Aposporic development of gametophyte in Sematophyllum subpinnatum (Brid.) E. Britton (Sematophyllaceae) from capsule wall

In the present study, an axenic culture of Sematophyllum subpinnatum (Brid.) E. Britton was attempted from spores. However, spores failed to germinate, but protonemata were seen arising from the diploid capsule wall cells by apospory. Few cells of the capsule wall turned green, and the protoplast divided, resulting in the extension of the protoplast as a germ tube, which developed into protonemata. Protonemata were less spreading, and adult gametophytes developed from these protonemata. The aposporic plants so developed were transferred to soil, and they showed normal growth but with decreased branching. No sex organs and sporophytes were seen. This is the first report of aposporic development of S. subpinnatum from the capsule wall.

De novo synthesized polyunsaturated fatty acids operate as both host immunomodulators and nutrients for Mycobacterium tuberculosis

Successful control of Mycobacterium tuberculosis (Mtb) infection by macrophages relies on immunometabolic reprogramming, where the role of fatty acids (FAs) remains poorly understood. Recent studies unraveled Mtb's capacity to acquire saturated and monounsaturated FAs via the Mce1 importer. However upon activation, macrophages produce polyunsaturated FAs (PUFAs), mammal-specific FAs mediating the generation of immunomodulatory eicosanoids. Here, we asked how Mtb modulates de novo synthesis of PUFAs in primary mouse macrophages and whether this benefits host or pathogen. Quantitative lipidomics revealed that Mtb infection selectively activates the biosynthesis of w6 PUFAs upstream of the eicosanoid precursor arachidonic acid (AA), via transcriptional activation of Fads2. Inhibiting FADS2 in infected macrophages impaired their inflammatory and antimicrobial responses but had no effect on Mtb growth in mice. Using a click-chemistry approach, we found that Mtb efficiently imports w6 PUFAs via Mce1 in axenic culture, including AA. Further, Mtb preferentially internalized AA over all other FAs within infected macrophages, by mechanisms partially depending on Mce1 and supporting intracellular persistence. Notably, IFNγ repressed de novo synthesis of AA by infected mouse macrophages and restricted AA import by intracellular Mtb. Together, these findings identify AA as a major FA substrate for intracellular Mtb, whose mobilization by innate immune responses is opportunistically hijacked by the pathogen and downregulated by IFNγ.

Transcriptional profiles of a foliar fungal endophyte (Pestalotiopsis, Ascomycota) and its endohyphal bacterium (Luteibacter, Gammaproteobacteria) in co-culture support sulfur exchange and growth regulation

Symbiosis with bacteria is widespread among eukaryotes, including fungi. Bacteria that live within fungal mycelia (endohyphal bacteria) occur in many plant-associated fungi, including diverse Mucoromycota and Dikarya. Pestalotiopsis sp. 9143 is a filamentous ascomycete isolated originally as a foliar endophyte of Platycladus orientalis (Cupressaceae). It is infected naturally with the endohyphal bacterium Luteibacter sp. 9143, which influences auxin and enzyme production by its fungal host. Previous studies have used transcriptomics to examine similar symbioses between endohyphal bacteria and root-associated fungi such as arbuscular mycorrhizal fungi and plant pathogens. However, currently there are no gene expression studies of endohyphal bacteria of Ascomycota, the most species-rich fungal phylum. We developed methods for assessing gene expression by Pestalotiopsis sp. and Luteibacter sp. when grown in co-culture and when each was grown axenically. Our assays showed that the density of Luteibacter sp. in co-culture was greater than in axenic culture, but the opposite was true for the Pestalotiopsis sp. Dual RNA-seq data demonstrate that growing in co-culture modulates developmental and metabolic processes in both the fungus and bacterium, potentially through changes in the balance of organic sulfur via methionine acquisition. Our analyses also suggest an unexpected, potential role of the bacterial type VI secretion system in symbiosis establishment, expanding current understanding of the scope and dynamics of fungal-bacterial symbioses.

Isolation and Culturing Axenic Microalgae: Mini–Review

Microalgae have several applications in nutraceuticals, pharmaceuticals, cosmetics, biofuel production, and bioremediation, among other fields. Isolation and purification are extremely important for obtaining axenic cultures of microalgae from different environments and crucial for their biotechnological applications, but it is not an easy task. In view of the above, it is fundamental to know the classical and advanced techniques and examples of how scientists from around the globe have applied such methods to isolate several genera and the impact of each step on successful algal purification. This review provides a brief and simple explanation of the methodology for sampling, growth, obtention of unialgal, and posterior axenic culture, which will facilitate the development of novel microalgae-related discoveries and applications for new researchers.

