Axenic Conditions Research Articles

Various dictyostelids from the environment can produce multilamellar bodies.

Multilamellar bodies (MLBs), structures composed of concentric membrane layers, are known to be produced by different protozoa, including species of ciliates, free-living amoebae, and Dictyostelium discoideum social amoebae. Initially believed to be metabolic waste, potential roles like cell communication and food storage have been suggested for D. discoideum MLBs, which could be useful for the multicellular development of social amoebae and as a food source. However, among dictyostelids, this phenomenon has only been observed with D. discoideum, and mainly with laboratory strains grown in axenic conditions. It was thought that other social amoebae may also produce MLBs. Four environmental social amoeba isolates were characterized. All strains belong to the Dictyostelium genus, including some likely to be Dictyostelium giganteum. They have distinctive phenotypes comprising their growth rate on Klebsiella aerogenes lawns and the morphology of their fruiting bodies. They all produce MLBs like those produced by a D. discoideum laboratory strain when grown on K. aerogenes lawns, as revealed by analysis using the H36 antibody in epifluorescence microscopy as well as by transmission electron microscopy. Consequently, this study shows that MLBs are produced by various dictyostelid species, which further supports a role for MLBs in the lifestyle of amoebae.

A Simple and Non-destructive Method for Chlorophyll Quantification of Chlamydomonas Cultures Using Digital Image Analysis.

Growing interest in the use of microalgae as a sustainable feedstock to support a green, circular, bio-economy has led to intensive research and development initiatives aimed at increasing algal biomass production covering a wide range of scales. At the heart of this lies a common need for rapid and accurate methods to measure algal biomass concentrations. Surrogate analytical techniques based on chlorophyll content use solvent extraction methods for chlorophyll quantification, but these methods are destructive, time consuming and require careful disposal of the resultant solvent waste. Alternative non-destructive methods based on chlorophyll fluorescence require expensive equipment and are less suitable for multiple sampling of small cultures which need to be maintained under axenic growth conditions. A simple, inexpensive and non-destructive method to estimate chlorophyll concentration of microalgal cultures in situ from digital photographs using the RGB color model is presented. Green pixel intensity and chlorophyll a, b and total chlorophyll concentration, measured by conventional means, follow a strong linear relationship (R2 = 0.985–0.988). In addition, the resulting standard curve was robust enough to accurately estimate chlorophyll concentration despite changes in sample volume, pH and low concentrations of bacterial contamination. In contrast, use of the same standard curve during nitrogen deprivation (causing the accumulation of neutral lipids) or in the presence of high quantities of bacterial contamination led to significant errors in chlorophyll estimation. The low requirement for equipment (i.e., a simple digital camera, available on smartphones) and widely available standard software for measuring pixel intensity make this method suitable for both laboratory and field-based work, particularly in situations where sample, qualified personnel and/or equipment is limited. By following the methods described here it should be possible to produce a standard curve for chlorophyll analysis in a wide range of testing conditions including different microalga cultures, culture vessel and photographic set up in any particular laboratory.

Dioxins/furans disturb the life cycle of the arbuscular mycorrhizal fungus, Rhizophagus irregularis and chicory root elongation grown under axenic conditions

Arbuscular mycorrhizal fungi (AMF)-assisted phytoremediation is a promising technology for sustainable removal of hazardous pollutants like dioxins/furans (PCDD/F) from the soil. However, little is known on AMF development in the presence of the persistent organic pollutants, PCDD/F. Thus, the present work aims at investigating the impact of increasing PCDD/F concentrations on the development of both partners of the symbiosis: the AMF, Rhizophagus irregularis and the chicory roots, Cichorium intybus L. grown under axenic conditions. Our results show that even R. irregularis spore germination is not affected by PCDD/F, it occurred mainly in linear way. However, root colonization, extra-radical hyphal elongation and sporulation are reduced by 40, 30, and 75%, respectively, at the highest PCDD/F concentration. In addition, while non-mycorrhizal root growth (length and dry weight) decreased at the highest PCDD/F concentration, no negative effect was observed on the dry weight of mycorrhizal roots. In conclusion, our findings show that although high PCDD/F concentrations disturb the main stages of R. irregularis development, the AMF remains able to fulfill its life cycle in the presence of PCDD/F. Moreover, the mycorrhizal inoculation protects the host plant against PCDD/F phytotoxicity. AMF could thus represent an interesting amendment option to assist phytoremediation of PCDD/F contaminated soils.

