Vestibular Rehabilitation as an Early Intervention in Athletes Who are Post-concussion: A Systematic Review.

Background: Sports-related concussions (SRC) are a common injury sustained by many athletes of all different age groups and sports. The current standard treatment is rest followed by aerobic activity. Minimal research has been done on the effects of vestibular rehabilitation for concussion treatment, especially in physical therapy practice. Objective: The purpose of this study was to examine the effects of early intervention of vestibular rehabilitation (VRT) on an athlete's time to return to play compared to rest alone. Study Design: Systematic Review. Methods: Two searches were conducted (August 2021 and January 2022) using databases: CINAHL complete, MEDLINE, PubMed, and Wiley online database. One hand search was performed to find relevant articles. Search terms included "vestibular rehabilitation" or "vestibular therapy" and "concussion" or "mild traumatic brain injury" or "mTBI" and "athletes" or "sports" or "athletics" or "performance", and "early interventions" or "therapy" or "treatment". Inclusion criteria were athletes with a SRC, incorporation of vestibular rehabilitation in athletes' recovery, and early vestibular intervention tools. Tools used to assess quality and risk of bias were the PEDro scale and the Oxford Centre for Evidence-Based Medicine 2011 Levels of Evidence. The PRISMA method for determining inclusion and exclusion criteria. Results: Eleven articles were included, six randomized control trials and five retrospective cohort studies. Various balance interventions, visual interventions utilizing vestibulo-ocular reflex (VOR), and cervical manual therapy were used during VRT for athletes' post-concussion. Incorporating visual interventions and cervical manual therapy into early rehabilitation significantly reduced symptoms and time to return to sport. However, balance interventions did not have a significant effect on reducing time to return to sport when used as a sole intervention. Conclusion: Addressing VRT deficits in the acute stages of a concussion may contribute to a quicker resolution of symptoms and a quicker return to sport. More research needs to be performed to determine the effectiveness of early intervention of VRT in concussion recovery. Level of Evidence: 1.

Development of Robust Tablet Formulations with Enhanced Drug Dissolution Profiles from Centrifugally-Spun Micro-Fibrous Solid Dispersions of Itraconazole, a BCS Class II Drug.

Fibre-based oral drug delivery systems are an attractive approach to addressing low drug solubility, although clear strategies for incorporating such systems into viable dosage forms have not yet been demonstrated. The present study extends our previous work on drug-loaded sucrose microfibres produced by centrifugal melt spinning to examine systems with high drug loading and investigates their incorporation into realistic tablet formulations. Itraconazole, a model BCS Class II hydrophobic drug, was incorporated into sucrose microfibres at 10, 20, 30, and 50% w/w. Microfibres were exposed to high relative humidity conditions (25 °C/75% RH) for 30 days to deliberately induce sucrose recrystallisation and collapse of the fibrous structure into powdery particles. The collapsed particles were successfully processed into pharmaceutically acceptable tablets using a dry mixing and direct compression approach. The dissolution advantage of the fresh microfibres was maintained and even enhanced after humidity treatment for drug loadings up to 30% w/w and, importantly, retained after compression into tablets. Variations in excipient content and compression force allowed manipulation of the disintegration rate and drug content of the tablets. This then permitted control of the rate of supersaturation generation, allowing the optimisation of the formulation in terms of its dissolution profile. In conclusion, the microfibre-tablet approach has been shown to be a viable method for formulating poorly soluble BCS Class II drugs with improved dissolution performance.

Where's the party? A mixed method study investigating characteristics, motivations, and harms of attending off-campus (over on-campus) parties.

ObjectiveWe examined the characteristics, motivations, and harms associated with celebrating events at off-campus parties (OCPs), including a particularly harmful off-campus block party compared to on-campus parties (CPs). Participants and Method: Survey with a representative sample of 491 students in Canada. Three focus groups with a total of 14 students who have attended the OCBP and one focus group with community members affected by the OCBP (n = 10). Results: 14.3% of students attended OCPs. OCPs were significantly more likely to be associated with harms. Focus groups revealed that students attend the OCBP as it is cheaper to consume alcohol compared to CPs. Improved music was the most common solution to improve CP. Community members identified 16 potential solutions to mitigate the harms and prevent the OCBP. Conclusion: Reducing costs and improving the experience may increase likelihood of students attending CPs and minimize harms of OCPs.