Effect of axenic culture and NAA in Vitro on masoyi (Cryptocarya massoy (Oken) Kosterm) seeds regeneration

The high valuable endemic commodities in Papua, Masoyi’s (Cryptocarya massoy) population facing great threat due to unsustainable harvest system. Generative propagation faces significant challenges due to seed characteristics and habitat conditions. Controlled conditions and the role of hormones have an important effect on generative growth. This study aimed to determine the influence of axenic culture with sterilization treatments Isothiazolone Biocide (IB) and 1-Naphtaleaneacetic Acid (NAA) in Murashige and Skoog (MS) medium on seed regeneration and to observe the development of seedlings at the acclimatization stage. The tissue culture method was used. The highest percentage of axenic cultures (57%) was obtained with 5% of BI. The germination rate of masoyi seeds was achieved by 100%. Furthermore, it showed varied responses depending upon concentrations of NAA, the addition of 1 ml l−1 NAA in MS medium is recommended. Acclimatization has been successfully carried out in the greenhouse (67% survival rate) and excellent seedlings growth at nursery (52.35 + 0.6 cm in height after one year transferred). The impact of the controlled conditions and the addition of NAA to axenic cultures in vitro increased the germination of masoyi seeds. Axenic culture and hormones were also important requirements for mass propagation of masoyi by tissue culture.

UPLC-QTOF-MS-Based Untargeted Study of Metabolites Secreted by Sclerotinia Sclerotiorum Pathogen Under Axenic Condition

The stem rot disease has emerged globally as a major disease threat to the productivity and seed quality of oilseed Brassica. The generalist causal pathogen Sclerotinia sclerotiorum (Lib.) de Bary shows large variability in their aggressiveness and pathogenicity. Revealing the metabolic profile and signaling components of the pathogen in host-pathogen interaction are fundamental in understanding host resistance to the disease. In this study, the metabolites released by the pathogenic strains of S. sclerotiorum under the axenic culture have been identified using the untargeted high-resolution UPLC-QTOF-ESI-MS/MS. The analysis of the ethyl acetate extracts of the S. sclerotiorum culture revealed ten major secondary metabolites namely, sclerin, sclerotinin-B, sclerone, melanin, bostrycoidin, botcinin-D, botcinin-A, gliovirin, scleramide, and botcinic acid. The later six metabolites are being reported for the first time in the culture extract of the S. sclerotiorum pathogen. The phylogenetic analysis based on the overlapping and unique informative peaks in the chromatograms, the six S. sclerotiorum strains were grouped into three major clads. The clustering based on metabolic profiles does not substantiate the diversity based on morphology or virulence differences on the host. The findings of the study signified the metabolites secreted under the axenic conditions are varies based on their growth and developmental stages and may not necessarily be the determining factors for their differential aggressiveness and virulence over the host.

Characterization of Variovorax Strain C6d Isolated from the Algae-bacteria Consortia

The rhizosphere microorganisms can form beneficial, pathogenic, or neutral relationships. These relationships can promote plant growth and productivity. Among them, a number of Variovorax isolates from the rhizosphere were isolated. Bacteria Variovorax strain C6d (AB552893) was isolated from the non axenic culture of Chlorella spp., C6. The cell was Gram-negative, motile, non-spore-forming, short and rod-shaped (0.5-1.0x1.5-2.0µm). Colonies were in white colour after 7 days on 10-fold diluted Nutrient Broth. The strain was able to tolerate NaCL to 1.0% but not to 4.0% of NaCl. It grew quite well at temperatures ranging from 10°C to 37°C, yet did not show any growth at 4°C and 42°C. The dominant isoprenoid quinone was ubiquinone 8 (Q8). The major fatty acid composition of this strain was summed feature 3, 16:0 and 18:1:w7. The DNA G+C content of strain C6d was 70.4 mol%.

