Degradation of biodegradable plastics by phytopathogenic smut fungi (2025)

Abstract

We evaluated biodegradable plastic (BP) degradation abilities of various smut fungi (Ustilaginomycotina), including 8 isolates from 6 species of Ustilaginales, 3 isolates from 2 species of Exobasidiales, 7 isolates from 5 species of Tilletiales, and 3 isolates from 3 species of Microbotryales belonging to the anther smut fungi (Pucciniomycotina). The BP-degrading abilities of these fungal isolates were compared to that of a known BP-degrader, Moesziomyces antarcticus JCM 10317. The BPs tested were polycaprolactone (PCL), polybutylene succinate adipate (PBSA), polybutylene succinate (PBS), and polylactic acid (PLA). BP-degrading ability was quantified by measuring the clear zones produced by fungi on BP-containing media at temperatures from 10 ℃ to 30 ℃. The experiments revealed 10 isolates from 7 species capable of degrading PBSA and PCL, and 7 isolates from 4 species capable of degrading PBS. No isolates degraded the PLA. Notably, the PBSA-degrading ability of one isolate of Ustilago trichophora was significantly higher than that of JCM 10317 at low temperature. However, JCM 10317 was superior at degrading PCL and PBS. Our results indicate that smut fungi are potentially valuable sources of isolates with superior BP-degrading ability.

1. Introduction

The basidiomycetous yeast strain JCM 10317, which was originally isolated from an Antarctic lake (Sugiyama et al., 1967), is recognized for its remarkable ability to degrade biodegradable plastics (BPs) (Kitamoto et al., 2011, 2018). This isolate, formerly classified as Pseudozyma antarctica (Goto, Sugiy. & Iizuka) Boekhout, has been reclassified as Moesziomyces antarcticus (Goto, Sugiy. & Iizuka) Q.M. Wang, Begerow, F.Y. Bai & Boekhout (Ustilaginaceae, Ustilaginales, Ustilaginomycotina) (Wang et al., 2015). This fungus was formerly considered a saprophyte that potentially degrades plant surface cuticles with a fatty acid polyester structure analogous to BPs (Kitamoto et al., 2011). However, Tanaka et al. (2019) discovered that the sexual state of this species is a smut fungus that parasitizes Echinochloa crus-galli (L.) P. Beauv. (Chloridoideae, Poaceae); and the asexual state obtained from smut spores collected in Japan degraded BP at levels similar to that of JCM 10317. Based on these findings, we hypothesize that asexual strains of various smut fungi with a life cycle similar to that of Mo. antarcticus will be able to degrade BPs. This suggests that BP-degrading fungal strains can potentially be obtained by germinating smut spores of phytopathogenic smut fungi.

BPs are increasingly being used in various applications, such as agricultural mulch films, plant pots, and packaging (bags, containers); and medical devices (sutures, drug delivery systems) (e.g. ). BPs are used to reduce environmental damage, minimize reliance on nonrenewable resources, and offer eco-friendly solutions for different industries (e.g. ; Silva et al., 2023). For BPs to be environmentally sustainable and widely applicable, they should be susceptible to degradation in various environments by microorganisms, including fungi (e.g. ). Some saprophytic fungi are known to produce extracellular enzymes that can effectively degrade BPs (e.g. Okal et al., 2023; Srikanth et al., 2022). Most of these fungi were obtained by chance from the environment during efforts to screen for plastic degrader (e.g. Černoša et al., 2024; Ekanayaka et al., 2022). However, the BP-degrading ability of fungi with specialized ecology, such as plant parasitic fungi, is mostly unknown, although some research has focused on the lignin-degrading fungi with the aim of using white rot fungi for plastic degradation (Bautista-Zamudio et al., 2023).

Smut fungi represent diverse groups of basidiomycetous plant pathogens that form sori containing teliospores (smut spores) in their host plants (Vánky, 2012). Numerous ultrastructural and molecular phylogenetic studies have clarified the taxonomic status of smut fungi and allied taxa (e.g. Kakishima, 2016; Wang et al., 2015). Currently, most smut fungi are classified under the orders Ustilaginales (Ustilaginomycetes, Ustilaginomycotina) and Tilletiales (Exobasidiomycetes, Ustilaginomycotina) (Begerow et al., 2014), and anther smut fungi are classified under the order Microbotryales (Microbotryomycetes, Pucciniomycotina) (Aime et al., 2014). Some fungal groups that do not produce sori and teliospores, such as Exobasidiales (Exobasidiomycetes), are also members of Ustilaginomycotina (Begerow et al., 2006). Many smut fungi, including Exobasidiales, assume a yeast-like form that produces uninucleate conidia (asexual spores) during their asexual stage. The ecological role of the yeast-like asexual stage of smut fungi is largely unknown; however, it is thought that some colonize plant surfaces by degrading the cuticle, and then eventually mate to form the plant-parasitic dikaryotic (sexual) stage.

