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Journal of Oleo ScienceCopyright ©2016 by Japan Oil Chemists’ Societydoi : 10.5650/jos.ess16067J. Oleo Sci. 65, (7) 621-627 (2016)

Biodegradable Plastic-degrading Activity of Various Species of ParaphomaMotoo Koitabashi1, Yuka Sameshima–Yamashita1, 2, Hideaki Koike3, Toyozo Sato4, Jouji Moriwaki5, Tomotake Morita6, Takashi Watanabe1, Shigenobu Yoshida1 and Hiroko Kitamoto1,＊

1 National Institute for Agro-Environmental Sciences (3-1-3 Kannondai Tsukuba, Ibaraki 305-8604, JAPAN)2 Research Fellow of the Japan Society for the Promotion of Science (1-8 Chiyoda-ku, Tokyo 102-8472, JAPAN)3 Bioprocess Research Institute, National Institute of Advanced Industrial Science and Technology (AIST) (1-1-1 Higashi, Tsukuba, Ibaraki

305-8566, JAPAN)4 Genetic Resources Center, National Institute of Agrobiological Sciences (2-1-2 Kannondai Tsukuba, Ibaraki 305-8602, JAPAN)5 Horticulture Research Division, NARO Kyushu Okinawa Agricultural Research Center (1823-1 Miimachi Kurume, Fukuoka 839-8503, JAPAN)6 Research Institute for Sustainable Chemistry, National Institute of Advanced Industrial Science and Technology (AIST) (1-1 Higashi, Tsukuba,

Ibaraki 305-8565, JAPAN)

1 INTRODUCTIONIn the cultivation of vegetables, covering the soil bed

with agricultural mulch films is an indispensable means of maintaining soil temperature and controlling weeds and pests. Generally, mulch film is made of nondegradable syn-thetic polymer. However, collection of the used film after harvest is labor-intensive, and disposal of the film is costly. To solve these problems, mulch films made of biodegrad-able plastics（BPs）have been developed that can be plowed into the ground with a tilling machine following use1）. However, the use of BP mulch film has not been widely adopted because of the difficulty in controlling the degrad-ability under various cultivation environments. A practical BP mulch film would have no significant decrease in per-formance during the planned useful lifetime, but would degrade rapidly after use. However, it is difficult to develop adequately degradable plastic films2）. Treatment with a BP-

＊Correspondence to: Hiroko Kitamoto, National Institute for Agro-Environmental Sciences, 3-1-3 Kannondai Tsukuba, Ibaraki 305-8604, JAPANE-mail: kitamoto@affrc.go.jpAccepted April 4, 2016 (received for review March 16, 2016)Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 onlinehttp://www.jstage.jst.go.jp/browse/jos/　　http://mc.manusriptcentral.com/jjocs

degrading enzyme is one means of accelerating the degra-dation of used BP mulch films. To obtain such enzymes, BP-degrading microorganisms have been isolated from the natural environment using selective agarose plates contain-ing emulsified poly（butylene succinate-co-adipate）（PBSA）. Microorganisms that can degrade emulsified PBSA produce a clear zone around the colony on the selection plate3）. However, the efficiency of isolation of microorganisms capable of degrading solid BPs is low.

With a focus on the similarities between the structures of the cuticle layer covering the leaves of plants and BPs, we isolated many types of yeast from the surface of rice plants and obtained powerful BP film-degrading enzymes4, 5）. In addition, the filamentous fungus B47-9 was selected from our collection of fungi isolated from gramineous plants as a high-BP film degrader6）. A B47-9 culture filtrate with BP-degrading activity was obtained. When the culture

Abstract: The fungal strain B47-9, isolated from barley, was previously selected as an effective degrader of various biodegradable plastic (BP) films such as poly(butylene succinate-co-adipate) (PBSA) and poly(butylene succinate) (PBS). The strain has not been identified based on mycological methods because it does not form fruiting bodies, which are the key to morphological identification. Here, we performed molecular phylogenetic analyses of the nuclear ribosomal RNA gene regions and their internal transcribed spacer region of B47-9 and related fungi. The results suggest that B47-9 is closely related to the genus Paraphoma. Investigation of the abilities of six strains belonging to the genus Paraphoma to degrade BPs indicated that all strains could degrade PBSA and PBS films to varying degrees. Based on our approach, we conclude that strain B47-9 is a species belonging to the genus Paraphoma.

Key words: biodegradable plastic film, Paraphoma, mulch film, phylogenetic analysis, poly (butylene succinate-co-adipate) (PBSA), poly (butylene succinate) (PBS)

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filtrate was sprayed over BP-based mulch film covering the soil bed in a pipe house, the film showed accelerated deg-radation7）.