Accumulation of 4-Deoxy-7-Hydroxytrichothecenes, but Not 4,7-Dihydroxytrichothecenes, in Axenic Culture of a Transgenic Nivalenol Chemotype Expressing the NX-type FgTri1 Gene

Fusarium graminearum species complex produces type B trichothecenes oxygenated at C-7. In axenic liquid culture, F. graminearum mainly accumulates one of the three types of trichothecenes, namely 3-acetyldeoxyinvalenol, 15-acetyldeoxyinvalenol, or mixtures of 4,15-diacetylnivalenol/4-acetylnivalenol, depending on each strain’s genetic background. The acetyl groups of these trichothecenes are slowly deacetylated to give deoxynivalenol (DON) or nivalenol (NIV) on solid medium culture. Due to the evolution of F. graminearum FgTri1, encoding a cytochrome P450 monooxygenase responsible for hydroxylation at both C-7 and C-8, new derivatives of DON, designated as NX-type trichothecenes, have recently emerged. To assess the risks of emergence of new NX-type trichothecenes, we examined the effects of replacing FgTri1 in the three chemotypes with FgTri1_NX chemotype, which encodes a cytochrome P450 monooxygenase that can only hydroxylate C-7 of trichothecenes. Similar to the transgenic DON chemotypes, the transgenic NIV chemotype strain accumulated NX-type 4-deoxytrichothecenes in axenic liquid culture. C-4 oxygenated trichothecenes were marginal, despite the presence of a functional FgTri13 encoding a C-4 hydroxylase. At present, outcrossing of the currently occurring NX chemotype with NIV chemotype strains of F. graminearum in the natural environment likely will not yield a new strain that produces a C-4 oxygenated NX-type trichothecene.

Comparative Genome Analysis of ‘Candidatus Phytoplasma luffae’ Reveals the Influential Roles of Potential Mobile Units in Phytoplasma Evolution

Phytoplasmas are insect-transmitted plant pathogens that cause substantial losses in agriculture. In addition to economic impact, phytoplasmas induce distinct disease symptoms in infected plants, thus attracting attention for research on molecular plant-microbe interactions and plant developmental processes. Due to the difficulty of establishing an axenic culture of these bacteria, culture-independent genome characterization is a crucial tool for phytoplasma research. However, phytoplasma genomes have strong nucleotide composition biases and are repetitive, which make it challenging to produce complete assemblies. In this study, we utilized Illumina and Oxford Nanopore sequencing technologies to obtain the complete genome sequence of ‘Candidatus Phytoplasma luffae’ strain NCHU2019 that is associated with witches’ broom disease of loofah (Luffa aegyptiaca) in Taiwan. The fully assembled circular chromosome is 769 kb in size and is the first representative genome sequence of group 16SrVIII phytoplasmas. Comparative analysis with other phytoplasmas revealed that NCHU2019 has an exceptionally repetitive genome, possessing a pair of 75 kb repeats and at least 13 potential mobile units (PMUs) that account for ∼25% of its chromosome. This level of genome repetitiveness is exceptional for bacteria, particularly among obligate pathogens with reduced genomes. Our genus-level analysis of PMUs demonstrated that these phytoplasma-specific mobile genetic elements can be classified into three major types that differ in gene organization and phylogenetic distribution. Notably, PMU abundance explains nearly 80% of the variance in phytoplasma genome sizes, a finding that provides a quantitative estimate for the importance of PMUs in phytoplasma genome variability. Finally, our investigation found that in addition to horizontal gene transfer, PMUs also contribute to intra-genomic duplications of effector genes, which may provide redundancy for neofunctionalization or subfunctionalization. Taken together, this work improves the taxon sampling for phytoplasma genome research and provides novel information regarding the roles of mobile genetic elements in phytoplasma evolution.

Evidence for Reversal of Immunosuppression by Homeopathic Medicine to a Predominant Th1-type Immune Response in BALB/c Mice Infected with Leishmania donovani

Background Visceral leishmaniasis (VL) is a neglected tropical disease that is fatal if treatment is not given. The available chemotherapeutic options are unsatisfactory, and so complementary therapies like homeopathy might be a promising approach. Methods A nosode from a pure axenic culture of Leishmania donovani was prepared and screened for its anti-leishmanial potential both in an in-vitro and an in-vivo experimental approach. Results Leishmania donovani amastigote promastigote nosode (LdAPN 30C) exhibited significant anti-leishmanial activity against the promastigote forms of Leishmania donovani and was found to be safe. A study conducted on VL-infected mice revealed that LdAPN 30C resolved the disease by modulating the host immune response toward the Th1 type through upregulating the pro-inflammatory cytokines (IFN-γ and IL-17) and inducing nitric oxide (NO) levels in the infected macrophages. The hepatic parasite load was also found to be significantly decreased. The nosode was found to be safe, as no histological alterations in the liver or kidney were observed in the animals treated with the LdAPN 30C. Conclusion This is the first study in which an axenic culture of Leishmania donovani has been used for the preparation of a homeopathic medication. The study highlights the anti-leishmanial and immunomodulatory potential of a homeopathic nosode in experimental VL.