Fungal Diversity of Rice Straw for Meju Fermentation

Abstract

Rice straw is closely associated with meju fermentation and it is generally known that the rice straw provides meju with many kinds of microorganisms. In order to elucidate the origin of meju fungi, the fungal diversity of rice straw was examined. Rice straw was collected from 12 Jang factories where meju are produced, and were incubated under nine different conditions by altering the media (MEA, DRBC, and DG18), and temperature ($15^{\circ}C$, $25^{\circ}C$, and $35^{\circ}C$). In total, 937 strains were isolated and identified as belonging to 39 genera and 103 species. Among these, Aspergillus, Cladosporium, Eurotium, Fusarium, and Penicillium were the dominant genera. Fusarium asiaticum (56.3%), Cladosporium cladosporioides (48.6%), Aspergillus tubingensis (37.5%), A. oryzae (31.9%), Eurotium repens (27.1%), and E. chevalieri (25.0%) were frequently isolated from the rice straw obtained from many factories. Twelve genera and 40 species of fungi that were isolated in the rice straw in this study were also isolated from meju. Specifically, A. oryzae, C. cladosporioides, E. chevalieri, E. repens, F. asiaticum, and Penicillium polonicum (11.8%), which are abundant species in meju, were also isolated frequently from rice straw. C. cladosporioides, F. asiaticum, and P. polonicum, which are abundant in the low temperature fermentation process of meju fermentation, were frequently isolated from rice straw incubated at $15^{\circ}C$ and $25^{\circ}C$, whereas A. oryzae, E. repens, and E. chevalieri, which are abundant in the high temperature fermentation process of meju fermentation, were frequently isolated from rice straw incubated at $25^{\circ}C$ and $35^{\circ}C$. This suggests that the mycobiota of rice straw has a large influence in the mycobiota of meju. The influence of fungi on the rice straw as feed and silage for livestock, and as plant pathogens for rice, are discussed as well.

Introduction

Rice straw is the most abundant agricultural residue in the world [24]. It is utilized as feed and bedding for animals, a source of biofuel, an additive of compost, and media for mushroom cultivation [1,8,15,24,26]. Diverse microorganisms such as bacteria, fungi, and yeast are present in the rice straw and these are utilized in various ways [8]. Silage is fermented by the colonizing microorganisms on rice straw, which aids in the process of digestion in the livestock [26]. The microorganisms on rice straw are used during the fermentation of meju and cheonggukjang [8,17]. Many fungi possess efficient enzymes and they are useful for food and feed industries [25].

Meju fermentation is closely associated with rice straw, as the rice straw is in direct contact with the soybeans in meju during the fermentation process, and it is known that the rice straw provides meju with various kinds of microorganisms [17]. The fungus on rice straw was examined by Lee et al. [15] in Korea and by Heral [7] in Egypt, but the isolation and identification of the fungi from rice straw in both studies were not comprehensive. The aims of this study were to examine the fungal diversity on the rice straw for meju fermentation, to compare the mycobiota inhabiting the rice straw and meju, and to presume the origin of fungi in meju. In addition, we were keen to document the basic information about the mycobiota for industrial usage of rice straw.

 

Materials and Methods

Rice straw samples for meju fermentation were collected from 12 Jang factories where meju are produced from November to December 2011. The geographical origin of the rice straw is listed in Table 1. After dividing the rice straw into three parts, the top, middle, and bottom, they were cut into pieces approximately 2 cm in length. Four pieces (2 pieces per plate) of each rice straw sample was incubated on nine different conditions using three media such as malt extract agar (MEA), dichloran rose bengal chloramphenicol agar (DRBC), and dichloran 18% glycerol agar (DG18) medium, and incubating at three different temperatures (15℃, 25℃, and 35℃) in the dark for 5-7 days. In total, 1,296 pieces of rice straw (12 samples; 3 parts, 3 temperatures, 3 media, and each 4 pieces) were used. The fungal colonies that grew on the rice straw or media were transferred to new MEA or DG18 media (for xerophilic fungi) and were further incubated for several days. After confirmation by microscopic examination, the fungi were transferred to MEA or DG18 slants, and were maintained at 4℃.