However, the strain B47-9 could not be identified. Al-though the taxonomy and identification of fungi are based largely on the morphological characteristics of their fruit-ing bodies and spores, B47-9 does not form such struc-tures. Comparison of the DNA sequence of the internal transcribed spacers ITS 1 and ITS 2 of nuclear ribosomal RNA genes and the 5.8S rDNA gene of B47-9 with those in the DNA Data Bank of Japan（DDBJ）indicated the highest sequence similarity to a strain of Paraphoma chrysanthe-micola6）. The genus Paraphoma was characterized mainly based on consistent thickness of the entire pycnidial wall composed of uniformly shaped and compact cells and well-differentiated setae densely spread over the pycnidial surface8）. Moreover, based on molecular phylogenic analy-sis of the small subunit 18S rDNA（SSU）and large subunit 28S rDNA（LSU）regions, Paraphoma was reconfirmed as a distinct genus9）.

The ITS region（the whole region spanning ITS1, 5.8S and ITS2）is the most frequently sequenced genetic marker of fungi, and have been proposed as universal fungal DNA barcodes that can be used to delineate species boundaries of many fungi10）. In this study, we examined the molecular relationship of strain B47-9 with the genus Paraphoma. Furthermore, the BP film degradation capabilities of six strains of the genus Paraphoma were evaluated.

2 EXPERIMENTAL PROCEDURES2.1 Fungal strain

Strain B47-9 was isolated from barley in May 2006 in Tsukuba, Ibaraki, Japan6）. B47-9 was deposited in the Na-tional Institute of Technology and Evaluation（NITE, Japan）and NITE Patent Microorganisms Depositary（accession number: NITE P-573）11）. Six strains belonging to the genus Paraphoma（Table 1）were deposited in the National Insti-tute of Agrobiological Sciences, Japan, （NIAS）Genebank.

2.2 Molecular phylogenetic analysesThe B47-9 genome was sequenced in our laboratory

using the Illumina platform Illumina Miseq System（Illumina K.K., Tokyo, Japan）, and the scaffold sequences were as-sembled using the sequence reads from both paired-end and mate pair libraries, which will be reported elsewhere. The sequences of SSU and LSU12） and ITS regions13） were identified by alignment of these genes from B47-9 against genomic scaffold sequences using FASTA version 36.3.6d14） with the default parameters without using a sequence mask. Multilocus analysis of sequence data of the SSU and LSU was performed. Target regions of the partial SSU, LSU rDNA, and ITS were analyzed using fungal specific primers NS1 and NS4 for partial SSU rDNA13）, LROR15）, and LR712） for partial LSU rDNA and ITS5 and ITS413）.

The phylogeny of the morphologically similar Coelomy-cetes was determined utilizing the sequence data of the SSU and LSU regions. The sequence data of the SSU and LSU genes for the 37 strains examined in this paper were revised by Aveskamp et al.16） and downloaded from the DDBJ/EMBL/GenBank databases. A phylogenetic tree was constructed from sequences of the SSU and LSU genes combined by the maximum likelihood（ML）method using shotgun searches with RAxML version 817）. The search was repeated until the maximum likelihood was identified. Base composition homogeneity tests were conducted using Kakusan418）. The reliability of the inferred tree was esti-mated by bootstrap analysis19） with 1000 replicates.

Sequence data of the ITS region for the 23 strains were also revised by Aveskamp et al.16） and downloaded from the DDBJ/EMBL/GenBank databases. The ITS region se-quences of six MAFF strains were obtained as described in our previous report6）. Phylogenetic analyses of the ITS region were performed for each dataset based on similar tree topologies obtained. Neighbor joining（NJ）distance analysis was conducted with MEGA Ver.520） with Kimura 2-parameter substitution models21）. The robustness of the trees was evaluated by 1000 bootstrap replications.

Table 1　Fungal strains degrading biodegradable plastics examined in this study.

