium citrinum
JN-HN-2-3	MH423999	MH423987	-	-	Penicillium citrinum
GN-HY-1-1	MH424001	MH423989	-	-	Penicillium crustosum
JN-YG-1-2	MH424000	MH423988	-	-	Penicillium crustosum
GW-GS-1-4	MH424002	MH423990	-	-	Penicillium neoechinulatum
JB-GC-1-1	MH424003	MH423991	-	-	Penicillium neoechinulatum
JN-GJ-3-2	MH424004	MH423992	-	-	Penicillium steckii
JB-IS-1-3	MH424006	MH423994	-	-	Penicillium steckii
CN-NS-2-2	MH424005	MH423993	-	-	Penicillium steckii
JB-NW-2-1	MH424007	MH423995	-	-	Penicillium steckii
JN-NJ-2-1	MH424008	MH423996	-	-	Penicillium steckii
Alternaria sp.
CN-GJ-3-2	MH423922	-	-	MH423917	Alternaria alternata
CN-GJ-3-1	MH423921	-	-	MH423916	Alternaria alternata
GB-GJ-2-2	MH423924	-	-	MH423919	Alternaria alternata
GB-GJ-2-3	MH423925	-	-	MH423920	Alternaria alternata
GB-GJ-2-1	MH423923	-	-	MH423918	Alternaria alternata
Fusarium sp.
GW-GN-2-3	-	-	MH423915	-	Fusarium equiseti
JB-IS-1-1	-	-	MH423914	-	Fusarium oxysporum
GN-US-1-2	-	-	MH423913	-	Fusarium proliferatum

The major fungal species in whole periods were Aspergillus spp. including A. flavus (28.2%), Cladosporium spp. (12.0%), Hyphopichia burtonii (9.2%), Penicillium spp. (8.5%), Mucor spp. (6.3%), Rhizopus spp. (6.3%), Cephaliophora tropica (3.5%), Alternaria alternata (3.5%), and Monascus sp. (2.8%) (Fig. 2B). Less commonly identified were Fusarium spp., Beauveria bassiana, Chaetomium globosum, Coprinellus sp., Irpex lacteus, Lichtheimia spp., Trichoderma spp., Byssochlamys spectabilis, Cochliobolus miyabeanus, Cunninghamella echinulata, Nigrospora oryzae, Mortierella oligospora, Polyporales sp., Rhizomucor pusillus, Talaromyces sp. and unidentified fungus. Among the isolates, two known mycotoxin-producing fungi, Aspergillus flavus (26 isolates) and Fusarium spp. (3 isolates) were identified.

The above results are consistent with a study which demonstrated that Aspergillus flavus is the major fungal contaminants of stored wheat in the presence of T. castaneum (Bosly and El-Banna, 2015). It is also consistent with another study on stored maize, where 10 species of fungi, Alternaria alternata, Aspergillus flavus, Aspergillus sp., Cladosporium sphaerospermum, Fusarium sp., Fusarium oxysporum, Penicillium sp., Mucor sp., Mucor racemosus and Rhizopus oryzae were isolated in the presence of T. castaneum (Simpanya et al., 2001).

In this study, we found the producer of aflatoxin, Aspergillus flavus, associated with T. castaneum, collected from16 RPCs (Gangneung (GN) in Gangwon (GW) province, Yeoju (YJ) in Gyeongii, Jincheon (JC), Chungju (CJ), and Boeun (BE) in Chungbuk (CB), Yeongdeok (YD), Gyeongju (GJ), Yecheon (YC) and Mungyeong (MG) in Gyeongbuk, Ulsan (US) and Hamyang (HY) in Gyeongnam (GN), Iksan (IK), Gochang (GC) and Namwon (NW) in Jeonbuk (JB), Yeonggwang (YG) and Naju (NJ) in Jeonnam) (Fig. 2C).

Other Aspergillus species were also found on beetles from 9 RPCs (Goseong (GS) in Gangwon province, Paju (PJ) and YJ in Gyeongii, Nonsan (NS) in Chungnam, GC in Jeonbuk, YG, NJ, Yeongam (YA) and Gurye (GR) in Jeonnam) (Fig. 2D, Table 1 and Table 2).

In addition, genus Penicillium, which is known to produce ochratoxin, was also isolated from the beetles collected in 10 RPCs (GS in Kangwon province, NS in Chungnam, Iksan (IK), GC, NS in Jeonbuk, HY in Gyeongnam, YG, NJ, Gangjin (GJ), and Haenam (HN) in Jeonnam). Another toxigenic genus, Alternaria alternata (Ostry, 2008), was found in Gongju (GJ) in Chungnam and Gyeongju (GJ) in Gyeongbuk province. Only three Fusarium species including Fusarium equiseti (Gangneung (GN) in Gangwon province), Fusarium oxysporum (Iksan (IK) in Jeonbuk), and Fusarium sp. (Ulsan (US) from Gyeongnam) were collected in 3 RPCs (Fig. 2E, Table 1). Other fungi were identified as saprophiles that proliferate on wood and debris in the facility.

The fungi Aspergillus spp., Penicillium spp., Fusarium spp., and Alternaria spp. are the major fungal species found in stored grains (Lee et al., 2011; Lee et al., 2014). More than 25% of stored grains worldwide have been reported to be contaminated with mycotoxins produced by these fungal species, and over 300 fungal metabolites have been reported to have toxicity on humans and animals (Galvano et al., 2001).

The genera Fusarium and Alternaria are known to mainly infect ears of cereal plants in the field, whereas the genera Aspergillus and Penicillium are contaminants of stored seeds, grains, and processed foods and produce mycotoxins (Adams, 1977). In particular, a number of harmful mycotoxins, such as deoxynivalenol (DON) and nivalenol (NIV), produced by Fusarium spp., and Aflatoxin produced by Aspergillus spp. are detected in stored grains (Lee et al., 2011; Lee et al., 2014; Son et al., 2011).

Both Aspergillus flavus and Fusarium spp. are known to produce mycotoxins but only Aspergillus flavus was found in this study. It is known that pests and fungi tend to co-occur in stored grains (Simpanya et al., 2001). It is necessary to investigate the distribution of pests and fungi in grain warehouses because pests promote the growth and propagation of fungi.

According to the studies on fungal and mycotoxin contamination of RPC grain samples, Aspergillus and Penicillium species were infrequently found nationwide but were particularly abundant in a few RPC samples (Lee et al., 2014). Alternaria, Nigrospora, and Epicoccum species were more consistently isolated at similar frequencies, whenever fungal contamination was detected. In accordance with the results from previous studies (Lee et al., 2014; Son et al), genera Aspergillus, Penicillium, Alternaria, and Nigrospora were identified from the T. castaneum collected at RPCs. Therefore, it is suspected that the red flour beetles are a potential vector for the transfer of toxigenic fungi and mycotoxins.

According to the study on mycotoxin contamination in different growth stages of rice (Nakaijima et al., 2008; Nash and Snyder, 1962), rice plants are always exposed to fungi and mycotoxins even before storage. So far, it has been reported that differences in temperature and humidity depending on the climate have a great influence on the growth of fungi and occurrence of mycotoxins (Russell et al., 2010). However, studies on the effect of temperature and humidity on pest-assisted mycotoxin production in stored grains are uncommon and remained to be investigated in the future. Our study shows that the storage pest, T. castaneum, could play an important role in transmission of fungi in stored rice in RPC and potentially contribute to mycotoxin contamination of rice.