Biodegradation of untreated plasticizers-free linear low-density polyethylene films by marine bacteria.

Polyethylene significantly contributes to marine plastic pollution. This study focuses on isolating bacteria from sea water and microplastic samples collected from the Tyrrhenian Sea and evaluating their ability to degrade virgin plasticizers-free linear low-density polyethylene (LLDPE) films. The isolates grew on the plastic film under aerobic conditions in shaken flasks leading to LLDPE mass losses of up to 2.597 ± 0.971 % after 60 days incubation. Biofilm formation on the film surface was confirmed by adhered protein quantification while film surface erosion and appearance of functional groups were revealed using SEM and FTIR analyses confirming biodegradation capabilities especially for isolates Bacillus velezensis MT9, Vreelandella venusta MT1 and Vreelandellatitanicae MT11. This is the first report on the biodegradation of plasticizers-free non pretreated LLDPE films by marine Bacillus sp. and Vreelandella sp.; most of the LLDPE biodegradation studies have been so far performed on plasticizer containing, pre-treated, or naturally weathered films.

Looking for peptides from rice starch processing by-product: Bioreactor production, anti-tyrosinase and anti-inflammatory activity, and in silico putative taste assessment.

One of the major challenges for the modern society, is the development of a sustainable economy also aiming at the valorization of agro-industrial by-products in conjunction with at a significant reduction of generated residues from farm to retail. In this context, the present study demonstrates a biotechnological approach to yield bioactive peptides from a protein fraction obtained as a by-product of the rice starch production. Enzymatic hydrolysis, with the commercial proteases Alcalase and Protamex, were optimized in bioreactor up to 2 L of volume. The two best digestates, selected with respect to peptide release and extract antioxidant capacity, were further fractionated (cut-offs of 10, 5, and 1 kDa) via cross-flow filtration. Amino acid composition indicated that most of the fractions showed positive nutritional characteristics, but a putative bitter taste. A fraction obtained with Alcalase enzyme (retentate 8 kDa) exerted anti-inflammatory potential, while the smaller molecular weight fractions (retentate 1-5 kDa and permeate < 1 kDa) were more active in tyrosinase inhibition. The latter were further sub-fractionated by size-exclusion chromatography. From the 15 most anti-tyrosinase sub-fractions, 365 peptide sequences were identified via liquid chromatography coupled with high resolution mass spectrometry. The present data support the possible exploitation of bioactive peptide from rice starch by-product as ingredients into food, nutraceutical, pharmaceutical, and cosmetic formulations.

Exhaustion of pentachlorophenol in soil microcosms with three Pseudomonas species as detoxification agents.

Pentachlorophenol (PCP) is a toxic compound, which is widely used as a wood preservative product and general biocide. It is persistent in the environment and has been classified as a persistent organic pollutant to be reclaimed in many countries. Bioremediation is an emerging approach to rehabilitating areas polluted by recalcitrant xenobiotics. In the present study, we evaluated the potential of three strains of Pseudomonas (P. putida S121, P. rhizophila S211, and P. fuscovagiceae S115) as bioremediation agents in depletion and detoxification of PCP in soil microcosms. PCP removal was effectively optimized using a central-composite experimental design and response surface methodology (RSM). The optimum conditions for maximum PCP removal yield (85 ± 5%) were: 500 mg/kg PCP concentration, 108 UFC/g soil inoculum size of each strain and 55 days incubation period. The bacterial strains, P. putida, P. rhizophila, and P. fuscovagiceae, showed good capability to tolerate and degrade PCP so that they could be successfully used in synergistic effect to treat PCP polluted soils.

An In-Vitro Evaluation of the Characteristics of Zein-Based Films for the Release of Lactobionic Acid and the Effects of Oleic Acid.

Lactobionic acid (LBA) is widely used in different industrial sectors owing to its biocompatibility characteristics as well as antioxidant and antimicrobial properties. In this study, mixtures of the protein zein with LBA and with the addition of oleic acid (OA) as a ternary system were investigated as drug delivery films for the release of LBA. The chosen combinations exploit the vast difference in water solubility between LBA and the other two components (zein and OA). DSC thermograms and dynamic mechanical spectra, alongside electron microscopy images, were used to describe the microstructural features of the films and were found to provide insights for the release of LBA from the two examined zein-based films immersed in an aqueous physiological solution. For both film systems, a burst release behavior was observed, followed by a rapid and total extraction of LBA. The required immersion time for the total extraction of LBA was greatly reduced when oleic acid was added to the precursor solution mixture for producing the films. The LBA released from the zein-based films was found to exhibit both the expected antioxidant properties as well as exerting bacteriostatic effects towards Escherichia coli and Staphylococcus epidermidis.