Strain Species Date of sampling Source locality Isolation sourceMAFF244948 Paraphoma chrysanthemicola April, 2014 Chikusei, Ibaraki Glycine soja

MAFF244949 Paraphoma chrysanthemicola April, 2014 Tsukuba, Ibaraki Glycine soja

MAFF244950 Paraphoma chrysanthemicola April, 2014 Tsukuba, Ibaraki Glycine soja

MAFF244952 Paraphoma radicina April, 2014 Tsukuba, Ibaraki Glycine soja

MAFF244953 Paraphoma radicina April, 2014 Tsukuba, Ibaraki Glycine soja

MAFF245116 Paraphoma chrysanthemicola July, 2014 Nayaro, Hokkaido Atractylodes lancea

B47-9 Unidentified May, 2006 Tsukuba, Ibaraki Hordeum vulgare

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2.3 BP �lm-degrading ability on agarose platesEvaluation of the BP film-degrading activities of six

Paraphoma strains and B47-9 was performed as described in our previous report6）. Two types of BP mulch film were used as the substrates: poly（butylene succinate）（PBS; Bi-onolle 1001G）and PBSA（Bionolle 3001G）, both of which have an average molecular weight of 20 to 25×104 and thickness of 20 μm and are colored black with carbon powder. These products were obtained from Showa Denko K. K.（Tokyo, Japan）.

The PBSA or PBS films were cut into squares measuring 2×2 cm, sterilized with 70％ ethanol, and air-dried on a clean bench. Four pieces of the cut films were placed on fungal minimum medium agarose plates side by side, forming a larger square with a distance of 2 mm between two adjacent cut films. The agarose grown with the test strain was sliced into cubes（2×2×2 mm）and inoculated at the center of the four cut films mounted in a dish. The cut films were removed from the agar plates after incuba-tion at 28℃ for 7 days; their images were scanned using a transparency scanner, and their degradation ratios were evaluated by comparing the luminance of the 4 cm2 area of the residual film with that of the fresh film as described previously6）.

3 RESULTS AND DISCUSSION3.1 Molecular phylogenetic analyses

The ML tree was calculated based on two gene sequenc-es of SSU and LSU（accession nos. LC126020 and LC126021, respectively）of the strain B47-9, with 37 strains downloaded from the DDBJ/EMBL/GenBank databases（Fig. 1）. In the phylogenetic tree, the strain B47-9 and P. chrysanthemicola, P. fimeti, and P. radicina formed a clade. The results indicated that B47-9 and P. chrysanthe-micola are closely related.

Six MAFF strains including four P. chrysanthemicola strains and two P. radicina strains were obtained from NIAS Genebank. The DNA sequences of their ITS region were analyzed and deposited in DDBJ as listed in Table 2. The phylogenic relationships of the strain B47-9 ITS region with those of MAFF strains and 23 strains obtained from the DDBJ/EMBL/GenBank databases were calculated based on NJ analysis（Fig. 2）. Based on these results, strain B47-9 and P. chrysanthemicola strains were also placed in the same clade. The results of NJ analysis with the ITS region were congruent with those of ML analysis of two combined loci, SSU and LSU（Fig. 1）. Both results showed that strain B47-9 grouped with the genus, Paraphoma including P. chrysanthemicola, P. radicina, and P. fimeti.

We previously named the BP-degrading enzyme of strain B47-9 “Paraphoma-related fungus cutinase-like enzyme

Fig. 1　 Maximum likelihood tree of SSU and LSU of strain B47-9 and species belonging to Paraphoma, Phoma, Pyrenochaeta, and related genera. Numbers on the branches represent the percentage of congruent clusters in bootstrap trials repeated 1000 times when the values were greater than 50％.

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（PCLE）” and demonstrated its ability to degrade various BP films, including PBS, PBSA, and poly（ε -caprolactone）, as well as poly（butylene adipate-co-terephthalate）（PBAT）and poly（DL-lactic acid）11）. Selection of suitable culture conditions using a jar fermenter resulted in a 20-fold in-crease in BP-degrading enzyme production by strain B47-

922）. For technical use of microorganisms, taxonomic infor-mation is needed to determine the influence of these or-ganisms on humans, animals, and the environment. Ac-cording to the taxonomic information, it may also be possible to obtain better BP-degrading enzyme producers from the same group. The strain B47-9 was isolated from

Table 2　Strain data of biodegradable plastics degraded fungi in this study.

Strain SpeciesDDBJ accesion no. Degradtion area (%)

ITS region PBSA PBSMAFF244948 Paraphoma chrysanthemicola LC126022 44.1 30.4MAFF244949 Paraphoma chrysanthemicola LC126023 34.9 35.0MAFF244950 Paraphoma chrysanthemicola LC126024 32.3 17.5MAFF244952 Paraphoma radicina LC126025 59.4 47.0MAFF244953 Paraphoma radicina LC126026 67.5 65.5MAFF245116 Paraphoma chrysanthemicola LC126027 6.8 0.4

B47-9 Unidentified AB693768 99.2 95.0

Fig. 2　 Neighbor joining tree of the rDNA-ITS region of strain B47-9 and species belonging to Paraphoma, Phoma, Pyrenochaeta, and related genera. Numbers on the branches represent the percentage of congruent clusters in bootstrap trials repeated 1000 times when the values were greater than 50％.

