Axenic Culture Conditions Research Articles

High-affinity iron uptake is required for optimal Epichloë festucae colonization of Lolium perenne and seed transmission.

Epichloë festucae uses a siderophore-mediated system to acquire iron, which is important to maintain endophyte-grass symbioses. Here we investigate the roles of the alternative iron acquisition system, reductive iron assimilation (RIA), via disruption of the fetC gene, which encodes a multicopper ferroxidase, either alone (i.e., ΔfetC) or in combination with disruption of the gene sidA, which encodes a siderophore biosynthesis enzyme (i.e., ΔfetC/ΔsidA). The phenotypic characteristics of these mutants were compared to ΔsidA and wild-type (WT) strains during growth under axenic culture conditions (in culture) and in symbiosis with the host grass, perennial ryegrass (in planta). Under iron deficiency, the colony growth rate of ΔfetC was slightly slower than that of WT, while the growth of ΔsidA and ΔfetC/ΔsidA mutants was severely suppressed. Siderophore analyses indicated that ΔfetC mutants hyperaccumulate ferriepichloënin A (FEA) at low iron concentrations and ferricrocin and FEA at higher iron concentrations. When compared to WT, all mutant strains displayed hyperbranching hyphal structures and a reduced ratio of Epichloë DNA to total DNA in planta. Furthermore, host colonization and vertical transmission through infection of the host seed were significantly reduced in the ΔfetC/ΔsidA mutants, confirming that high-affinity iron uptake is a critical process for Epichloë transmission. Thus, RIA and siderophore iron uptake are complementary systems required for the maintenance of iron metabolism, fungal growth, and symbiosis between E. festucae and perennial ryegrass.

Insights Into Manganese Solubilizing Bacillus spp. for Improving Plant Growth and Manganese Uptake in Maize.

Manganese (Mn) is an essential micronutrient for plant growth that is involved in the structure of photosynthetic proteins and enzymes. Mn deficiency is widespread mainly in dry, calcareous, and sandy soil, which leads to a significant decrease in crop yield. Mn-reducing bacteria promote the solubilization of Mn minerals, thus increasing Mn availability in soil. The present study aimed to assess the Mn solubilizing ability and plant growth-promoting potential of Bacillus spp. strains for maize plants with insoluble Mn compounds. Several Mn-solubilizing bacterial (MSB) strains were isolated from the maize rhizosphere using nutrient agar media amended with 50 mM MnO2. These strains were screened based on qualitative and quantitative solubilization of Mn, phosphorus, potassium, and zinc and production of ammonia. The majority of MSB strains were positive for catalase, protease, amylase, and oxidase activity, while more than 60% of tested strains were positive for lipase activity, and the production of indole-3-acetic acid and siderophores. Forty-five percent of the tested strains also showed solubilization of potassium. All the MSB strains were evaluated for their ability to promote plant growth and Mn uptake in the presence of MnO2 under axenic sand culture conditions. The results revealed that inoculation with MSB strains under sand culture significantly improved the growth of maize seedlings except for strains ASH7, ASH10, and ASH12. Comparatively, strains ASH6, ASH11, ASH19, ASH20, and ASH22 demonstrated a better increase in plant growth, fresh and dry biomass, and Mn uptake in roots and shoots than the other strains tested. All of these strains were identified as Bacillus spp. through 16S rRNA partial gene sequencing. Maize inoculation with these selected identified MSB strains also resulted in an increase in maize growth and nutrient uptake in maize roots and shoots under soil culture conditions in the presence of native soil Mn. The current study highlights the importance of MSB strain inoculation which could be a potential bioinoculants to promote plant growth under Mn deficiency.

Growth of ‘Candidatus Liberibacter asiaticus’ in a host-free microbial culture is associated with microbial community composition

