Motoo Koitabashi

Phyllosphere yeasts rapidly break down biodegradable plastics

The use of biodegradable plastics can reduce the accumulation of environmentally persistent plastic wastes. The rate of degradation of biodegradable plastics depends on environmental conditions and is highly variable. Techniques for achieving more consistent degradation are needed. However, only a few microorganisms involved in the degradation process have been isolated so far from the environment. Here, we show that Pseudozyma spp. yeasts, which are common in the phyllosphere and are easily isolated from plant surfaces, displayed strong degradation activity on films made from poly-butylene succinate or poly-butylene succinate-co-adipate. Strains of P. antarctica isolated from leaves and husks of paddy rice displayed strong degradation activity on these films at 30°C. The type strain, P. antarctica JCM 10317, and Pseudozyma spp. strains from phyllosphere secreted a biodegradable plastic-degrading enzyme with a molecular mass of about 22 kDa. Reliable source of biodegradable plastic-degrading microorganisms are now in our hands.

Thirteen novel deoxynivalenol‐degrading bacteria are classified within two genera with distinct degradation mechanisms

The mycotoxin deoxynivalenol (DON), a secondary metabolite produced by species of the plant pathogen Fusarium, causes serious problems in cereal crop production because of its toxicity towards humans and livestock. A biological approach for the degradation of DON using a DON-degrading bacterium (DDB) appears to be promising, although information about DDBs is limited. We isolated 13 aerobic DDBs from a variety of environmental samples, including field soils and wheat leaves. Of these 13 strains, nine belonged to the Gram-positive genus Nocardioides and other four to the Gram-negative genus Devosia. The degradation phenotypes of the two Gram types were clearly different; all washed cells of the 13 strains degraded 100 μg mL(-1) DON to below the detection limit (0.5 μg mL(-1)), but the conditions inducing the DON-degrading activities differed between the two Gram types. The HPLC profiles of the DON metabolites were also distinct between the two genera, although all strains produced 3-epi-deoxynivalenol. The Gram-positive strains showed DON assimilation in media containing DON as a carbon source, whereas the Gram-negatives did not. Our results suggest that aerobic DDBs are distributed within at least two phylogenetically restricted genera, suggesting independent evolution of the DON-degradation mechanisms.

Bacterial Cytochrome P450 System Catabolizing the Fusarium Toxin Deoxynivalenol

ABSTRACT Deoxynivalenol (DON) is a natural toxin of fungi that cause Fusarium head blight disease of wheat and other small-grain cereals. DON accumulates in infected grains and promotes the spread of the infection on wheat, posing serious problems to grain production. The elucidation of DON-catabolic genes and enzymes in DON-degrading microbes will provide new approaches to decrease DON contamination. Here, we report a cytochrome P450 system capable of catabolizing DON in Sphingomonas sp. strain KSM1, a DON-utilizing bacterium newly isolated from lake water. The P450 gene ddnA was cloned through an activity-based screening of a KSM1 genomic library. The genes of its redox partner candidates (flavin adenine dinucleotide [FAD]-dependent ferredoxin reductase and mitochondrial-type [2Fe-2S] ferredoxin) were not found adjacent to ddnA; the redox partner candidates were further cloned separately based on conserved motifs. The DON-catabolic activity was reconstituted in vitro in an electron transfer chain comprising the three enzymes and NADH, with a catalytic efficiency (k cat/Km ) of 6.4 mM−1 s−1. The reaction product was identified as 16-hydroxy-deoxynivalenol. A bioassay using wheat seedlings revealed that the hydroxylation dramatically reduced the toxicity of DON to wheat. The enzyme system showed similar catalytic efficiencies toward nivalenol and 3-acetyl deoxynivalenol, toxins that frequently cooccur with DON. These findings identify an enzyme system that catabolizes DON, leading to reduced phytotoxicity to wheat.