Expanding the molecular toolbox for Zygnematophyceae – transient genetic transformation of the desmid Micrasterias radians var. evoluta

ABSTRACT In the past, various species of the unicellular algal genus Micrasterias (Zygnematophyceae) have been used for research in the fields of cell biology and physiology. Relatively large cell size, highly differentiated cell shape and a remarkable evolutionary position make Micrasterias interesting, especially for cytomorphogenetic studies. However, within this genus a model organism enabling molecular research has not yet been established. Micrasterias radians var. evoluta (W.B.Turner) Krieger allows easy culturing under axenic conditions and performs its whole life cycle in vitro thus fulfilling two important prerequisites for a model organism. In this work resources allowing transient expression of transgenes were developed. First, axenic lines were obtained by the treatment of zygospores with a cocktail of antibiotics followed by germination and regeneration. In order to allow transgene expression we isolated the 5′ -flanking region of the M. radians var. evoluta Actin1 gene (MrACT1) and fused it to the green fluorescent protein (GFP). A higher promoter activity compared with various heterologous promoters regarding GFP expression was observed. Transient transgene expression was achieved by polyethylene glycol (PEG)-mediated protoplast transformation, yielding a rate of 3.9% transformed cells per surviving protoplasts. Transgene expression was also achieved by particle bombardment of vegetative cells. Employing the established protocol, a Lifeact-GFP fusion protein for labelling F-actin was expressed, allowing visualization of the actin cytoskeleton in M. radians var. evoluta.

Maize growth and yield promoting endophytes isolated into a legume root nodule by a cross-over approach

Rhizobia can colonize the roots of grasses in the Brazilian semiarid region; however, studies to isolate these bacteria for growth promotion of maize via unconventional isolation approaches have not been conducted. This study aimed to isolate, characterize (biochemically and molecularly) and assess the potential of cowpea nodulating/maize associated rhizobia for plant growth promotion. Extracts of surface-disinfected maize root were inoculated onto cowpea seeds in axenic conditions. Rhizobia were isolated and classified by sequencing of the 16 S rRNA and nodC genes, and recA and atpD were specifically assayed for Bradyrhizobium. The in vitro ability of rhizobia to produce auxin and siderophores, solubilize calcium phosphate, exhibit antagonism against the growth of the pathogen Fusarium verticillioides, and undergo endophytic colonization were evaluated. The ability of rhizobia to promote the growth of both potted cowpea and maize and to promote the growth and yield of maize under field conditions was also evaluated. Thirteen isolates were obtained, including six Rhizobium, four Agrobacterium, and three Bradyrhizobium isolates, which exhibited high genetic variability and low similarity with the type strains. All bacteria showed at least one plant growth promotion mechanism. Six bacteria stood out regarding the promotion of maize growth, and one bacterium showed the best performance in cowpea. The results of the field assay with maize indicated that Bradyrhizobium sp. ESA 114 and Rhizobium sp. ESA 116 induced the same maize grain yields as those observed in plants inoculated with the commercial strain of Azospirillum brasilense Ab-V5. Maize harbors a community of putative facultative endophytic rhizobia that are genetically diverse and have the potential to be used in inoculant production.

Rhizobium rhizogenes-mediated transformation of Rhodiola rosea leaf explants

ABSTRACT Rhodiola rosea L. is an endangered medicinal plant distributed in mountains and in high latitude regions. For its conservation, sustainable methods for the obtaining of its bioactive compounds must be developed. This work hypothesized that leaf, stem and rhizome explants of R. rosea from different geographical origins respond differently to inoculation with Rhizobium rhizogenes agropine strain ATCC43057. The objective was to generate R. rosea hairy roots (HRs) containing rol-genes. These HRs could be cultivated under axenic conditions for the extraction of the medical compounds rosavinoids and salidroside. Hereby, production of bioactive compounds could be improved per plant biomass. Thirteen R. rosea accessions of Alpine, Scandinavian, Nordic Gene Bank (NGB) and Russian origins were compared for their explant survival and HR formation. Significant differences were observed among plants from different geographical origins, where the NGB leaf explants exhibited up to 70% of HR formation and the Russian accessions did not exhibit HRs at all. Moreover, maintaining explants in light conditions after R. rhizogenes inoculation resulted in higher explant survival and HR formation rate (35%) when compared with explants kept in darkness (9%). Taken together, an efficient HR formation in roseroot by inoculation of R. rhizogenes following culturing in light was reported as a required step. This work represents a stepping-stone to R. rosea HR cultivation in bioreactors as well as regenerating whole plants. Hence, it is initiating a novel route towards high-throughput production of bioactive compounds as well preventing depletion of natural roseroot populations.