'Candidatus Liberibacter asiaticus' ('Ca. L. asiaticus'), the suspected causative agent of citrus greening disease, is one of many phloem-restricted plant pathogens that have not been isolated and grown in an axenic culture. In this study, infected Asian citrus psyllids were used to prepare a host-free source of 'Ca. L. asiaticus'. Host-free mixed microbial cultures of 'Ca. L. asiaticus' were grown in the presence of various antibiotic treatments to alter the composition of the microbial communities. Our hypothesis was that the presence of selected antibiotics would enhance or reduce the presence of 'Ca. L. asiaticus' in a host-free culture composed of a mixed bacterial population through changes in the microbial community structure. We determined how 'Ca. L. asiaticus' growth changed with the various treatments. Treatment with vancomycin (50 μg/mL), streptomycin (0.02 μg/mL), or polymyxin B (4 μg/mL) was associated with an increased abundance of 'Ca. L. asiaticus' of 7.35 ± 0.27, 5.56 ± 0.15, or 4.54 ± 0.83 fold, respectively, compared to untreated mixed microbial cultures, while treatment with 100 μg/mL vancomycin; 0.5, 1, or 2 μg/mL streptomycin; or 0.5 μg/mL of polymyxin B was associated with reduced growth. In addition, the growth of 'Ca. L. asiaticus' was associated with the microbial community composition of the mixed microbial cultures. A positive relationship between the presence of the Pseudomonadaceae family and 'Ca. L. asiaticus' growth was observed, while the presence of 'Ca. L. asiaticus' was below the detection limit in cultures that displayed high abundances of Bacillus cereus. Our findings offer strategies for developing effective axenic culture conditions and suggest that enrichment of the Bacillaceae family could serve as a paratransgenic approach to controlling citrus greening disease.

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.

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.

Ectomycorrhizae formed by three Japanese truffle species (Tuber japonicum, T. longispinosum, and T. himalayense) on indigenous oak and pine species.

Modern truffle cultivation systems started in Europe in the early 1970s and are now successfully used for several European truffles throughout the world. However, systems for indigenous novel truffle species need to be developed in several regions, especially where truffle cultivation has not been attempted so far, such as in Japan. Recently, two new and one known truffle species that are expected to be edible were reported from Japan: Tuber japonicum, T. longispinosum, and T. himalayense. Here, we conducted mycorrhization trials between these three truffle species and four native tree species in Japan (Quercus acutissima, Q. phillyraeoides, Q. serrata, and Pinus densiflora) using spore suspension and trap-plant seedling techniques under axenic pot culture conditions to understand their potential host ranges and mycorrhizal morphologies and to determine whether these inoculation methods are applicable for mycorrhization of Japanese truffles with native host plants. Of the 12 combinations, nine were successful for mycorrhization, including both oak and pine trees. The T. japonicum mycorrhiza was characterized by short, needle-shaped cystidia without septa, whereas the two black truffles, T. longispinosum and T. himalayense, were indistinguishable from each other because they shared the same morphological and anatomical characters such as brownish, long cystidia with right angle ramification. These features were similar to related black truffle species. The results of the present study indicate that the inoculation method used for European truffles can also be applied for mycorrhization between Japanese truffle species and compatible native pine and/or oak hosts in Japan.

ROS regulate differentiation of visceralizing Leishmania species into the virulent amastigote form.

Leishmania virulence and disease development critically depends on the ability of Leishmania promastigotes to infect, differentiate into amastigote forms and replicate inside mammalian host macrophages. Understanding changes associated with amastigote differentiation in axenic culture conditions is key to identifying virulence factors. Here we compared efficiency of the conventional pH-temperature-dependent shift method to induce amastigote differentiation with the recently identified trigger for differentiation mediated by mitochondrial reactive oxygen species (ROS). Using two different visceral leishmaniasis species, L. infantum and. L. donovani, we show that ROS-generating methods such as iron deprivation or exposure to sub-lethal concentrations of H2O2 or menadione are significantly more effective in promoting promastigoteamastigote differentiation than the low pH-high temperature shift, leading to higher survival rates, morphological changes and gene expression patterns characteristic of the amastigote stage. Notably, both H2O2 and menadione-mediated differentiation did not require up-regulation of the mitochondrial electron transport chain (ETC)-associated protein p27, suggesting that treatment with oxidants bypasses the necessity to upregulate mitochondrial activity, a precondition for mROS generation. Our findings confirm that ROS-induced differentiation occurs in multiple Leishmania species, including the medically important visceralizing species, and provide mechanistic rationale for earlier reports demonstrating markedly increased virulence of L. infantum promastigotes pre-treated with oxidative reagents.