In vitro and in vivo biological assessment of dual drug-loaded coaxial nanofibers for the treatment of corneal abrasion.

The treatment of corneal abrasion currently involves the topical administration of antibiotics, with moxifloxacin HCl (0.5% w/v) eye drops being one of the most widely used treatments. Our previous work (Tawfik et al., 2020) involved the development of coaxial poly-lactic-co-glycolic acid (PLGA) and polyvinylpyrrolidone (PVP) nanofibers loaded with the antibiotic moxifloxacin HCl and the anti-scarring agent pirfenidone in the core (PVP) and shell (PLGA) respectively, with a view to the system comprising an ocular insert for the combination therapy of corneal abrasion. In this study, we examine the antimicrobial, anti-scarring and pharmacokinetic properties of the fibers alongside consideration of their toxicity and propensity for irritation. Minimum inhibitory concentration and zone of inhibition studies against S. aureus and P. aeruginosa were performed, while fibroblast cell viability and α-smooth muscle actin (α-SMA, a biomarker for scar formation) were measured using MTT and Western Blot assays, respectively. Pharmacokinetic studies and efficacy against infection were performed using a rabbit model, while ocular irritancy was assessed using the Draize test. The studies demonstrated that the antimicrobial activity of the moxifloxacin HCl was preserved following encapsulation into the nanofibers, while the downregulation of α-SMA was demonstrated using concentrations below the IC20 values (concentration required to decrease corneal fibroblast viability by no more than 20%). The pharmacokinetic study showed retention and sustained release of the moxifloxacin HCl over a 24-hour period, in contrast to equivalent eye drops which required four times daily dosing. Evidence of low level (according to the MMTS scale) irritation was detected for the nanofiber systems. Overall, the study has demonstrated that the dual drug-loaded nanofiber system shows potential for once daily dosing as an ocular insert for the treatment of corneal abrasion.

A Potential Alternative Orodispersible Formulation to Prednisolone Sodium Phosphate Orally Disintegrating Tablets.

The orally disintegrating tablet (ODT) has shown vast potential as an alternative oral dosage form to conventional tablets wherein they can disintegrate rapidly (≤30 s) upon contact with saliva fluid and should have an acceptable mouthfeel as long as their weight doesn't exceed 500 mg. However, owing to the bitterness of several active ingredients, there is a need to find a suitable alternative to ODTs that maintains their features and can be taste-masked more simply and inexpensively. Therefore, electrospun nanofibers and solvent-cast oral dispersible films (ODFs) are used in this study as potential OD formulations for prednisolone sodium phosphate (PSP) that is commercially available as ODTs. The encapsulation efficiency (EE%) of the ODFs was higher (≈100%) compared to the nanofibers (≈87%), while the disintegration time was considerably faster for the electrospun nanofibers (≈30 s) than the solvent-cast ODFs (≈700 s). Hence, accelerated release rate of PSP from the nanofibers was obtained, due to their higher surface area and characteristic surface morphology that permitted higher wettability and thus, faster erosion. Taste-assessment study using the electronic-tongue quantified the bitterness threshold of the drug and its aversiveness concentration (2.79 mM). Therefore, a taste-masking strategy would be useful when further formulating PSP as an OD formulation.

Development and Evaluation of Feline Tailored Amlodipine Besylate Mini-Tablets Using L-lysine as a Candidate Flavouring Agent.