Cryopreservation of Chlorella vulgaris Using Different Cryoprotectant Agents

The objective of this study is to evaluate the cryopreservation of Chlorella vulgaris using different substances. The C. vulgaris was cultured in medium MH, the microalgae were grown under a 12:12 h light: dark photoperiod, illumination with 40 W led lamps, and a controlled temperature of 28±1 ºC. C.vulgaris was cultured for 15 days and the culture was aliquoted into 3-mL cryogenic tubes. The 3-mL aliquot was centrifuged, the supernatant was discarded, and the pellet was resuspended in different cryoprotectant solutions, T1-PVS1, T2-PVS2, T3-PVS2 (1% phloroglucinol), T4 (2 M glycerol), and T5 (5% methanol). The samples were rapidly frozen in liquid nitrogen (-196 °C) and analyzed after 15, 150, and 300 days of freezing. Cell viability was determined in cultures grown for 20 days. The only effective treatment was T5, which promoted the growth of thawed cultures in both solid and liquid media. After 15 days of freezing in liquid nitrogen and 20 days of culture growth, the number of viable and nonviable cells was 3.42±0.72 × 107 and 0.06±0.009 × 107, respectively, and viability was 98.2%. Similar values were obtained after 150 and 300 days of freezing: 2.17±0.15 × 107 and 2.35±0.18 × 107 viable cells, 0.05±0.02 × 107 and 0.10±0.02 × 107 nonviable cells, and viability of 97.6% and 95.8%, respectively. The cryopreservation protocol for microalgae C. vulgaris using 5% methanol was effective; therefore, it is possible to maintain this strain under axenic conditions in liquid nitrogen for long periods.

Siderophores in plant root tissue: Tagetes patula nana colonized by the arbuscular mycorrhizal fungus Gigaspora margarita.

More than 70% of vascular plant species live in symbiosis with arbuscular mycorrhizal (AM) fungi. In addition to other effects this symbiosis is known for its significance for plant nutrition including iron. Fungal iron mobilization from soil is commonly dependent on siderophores. This study reports on a search for such iron-chelators in root tissue of Tagetes patula nana var. plena colonized by Gigaspora margarita. The AM colonized plants and uninoculated controls were grown under strictly axenic conditions. HPLC analyses of aqueous extracts from plant roots have provided clear evidence for the presence of a rhizoferrin type siderophore, named glomuferrin, in root tissue of mycorrhizal seedlings. Results from HPLC analytical work are seconded by molecular biological data: A BLASTp search revealed that the AM fungal species Gigaspora rosea, Rhizophagus irregularis (formerly Glomus intraradices), Glomus cerebriformis and Diversispora epigea encode a non-ribosomal peptide synthetase (NRPS)-independent siderophore synthase (NIS), which is homologous to the rhizoferrin synthetase of Rhizopus delemar. Thus this study indicates that the biosynthesis of rhizoferrin type siderophores such as glomuferrin (= bis-imidorhizoferrin) may be widespread in the AM symbiosis.

Production of algal biomass for its biochemical profile using slaughterhouse wastewater for treatment under axenic conditions

Slaughterhouse produce large amount of wastewater, containing high pollutant load in terms of protein, fats and meat pieces, might lead to source of non-point contamination. Various concentrations (25%, 50%, 75%, and 100%) of slaughterhouse wastewater were used to increase the algal biomass production, pollutants removal and biochemical profile analysis under controlled conditions of C. pyrenoidosa. Results showed that the maximum biomass yield 430 mg L−1 was achieved at 50% concentration of wastewater to other concentration of wastewater. Direct relation was observed in between pollution load and nutrient load of SHWW with biochemical profile of C. pyrenoidosa. The COD/BOD ratio (1.9) was found to be significant on the scale of degradability by algal biomass. Sufficient nutrient removal efficiencies (23–42%, 18–48%) and pollutant load efficiencies (17–31%, 7–29%) were observed. Findings showed that slaughterhouse wastewater is rich in nutrients, which can be utilized for algal biomass production and wastewater remediation for future endeavors.