For the molecular identification of the various fungi, genomic DNA was extracted using the DNeasy Plant Mini Kit (69106; Qiagen, Hilden, Germany) according to the method of Kim et al. [13]. The selection of the sequencing region depended on the genera; the partial β-tubulin gene using bt2a and bt2b primers [6] for Aspergillus, Eurotium, and Penicillium, the partial actin gene using act-512F and act-783R primers [2] for Cladosporium, the partial elongation factor 1-alpha gene using EF1 and EF2 primers [5] for Fusarium, and the internal transcribed spacer of nuclear ribosomal DNA (rDNA-ITS) using ITS1 and ITS4 primers [28] for the other genera. In addition, morphological characters of strains were analyzed for confirmation of molecular identification, according to methods by Pitt and Hocking [19] and Samson et al. [20].

 

Results and Discussion

A total of 937 strains were isolated from 1,296 pieces of rice straw and were identified as belonging to 39 genera and 103 species (Table 2). The genera Aspergillus, Cladosporium, Eurotium, Fusarium, and Penicillium were the dominant fungi (occurrence more than 100 in 1,296 pieces) in rice straw, followed by the genera Phoma, Epicoccum, Lichtheimia, Nigrospora, Chaetomium, Bionectria, and Trichothecium (occurrence more than 30/1,296 pieces). Cochliobolus and Coprinellus were infrequently isolated (occurrence less than 30/1,296 pieces) but were isolated from 7 Jang factories.

Table 1.aThe percentage indicates rice straw pieces from which the species were isolated, out of 108 pieces (3 temperatures, 3 media, 3 parts, and 4 pieces) of rice straw.

Occurrences of fungi from rice straw were dependent on the isolation media used (Table 2). The most diverse species were isolated on DRBC, followed by DG18. Aspergillus (except Eurotium) and Cladosporium were isolated from rice straw cultured on all the three media, MEA, DRBC, and DG18. Eurotium, Schizophyllum, Syncephalastrum, and Penicillium were more frequently isolated on DG18, but the other genera were more frequently isolated on MEA and/or DRBC. Specifically, Beauveria, Bionectria, Coprinellus, Hypochnicium, Khuskia, Rhizomucor, Talaromyces, and Verticillium were not isolated on DG18. Occurrence of fungi from rice straw was also dependent on incubation temperatures (Table 2). The most diverse species were isolated from samples incubated at 25℃, but some fungi were isolated only from samples incubated at 15℃ or 35℃. However, the mycobiota on rice straw did not differ much according to collecting regions and parts of rice straw (Table 2). Therefore, the isolation frequency of the fungal species from the rice straw was counted based on the optimum media and temperature in this study (Table 2, fourth column). The total pieces of rice straw were 144 (12 samples, 3 parts, and each 4 pieces) on optimum isolating condition for every species.

Fourteen species of Aspergillus were isolated (Table 2). A. tubingensis and A. oryzae were isolated from rice straw obtained from 10 factories with a 37.5% frequency (54/144 pieces) and 31.9% (46/144) under optimum isolating conditions, respectively. Many Aspergillus species showed higher frequency in samples incubated at 35℃ than at 25℃ or 15℃. Among the six Cladosporium species (Table 2) isolated, the most frequent species was C. cladosporioides (48.6%), followed by C. pseudocladosporioides. Cladosporium strains were frequently isolated from rice straw samples cultured at either 15℃ or 25℃. In the case of Eurotium, eight species (Table 2) were isolated from rice straw. E. repens (27.1%) was the most frequent species, followed by E. chevalieri (25%), E. herbariorum (22.9%), E. rubrum (16%), and E. amstelodami (15.3%). Eurotium was isolated with a higher frequency at 25℃ or 35℃, than at 15℃. Fifteen species of Penicillium were isolated from rice straw (Table 2) and the common species were P. polonicum (11.8%), P. steckii (14.6%), and Penicillium sp. (10.4%). P. polonicum was present in the highest frequency in samples of rice straw incubated at 15℃, whereas P. steckii was the predominant species in samples incubated at 25℃. Penicillium is generally known as psychrophilic fungi [21]. Eight species of Fusarium were isolated from rice straw and the predominant species was F. asiaticum (11 factories with 56.3% frequency), which showed higher frequency at 15℃ and 25℃. Among the other genera, Epicoccum nigrum (14.6%), Lichtheimia corymbifera (13.9%), and Phoma sp. (13.9%) were also isolated with relatively high frequency. In this study, the genera Aspergillus, Cladosporium, and Eurotium were isolated from the rice straw obtained from all the 12 factories. Aspergillus oryzae, A. tubingensis, Cladosporium cladosporioides, Eurotium chevalieri, E. repens, and F. asiaticum were consistently isolated from many factories and with high frequency, and these species could be the typical species on rice straw.