Felines may find orally administered medicines unpalatable, thus presenting a problem in the treatment of chronic conditions such as hypertension, a commonly diagnosed condition in felines requiring daily administration of medication. A pertinent example is amlodipine besylate, formulations of which are known to be poorly tolerated by cats. There is therefore a need to develop feline-specific delivery approaches that are both simple to administer and mask the taste of the drug, thereby enhancing the owner's commitment to treatment and the associated therapeutic outcome for the companion animal. In addition, it is helpful to develop accessible and reproducible means of assessing taste for pre-clinical selection, hence the use of recently developed taste biosensor systems for veterinary applications is an area of interest. This study focuses on developing feline-specific amlodipine besylate formulations by improving the taste using a suitable flavouring agent while reducing dosage form size to a 2 mm diameter mini-tablet. The choice of L-lysine as a flavouring agent was based on the dietary and taste preference of cats. The impact of L-lysine on the taste perception of the formulation was evaluated using a biosensor system (E-tongue) fitted with sensors sensitive to bitter tastes. The results showed L-lysine successfully masked bitterness, while the drug release studies suggest that it has no impact on drug dissolution. In addition, tableting parameters such as tablet mass uniformity, content uniformity, tablet diameter, thickness and hardness were all satisfactory. The present study suggests that amlodipine besylate mini-tablets containing L-lysine could improve the palatability and in turn support product acceptability and ease of administration. These data could have an impact on orally administered medicines for cats and other veterinary species through product differentiation and competitive advantage in the companion animal market sector. The study also outlines the use of the electronic tongue as a tool for formulation selection in the veterinary field.

Dual drug-loaded coaxial nanofibers for the treatment of corneal abrasion.

Corneal abrasion is a scratch wound on the surface of the anterior segment of the eye, which can predispose a patient to corneal infection and scarring, particularly if the cut penetrates to the deep corneal layers. Here we investigate a novel approach to co-administer an anti-scarring agent and an antibiotic, both being incorporated into one dosage form so as to accelerate wound closure and to treat any associated infection. More specifically, we have used electrospun fibers as a means of incorporating the two drugs into distinct compartments via coaxial electrospinning. Samples were characterised using a range of imaging, spectroscopic and thermal methods, while an HPLC assay has been developed to allow measurement of the concentration of both drug components in both the initial fibers and on release. Fibers loaded with pirfenidone in the hydrophobic polymer, PLGA, as the outer layer and moxifloxacin in the hydrophilic polymer PVP as the inner layer were successfully prepared, with smooth and non-porous surfaces and a mean diameter of circa 630 nm. TEM image demonstrated clear distinctive layers (a core and a shell), suggesting the successful preparation of the drug-loaded coaxial fibers, supported by HPLC entrapment studies, while fluorescence microscopy confirmed the presence of the moxifloxacin within the fibers. The fibers were capable of extending the release of both drugs, hence raising the possibility of a single daily dose of the drug-loaded coaxial fibers for the treatment of corneal abrasion.

Design and Characterization of Cyclosporine A-Loaded Nanofibers for Enhanced Drug Dissolution.

Despite widespread use as an immunosuppressant, the therapeutic efficacy of the undecapeptide cyclosporine A (CyA) is compromised when given by the oral route because of the innate hydrophobicity of the drug molecule, potentially leading to poor aqueous solubility and bioavailability. The aim of this study was to develop and characterize nanofibers based on the water-miscible polymer polyvinylpyrrolidone (PVP), incorporating CyA preloaded into polymeric surfactants so as to promote micelle formation on hydration; therefore, this approach represents the novel combination of three dissolution enhancement methodologies, namely solid dispersion technology, micellar systems, and nanofibers with enhanced surface area. The preparation of the nanofibers was performed in two steps. First, mixed micelles composed of the water-soluble vitamin E derivative d-α-tocopheryl poly(ethylene glycol) 1000 succinate and the amphiphilic triblock polymer Pluronic F127 (Poloxamer 407) were prepared. The micelles were characterized in terms of size, surface charge, drug loading, and encapsulation efficiency using transmission electron microscopy, dynamic light scattering, Fourier-transform infrared spectroscopy, high-performance liquid chromatography, and scanning electron and atomic force microscopy analysis. Nanofibers composed of PVP and the drug-loaded surfactant system were then prepared via electrospinning, with accompanying thermal, spectroscopic, and surface topological analysis. Dissolution studies indicated an extremely rapid dissolution profile for the fibers compared to the drug alone, while wettability studies also indicated a marked decrease in contact angle compared to the drug alone. Overall, the new approach appears to offer a viable means for considerably improving the dissolution of the hydrophobic peptide CyA, with associated implications for improved oral bioavailability.