Comparative Ergot Alkaloid Elaboration by Selected Plectenchymatic Mycelia of Claviceps purpurea through Sequential Cycles of Axenic Culture and Plant Parasitism.

Ergot alkaloids have an established place in plant pathology and toxicology. As pharmaceuticals, their sourcing is via natural or managed agricultural occurrence of sclerotia of Claviceps purpurea (Fr.) Tul. or through industrial fermentation processes with other Claviceps. The key factor for biosynthesis is differentiation of a particular mycelial anatomy. Previous study of these fungi from two disparate English grass genera, Spartina and Phragmites, has shown that only mycelia expressing a plectenchymatic sclerotium-like anatomy in specific axenic culture conditions elaborated ergot alkaloids, and then only as far as lysergic acid. The present report describes sequential cycles of axenic and parasitic cultivation for wild isolates from Dactylis and Alopecurus with intervention of a single ascospore step. This confirms the homozygous character of C. purpurea and defines several potential experimental axenic and parasitic conditions within the species for comparing genomic aspects of partial or full biosynthesis of cyclic tri-peptide alkaloids. Whereas Alopecurus ergot isolates readily parasitized rye, use of Dactylis isolates as inoculum for rye ovaries failed to cause the usual sphacelial fructification but supported growth of exceptionally thin sclerotia, sometimes two in a floret, with low alkaloid content attributed to reduced medullary component. However, after two cycles of axenic and rye-parasitic cultivation, and consistent re-selection of the plectenchymatic character in axenic mycelia, typical growth of ergot sclerotia occurred on rye. Caution thus seems necessary in tests for putative host specificity in any taxonomic realignments within the classical concept of C. purpurea. A Dactylis ergot isolate was also uniquely shown to parasitise the plumule of germinating rye seeds confirming the susceptibility of apical tissues. A key biosynthetic feature of a mycelial glyceride oil, rich in ricinoleic acid, as a prelude to axenic and parasitic formation of ergot alkaloids by C. purpurea is emphasised.

Genetically diverse lentil- and faba bean-nodulating rhizobia are present in soils across Central and Southern Ethiopia.

In total 196 bacterial isolates were obtained from root nodules of lentil (Lens culinaris) and faba bean (Vicia faba) grown on soil samples collected from 10 different sites in central and southern parts of Ethiopia. All isolates were identified as members of the genus Rhizobium by using recA gene sequence analysis. In the recA phylogenetic tree 195 rhizobial strains were classified into nine genospecies. The phylogeny of symbiotic genes nodC and nifH revealed five and six distinct groups respectively, largely dominated by symbiovar viciae. A multivariate analysis showed that environmental variables of the sampling sites considered in this study had more effect on the distribution and composition of the genospecies than the host legumes of the strains. Twenty representative strains, selected based on their isolation site, host plant and nodC group, were able to nodulate all lentil, faba bean, field pea (Pisum abyssinicum) and grass pea (Lathyrus sativus) plants in a greenhouse test in axenic conditions. The majority of the rhizobial strains were effective nitrogen-fixing symbionts for all tested legumes, indicating their potential to serve as broad host-range inoculants in agriculture. The present work suggests the presence of taxonomically and symbiotically diverse rhizobial species for legumes in the Viciae tribe in Ethiopia.

Macroalgal-bacterial interactions: identification and role of thallusin in morphogenesis of the seaweed Ulva (Chlorophyta).

Macroalgal microbiomes have core functions related to biofilm formation, growth, and morphogenesis of seaweeds. In particular, the growth and development of the sea lettuce Ulva spp. (Chlorophyta) depend on bacteria releasing morphogenetic compounds. Under axenic conditions, the macroalga Ulva mutabilis develops a callus-like phenotype with cell wall protrusions. However, co-culturing with Roseovarius sp. (MS2) and Maribacter sp. (MS6), which produce various stimulatory chemical mediators, completely recovers morphogenesis. This ecological reconstruction forms a tripartite community which can be further studied for its role in cross-kingdom interactions. Hence, our study sought to identify algal growth- and morphogenesis-promoting factors (AGMPFs) capable of phenocopying the activity of Maribacter spp. We performed bioassay-guided solid-phase extraction in water samples collected from U. mutabilis aquaculture systems. We uncovered novel ecophysiological functions of thallusin, a sesquiterpenoid morphogen, identified for the first time in algal aquaculture. Thallusin, released by Maribacter sp., induced rhizoid and cell wall formation at a concentration of 11 pmol l−1. We demonstrated that gametes acquired the iron complex of thallusin, thereby linking morphogenetic processes with intracellular iron homeostasis. Understanding macroalgae–bacteria interactions permits further elucidation of the evolution of multicellularity and cellular differentiation, and development of new applications in microbiome-mediated aquaculture systems.