Degradation of biodegradable plastic mulch films in soil environment by phylloplane fungi isolated from gramineous plants

To improve the biodegradation of biodegradable plastic (BP) mulch films, 1227 fungal strains were isolated from plant surface (phylloplane) and evaluated for BP-degrading ability. Among them, B47-9 a strain isolated from the leaf surface of barley showed the strongest ability to degrade poly-(butylene succinate-co-butylene adipate) (PBSA) and poly-(butylene succinate) (PBS) films. The strain grew on the surface of soil-mounted BP films, produced breaks along the direction of hyphal growth indicated that it secreted a BP-degrading enzyme, and has directly contributing to accelerating the degradation of film. Treatment with the culture filtrate decomposed 91.2 wt%, 23.7 wt%, and 14.6 wt% of PBSA, PBS, and commercially available BP polymer blended mulch film, respectively, on unsterlized soil within 6 days. The PCR-DGGE analysis of the transition of soil microbial community during film degradation revealed that the process was accompanied with drastic changes in the population of soil fungi and Acantamoeba spp., as well as the growth of inoculated strain B47-9. It has a potential for application in the development of an effective method for accelerating degradation of used plastics under actual field conditions.

New biocontrol method for parsley powdery mildew by the antifungal volatiles-producing fungus Kyu-W63

Biocontrol of parsley powdery mildew, caused by Oidium sp., was examined for 3 years under greenhouse conditions using a filamentous fungus, strain Kyu-W63, which produces antifungal volatiles. Kyu-W63 was cultured on potato-dextrose agar in sterile polycarbonate pots and then placed at 30-cm intervals in the center of each ridge in 1998, 1999, and 2000. This Kyu-W63 treatment significantly inhibited disease severity compared to control plots.

Antifungal substances produced by fungal strain Kyu-W63 from wheat leaf and its taxonomic position

Fungal strain Kyu-W63 from a wheat leaf suppressed the development of wheat powdery mildew caused by Blumeria graminis f. sp. tritici. Nuclear magnetic resonance analysis revealed that Kyu-W63 produced two volatile substances: 5-pentyl-2-furaldehyde and 5-(4-pentenyl)-2-furaldehyde. Although the two are known to be nematicidal substances, the antifungal activity of 5-(4-pentenyl)-2-furaldehyde was first confirmed in the present study. Culture experiments revealed that synthesized 5-pentyl-2-furaldehyde had antifungal activity against three other pathogens – Fusarium oxysporum f. sp. lycopersici, Colletotrichum fragariae, Botrytis cinerea – with a minimum inhibitory concentration of 2–4 µg/ml. The two fractionated substances were also inhibitory against C. fragariae. Because the absence of spores in strain Kyu-W63 made its identification difficult, the sequence in the rDNA internal transcribed spacer (ITS) region of Kyu-W63 was compared with that of Irpex lacteus, which is known to produce these substances. Strain Kyu-W63 completely agreed with I. lacteus in terms of the ITS sequences, so strain Kyu-W63 was inferred to be I. lacteus. Two other strains of I. lacteus tested also produced the two furaldehydes and suppressed the growth of F. oxysporum f. sp. lycopersici. Both Kyu-W63 and additional strains produced the two furaldehydes on potato dextrose agar but not on water agar.

Xylose induces the phyllosphere yeast Pseudozyma antarctica to produce a cutinase-like enzyme which efficiently degrades biodegradable plastics

There is a need to speed up the degradation of used agricultural mulch films that are made of biodegradable plastics (BPs) in the field. Treating them with BP-degrading enzymes could be a solution to this problem. A cutinase-like enzyme of yeast Pseudozyma antarctica (PaE) has wide specificity of BPs degradation, but needs to be produced efficiently. Here we report that the production of PaE by P. antarctica can be increased by using xylose as carbon source. BP-degradation activity was analyzed using a polybutylene succinate-co-adipate (PBSA) emulsion as the substrate. Strain P. antarctica GB-4(1)W was found to be the best PaE producer among the tested strains. Using a 5-L jar fermentor with xylose fed-batch cultivation, high PaE productivity could be maintained and about 21 U/ml of PaE was obtained in 120 h. This amount was 100 times higher than the amount that we obtained previously (0.21 U/ml by flask cultivation using glycerol as carbon source). Under repeated xylose fed-batch cultivation with 24 h intervals, the maximum PaE production rate (0.34 U/ml/h) was maintained and the highest PaE productivity (28,000 U/2 L/d) was repeatedly obtained for 7 intervals. The activity of filtered jar-culture (crude PaE) was stable over 12 weeks at 4°C. Commercially available BP mulch films (20 μm thickness, cut into 1-cm-squares) were completely degraded by submerging them in crude PaE (2 U/ml) at 30°C in 24 h. These results indicated that concentrated PaE can rapidly degrade the strength of BP mulch films in the field so that they do not interfere with plowing.