Mediterranean Sea bacteria as a potential source of long-chain polyunsaturated fatty acids.

Long-chain polyunsaturated fatty acids (LC-PUFAs), including EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid) are important nutritional ingredients in fish feed. So far, fish oil has been used as a main source of LC-PUFAs; however, the limited global supply of fish oil is not able to meet the demand of the growing aquaculture sector. Hence, sustainability of aquaculture industry could be supported by searching alternative sources of these compounds. Marine microorganisms represent a sustainable and stable supply source of LC-PUFAs. A collection of 209 bacterial isolates obtained from sediment samples recovered in the Mediterranean Sea was screened in order to select new LC-PUFAs producers. Among 95 putative producers selected based on colourimetric screening, 31 quickly growing were selected for further studies. The detection of LC-PUFAs was confirmed from 15 isolates belonging to the genera Marinobacter, Halomonas and Thalassospira by GC-FID analysis. Among them, the isolate Marinobacter sp. G16.20 was found to be a potentially high LC-PUFA producer exhibiting relatively high levels of DHA in particular (maximum productivity of 1.85 ± 0.371 mg/g, representing 45.89% of the total fatty acids detected and identified). Microorganisms belonging to the genera reported in this study showed biotechnological traits interesting for their potential future application in aquaculture.

Biodegradation of polyvinyl chloride plastic films by enriched anaerobic marine consortia.

Plastics remarkably contribute to marine litter, which is raising serious concerns. Currently, little is known about the fate of most plastics entering the marine environment and their potential biodegradation rate and extent under anoxic conditions. In this work, biodegradation of polyvinyl chloride (PVC) films by consortia enriched from marine samples (litter and water) was evaluated in anaerobic microcosms. After 7 months, three microcosms showed dense biofilms on plastic surfaces, gravimetric weight losses up to 11.7 ± 0.6%, marked decreases in thermal stability and average molecular weight of the polymer, suggesting microbial attack towards polymer chains. After 24 months, further three consortia showed the same abilities. Microbial communities analyzed at month 24 included taxa closely related to those previously reported as halogenated organic compounds degraders. The study is the first report on PVC biodegradation by marine anaerobic microbes and provides insights on potential biodegradation of the plastic film introduced into the sea by native microbes.

Biodegradation of oil-based plastics in the environment: Existing knowledge and needs of research and innovation.

The production of synthetic oil-based plastics has led to the accumulation of huge amounts of the plastic waste in the environment, especially in the marine system, very often the final sink for many types of conventional wasted plastics. In particular, (micro)plastics account for the majority of litter items in the marine environment and a high percentage of such litter is originating from land sources. Attempts to mitigate the harmful effects of conventional plastics such as the development of novel management strategies together with the gradual substitution of them with biodegradable (bio)plastics are representing future solutions. However, high amounts of conventional plastics have been accumulating in the environment since several years. Although many studies reported on their potential biodegradation by microbes in and from terrestrial environments, very little is known about the biodegradability of these plastics in freshwater systems and only recently more reports on their biodegradation by marine microorganisms/in marine environment were made available. In this review, we first provide a summary of the approaches applied for monitoring and assessing conventional plastics biodegradation under defined conditions. Then, we reviewed historical and recent findings related to biodegradation of four major plastics produced in European Union (EU), i.e. Polyethylene, Polyvinyl Chloride, Polypropylene and Polystyrene, in terrestrial and aquatic environments and by pure and mixed microbial cultures obtained from them.

Polyvinyl chloride biodegradation by Pseudomonas citronellolis and Bacillus flexus.