Table 2.aThe number indicates factories from which the species were isolated. bThe species were isolated from meju, ***with high frequency, **medium frequency, or *low frequency. cThe number indicates the incidence of each species on optimum isolating condition from 144 pieces (12 samples, 3 parts, and each 4 pieces) of rice straw. dNumbers 15, 25, and 35 indicate the temperature at which the samples were incubated, and the letters M, R, and G indicate the media MEA, DRBC, and DG18 used for the culture, respectively. eThe number indicates rice straw pieces from which the species were isolated, out of 432 pieces (12 samples, 3 temperatures, 3 parts, and 4 pieces) of rice straw. fThe number indicates rice straw pieces from which the species were isolated, out of 432 pieces (12 samples, 3 media, 3 parts, and 4 pieces) of rice straw. gThe number indicates rice straw pieces from which the species were isolated, out of 432 pieces (12 samples, 3 media, 3 temperatures, and 4 pieces) of rice straw. hThe RDA numbers are the DNA sequence accession number of Korean Agricultural Culture Collection (KACC). Readers can access the sequence from information of the corresponding KACC No. in the KACC homepage (http://www.genebank.go.kr).

Lee et al. [15] isolated 16 genera and 25 species from rice straw collected from 11 different sites in Korea. Curvularia intermedia was the most frequent fungi, followed by Alternaria alternata, Dothideomycete sp., Cladosporium sp., Nigrospora oryzae, Aspergillus niger, Fusarium graminearum, Mucor circinelloides, Gaeumannomyces graminis, var. graminis, Penicillium oxalicum, Trichoderma harzianum, and Fusarium proliferatum. Of these, 6 genera and 10 species such as Aspergillus niger, A. ochraceus, A. versicolor, Cochliobolus miyabeanus, Epicoccum nigrum, Fusarium graminearum (F. asiaticum in this study), Fusarium oxysporum, Mucor circinelloides, Nigrospora oryzae, and Penicillium oxalicum were also isolated in this study. Among the dominant species reported by Lee et al. [15], Alternaria alternata, Curvularia intermedia, F. proliferatum, Gaeumannomyces graminis var. graminis, and Trichoderma harzianum were not isolated in this study. On the other hand, the dominant fungi isolated from the rice straw in this study, Aspergillus oryzae, A. tubingensis, Cladosporium cladosporioides, Eurotium chevalieri, and E. repens were not isolated in the study of Lee et al. [15].

In the study carried out by Helal [7], 30 genera and 64 species were isolated from rice straw collected from the Sharkia Province in Egypt. Aspergillus terreus, A. niger, A. fumigatus, A. flavus, Mucor racemosus, Myrothecium roridum, and Trichoderma koningii were the frequently isolated species. Among the fungi reported by Helal [7], 12 genera and 21 species, including Aspergillus flavus (A. oryzae or A. flavus in this study), A. niger, A. terreus, Cladosporium cladosporioides, and Epicoccum nigrum, were also isolated from this study. However, Mucor racemosus, Myrothecium roridum, and Trichoderma koningii, which were the dominant species in the study of Helal [7], were not isolated in this study. On the other hand, Aspergillus tubingensis, Cladosporium pseudocladosporioides, Eurotium spp., Fusarium asiaticum, F. incarnatum, Lichtheimia corymbifera, Penicillium polonicum, and P. steckii were not reported from the rice straw samples in the study of Helal [7].

The difference in mycoflora between this and the other studies [7, 15] could probably be due to the different collection period of the rice straw and the isolation media used. Lee et al. [15] collected rice straw shortly after harvest, whereas this study collected in the winter season, 2-4 months after harvest. Therefore, the rice straw used in this study was drier and more decomposed than that of Lee et al. [15]. Xerophilic and dry resistant fungi were isolated more in this study. Helal [7] and Lee et al. [15] did not isolate Eurotium, as they used only MEA or Czapek’s agar, respectively, but did not use DG18 or DRBC. Eurotium does not grow on MEA or Czapek’s agar, but grows well on DG18 [21]. DG18 and DRBC, which were used in this study, are the recommended media for isolation and enumeration of fungi from foods and feeds [21]. Lee et al. [15] used morphological characters and rDNA-ITS for identification, whereas Helal [7] used only morphological characters for identification. Nowadays, many fungi are not able to be clearly identified by morphological character and/or rDNA-ITS sequence [22]. Specifically, A. niger/tubingensis and A. sydowii/versicolor cannot be distinguished by rDNA-ITS, but the β-tubulin sequence can distinguish them [18, 23].