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barley. Although P. chrysanthemicola was originally iso-lated from Chrysanthemum morifolium9）, P. chrysanthe-micola MAFF244948, 244949, and 244950 examined here were isolated from Glycine soja, and MAFF245116 was isolated from Atractylodes lancea. Paraphoma radicina MAFF 244952 and MAFF 244953 examined here were iso-lated from Glycine soja. Therefore, all of the Paraphoma strains tested were isolated from plants. With the excep-tion of MAFF 245116, all of the source plants were collect-ed within Ibaraki prefecture, Japan.

3.2 Evaluation of enzyme-based BP mulch �lm degradationBecause strain B47-9 has strong BP degradation ability,

the capabilities of six MAFF Paraphoma strains were

tested. Similar to strain B47-9, all of the MAFF strains tested showed the ability to degrade emulsified PBSA on selective agarose plates. In addition, all of the tested strains degraded films composed of PBSA and PBS（Fig. 3）. Strain B47-9 reduced the area of PBSA film by 99.2％ and that of the PBS film by 95％（Table 2）. On the other hand, the other strains degraded PBSA film by 6.8％ to 67.5％ and PBS film by 0.4％ to 65.5％（Table 2）. When we previ-ously tested 1227 strains of phylloplane fungi of various morphologies, only 55 strains（4.5％）were selected as capable of degrading emulsified PBSA6）. Of these 55 strains, 43（78.2％）and 37（67.3％）degraded PBSA and PBS, respectively, on the same selective agarose plates6）. The observation that all of the strains belonging to the

Fig. 3　 Images of degraded PBSA films（left）and PBS films（right）removed from agarose plates with colonies of the fungal strains examined. a: MAFF244948, b: MAFF244949, c: MAFF244950, d: MAFF244952, e: MAFF244953, f: MAFF245116, g: B47-9, h: Control.

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genus Paraphoma investigated in this study had the ability to degrade BP was of great interest as potential sources of strong BP degraders. In future, we intent to in-vestigate the capacities of fungal strains belonging to the genera Phoma and Pyrenochaeta, which are closely related to the genus Paraphoma, to degrade polymeric compounds.

4 ConclusionFor establishment of a technique to accelerate degrada-

tion of used BP agricultural mulch films using BP-degrad-ing filamentous fungi, we found the source of strong de-grader. Practical application of these microorganisms to large fields will lead to the development of low-cost, envi-ronmentally friendly techniques.

AcknowledgmentsWe thank Showa Denko K. K. for generously supplying

Bionolle materials. This work was partially supported fi-nancially by the Science and Technology Research Promo-tion Program 25017A for Agriculture, Forestry, Fisheries and Food Industry, and in part by the Japan Society for the Promotion of Science KAKENHI Grant Number 25450502.

References1） Brodhangen, M.; Peyron, M.; Miles, C.; Inglis, D. A.

Biodegradable plastic agricultural mulches and key features of microbial degradation. App. Microbiol. Biotechnol. 99, 1039-1056（2015）.

2） Kyrikou, I.; Briassoulis, D. Biodegradation of agricul-tural plastic films: A Critical Review. J. Polym. Envi-ron. 15, 125-150（2007）.

3） Uchida, H.; Nakajima-Kambe, T.; Shigeno-Akutsu, Y.; Nomura, N.; Tokiwa, Y.; Nakahara, T. Properties of a bacterium which degrades solid poly（tetramethylene succinate）-co-adipate, a biodegradable plastic. FEMS Microbiol. Lett. 189, 25-29（2000）.

4） Kitamoto, H. K.; Shinozaki, Y.; Cao, X. H.; Morita, T.; Konishi, M.; Tago, K.; Kajiwara, H.; Koitabashi, M.; Yo-shida, S.; Watanabe, T.; Sameshima-Yamashita, Y.; Nak-ajima-Kambe, T.; Tsushima, S. Phyllosphere yeasts rapidly break down biodegradable plastics. AMB Ex-press 1, 44（2011）.