In vitro drug release from acetylated high amylose starch-zein films for oral colon-specific drug delivery.

This study describes the preparation of free films of zein with and without acetylated high amylose maize starch (HAS) and their corresponding coated tablets as a novel approach to colonic drug delivery. We hypothesise that the embedding of a digestible starch component within the inert zein would allow the film to remain intact until the large intestine is reached. Free films of zein alone and starch/zein were prepared and characterized. SEM and AFM images of film surface showed that films were morphologically inhomogeneous, particularly at lower HAS/Zein ratios; however, nanothermal analysis data suggested that these differences in appearance within the same film are not compositional differences. Moreover, FT-IR could detect no molecular interaction between the two polymers. Paracetamol tablets were coated with HAS/Zein aqueous based coatings of different compositions to a TWG of 20%. Drug release from zein alone and 1:5 HAS/Zein coated tablets under upper gastrointestinal conditions (pH 1.2, pH 6.8 with pepsin and pancreatin included) was very similar (for example approximately 12% and 14% of the drug was released, respectively, after 6 h in a sequential in vitro test), suggesting that release in this region is limited and is not influenced by the presence of HAS in the ratio to zein under study. Studies using an in vitro colon model showed that under simulated colonic conditions, the drug release was significantly (p < 0.05) more rapid from 1:5 HAS/Zein, compared to the zein alone coating formulation. These data therefore support the potential use of zein-starch mixed films for colonic targeting purposes.

Structural plasticity in root-fungal symbioses: diverse interactions lead to improved plant fitness.

Root-fungal symbioses such as mycorrhizas and endophytes are key components of terrestrial ecosystems. Diverse in trophy habits (obligate, facultative or hemi-biotrophs) and symbiotic relations (from mutualism to parasitism), these associations also show great variability in their root colonization and nutritional strategies. Specialized interface structures such as arbuscules and Hartig nets are formed by certain associations while others are restricted to non-specialized intercellular or intracellular hyphae in roots. In either case, there are documented examples of active nutrient exchange, reinforcing the fact that specialized structures used to define specific mycorrhizal associations are not essential for reciprocal exchange of nutrients and plant growth promotion. In feremycorrhiza (with Austroboletus occidentalis and eucalypts), the fungal partner markedly enhances plant growth and nutrient acquisition without colonizing roots, emphasizing that a conventional focus on structural form of associations may have resulted in important functional components of rhizospheres being overlooked. In support of this viewpoint, mycobiome studies using the state-of-the-art DNA sequencing technologies have unearthed much more complexity in root-fungal relationships than those discovered using the traditional morphology-based approaches. In this review, we explore the existing literature and most recent findings surrounding structure, functioning, and ecology of root-fungal symbiosis, which highlight the fact that plant fitness can be altered by taxonomically/ecologically diverse fungal symbionts regardless of root colonization and interface specialization. Furthermore, transition from saprotrophy to biotrophy seems to be a common event that occurs in diverse fungal lineages (consisting of root endophytes, soil saprotrophs, wood decayers etc.), and which may be accompanied by development of specialized interface structures and/or mycorrhiza-like effects on plant growth and nutrition.

Tomato CYCLOPS/IPD3 is required for mycorrhizal symbiosis but not tolerance to Fusarium wilt in mycorrhiza-deficient tomato mutant rmc.

Mycorrhizal symbiosis requires several common symbiosis genes including CYCLOPS/IPD3. The reduced mycorrhizal colonisation (rmc) tomato mutant has a deletion of five genes including CYCLOPS/IPD3, and rmc is more susceptible to Fusarium wilt than its wild-type parental line. This study investigated the genetic defects leading to both fungal interaction phenotypes and whether these were separable. Complementation was performed in rmc to test the requirement for CYCLOPS/IPD3 in mycorrhiza formation and Fusarium wilt tolerance. Promoter analysis via GFP expression in roots was conducted to determine the role of native regulatory elements in the proper functioning of CYCLOPS/IPD3. CYCLOPS/IPD3 regulated by its native promoter, but not a 2×35S promoter, restores mycorrhizal association in rmc. GFP regulated by the 2×35S promoter is not expressed in epidermal cells of roots, indicating that expression of CYCLOPS/IPD3 in these cells is required for colonisation by the fungi utilised in this research. CYCLOPS/IPD3 did not restore Fusarium wilt tolerance, however, showing that the genetic requirements for mycorrhizal association and Fusarium wilt tolerance are different. Our results confirm the expected role of CYCLOPS/IPD3 in mycorrhizal symbiosis and suggest that Fusarium tolerance is conferred by one of the other four genes affected by the deletion.