Evaluation of DNA Methylation Changes by CRED–RA Analysis Following Prednisone Treatment of Endophyte, Fusarium oxysporum

Endophytes that represent a sub-set of plant resident microbes are a reservoir of bioactive metabolites. Many of the secondary metabolite biosynthetic gene clusters of endophytes are silent under axenic culture conditions. Epigenetic reprogramming of such cryptic pathways is possible by use of small molecule modulators like prednisone. Methylation changes induced by prednisone, a hypomethylating epigenetic modulator were studied in endophytic Fusarium oxysporum. CRED-RA analysis following exposure to non-cytotoxic dose (300µM) revealed prednisone as effective in inducing non-methylation and semi-methylation pattern while inhibiting full-methylation of the genome. Effectiveness of prednisone as a DNA methyl transferase inhibitor can be explored in future to study alterations in secondary metabolite gene expression profile in endophytic F. oxysporum.

Cryptic Chemical Communication: Secondary Metabolic Responses Revealed by Microbial Co-culture.

Microbial secondary metabolites (SMs) have long been viewed as a significant source of novel pharmaceutical and agrochemical molecules. With the increasing availability of genomic data, numerous biosynthetic gene clusters (BGCs) have been discovered. Despite the presence of tens of thousands of BGCs that can theoretically produce extremely diverse SMs, many gene clusters remain in a silent state under axenic culture conditions. Co-culture is a promising research approach as it stimulates the expression of cryptic BGCs to produce novel metabolites and also mimics natural interspecies interactions in a laboratory environment. In recent years, the roles of SMs in microbial communication have caught the attention of researchers and our understanding of microbes and their production of remarkable SMs has improved. SMs may be extensively involved in a variety of communication events among microorganisms. We herein summarize certain representative findings in the field of chemical communication involving SMs in co-culture systems.

Endophytic Fungi of Native Salvia abrotanoides Plants Reveal High Taxonomic Diversity and Unique Profiles of Secondary Metabolites.

Endophytic fungi are often embedded in their host’s metabolic networks, which can result in alterations of metabolite production and higher amounts of active compounds in medicinal plants. This study reports the occurrence, diversity, and secondary metabolite profiles of endophytic fungi isolated from Salvia abrotanoides plants obtained from three geographically distinct sites in Iran. A total of 56 endophytic fungi were isolated from roots and leaves of S. abrotanoides; site-specificity and root-dominated colonization was found to be a general characteristic of the endophytes. Based on molecular identification, the endophytic fungi were classified into 15 genera. Mycelial extracts of these isolates were subjected to high-resolution mass spectrometry analyses and revealed a broad spectrum of secondary metabolites. Our results demonstrated that Penicillium canescens, P. murcianum, Paraphoma radicina, and Coniolariella hispanica are producers of cryptotanshinone, which is a main bioactive compound of S. abrotanoides. Moreover, it was shown that it can be produced independent of the host plant. The effect of exogenous gibberellin on S. abrotanoides and endophytic fungi was shown to have a positive effect on increasing the cryptotanshinone production in the plant as well as in endophytic fungi cultivated under axenic conditions. Our findings provide further evidence that endophytic fungi play an important role in the production plant bioactive metabolites. Moreover, they provide an exploitable basis to increase cryptotanshinone production in S. abrotanoides.

Effects of plant growth regulators on in vitro seed germination and seedling development of Enhalus acoroides, a tropical seagrass

The effects of plant growth regulators (PGRs) on seed germination and seedling development of Enhalus acoroides were investigated under axenic conditions. Seeds were cultured in sterile liquid medium supplemented with different concentrations of 6-benzyladenine (BA) × α-naphthylacetic acid (NAA) and N-phenyl-N’-(1,2,3-thidiazol-5-yl) urea (TDZ) × 2,4-dichlorophenoxyacetic acid (2,4-D). All seeds fully germinated (100% germination rates) within 1 d on liquid media without PGRs, while germination of seeds in liquid media supplemented with PGRs was delayed with 94–100% germination within 2–7 d. Embryogenic callus was obtained after 2 wk when cultured in liquid medium supplemented with 18 μM BA and 160 μM NAA. The addition of TDZ and 2,4-D inhibited root emergence. The results showed a significant effect of the culture medium on seedling development, but not on seed germination.