Specificity of Pseudomonas isolates on healthy and Fusarium head blight-infected spikelets of wheat heads.

The specificity of culturable bacteria on healthy and Fusarium head blight (FHB)-infected spikelets of wheat heads was investigated to find a candidate of biocontrol agents against FHB. The bacterial genus Pseudomonas was commonly isolated from the tissues, and phylogenetic analysis using 16S ribosomal RNA gene sequences of isolates of the genera revealed that particular phylogenetic groups in the genus specifically inhabited either healthy or infected spikelet tissues. The specificity of each group was suggested to be due to differences in the ability to form biofilms and colonize spikelet tissues; isolates originated from healthy spikelets formed biofilms on polyvinyl chloride microplate wells and highly colonized the spikelet tissues. Other bacterial groups obtained from FHB-infected spikelets less formed biofilms and attached with low densities on the spikelet tissues. Their colonization on the tissues, however, was promoted when co-inoculated with the causal pathogenic fungus, Fusarium graminearum, and several isolates were observed to smash the mycelia in vivo. Moreover, based on results of in vitro mycelial growth inhibition activity, the diseased tissue-originated isolates were verified to have a negative effect on the fungal growth. These results suggest that Pseudomonas isolates obtained from infected spikelet tissues were highly associated with the FHB pathogen and have potential as candidates for biological control against FHB.

Aromatic Substances Inhibiting Wheat Powdery Mildew Produced by a Fungus Detected with a New Screening Method for Phylloplane Fungi

Microorganisms isolated from wheat leaf surfaces were screened for inhibition of wheat powdery mildew. A new screening method, in which wheat leaves were inoculated with Blumeria graminis f. sp. tritici and incubated with the cultured microorganisms under non-contact conditions, was developed in the present study. Using this method, 10 phylloplane fungi that inhibited wheat powdery mildew were selected from 408 microorganisms isolated from wheat leaf surfaces. Among these 10 strains, a fungus designated as Kyu-W63 had an especially strong inhibitory effect. Kyu-W63 produced white colonies without spores when cultivated on PDA. Kyu-W63 had a strong aromatic odor when being cultured. Wheat powdery mildew was suppressed even though a membrane filter with a pore size of 0.45 μm was placed between the mycelial colony and wheat leaf segment. However, when activated charcoal was introduced, Kyu-W63 did not inhibit growth of B. graminis. It was presumed that volatile substances were involved in the inhibitory effect of Kyu-W63. GC-MS analysis was used to identify two substances produced by Kyu-W63 with molecular weights of 164 and 166. Kyu-W63 also inhibited the in vitro growth of four plant pathogenic fungi other than B. graminis.

Production of a biodegradable plastic-degrading enzyme from cheese whey by the phyllosphere yeast Pseudozyma antarctica GB-4(1)W

Cheese whey is a by-product of cheese production and has high concentrations of lactose (about 5%) and other nutrients. Pseudozyma antarctica produces a unique cutinase-like enzyme, named PaE, that efficiently degrades biodegradable plastics. A previous study showed that a combination of 1% oil and 0.5% lactose increased cutinase-like enzyme production by another species of yeast. In this study, to produce PaE from cheese whey, we investigated the effects of soybean oil on PaE production (expressed as biodegradable plastic-degrading activity) by P. antarctica growing on lactose or cheese whey. In flask cultures, the final PaE activity was only 0.03 U/ml when soybean oil was used as the sole carbon source, but increased to 1.79 U/ml when a limited amount of soybean oil (under 0.5%) was combined with a relatively high concentration of lactose (6%). Using a 5-L jar fermentor with lactose fed-batch cultivation and periodic soybean oil addition, about 14.6 U/ml of PaE was obtained after 5 days of cultivation. When the lactose was replaced with cheese whey, PaE production was 10.8 U/ml after 3 days of cultivation.