Field studies on the deterioration of microplastic films from ultra-thin compostable bags in soil.

In recent years, some countries have replaced single-use plastic bags with bags manufactured from compostable plastic film that can be used for collecting food wastes and composted together with the waste. Because industrial compost contains undeteriorated fragments of these bags, application to field soil is a potential source of small-sized residues from these bags. This study was undertaken to examine deterioration of these compostable film microplastics (CFMPs) in field soil at three different localities in Italy. Deterioration of CFMPs did not exceed 5.7% surface area reduction during the 12-month experimental period in two sites located in Northern Italy. More deterioration was observed in the Southern site, with 7.2% surface area reduction. Deterioration was significantly increased when fields were amended with industrial compost (up to 9.6%), but not with home compost. Up to 92.9% of the recovered CFMPs were associated with the soil fungus Aspergillus flavus, with 20.1%-71.2% aflatoxin-producing isolates. Application of industrial compost resulted in a significant increase in the percentage of CFMPs associated with A. flavus. This observation provides an argument for government regulation of accumulation of CFMPs and elevation of hazardous fungi levels in agricultural soils that receive industrial compost.

Mellein: Production in culture by Macrophomina phaseolina isolates from soybean plants exhibiting symptoms of charcoal rot and its role in pathology.

Macrophomina phaseolina (Mp) is a fungal pathogen proposed to enter host roots by releasing toxins that induce local necrosis in roots allowing entry of hyphae. Mp is reported to produce several potent phytotoxins, including (-)-botryodiplodin and phaseolinone, but isolates that do not produce these phytotoxins retain virulence. One hypothesis explaining these observations is that some Mp isolates may produce other unidentified phytotoxin(s) responsible for virulence. A previous study of Mp isolates from soybean found 14 previously unreported secondary metabolites using LC-MS/MS, including mellein, which has various reported biological activities. This study was conducted to investigate the frequency and amounts of mellein produced in culture by Mp isolates from soybean plants exhibiting symptoms of charcoal rot and to investigate the role of mellein in any observed phytotoxicity. LC-MS/MS analysis of cell-free culture filtrates (CCFs) from 89 Mp isolates revealed that 28.1% produced mellein (49-2,203 µg/L). In soybean seedlings in hydroponic culture, Mp CCFs diluted to 25% (vol/vol) in hydroponic growth medium induced phytotoxic symptoms with frequencies of 73% chlorosis, 78% necrosis, 7% wilting, and 16% death, and at 50% (vol/vol) induced phytotoxicity with frequencies of 61% chlorosis, 82% necrosis, 9% wilting, and 26% death. Commercially-available mellein (40-100 µg/mL) in hydroponic culture medium induced wilting. However, mellein concentrations in CCFs exhibited only weak, negative, insignificant correlations with phytotoxicity measures in soybean seedlings, suggesting that mellein does not contribute substantially to observed phytotoxic effects. Further investigation is needed to determine if mellein plays any role in root infection.

Pigment Produced by Glycine-Stimulated Macrophomina Phaseolina Is a (-)-Botryodiplodin Reaction Product and the Basis for an In-Culture Assay for (-)-Botryodiplodin Production.

An isolate of Macrophomina phaseolina from muskmelons (Cucumis melo)was reported by Dunlap and Bruton to produce red pigment(s) in melons and in culture in the presence of added glycine, alanine, leucine, or asparagine in the medium, but not with some other amino acids and nitrogen-containing compounds. We explored the generality and mechanism of this pigment production response using pathogenic M. phaseolina isolates from soybean plants expressing symptoms of charcoal rot disease. A survey of 42 M. phaseolina isolates growing on Czapek-Dox agar medium supplemented with glycine confirmed pigment production by 71% of isolates at the optimal glycine concentration (10 g/L). Studies in this laboratory have demonstrated that some pathogenic isolates of M. phaseolina produce the mycotoxin (-)-botryodiplodin, which has been reported to react with amino acids, proteins, and other amines to produce red pigments. Time course studies showed a significant positive correlation between pigment and (-)-botryodiplodin production by selected M. phaseolina isolates with maximum production at seven to eight days. Pigments produced in agar culture medium supplemented with glycine, beta-alanine, or other amines exhibited similar UV-vis adsorption spectra as did pigments produced by (±)-botryodiplodin reacting in the same agar medium. In a separate study of 39 M. phaseolina isolates, red pigment production (OD520) on 10 g/L glycine-supplemented Czapek-Dox agar medium correlated significantly with (-)-botryodiplodin production (LC/MS analysis of culture filtrates) in parallel cultures on un-supplemented medium. These results support pigment production on glycine-supplemented agar medium as a simple and inexpensive in-culture method for detecting (-)-botryodiplodin production by M. phaseolina isolates.

First Report of the Production of Mycotoxins and Other Secondary Metabolites by Macrophomina phaseolina (Tassi) Goid. Isolates from Soybeans (Glycine max L.) Symptomatic with Charcoal Rot Disease.

Macrophomina phaseolina (Tassi) Goid., the causal agent of charcoal rot disease of soybean, is capable of causing disease in more than 500 other commercially important plants. This fungus produces several secondary metabolites in culture, including (-)-botryodiplodin, phaseolinone and mellein. Given that independent fungal isolates may differ in mycotoxin and secondary metabolite production, we examined a collection of 89 independent M. phaseolina isolates from soybean plants with charcoal rot disease using LC-MS/MS analysis of culture filtrates. In addition to (-)-botryodiplodin and mellein, four previously unreported metabolites were observed in >19% of cultures, including kojic acid (84.3% of cultures at 0.57-79.9 µg/L), moniliformin (61.8% of cultures at 0.011-12.9 µg/L), orsellinic acid (49.4% of cultures at 5.71-1960 µg/L) and cyclo[L-proline-L-tyrosine] (19.1% of cultures at 0.012-0.082 µg/L). In addition, nine previously unreported metabolites were observed at a substantially lower frequency (<5% of cultures), including cordycepin, emodin, endocrocin, citrinin, gliocladic acid, infectopyron, methylorsellinic acid, monocerin and N-benzoyl-L-phenylalanine. Further studies are needed to investigate the possible effects of these mycotoxins and metabolites on pathogenesis by M. phaseolina and on food and feed safety, if any of them contaminate the seeds of infected soybean plants.