Co-inoculation of selected nodule endophytic rhizobacterial strains with Rhizobium tropici promotes plant growth and controls damping off in common bean

Production of common bean (Phaseolus vulgaris) is limited by the occurrence of damping off (rhizoctoniosis), which is caused by the fungus Rhizoctonia solani. However, the co-inoculation of plant growth-promoting rhizobacteria (PGPR) involved in biological control along with diatomic nitrogen (N2)-fixing rhizobia can enhance N nutrition and increase production. In this context, finding microorganisms with synergistic effects that perform these two roles is of fundamental importance to ensure adequate yield levels. The aim of this study was to evaluate the effects of co-inoculation of nodule endophytic strains of the genera Bacillus, Paenibacillus, Burkholderia, and Pseudomonas with Rhizobium tropici CIAT 899, an N2-fixing rhizobial strain, on the biocontrol of damping off and growth promotion in common bean plants. Greenhouse experiments were conducted under axenic conditions using the common bean cultivar Pérola. The first experiment evaluated the potential of the 14 rhizobacterial strains, which were inoculated alone or in combination with CIAT 899, for the control of R. solani. The second experiment evaluated the ability of these 14 rhizobacterial strains to promote plant growth with three manners of N supply: co-inoculation with CIAT 899 at low mineral N supply (5.25 mg N mL−1), low mineral N supply (5.25 mg N mL−1), and high mineral N supply (52.5 mg N mL−1). The use of rhizobacteria combined with rhizobia contributed in a synergistic manner to the promotion of growth and the control of damping off in the common bean. Co-inoculation of the strains UFLA 02-281/03-18 (Pseudomonas sp.), UFLA 02-286 (Bacillus sp.), and UFLA 04-227 (Burkholderia fungorum) together with CIAT 899 effectively controlled damping off. For the common bean, mineral N supply can be replaced by the co-inoculation of CIAT 899 with plant growth-promoting strains UFLA 02-281/02-286/02-290/02-293. Nodule endophytes UFLA 02-281/02-286 are promising for co-inoculation with CIAT 899 in the common bean, promoting synergy with rhizobial inoculation and protection against disease.

The microbiota of Drosophila suzukii influences the larval development of Drosophila melanogaster.

Microorganisms play a central role in the biology of vinegar flies such as Drosophila suzukii and Drosophila melanogaster: serving as a food source to both adults and larvae, and influencing a range of traits including nutrition, behavior, and development. The niches utilized by the fly species partially overlap, as do the microbiota that sustain them, and interactions among these players may drive the development of crop diseases. To learn more about how the microbiota of one species may affect the other, we isolated and identified microbes from field-caught D. suzukii, and then characterized their effects on D. melanogaster larval development time in the laboratory. We found that the D. suzukii microbiota consistently included both yeasts and bacteria. It was dominated by yeasts of the genus Hanseniaspora, and bacteria from the families Acetobacteraceae and Enterobacteriaceae. Raising D. melanogaster under gnotobiotic conditions with each microbial isolate individually, we found that some bacteria promoted larval development relative to axenic conditions, but most did not have a significant effect. In contrast, nearly all the yeasts tested significantly accelerated larval development. The one exception was Starmerella bacillaris, which had the opposite effect: significantly slowing larval developmental rate. We investigated the basis for this effect by examining whether S. bacillaris cells could sustain larval growth, and measuring the survival of S. bacillaris and other yeasts in the larval gut. Our results suggest S. bacillaris is not digested by D. melanogaster and therefore cannot serve as a source of nutrition. These findings have interesting implications for possible interactions between the two Drosophilia species and their microbiota in nature. Overall, we found that microbes isolated from D. suzukii promote D. melanogaster larval development, which is consistent with the model that infestation of fruit by D. suzukii can open up habitat for D. melanogaster. We propose that the microbiome is an important dimension of the ecological interactions between Drosophila species.