D-Allulose, a stereoisomer of d-fructose, extends Caenorhabditis elegans lifespan through a dietary restriction mechanism: A new candidate dietary restriction mimetic

Dietary restriction (DR) is an effective intervention known to increase lifespan in a wide variety of organisms. DR also delays the onset of aging-associated diseases. DR mimetics, compounds that can mimic the effects of DR, have been intensively explored. d-Allulose (d-Alu), the C3-epimer of d-fructose, is a rare sugar that has various health benefits, including anti-hyperglycemia and anti-obesity effects. Here, we report that d-Alu increased the lifespan of Caenorhabditis elegans both under monoxenic and axenic culture conditions. d-Alu did not further extend the lifespan of the long-lived DR model eat-2 mutant, strongly indicating that the effect is related to DR. However, d-Alu did not reduce the food intake of wild-type C. elegans. To explore the mechanisms of the d-Alu longevity effect, we examined the lifespan of d-Alu-treated mutants deficient for nutrient sensing pathway-related genes daf-16, sir-2.1, aak-2, and skn-1. As a result, d-Alu increased the lifespan of the daf-16, sir-2.1, and skn-1 mutants, but not the aak-2 mutant, indicating that the lifespan extension was dependent on the energy sensor, AMP-activated protein kinase (AMPK). d-Alu also enhanced the mRNA expression and enzyme activities of superoxide dismutase (SOD) and catalase. From these findings, we conclude that d-Alu extends lifespan by increasing oxidative stress resistance through a DR mechanism, making it a candidate DR mimetic.

Callus culture and in vitro production of anthraquinone in Gynochthodes umbellata (L.) Razafim. & B. Bremer (Rubiaceae)

Gynochthodes umbellata is a rare medicinal plant used in traditional systems of medicine for curing various ailments. It is a woody climber belonging to the family Rubiaceae. Its over collection from the wild population resulted the rapid depletion of this species. There is an urgent need to conserve this species and also establish an alternative system for the production of bioactive compound in vitro without harming the wild population. The present investigation was carried out to analyse secondary metabolite (anthraquinone) production in callus cultures of G. umbellata. In vitro production of anthraquinone through callus cultures of G. umbellata was standardized using in vitro leaves, derived from the nodal explant cultures maintained in Murashige and Skoog solid medium containing 2mg/l 6-benzylaminopurine and 3% sucrose. Leaves were harvested from the healthy shoots of axenic cultures and culture conditions were optimised for the establishment of callus. In vitro derived leaves (1–2cm2) were inoculated onto Murashige and Skoog medium containing different individual concentrations of 2, 4-Dichlorophenoxyacetic acid (0.5–5mg/l), indole-3-butyric acid (0.5–5mg/l), indole-3-acetic acid (0.5–5mg/l) and 1-naphthaleneacetic acid (0.5–5mg/l). Of the different auxins tried for callus induction, 2, 4-Dichlorophenoxyacetic acid was found to be the most suitable auxin with different concentrations for the induction of friable yellow callus. 1-Naphthaleneacetic acid doesn’t show any callus formation while indole-3-butyric acid and indole-3-acetic acid produced greenish white compact callus. A maximum fresh weight (1.7953±0.61g) and dry weight (0.1149±0.04g) of the friable yellow callus and highest amount of anthraquinone content (18.1875±0.58mg/g dw) were recorded in MS medium supplemented with 1mg/l 2, 4-Dichlorophenoxyacetic acid. From the present study it revealed that the callus culture is an effective method for the production of anthraquinone in vitro in G. umbellata. Anthraquinone production through in vitro leaf segment derived callus culture is a viable system for the production of this pharmacologically and industrially important natural plant pigment.

Fungal endophyte protects Atractylodes lancea from root rot caused by Fusarium oxysporum

Endophytic fungi, which stimulate a variety of defence reactions in host plants without causing visible disease symptoms, have been isolated from almost every plant. However, beneficial interactions between fungal endophytes and pathogens from the same habitat remain largely unknown. An inoculation of Atractylodes lancea plantlets with Gilmaniella sp. AL12 (AL12) prior to infection with Fusarium oxysporum prevented the necrotization of root tissues and plant growth retardation commonly associated with fusarium root rot. Quantification of F. oxysporum infections using real‐time PCR revealed a correlation between root rot symptoms and the relative amount of fungal DNA. Pretreatment with AL12 reduced the accumulation of reactive oxygen species stimulated by F. oxysporum. An in vitro analysis of their interactions under axenic culture conditions showed AL12 could inhibit F. oxysporum growth. Additionally, F. oxysporum infections were shown to decrease salicylic acid (SA) production compared with control plantlets. SA biosynthesis inhibitors, 2‐aminoindan‐2‐phosphonic acid and paclobutrazol, abolished the inhibition of F. oxysporum growth in A. lancea even after inoculation with AL12. The results indicated that the fungal endophyte protected A. lancea not only by direct antibiosis, but also by reversing the F. oxysporum‐mediated suppression of SA production.