In this study, we hypothesized that smut fungi are BP degraders. BP degradation experiments were conducted at 25 ℃ using isolates from three representative groups of classical concept smut fungi: Ustilaginales, Tilletiales, and Microbotryales, as well as some isolates of Exobasidiales, which are not traditionally considered as smut fungi. The next BP degradation experiments were conducted from 10 ℃ to 30 ℃ using selected isolates because fungi capable of degrading BPs at lower temperatures are more advantageous for field applications. The aim of our study was to obtain fungi with high BP degradation abilities, which we accomplished by evaluating the BP-degrading abilities of selected isolates of smut fungi at various temperatures.

2. Materials and methods

2.1. Biodegradable plastics and media

We used the following polyester-type BPs in our experiments: poly(ε-caprolactone) (PCL: FUJIFILM Wako Pure Chemical, Osaka, Japan), poly(butylene succinate)-co-(butylene adipate) (PBSA: bionolle® #3001, Showa Highpolymer Co. Ltd., Tokyo, Japan), poly(butylene succinate) (PBS: bionolle® #1001, Showa Highpolymer Co. Ltd.), and poly(lactic acid) (PLA: Standard-Testpiece, Kanagawa, Japan).

We first prepared an overlay medium containing a BP emulsion for the BP degradation experiments. The composition of this medium is slightly modified from the one used by Kitamoto et al. (2011). Briefly, the tested BP was dissolved by shaking 0.5 g in 10-15 mL of dichloromethane in a capped 50-mL centrifuge bottle for 1 h. After transferring the dissolved BP in a 200-mL beaker, we added 500 µL of 2% N-lauroylsarcosine sodium and 25 mL of distilled water. The mixture was sonicated using an ultrasonic homogenizer (Vibra-Cell VC-750; Sonics & Materials Inc., Newtown, CT, USA) for 5 min. After sonication, the mixture was stirred at 80 ℃ for 5-15 min using a magnetic stirrer in a fume hood to evaporate the dichloromethane. The volume of the BP emulsion was then raised to 100 mL by gradually adding hot distilled water with stirring. We then added 0.2 g of sodium dihydrogen phosphate, 0.05 g of magnesium sulfate heptahydrate, and 0.02 g of potassium dihydrogen phosphate while stirring the emulsion. The pH of the BP emulsion was adjusted to 7.0 by gradually adding 1% sodium hydroxide solution using a pipette and a pH meter. Finally, 1.5 g of agar powder was added to the emulsion and stirred until dissolved. The prepared BP emulsion medium was dispensed into Petri dishes (58 mm in diameter, 15 mm depth; 3 mL per dish) and allowed to solidify. A carbon-deficient medium (consisting of 0.67 g Difco Yeast Nitrogen Base without amino acids, 1.5 g agar powder and 100 mL distilled water) was prepared and overlaid on the BP emulsion medium (3 mL per dish) and solidified.

2.2. Fungal cultures

The 28 isolates from 16 fungal species that were used in this study are listed in Table 1. The reference isolate, JCM 10317, was obtained from JCM (Japan Collection of Microorganisms, RIKEN BioResource Research Center, Ibaraki, Japan), while the isolates of Exobasidium camelliae Shirai and Tilletia horrida Takah. were obtained from MAFF (Ministry of Agriculture, Forestry & Fisheries, Ibaraki, Japan). The rest of the isolates were isolated by our laboratory (Fig. 1). We reported using the isolates of Macalpinomyces spermophorus (Berk. & M.A. Curtis ex de Toni) Vánky, Mo. antarcticus, Neovossia moliniae (Thüm.) Körn., Pilocintractia fimbristylidicola (Pavgi & Mundk.) Vánky, Sporisorium manilense (Syd. & P. Syd.) Vánky, Tilletia arundinellae L. Ling, Tilletia vittata (Berk.) Mundk., and Ustilago phragmitis L. Ling previously (Tanaka 2021; ; Tanaka et al., 2019). The procedures we used to identify and culture the other fungal species are described in Tanaka (2021). The isolates of Kordyana sp. were cultured from basidiospores, while the isolates of Microbotryum spp., Sphacelotheca hydropiperis (Schumach.) de Bary, Tilletia barclayana (Bref.) Sacc. & P. Syd., and Ustilago trichophora (Link) Kunze were each grown from a single smut spore. Our procedures for light and scanning electron microscopy were described in Tanaka et al. (2019).