The accumulation of high amounts of petroleum-derived plastics in the environment has raised ecological and health concerns. The aim of this work was to study the biodegradative abilities of five bacterial strains, namely Pseudomonas chlororaphis, Pseudomonas citronellolis, Bacillus subtilis, Bacillus flexus and Chelatococcus daeguensis, towards polyethylene, polypropylene, polystyrene and polyvinyl chloride films under aerobic conditions. Preliminary screening resulted in the selection of P. citronellolis and B. flexus as potential PVC film degraders. Both strains were able to form a biofilm on the plastic film surface and to cause some modifications to the FTIR spectra of biomass-free PVC films. The two strains were then used to set up a PVC film biodegradation assay in 2-liter flasks. After 45 days incubation, fragmentation of the film was observed, suggesting that PVC biodegradative activity took place. Gel permeation chromatography analysis showed a reduction in average molecular weight of 10% for PVC incubated with P. citronellolis, with PVC polymer chains apparently attacked. Based on these results, the P. citronellolis strain was selected for biodegradation assays of two waste PVC films, used either nonsterile or subjected to ethanol sterilization. Chemical analyses on the incubated films confirmed the biodegradation of waste PVC plastics as shown by a gravimetric weight loss of up to about 19% after 30 days incubation. In summary, this work reports the biodegradation of PVC films by P. citronellolis and B. flexus. Both strains were shown to act mainly against PVC additives, exhibiting a low biodegradation rate of PVC polymer.

Genome analysis provides insights into crude oil degradation and biosurfactant production by extremely halotolerant Halomonas desertis G11 isolated from Chott El-Djerid salt-lake in Tunisian desert.

Here, we report the genomic features and the bioremediation potential of Halomonas desertis G11, a new halophilic species which uses crude oil as a carbon and energy source and displays intrinsic resistance to salt stress conditions (optimum growth at 10% NaCl). G11 genome (3.96 Mb) had a mean GC content of 57.82%, 3622 coding sequences, 480 subsystems and 64 RNA genes. Annotation predicted 38 genes involved in osmotic stress including the biosynthesis of osmoprotectants glycine-betaine, ectoine and osmoregulated periplasmic glucans. Genome analysis revealed also the versatility of the strain for emulsifying crude oil and metabolizing hydrocarbons. The ability of G11 to degrade crude oil components and to secrete a glycolipid biosurfactant with satisfying properties was experimentally confirmed and validated. Our results help to explain the exceptional capacity of G11 to survive at extreme desertic conditions, and highlight the metabolic features of this organism that has biotechnological and ecological potentialities.

Bacterial polyextremotolerant bioemulsifiers from arid soils improve water retention capacity and humidity uptake in sandy soil.

BACKGROUND: Water stress is a critical issue for plant growth in arid sandy soils. Here, we aimed to select bacteria producing polyextremotolerant surface-active compounds capable of improving water retention and humidity uptake in sandy soils. RESULTS: From Tunisian desert and saline systems, we selected eleven isolates able to highly emulsify different organic solvents. The bioemulsifying activities were stable with 30% NaCl, at 4 and 120 °C and in a pH range 4-12. Applications to a sandy soil of the partially purified surface-active compounds improved soil water retention up to 314.3% compared to untreated soil. Similarly, after 36 h of incubation, the humidity uptake rate of treated sandy soil was up to 607.7% higher than untreated controls. CONCLUSIONS: Overall, results revealed that polyextremotolerant bioemulsifiers of bacteria from arid and desert soils represent potential sources to develop new natural soil-wetting agents for improving water retention in arid soils.

Root bacterial endophytes confer drought resistance and enhance expression and activity of a vacuolar H+ -pumping pyrophosphatase in pepper plants.

It has been previously shown that the transgenic overexpression of the plant root vacuolar proton pumps H+ -ATPase (V-ATPase) and H+ -PPase (V-PPase) confer tolerance to drought. Since plant-root endophytic bacteria can also promote drought tolerance, we hypothesize that such promotion can be associated to the enhancement of the host vacuolar proton pumps expression and activity. To test this hypothesis, we selected two endophytic bacteria endowed with an array of in vitro plant growth promoting traits. Their genome sequences confirmed the presence of traits previously shown to confer drought resistance to plants, such as the synthesis of nitric oxide and of organic volatile organic compounds. We used the two strains on pepper (Capsicuum annuum L.) because of its high sensitivity to drought. Under drought conditions, both strains stimulated a larger root system and enhanced the leaves' photosynthetic activity. By testing the expression and activity of the vacuolar proton pumps, H+ -ATPase (V-ATPase) and H+ -PPase (V-PPase), we found that bacterial colonization enhanced V-PPase only. We conclude that the enhanced expression and activity of V-PPase can be favoured by the colonization of drought-tolerance-inducing bacterial endophytes.