From the traditional Korean meju, which is made by using rice straw, 26 genera and 101 species of fungi were isolated [9-12,14]. Of these, 12 genera and 40 species were also isolated from rice straw. Aspergillus oryzae, Mucor circinelloides, M. racemosus, Penicillium polonicum, Eurotium repens, Scopulariopsis brevicaulis, P. solitum, E. chevalieri, Lichtheimia ramosa, Fusarium asiaticum, and Cladosporium cladosporioides were the most frequently isolated from traditional Korean meju. Of the above, Aspergillus oryzae (31.9% of rice straw), Eurotium chevalieri (25.0%), E. repens (27.1%), Fusarium asiaticum (56.3%) Penicillium polonicum (11.8%), and Cladosporium cladosporioides (48.6%) were also frequently isolated from rice straw. C. cladosporioides, F. asiaticum, and P. polonicum, which are abundant in the low temperature fermentation process of meju fermentation, were frequently isolated from rice straw incubated at 15℃ and 25℃ in this study. A. oryzae, E. repens, and E. chevalieri, which are abundant in the high temperature fermentation process of meju fermentation, were frequently isolated from rice straw incubated at 25℃ and 35℃. This suggests that the rice straw could provide meju with diverse fungi during its fermentation process. Indeed, it has been observed that many fungi are transferred from rice straw to meju. The fungi, C. cladosporioides and F. asiaticum, which could be transferred from rice straw to meju during the low temperature fermentation process, are not regarded as ferment fungi, but are regarded as contaminants of meju. On the other hand, A. oryzae, E. repens, and E. chevalieri, which could be transferred into meju from rice straw during the high temperature fermentation process, are generally regarded as good ferment fungi on meju. In other words, rice straw might provide meju with contaminant fungi during the low temperature fermentation process and with beneficial fungi during the high temperature fermentation process. Therefore, it might be a good method to use rice straw during only high temperature fermentation, in order not to provide meju with contaminant fungi, but to provide meju with beneficial fungi, if we consider only the fungal perspective.

The most frequent fungi on rice straw, Fusarium asiaticum (56.3%), is a causal pathogen of Fusarium blight on rice. Cladosporium cladosporioides (48.6%), the second most frequent species on rice straw, has been reported as the causal pathogen of ear blight on rice [27]. In addition, Fusarium incarnatum (ear blight) (11.1%), Cochliobolus lunatus (ear blight) (0.7%), Khuskia oryzae (ear blight) (2.1%), F. fujikuroi (Bakanae disease) (9.0%), Nigrospora oryzae (Brown spot) (9.0%), and Cochliobolus miyabeanus (Heliminthosporium blight) (2.8%) [27] were also isolated from rice straw. This information could be useful to understand the mechanism of disease occurrence on rice.

Sung et al. [26] detected mycotoxins from 42% of rice straw bale silage. Ochratoxin A, deoxynivalenol, and zearalenone were detected, but aflatoxin (B1, B2, G1, G2) and fumonisin (B1, B2) were not detected [26]. Aspergillus ochraceus and A. westerdijkiae, which were isolated quite frequently in this study, could be causal fungi for the ochratoxin, and Fusarium asiaticum, which was also one of the most frequent species, also could be a causal fungus for deoxynivalenol and zearalenone [26]. However, Aspergillus flavus, which was isolated with 3.5% frequency in this study, did not produce a detectable quantity of aflatoxin in the study of Sung et al. [26].

On the other hand, enzymes such as amylase, xylanase, and protease that are produced by fungi that are found on the rice straw are useful in silage preparation [16]. In addition, the fungi isolated from rice straw, Aspergillus oryzae (Koji), Eurotium herbariorum (Katsuobushi), E. repens (Katsuobushi), Penicillium chrysogenum (animal food), Rhizopus arrhizus (Kaffir Beer), and Rhizopus oryzae (Lao-chao, biodiesel fuel), are used widely in various industries [3,4]. Therefore, the documentation of fungal diversity and their frequency in rice straw in this study can provide the basic information for usage of rice straw for various industries.

Strains isolated in this study are preserved in the Korean Agricultural Culture Collection (KACC) (http://www.genebank.go.kr) for future research and these are accessible to other researchers.