We prepared each test fungal culture by first culturing each fungus in 100 mL of potato dextrose broth (PDB; Formedium, Norfolk, UK) in a 200-mL elementary flask at 25 ℃ for a few days. The precultured fungi were transferred to a 50-mL centrifuge tube and precipitated by centrifugation (5000 rpm or 15000 rpm), followed by suspension in sterile distilled water. For fungi with no yeast-like growth stage, the mycelia were cut using a Polytron homogenizer (PT 2100; Kinematica AG, Malters, Switzerland). The fungal concentration was calculated using a Thoma hemocytometer and adjusted to 60000-80000 fungal cells/µL by diluting with distilled water.

2.3. Biodegradable plastic degradation experiment

We quantified the BP degradation ability of the fungi at various temperatures by measuring the clear zone area formed in the overlay medium by the inoculated fungi (Fig. 2). First, Whatman glass fiber filters (0.26 mm thick, pore size 1.6 µm; GE Healthcare Life Science, Buckinghamshire, UK) were punched into 6-mm diameter disks, autoclaved, and thoroughly dried. Then, 10 µL of the prepared fungal suspensions were inoculated onto the glass filter disks placed at three locations on the overlay medium. The Petri dishes were sealed and incubated in the dark at 10, 15, 20, 25, and 30 ℃ for two wk. The filter disks were gently washed before measuring the clear zone area using ImageJ v1.53 software (National Institute of Health, MD, USA).

2.4. Statistical analysis

We used Welch's t-test to compare the clear zone area of each test isolated to that of JCM 10317 grown at the same temperature. A one-tailed test was employed, and the significance level (α) was set at 0.05. All data are presented as the mean of six areas, and error bars represent 95% confidence intervals (Fig. 3).

3. Results

We initially investigated the BP-degrading ability of the phytopathogenic smut fungi at 25 ℃ (Table 1). This was accomplished by measuring the clear zones formed by the inoculated fungi in the overlay medium containing the BP emulsion as the sole carbon source. The clear zones were formed as the inoculated fungus degraded and used BPs in the medium as its carbon source (Fig. 2). Results of this experiment revealed that out of the 28 isolates from the 16 species tested, 16 isolates from 7 species degraded PCL and PBSA, while 13 isolates from 4 species degraded PBS (Table 1). None of the tested fungi degraded PLA. Among the BP-degrading fungi, Ma. spermophorus, Mo. antarcticus, U. trichophora, and U. phragmitis exhibited yeast-like growth. In contrast, Kordyana sp., N. moliniae and P. fimbristylidicola formed aggregates during precultivation. Because the aggregates were difficult to use even after homogenization, two species (Kordyana sp., and P. fimbristylidicola) were excluded from subsequent experiments.

We compared the BP-degrading abilities of eight isolates of Mo. antarcticus, one isolate each of U. trichophora, U. phragmitis, Ma. spermophorus, and N. moliniae at different temperatures (Fig. 3). Among them, U. phragmitis, Ma. spermophorus, and N. moliniae were excluded from the PBS degradation experiment, because the above tests showed that they did not have PBS degrading ability. Our results show that U. trichophora degraded PCL significantly more than JCM 10317 at 15 ℃; however, JCM 10317 demonstrated the highest PCL degradation at 20-30 ℃. Meanwhile, U. trichophora degraded PBSA significantly more than JCM 10317 at 10-30 ℃. Although JCM 10317 performed the maximum level of PBSA degradation at 30 ℃, U. trichophora performed the maximum level of PBSA degradation at 20 ℃. Furthermore, U. trichophora grown at 15 ℃ outperformed JCM 10317 at 30 ℃. We observed variations in PBSA-degrading ability among Mo. antarcticus isolates. Among them, MAFF 246415 outperformed JCM 10317 at 10-20 ℃; NBRC 116704 outperformed JCM 10317 at 10 ℃, 20 ℃, and 25 ℃. The PBS-degrading ability of U. trichophora was also significantly better than that of JCM 10317 at 10 ℃ and 15 ℃.