Assessment of genetic diversity and bioremediation potential of pseudomonads isolated from pesticide-contaminated artichoke farm soils.

A total of 68 dimethoate and pentachlorophenol-tolerant rhizobacteria, isolated from a pesticide-contaminated agricultural soil, have been identified and typed by means of 16S-23S rRNA internal transcribed spacers analysis (ITS-PCR), 16S rRNA gene sequencing and by repetitive extragenic palindromic (BOX-PCR). The majority of bacterial isolates (84.31%) belonged to Proteobacteria (with a predominance of Gammaproteobacteria, 72.54%), while the remaining isolates were affiliated with Firmicutes (9.80%), Bacteroidetes (1.96%) and Actinobacteria (3.92%). The pesticide-tolerant bacterial isolates belonged to 11 genera, namely Pseudomonas, Bacillus, Acinetobacter, Flavobacterium, Comamonas, Achromobacter, Rhodococcus, Ochrobactrum, Aquamicrobium, Bordetella and Microbacterium. Within the well-represented genus Pseudomonas (n = 36), the most common species was Pseudomonas putida (n = 32). The efficacy of the selected strain, Pseudomonas putida S148, was further investigated for biodegradation of pentachlorophenol (PCP) in minimal medium, when used as a sole carbon and energy source. At an initial concentration of 100 mg/L, P. putida S148 degraded 91% of PCP after 7 days. GC-MS analyses revealed the formation of tetrachlorohydroquinone, tri- and di-chlorophenols as biodechlorination products in PCP remediation experiments. The toxicity estimation showed that 50% lethal concentration (LC50) and 50% growth inhibition concentration (IGC50) obtained values for the major identified compounds (2,3,4,6 tetrachlorophenol, 2,3,5,6 tetrachlorophenol and tetrachlorohydroquinone) were higher than those estimated for the PCP indicating that the metabolites are less toxic than the original compound for those specific organisms. S148 strain could be added to pesticide-contaminated agricultural soils as a bacterial inoculant for its potential to improve soil quality.

Pseudomonas rhizophila S211, a New Plant Growth-Promoting Rhizobacterium with Potential in Pesticide-Bioremediation.

A number of Pseudomonas strains function as inoculants for biocontrol, biofertilization, and phytostimulation, avoiding the use of pesticides and chemical fertilizers. Here, we present a new metabolically versatile plant growth-promoting rhizobacterium, Pseudomonas rhizophila S211, isolated from a pesticide contaminated artichoke field that shows biofertilization, biocontrol and bioremediation potentialities. The S211 genome was sequenced, annotated and key genomic elements related to plant growth promotion and biosurfactant (BS) synthesis were elucidated. S211 genome comprises 5,948,515 bp with 60.4% G+C content, 5306 coding genes and 215 RNA genes. The genome sequence analysis confirmed the presence of genes involved in plant-growth promoting and remediation activities such as the synthesis of ACC deaminase, putative dioxygenases, auxin, pyroverdin, exopolysaccharide levan and rhamnolipid BS. BS production by P. rhizophila S211 grown on olive mill wastewater based media was effectively optimized using a central-composite experimental design and response surface methodology (RSM). The optimum conditions for maximum BS production yield (720.80 ± 55.90 mg/L) were: 0.5% (v/v) inoculum size, 15% (v/v) olive oil mill wastewater (OMWW) and 40°C incubation temperature at pH 6.0 for 8 days incubation period. Biochemical and structural characterization of S211 BS by chromatography and spectroscopy studies suggested the glycolipid nature of the BS. P. rhizophila rhamnolipid was stable over a wide range of temperature (40-90°C), pH (6-10), and salt concentration (up to 300 mM NaCl). Due to its low-cost production, emulsification activities and high performance in solubilization enhancement of chemical pesticides, the indigenous BS-producing PGPR S211 could be used as a promising agent for environmental bioremediation of pesticide-contaminated agricultural soils.

Potential use of ricotta cheese whey for the production of lactobionic acid by Pseudomonas taetrolens strains.