A Novel Tomato Fusarium Wilt Tolerance Gene.

The reduced mycorrhizal colonization (rmc) tomato mutant is unable to form mycorrhiza and is more susceptible to Fusarium wilt compared with its wild-type isogenic line 76R. The rmc mutant has a chromosomal deletion affecting five genes, one of which is similar to CYCLOPS. Loss of this gene is responsible for non-mycorrhizality in rmc but not enhanced Fusarium wilt susceptibility. Here, we describe assessment of a second gene in the rmc deletion, designated Solyc08g075770 that is expressed in roots. Sequence analyses show that Solyc08g075770 encodes a small transmembrane protein with putative phosphorylation and glycosylation sites. It is predicted to be localized in the plasma membrane and may function in transmembrane ion transport and/or as a cell surface receptor. Complementation and knock-out strategies were used to test its function. Some putative CRISPR/Cas-9 knock-out transgenic events exhibited Fusarium wilt susceptibility like rmc and some putative complementation lines were 76R-like, suggesting that the tomato Solyc08g075770 functions in Fusarium wilt tolerance. This is the first study to demonstrate that Solyc08g075770 is the contributor to the Tfw locus, conferring tolerance to Fusarium wilt in 76R which was lost in rmc.

Investigating the physical stability of repackaged medicines stored into commercially available multicompartment compliance aids (MCAs).

BACKGROUND: Compliance aids are devices which have been developed and are currently used to assist individuals in their medicines management. The use of compliance aids involves the transfer of medicines from the manufacturers' original packaging and repackaged into an multicompartment compliance aid (MCA). MCAs do not guarantee the same level of protection compared to manufacturer's original packaging. OBJECTIVE: The aim of this study was to investigate the stability profile of atenolol, aspirin and lansoprazole dosage forms repackaged together in two different commercially available MCAs. METHODS: In a laboratory in the United Kingdom, the physical stability of the formulations repackaged into two commercially available brands of MCAs was evaluated. After 8 weeks of storage (under controlled ambient conditions), changes in the disintegration (tablets only) and dissolution properties (all formulations) were examined in accordance with British Pharmacopoeia (BP) specifications. KEY FINDINGS: Findings from this study confirm that changes in solid-dosage form quality are observed when repackaged into MCAs compared to manufacturers packaging resulting in differences in in-vitro dissolution performance. However, even with these changes, overall product performance was acceptable and within BP specifications. CONCLUSION: There is a need for greater collaboration in this area between manufacturers, hospital and community pharmacists, academics and policymakers to increase the data available on the physical stability and in turn performance of medicines repackaged into MCAs.

Plant-expressed Hepatitis B core antigen virus-like particles: Characterization and investigation of their stability in simulated and pig gastro-intestinal fluids.

Virus-like particles (VLPs) are potential oral vaccine candidates, as their highly compact structure may allow them to withstand the harsh conditions of the gastro-intestinal (GI) environment. Hepatitis B core antigen (HBcAg) is an immunogenic protein that assembles into 30 or 34nm diameter VLPs. Here, the stabilities of both the HBcAg polypeptide itself and the three-dimensional structure of the VLPs upon exposure to in vitro and ex vivo simulated gastric and intestinal fluids were investigated. Plant-expressed HBcAg VLPs were efficiently purified by sucrose density gradient and characterized. The purified VLPs did not show major chemical or physical instability upon exposure to the low pH conditions typically found in the stomach; however, they completely agglomerated upon acidification and subsequent pH neutralization. The HBcAg polypeptide was highly digested upon exposure to pepsin in simulated gastric fluids. HBcAg appeared more stable in both simulated and ex vivo intestinal fluids, where despite a partial digestion of the HBcAg polypeptide, the VLPs maintained their most immunogenic epitopes and their particulate conformation. These results suggest that HBcAg VLPs are likely to be unstable in gastric fluids, yet if the gastric instability could be bypassed, they could maintain their particulate structure and immunogenicity in intestinal fluids.