4. Discussion

We investigated the BP-degrading abilities of phytopathogenic smut fungi, encompassing 6 species of Ustilaginales, 2 species of Exobasidiales, 5 species of Tilletiales, and 3 species of Microbotryales. Among these, five species of Ustilaginales, one species of Exobasidiales, and one species of Tilletiales demonstrated BP-biodegrading ability. All isolates of these BP-degrading species degraded PBSA and PCL, while only four species degraded PBS. None of the tested isolates degraded PLA, which aligns with the findings by Urbanek et al. (2017) showing that PBSA is the BP that is most readily degraded by fungi, while PLA is the least degradable. Our results confirm our hypothesis that smut fungi other than Mo. antarcticus are BP degraders.

The observed variation in BP-degrading abilities among the tested smut fungi is likely related to the enzymatic abilities of each species. Notably, some smut fungi were unable to degrade BP, which may provide insights into their lifecycle and infection strategies. Typically, smut fungi assume a yeast-like state on the plant surface or in the soil during their asexual phase (e.g. Jiang et al., 2024). During this state, they do not actively penetrate the plant tissue. The inability of some fungal species to degrade BP suggests that they do not need to degrade the plant's surface cuticle during this phase. Further research is necessary to elucidate the precise role of these abilities in the lifecycle and infection processes of smut fungi.

We identified certain smut fungal isolates capable of degrading BPs at low temperatures; such isolates offer several advantages for BP degradation. For instance, utilizing these isolates will allow BP-based agricultural materials to be used in cooler climate zones and during cold seasons. The temperature-dependent variations in BP degradation efficiency observed among various fungal species likely reflect their respective optimal growth temperatures, which previous studies demonstrated on psychrophilic and psychrotolerant fungi (Margesin et al., 2007). Future studies should focus on optimizing BP degradation processes at low-temperatures and exploring their applications in various agricultural contexts, particularly in regions and seasons with lower temperatures.

One U. trichophora isolate showed the highest PBSA degradation ability, while Mo. antarcticus isolates exhibited the highest degradation abilities for PCL and PBS. To the best of our knowledge, this is the first report describing the high PBSA degradation ability of U. trichophora. The high BP degradation ability of Mo. antarcticus has already been reported. The optimum BP degradation temperature of U. trichophora is lower than that of Mo. antarcticus, suggesting that U. trichophora will be able to degrade PBSA even in low-temperature environments. This observation is consistent with previous research showing that the efficiency of microbial BP degradation is influenced by various environmental factors, among which temperature is a crucial determinant of both microbial growth and enzymatic processes (). Additionally, the yeast-like state of U. trichophora (which is similar to that of Mo. antarcticus) offers potential advantages for practical applications. Compared to mycelia, the yeast-like forms of fungi are generally easier to handle in practical applications; therefore, such fungi can facilitate BP degradation in various contexts across different temperature ranges.

We observed temperature-dependent variations in the BP-degrading ability among our Mo. antarcticus isolates, suggesting that similar variations may exist among the isolates of other smut fungi. Furthermore, we expanded the number of species of smut fungi that are capable of BP degradation. The inclusion of more species and isolates in future studies can potentially lead to the discovery of smut fungi with higher BP-degrading abilities. Smut fungi-related yeast-like fungi such as Pseudozyma, Meira, Microstroma, and Tilletiopsis are easily isolated from the phylloplane or soil as basidiomycetous yeasts (Sampaio, 2004). However, the true potential for BP degradation research lies in the sexual stages of smut fungi. On the one hand, these sexual morphs are difficult to collect because they only appear during specific periods and are associated with particular host plants. Conversely, many smut fungi are known only by their sexual morph. Although these fungi are difficult to collect and identify, accessing their sexual state provides a valuable resource for selecting BP-degrading isolates. This approach complements existing methods to screen for plastic degrader because it accesses genetic diversity that is not easily available through environmental sampling of asexual morphs. Such an approach can expand our range of BP-degrading microorganisms.

Disclosure

The authors declare no conflict of interest associated with this manuscript.

Acknowledgments

This study was supported by grant G-2019-1-012 from the Institute for Fermentation, Osaka (IFO). We would like to thank Mr. Ryunosuke Ishii, Mr. Kento Matsuura, Mr. Tatsuya Suga, and Ms. Chise Ohno for conducting the preliminary tests that lead to this research.

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