Lactobionic acid (LBA) is a fine chemical largely applied in the food, chemical, cosmetics and pharmaceutical industries. Here, its production from ricotta cheese whey (RCW), or scotta, the main by-product obtained from ricotta cheese production process and currently employed mainly for cattle feed, was evaluated. Among seven bacterial species tested, only two Pseudomonas taetrolens strains were selected after preliminary screening in shake-flasks. When autoclaved RCW was used, a lactobionic acid titer of 34.25 ±â€¯2.86 g/l, with a conversion yield (defined as mol LBA/mol of consumed lactose%) of up to 85 ±â€¯7.0%, was obtained after 48 h of batch fermentation in 3 L stirred tank bioreactor. This study is a preliminary investigation on the potential industrial use of scotta as a substrate for bacterial growth and lactobionic acid production that details the possible biotechnological valorization pathways and feasibility of the process.

Marinobacter sp. from marine sediments produce highly stable surface-active agents for combatting marine oil spills.

BACKGROUND: The application of chemical dispersants as a response to marine oil spills is raising concerns related to their potential toxicity also towards microbes involved in oil biodegradation. Hence, oil spills occurring under marine environments necessitate the application of biodispersants that are highly active, stable and effective under marine environment context. Biosurfactants from marine bacteria could be good candidates for the development of biodispersant formulations effective in marine environment. This study aimed at establishing a collection of marine bacteria able to produce surface-active compounds and evaluating the activity and stability of the produced compounds under conditions mimicking those found under marine environment context. RESULTS: A total of 43 different isolates were obtained from harbor sediments. Twenty-six of them produced mainly bioemulsifiers when glucose was used as carbon source and 16 were biosurfactant/bioemulsifiers producers after growth in the presence of soybean oil. Sequencing of 16S rRNA gene classified most isolates into the genus Marinobacter. The produced emulsions were shown to be stable up to 30 months monitoring period, in the presence of 300 g/l NaCl, at 4 °C and after high temperature treatment (120 °C for 20 min). The partially purified compounds obtained after growth on soybean oil-based media exhibited low toxicity towards V. fischeri and high capability to disperse crude oil on synthetic marine water. CONCLUSIONS: To the best of our knowledge, stability characterization of bioemulsifiers/biosurfactants from the non-pathogenic marine bacterium Marinobacter has not been previously reported. The produced compounds were shown to have potential for different applications including the environmental sector. Indeed, their high stability in the presence of high salt concentration and low temperature, conditions characterizing the marine environment, the capability to disperse crude oil and the low ecotoxicity makes them interesting for the development of biodispersants to be used in combatting marine oil spills.

Biotechnological applications of extremophiles, extremozymes and extremolytes.

In the last decade, attention to extreme environments has increased because of interests to isolate previously unknown extremophilic microorganisms in pure culture and to profile their metabolites. Microorganisms that live in extreme environments produce extremozymes and extremolytes that have the potential to be valuable resources for the development of a bio-based economy through their application to white, red, and grey biotechnologies. Here, we provide an overview of extremophile ecology, and we review the most recent applications of microbial extremophiles and the extremozymes and extremolytes they produce to biotechnology.

Halo-alkalitolerant and thermostable cellulases with improved tolerance to ionic liquids and organic solvents from Paenibacillus tarimensis isolated from the Chott El Fejej, Sahara desert, Tunisia.

The wide number of industrial processes applying cellulases highlights the importance of discovering robust enzymes able to work under harsh conditions. In this study, carboxymethyl cellulase (CMCase) activity of Paenibacillus tarimensis was characterized. A high activity was observed in pH range 3.0-10.5 and 9 mM-5 M NaCl. In high salt buffer at 80°C, >80% and >76% of relative activity was retained at 20% of the ionic liquids (ILs) [EMIM]Ac and [BMIM]Cl; while >40% was detected with 40% [BMIM]Cl. Five CMCases were detected by renaturing SDS-PAGE. Their activity was retained in presence of 1.7 up to 5 M NaCl (for CMC1) or 4.6 M KCl; 5% organic solvents or 10 mM bivalent ions, EDTA and heavy metals; under neutral and halo-alkaline conditions. These cellulases stabile and highly functional under harsh conditions are promising candidates for application in detergents, textiles, paper/pulp industry; and simultaneous ILs treatment-saccharification of lignocellulose.

An investigation of horizontal transfer of feed introduced DNA to the aerobic microbiota of the gastrointestinal tract of rats.