Screening of biologically active monosaccharides: growth inhibitory effects of d-allose, d-talose, and l-idose against the nematode Caenorhabditis elegans.

We compared the growth inhibitory effects of all aldohexose stereoisomers against the model animal Caenorhabditis elegans. Among the tested compounds, the rare sugars d-allose (d-All), d-talose (d-Tal), and l-idose (l-Ido) showed considerable growth inhibition under both monoxenic and axenic culture conditions. 6-Deoxy-d-All had no effect on growth, which suggests that C6-phosphorylation by hexokinase is essential for inhibition by d-All.

Establishment of Anthoceros agrestis as a model species for studying the biology of hornworts.

BackgroundPlants colonized terrestrial environments approximately 480 million years ago and have contributed significantly to the diversification of life on Earth. Phylogenetic analyses position a subset of charophyte algae as the sister group to land plants, and distinguish two land plant groups that diverged around 450 million years ago – the bryophytes and the vascular plants. Relationships between liverworts, mosses hornworts and vascular plants have proven difficult to resolve, and as such it is not clear which bryophyte lineage is the sister group to all other land plants and which is the sister to vascular plants. The lack of comparative molecular studies in representatives of all three lineages exacerbates this uncertainty. Such comparisons can be made between mosses and liverworts because representative model organisms are well established in these two bryophyte lineages. To date, however, a model hornwort species has not been available.ResultsHere we report the establishment of Anthoceros agrestis as a model hornwort species for laboratory experiments. Axenic culture conditions for maintenance and vegetative propagation have been determined, and treatments for the induction of sexual reproduction and sporophyte development have been established. In addition, protocols have been developed for the extraction of DNA and RNA that is of a quality suitable for molecular analyses. Analysis of haploid-derived genome sequence data of two A. agrestis isolates revealed single nucleotide polymorphisms at multiple loci, and thus these two strains are suitable starting material for classical genetic and mapping experiments.ConclusionsMethods and resources have been developed to enable A. agrestis to be used as a model species for developmental, molecular, genomic, and genetic studies. This advance provides an unprecedented opportunity to investigate the biology of hornworts.

The endophytic fungus Piriformospora indica protects wheat from fusarium crown rot disease in simulated UK autumn conditions

The root endophytic fungus Piriformospora indica (Sebacinacea) forms mutualistic symbioses with a broad range of host plants, increasing their biomass production and resistance to fungal pathogens. This study evaluated the effect of P. indica on fusarium crown rot disease of wheat, under in vitro and glasshouse conditions. Interaction of P. indica and Fusarium isolates under axenic culture conditions indicated no direct antagonistic activity of P. indica against Fusarium isolates. Seedlings of wheat were inoculated with P. indica and pathogenic Fusarium culmorum or F. graminearum and grown in sterilized soil‐free medium or in a non‐sterilized mix of soil and sand. Fusarium alone reduced emergence and led to visible browning and reduced root growth. Roots of seedlings in pots inoculated with both Fusarium isolates and P. indica were free of visible symptoms; seed emergence and root biomass were equivalent to the uninoculated. DNA was quantified by real‐time polymerase chain reaction (qPCR). The ratio of FusariumDNA to wheat DNA rose rapidly in the plants inoculated with Fusarium alone; isolates and species were not significantly different. Piriformospora indica inoculation reduced the ratio of Fusarium to host DNA in the root systems. The reduction increased with time. The ratio of P. indica to wheat DNA initially rose but then declined in root systems without Fusarium. With Fusarium, the ratio rose throughout the experiment. The absolute amount of FusariumDNA in root systems increased in the absence of P. indica but was static in plants co‐inoculated with P. indica.