5） Watanabe, T.; Suzuki, K.; Shinozaki, Y.; Yarimizu, T.; Yoshida, S.; Sameshima-Yamashita, Y.; Koitabashi, M.; Kitamoto, H.K. A UV-induced mutant of Cryptococcus flavus GB-1 with increased production of a biodegrad-able plastic-degrading enzyme. Process Biochem. 50,

1718-1724（2015）.6） Koitabashi, M.; Noguchi, M. T.; Sameshima-Yamashita,

Y.; Hiradate, S.; Suzuki, K.; Yoshida, S.; Watanabe, T.; Shinozaki, Y.; Tsushima, S.; Kitamoto, H. K. Degrada-tion of biodegradable plastic mulch films in soil envi-ronment by phylloplane fungi isolated from gramine-ous plants. AMB Express 2, 40（2012）.

7） Koitabashi, M.; Sameshima-Yamashita, Y.; Watanabe, T.; Shinozaki, Y.; Kitamoto, H. Phylloplane fungal enzyme accelerate decomposition of biodegradable plastic film in agricultural settings. Japan Agricultural Research Quarterly 50, 229-234（2016）.

8） Morgan-Jones, G.; White J. F.; Studies in the genus Phoma III. Paraphoma, a new genus to accommodate Phoma radicina. Mycotaxon 18, 57-65（1983）.

9） Gruyter, J.; Woudenberg, J. H.; Aveskamp, M. M., Verk-ley, G. J.; Groenewald, J. Z.; Crous, P. W. Systematic reappraisal of species in Phoma section Paraphoma, Pyrenochaeta and Pleurophoma. Mycologia 102, 1066-1081（2010）.

10） Begerow, D.; Nilsson, H.; Unterseher, M.; Maier, W. Current state and perspectives of fungal DNA barcod-ing and rapid identification procedures. Appl. Micro-biol. Biotechnol. 87, 99-108（2010）.

11） Suzuki, K.; Noguchi, M. T.; Shinozaki, Y.; Koitabashi, M.; Sameshima-Yamashita, Y.; Yoshida, S.; Fujii, T.; Kita-moto, H. K. Purification, characterization, and cloning of the gene for a biodegradable plastic-degrading en-zyme from Paraphoma-related fungal strain B47-9. Appl. Microbiol. Biotechnol. 98, 4457-4465（2014）.

12） Vilgalys, R.; Hester, M. Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. J. Bacteriol.172, 4238-4246（1990）.

13） White, T. J.; Bruns, T.; Lee, S.; Taylor, J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR Protocols: a guide to meth-ods and applications（Innis MA, Gelfand DH, Sninsky JJ, White TJ, eds）. Academic Press, San Diego, Cali-fornia, U.S.A. 315-322（1990）.

14） Pearson, W. R.; Lipman, D. J. Improved tools for bio-logical sequence comparison. Proc. Natl. Acad. Sci. 85, 2444-2448（1988）.

15） Rehner, S. A.; Samuels, G. J. Taxonomy and phylogeny of Gliocladium analysed from nuclear large subunit ribosomal DNA sequences. Mycological Res. 98, 625-634（1994）.

16） Aveskamp, M. M.; de Gruyter, J.; Woudenberg, J. H. C.; Verkley, G. J. M.; Crous, P. W. Highlights of the Didy-mellaceae: A polyphasic approach to characterise Phoma and related pleosporalean genera. Stud. My-col. 65, 1-60（2010）.

17） Stamatakis, A. RAxML Version 8: A tool for phyloge-netic analysis and post-analysis of large phylogenies.

Biodegradable plastic-degrading activity of various species of Paraphoma

J. Oleo Sci. 65, (7) 621-627 (2016)

627

Bioinformatics 30, 1312-1313（2014）.18） Tanabe, A. S. Kakusan4 and Aminosan: two programs

for comparing nonpartitioned, proportional and sepa-rate models for combined molecular phylogenetic analyses of multilocus sequence data. Mol. Ecol. Res. 11, 914-921（2011）.

19） Felsenstein, J. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783-791（1985）.

20） Tamura, K.; Peterson, D.; Peterson, N.; Stecher G.; Nei, M.; Kumar, S. MEGA5: Molecular evolutionary ge-netics analysis using maximum likelihood, evolution-

ary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, 2731-2739（2011）.

21） Kimura, M. A simple method for estimating evolution-ary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16, 111-120（1980）.

22） Sameshima-Yamashita, Y.; Koitabashi, M.; Tsuchiya, W.; Suzuki, K.; Watanabe, T.; Shinozaki, Y.; Yamamoto-Tamura, K.; Yamazaki, T.; Kitamoto, H. Enhancement of biodegradable plastic-degrading enzyme production from Paraphoma-like fungus, strain B47-9. J. Oleo. Sci. 16, 257-262（2016）.