Microfibrous Solid Dispersions of Poorly Water-Soluble Drugs Produced via Centrifugal Spinning: Unexpected Dissolution Behavior on Recrystallization.

Temperature-controlled, solvent-free centrifugal spinning may be used as a means of rapid production of amorphous solid dispersions in the form of drug-loaded sucrose microfibers. However, due to the high content of amorphous sucrose in the formulations, such microfibers may be highly hygroscopic and unstable on storage. In this study, we explore both the effects of water uptake of the microfibers and the consequences of deliberate recrystallization for the associated dissolution profiles. The stability of sucrose microfibers loaded with three selected BCS class II model drugs (itraconazole (ITZ), olanzapine (OLZ), and piroxicam (PRX)) was investigated under four different relative humidity conditions (11, 33, 53, and 75% RH) at 25 °C for 8 months, particularly focusing on the effect of the highest level of moisture (75% RH) on the morphology, size, drug distribution, physical state, and dissolution performance of microfibers. While all samples were stable at 11% RH, at 33% RH the ITZ-sucrose system showed greater resistance against devitrification compared to the OLZ- and PRX-sucrose systems. For all three samples, the freshly prepared microfibers showed enhanced dissolution and supersaturation compared to the drug alone and physical mixes; surprisingly, the dissolution advantage was largely maintained or even enhanced (in the case of ITZ) following the moisture-induced recrystallization under 75% RH. Therefore, this study suggests that the moisture-induced recrystallization process may result in considerable dissolution enhancement compared to the drug alone, while overcoming the physical stability risks associated with the amorphous state.

Development of micro-fibrous solid dispersions of poorly water-soluble drugs in sucrose using temperature-controlled centrifugal spinning.

Solid dispersion technology represents a successful approach to addressing the bioavailability issues caused by the low aqueous solubility of many Biopharmaceutics Classification System (BCS) Class II drugs. In this study, the use of high-yield manufacture of fiber-based dispersion is explored as an alternative approach to monolith production methods. A temperature-controlled solvent-free centrifugal spinning process was used to produce sucrose-based microfibers containing the poorly water-soluble drugs olanzapine and piroxicam (both BCS Class II); these were successfully incorporated into the microfibers and the basic characteristics of fiber diameter, glassy behavior, drug loading capacity and drug-sucrose interaction assessment were measured. Scanning electron microscopy revealed that bead-free drug-loaded microfibers with homogenous morphology and diameter in the range of a few micrometers were prepared using our process. Differential scanning calorimetric and X-ray diffraction analyses showed that both drug and carrier were present in the amorphous state in the microfibers, although in the case of piroxicam-loaded microfibers, the presence of small amounts of crystalline drug was observed under polarized light microscopy and in Fourier transform infrared spectra. Drug dissolution performance was evaluated under both sink and non-sink conditions and was found to be significantly enhanced compared to the corresponding crystalline physical mixtures and pure drugs, with evidence of supersaturation behavior noted under non-sink conditions. This study has demonstrated that microfiber-based dispersions may be manufactured by the centrifugal spinning process and may possess characteristics that are favorable for the enhanced dissolution and oral absorption of drugs.

Sensitivity of jarrah (Eucalyptus marginata) to phosphate, phosphite, and arsenate pulses as influenced by fungal symbiotic associations.