BACKGROUND: Horizontal gene transfer through natural transformation of members of the microbiota of the lower gastrointestinal tract (GIT) of mammals has not yet been described. Insufficient DNA sequence similarity for homologous recombination to occur has been identified as the major barrier to interspecies transfer of chromosomal DNA in bacteria. In this study we determined if regions of high DNA similarity between the genomes of the indigenous bacteria in the GIT of rats and feed introduced DNA could lead to homologous recombination and acquisition of antibiotic resistance genes. RESULTS: Plasmid DNA with two resistance genes (nptI and aadA) and regions of high DNA similarity to 16S rRNA and 23S rRNA genes present in a broad range of bacterial species present in the GIT, were constructed and added to standard rat feed. Six rats, with a normal microbiota, were fed DNA containing pellets daily over four days before sampling of the microbiota from the different GI compartments (stomach, small intestine, cecum and colon). In addition, two rats were included as negative controls. Antibiotic resistant colonies growing on selective media were screened for recombination with feed introduced DNA by PCR targeting unique sites in the putatively recombined regions. No transformants were identified among 441 tested isolates. CONCLUSIONS: The analyses showed that extensive ingestion of DNA (100 µg plasmid) per day did not lead to increased proportions of kanamycin resistant bacteria, nor did it produce detectable transformants among the aerobic microbiota examined for 6 rats (detection limit < 1 transformant per 1,1 × 10(8) cultured bacteria). The key methodological challenges to HGT detection in animal feedings trials are identified and discussed. This study is consistent with other studies suggesting natural transformation is not detectable in the GIT of mammals.

The stability and degradation of dietary DNA in the gastrointestinal tract of mammals: implications for horizontal gene transfer and the biosafety of GMOs.

The fate of dietary DNA in the gastrointestinal tract (GIT) of animals has gained renewed interest after the commercial introduction of genetically modified organisms (GMO). Among the concerns regarding GM food, are the possible consequences of horizontal gene transfer (HGT) of recombinant dietary DNA to bacteria or animal cells. The exposure of the GIT to dietary DNA is related to the extent of food processing, food composition, and to the level of intake. Animal feeding studies have demonstrated that a minor amount of fragmented dietary DNA may resist the digestive process. Mammals have been shown to take up dietary DNA from the GIT, but stable integration and expression of internalized DNA has not been demonstrated. Despite the ability of several bacterial species to acquire external DNA by natural transformation, in vivo transfer of dietary DNA to bacteria in the intestine has not been detected in the few experimental studies conducted so far. However, major methodological limitations and knowledge gaps of the mechanistic aspects of HGT calls for methodological improvements and further studies to understand the fate of various types of dietary DNA in the GIT.

'Candidatus Liberibacter europaeus' sp. nov. that is associated with and transmitted by the psyllid Cacopsylla pyri apparently behaves as an endophyte rather than a pathogen.

'Candidatus Liberibacter spp.' cause serious plant diseases. 'Candidatus Liberibacter asiaticus', 'Ca. L. americanus' and 'Ca. L. africanus' are the aetiological agents of citrus greening (Huanglongbing) in Asia, America and Africa. 'Candidatus Liberibacter solanacearum' causes diseases in Solanaceae in America and New Zealand. All four species are vectored by psyllid insects of different genera. Here, we show that the pear psyllid pest Cacopsylla pyri (L.) hosts a novel liberibacter species that we named 'Ca. Liberibacter europaeus'. It can bloom to high titres in the psyllid host, with more than 10(9) 16S rRNA gene copies per individual. Fluorescent in situ hybridization experiments showed that 'Ca. L. europaeus' is present in the host midgut lumen, salivary glands and Malpighian tubules. 'Candidatus L. europaeus' has a relatively high prevalence (> 51%) in C. pyri from different areas in the Piedmont and Valle d'Aosta regions in Italy and can be transmitted to pear plants in experimental transmission trials. However, even though high titres of the bacterium (more than 10(8) 16S rRNA gene copies g(-1) of pear plant tissue) could be detected, in the pear tissues no specific disease symptoms could be observed in the infected plants over a 6-month period. Despite liberibacters representing potential quarantine organisms, 'Ca. L. europaeus', first described in Italy and Europe, apparently behaves as an endophyte rather than a pathogen.