Overexpression of Specific Cysteine Peptidases Confers Pathogenicity to a Nonpathogenic Entamoeba histolytica Clone

ABSTRACTCysteine peptidases (CPs) of Entamoeba histolytica are considered to be important pathogenicity factors. Previous studies have found that under standard axenic culture conditions, only four (ehcp-a1, ehcp-a2, ehcp-a5, and ehcp-a7) out of 35 papain-like ehcp genes present in the E. histolytica genome are expressed at high levels. Little is known about the expression of CPs in E. histolytica during amoebic liver abscess (ALA) formation. In the current study, a quantitative real-time PCR assay was developed to determine the expression of the various ehcp genes during ALA formation in animal models. Increased expression of four ehcp genes (ehcp-a3, -a4, -a10, and -c13) was detected in the gerbil and mouse models. Increased expression of another three ehcp genes (ehcp-a5, -a6, and -a7) was detected in the mouse model only, and two other ehcp genes (ehcp-b8 and -b9) showed increased expression in the gerbil model only. Trophozoites of the nonpathogenic E. histolytica HM-1:IMSS clone A1, which was unable to induce ALAs, were transfected with vectors enabling overexpression of those CPs that are expressed at high levels under culture conditions or during ALA formation. Interestingly, overexpression of ehcp-b8, -b9, and -c13 restored the pathogenic phenotype of the nonpathogenic clone A1 whereas overexpression of various other peptidase genes had no effect on the pathogenicity of this clone.

Intrinsic susceptibility of Giardia duodenalis assemblage subtypes AI, AII, B and EIII for nitric oxide under axenic culture conditions

The antigiardial effects of nitric oxide (NO·) have been reported in vitro, but only for assemblage A(I) lab strains. This study investigated the intrinsic NO· susceptibility of different assemblage subtypes. The susceptibility (IC₅₀) for NO· released by MAHMA NONOate was studied for three lab (WB, G1 and GS/M-83-H7) and six field isolates of assemblage subtypes A(I), A(II), B and E(III). Tests were performed in phosphate-buffered saline supplemented with L-cysteine HCl, trypticase peptone, powder bovine bile and 20% inactivated foetal calf serum (for assemblages A and E) or human serum (for assemblage B), adjusted to pH 7.3, to support adequate trophozoite survival. Flow cytometry with fluorescein diacetate and propidium iodide as viability indicators was used to determine trophozoite viability. This study indicated that the NO· susceptibilities of assemblage A lab and field strains (subtypes A(I) and A(II)) were fully comparable, indicating that the NO· susceptibility of the lab strains remained representative for their genotype. The trophozoites of assemblages B and E(III) showed comparable NO· susceptibilities that were markedly higher than the susceptibilities of assemblage subtypes A(I) and A(II). This study suggests a role for the assemblage subtype in defining NO· susceptibilities. The underlying mechanisms still need to be elucidated, but assemblage-linked differences in the expression of the genes coding for flavohemoglobin or A-type flavoprotein may certainly deserve further attention.

Variation in Sodium Chloride Resistance of Cenococcum geophilum and Suillus granulatus Isolates in Liquid Culture

We studied the resistance of Cenococcum geophilum and Suillus granulatus isolates to NaCl during growth under axenic culture conditions. C. geophilum isolates displayed variations in NaCl resistance; mycelial growth of most isolates was inhibited above 200 mM. All isolates of S. granulatus were tolerant to high NaCl content.

Growth responses to and accumulation of mercury by ectomycorrhizal fungi

Heavy metals have been shown to negatively affect the growth of ectomycorrhizal fungi (ECMF). In addition, ECMF have been shown to accumulate heavy metals and to protect host trees from metal toxicity. However, specific literature on the interactions between ECMF and mercury (Hg) is scant. This paper describes the responses of ECMF to Hg in axenic culture conditions. Six ECMF from an area with no known history of direct Hg contamination were tested to determine their sensitivity to Hg. ECMF were incubated on solid medium amended with Hg (0–50 μM) as HgCl 2 and the effect of Hg on radial growth was determined. The effect of preexposure cultivation on Hg sensitivity, the effect of Hg on biomass production, and the ability to accumulate Hg were determined for four of the ECMF. At micromolar concentrations, Hg significantly inhibited the radial growth rate of ECMF. This inhibitory effect was lessened in some ECMF when an established colony was exposed to Hg. Mercury lowered biomass production by some ECMF, and ECMF accumulate Hg from a solid growth substrate in direct relation to the amount of Hg added to the media. Possible implications for ECMF communities in Hg-impacted areas are discussed.