Effects of the fungus Pestalotiopsis maculans (Ascomycota: Amphisphaeriales) on the gametophytic development of the fern Lygodium venustum (Lygodiaceae)

Lygodium sp. is a climbing fern that occurs in open forests, settling frequently in areas with disturbed vegetation. In Argentina, Lygodium venustum inhabits in the protected area "Moconá" Provincial Park, in few and small populations. In-vitro cultivation of spores is a strategy for ex-situ conservation of species with reduced or threatened populations. Usually, fern spores need to be surface sterilized before sowing, to avoid the development of unwanted microorganisms. As part of our project focused on the conservation of native fern species in Argentina, the aim of this work was to evaluate a propagation protocol for L. venustum through in-vitro cultivation of spores, employing a standard procedure of disinfection, to investigate its effect on spore germination and the subsequent gametophyte as well as its response by the inoculation with a fungus. A batch of spores was previously treated with 10% (v/v) solution of NaOCl during 2 min and then sown in Dyer agar medium supplemented with 1% Nystatin (T1). The petri dishes were incubated in a growth chamber under white illumination (28μmol m-2 s-1) with a photoperiod of 12 hr and a temperature of 22±2°C. Another set of spores without previous disinfection was sown on Dyer agar medium and under the same conditions and used as a control (T0). Spore germination and gametophyte and sporophyte development were monitored through periodical observations under stereoscopic and light microscopes. As during cultures of non-disinfected spores a fungus developed in association with L. venustum gametophytes, its conidia were morphologically characterized as well as those obtained from the black spots present on the sporophyte leaves. A conidial suspension was applied to 20 days-old gametophytes obtained in axenic conditions to evaluate the appearance of any symptom. In both treatments (T1 and T0), spores germinated at 7 days after sowing. The pattern of spore germination followed the Anemia-type, unlike the Vittaria-type found by other researchers. Untreated spores showed a higher mean percentage of germination than those treated (86 and 72 % in 30 days, respectively). In T0 gametophytes grew in association with a fungus, whose conidia had the morphological characteristics of Pestalotiopsis maculans, as occurred with the conidia obtained from the black spots of L. venustum leaves. The association of P. maculans with L. venustum is reported here for the first time. The gametophytes developed in T0 and those inoculated with P. maculans became necrotic and died before producing gametangia, suggesting a pathogenic role of the fungus. The elevated percentage of spore germination registered in T0 could also indicate some effect of P. maculans or its metabolites in the promotion of spore germination. Gametophytes developed in T1 became bisexual or unisexual male, which points to the presence of some antheridiogen that induces the production of antheridia on smaller or slower-growing gametophytes. The sporophytes arose after 4 months of spore sown in T1, pointing out the methodology employed here suitable for the ex-situ conservation of L. venustum.

Sensitivity of live microalgal aquaculture feed to singlet oxygen-based photodynamic therapy

Highly nutritional microalgal species are extensively used in aquaculture as live feedstock. Due to difficulties in maintaining microalgae in axenic conditions, they represent a potential pathogen carrier and disease vector in aquaculture ponds. Photodynamic therapy (PDT) via singlet oxygen (1O2) production is a promising sterilization technique in aquaculture. Here, we report on the sensitivity of aquaculture-relevant microalgae towards 1O2 generated by the cationic photosensitizer TMPyP. Possible PDT sterilization protocols of contaminated microalgae cultures were evaluated using the luminescent bacterium Vibrio campbellii ISO7 as a model aquaculture pathogen. Species-specific sensitivity of microalgae to TMPyP-mediated PDT was demonstrated and found to be strongly influenced by the nature and architecture of their respective cell wall. While cytotoxicity was not evident against Nannochloropsis oculata, toxicity of 1O2 was dose-, time- and light activation-dependent against Tisochrysis lutea, Tetraselmis chui, Chaetoceros muelleri and Picochlorum atomus. The 1O2-resilient N. oculata was sterilized when incubated under light in the presence of V. campbellii ISO7 (up to 107 CFU mL−1) and 20 μM TMPyP; hence, TMPyP-based PDT sterilization of N. oculata could be suitable for aquaculture hatcheries. This study also suggests that PDT using cationic porphyrins such as TMPyP holds potential as an algicidal treatment in aquaria and aquaculture systems (but more research using opportunistic and toxic species is needed for confirmation).