Screening Leishmania donovani‐specific genes required for visceral infection

Comparison of the Leishmania infantum genome with Leishmania braziliensis and Leishmania major genomes has identified 25 L. infantum species-specific genes that are absent or pseudogenes in L. major and L. braziliensis. To determine whether these L. infantum species-specific genes are involved in visceral Leishmania infection, we cloned the orthologues of 14 L. infantum species-specific genes from the genetically closely related Leishmania donovani and introduced them into L. major. Two of these L. donovani species-specific genes were found to significantly increase L. major survival in visceral organs in BALB/c mice. One (orthologue of LinJ28_V3.0340; Ld2834) of these two genes was further investigated. The L. donovani Ld2834 null mutants displayed dramatically reduced virulence in BALB/c mice and were unable to survive in axenic amastigote culture conditions arguing that Ld2834 plays a crucial role in enabling L. donovani survive at the increased temperature typically associated with visceral organs. Ld2834 encodes a 50 kDa protein that is localized in the cytoplasma and has no significant sequence similarity with other known genes. This study validates the importance of comparative genomics for understanding Leishmania species pathology and argues that Leishmania species-specific genes play important roles in tissue tropism and virulence.

Australasian Huperzia as potential sources of Huperzine A and B

The Lycopodiaceae is an ancient and cosmopolitan family of fern allies that include an estimated 35 Huperzia species occurring throughout the South-East Asian and Australian region. Thirteen species naturally occur in Australia and are found mainly in the tropical rainforests of far north Queensland. Over the past decade, there has been renewed interest in Huperzia and their respective Huperzine alkaloid concentrations following the discovery of Huperzine A (HupA) and Huperzine B (HupB) alkaloids in H. serrata (Thunb. ex Murray) Trevis. Both alkaloids are of pharmaceutical interest since they are highly selective and potent reversible inhibitors of acetylcholine esterase. Huperzine alkaloid concentrations of Australasian Huperzia have not been well documented, and no prior studies have been undertaken to investigate the amenability of Australasian Huperzia to alternative propagation techniques such as axenic culture. This research presents an extensive screen of 16 Australasian Huperzia species to investigate their Huperzine alkaloid concentrations. HupA (0.032 to 1.012 mg g-1 DW) was detected in ten out of the sixteen Huperzia species examined, while HupB (0.008 to 0.339 mg g-1 DW) was detected in eight. From this extensive study, H. elmeri (Herter) Holub was observed as the species with the greatest potential to yield high Huperzine-containing individuals. In addition, the screen established that Australasian Huperzia generally contain higher HupA levels than H. serrata, the main source of commercial HupA, which on average only contains 0.082 mg g-1 DW HupA. The fractionation and spectrometric analysis of alkaloids as part of the screen led to the discovery of three Huperzine alkaloids co-occurring within the same plant: HupA, HupB and Huperzine C, isolated from an individual of an Australian H. carinata (Desv. Ex Poir.) Trevis. The potential of establishing axenic cultures of Australasian Huperzia was also investigated in this research. Actively growing axenic cultures of H. carinata, H. squarrosa (C.Forster) Trevisan, H. phlegmaria (L.) Rothm. and H. phlegmarioidies (Gaudich.) Rothm., together with callus and cell suspension cultures of H. carinata and H. phlegmaria, were successfully established. The results suggest that culturing in total darkness is essential to allow for optimal callus and cell suspension growth. In addition, this study also investigated the possibilities of germinating various Huperzia spores, by both symbiotic and asymbiotic means. Germination of H. squarrosa spores was achieved by both symbiotic and asymbiotic means, and was only observed in cultures which were kept in the dark, implying that there is a form of photo-inhibition mechanism preventing spores from germinating when they are exposed to light. Beneficial effects of various types of spore treatments prior to sowing, in terms of increased spore germination was also observed. In conclusion, the results presented suggest that Australasian Huperzia are indeed a potentially valuable resource for Huperzine alkaloids. The investigations into the conditions required for the successful introduction and maintenance of Australasian Huperzia in axenic culture has also further extended our understanding of these plants, and their amenability towards axenic culture conditions as a means of alternative propagation.