Many plant species adapted to P-impoverished soils, including jarrah (Eucalyptus marginata), develop toxicity symptoms when exposed to high doses of phosphate (Pi) and its analogs such as phosphite (Phi) and arsenate (AsV). The present study was undertaken to investigate the effects of fungal symbionts Scutellospora calospora, Scleroderma sp., and Austroboletus occidentalis on the response of jarrah to highly toxic pulses (1.5 mmol kg(-1) soil) of Pi, Phi, and AsV. S. calospora formed an arbuscular mycorrhizal (AM) symbiosis while both Scleroderma sp. and A. occidentalis established a non-colonizing symbiosis with jarrah plants. All these interactions significantly improved jarrah growth and Pi uptake under P-limiting conditions. The AM fungal colonization naturally declines in AM-eucalypt symbioses after 2-3 months; however, in the present study, the high Pi pulse inhibited the decline of AM fungal colonization in jarrah. Four weeks after exposure to the Pi pulse, plants inoculated with S. calospora had significantly lower toxicity symptoms compared to non-mycorrhizal (NM) plants, and all fungal treatments induced tolerance against Phi toxicity in jarrah. However, no tolerance was observed for AsV-treated plants even though all inoculated plants had significantly lower shoot As concentrations than the NM plants. The transcript profile of five jarrah high-affinity phosphate transporter (PHT1 family) genes in roots was not altered in response to any of the fungal species tested. Interestingly, plants exposed to high Pi supplies for 1 day did not have reduced transcript levels for any of the five PHT1 genes in roots, and transcript abundance of four PHT1 genes actually increased. It is therefore suggested that jarrah, and perhaps other P-sensitive perennial species, respond positively to Pi available in the soil solution through increasing rather than decreasing the expression of selected PHT1 genes. Furthermore, Scleroderma sp. can be considered as a fungus with dual functional capacity capable of forming both ectomycorrhizal and non-colonizing associations, where both pathways are always accompanied by evident growth and nutritional benefits.

Ecto- and arbuscular mycorrhizal symbiosis can induce tolerance to toxic pulses of phosphorus in jarrah (Eucalyptus marginata) seedlings.

In common with many plants native to low P soils, jarrah (Eucalyptus marginata) develops toxicity symptoms upon exposure to elevated phosphorus (P). Jarrah plants can establish arbuscular mycorrhizal (AM) and ectomycorrhizal (ECM) associations, along with a non-colonizing symbiosis described recently. AM colonization is known to influence the pattern of expression of genes required for P uptake of host plants and our aim was to investigate this phenomenon in relation to P sensitivity. Therefore, we examined the effect on hosts of the presence of AM and ECM fungi in combination with toxic pulses of P and assessed possible correlations between the induced tolerance and the shoot P concentration. The P transport dynamics of AM (Rhizophagus irregularis and Scutellospora calospora), ECM (Scleroderma sp.), non-colonizing symbiosis (Austroboletus occidentalis), dual mycorrhizal (R. irregularis and Scleroderma sp.), and non-mycorrhizal (NM) seedlings were monitored following two pulses of P. The ECM and A. occidentalis associations significantly enhanced the shoot P content of jarrah plants growing under P-deficient conditions. In addition, S. calospora, A. occidentalis, and Scleroderma sp. all stimulated plant growth significantly. All inoculated plants had significantly lower phytotoxicity symptoms compared to NM controls 7 days after addition of an elevated P dose (30 mg P kg(-1) soil). Following exposure to toxicity-inducing levels of P, the shoot P concentration was significantly lower in R. irregularis-inoculated and dually inoculated plants compared to NM controls. Although all inoculated plants had reduced toxicity symptoms and there was a positive linear relationship between rank and shoot P concentration, the protective effect was not necessarily explained by the type of fungal association or the extent of mycorrhizal colonization.

A novel plant-fungus symbiosis benefits the host without forming mycorrhizal structures.

• Most terrestrial plants form mutually beneficial symbioses with specific soil-borne fungi known as mycorrhiza. In a typical mycorrhizal association, fungal hyphae colonize plant roots, explore the soil beyond the rhizosphere and provide host plants with nutrients that might be chemically or physically inaccessible to root systems. • Here, we combined nutritional, radioisotopic ((33)P) and genetic approaches to describe a plant growth promoting symbiosis between the basidiomycete fungus Austroboletus occidentalis and jarrah (Eucalyptus marginata), which has quite different characteristics. • We show that the fungal partner does not colonize plant roots; hyphae are localized to the rhizosphere soil and vicinity and consequently do not transfer nutrients located beyond the rhizosphere. Transcript profiling of two high-affinity phosphate (Pi) transporter genes (EmPHT1;1 and EmPHT1;2) and hyphal-mediated (33)Pi uptake suggest that the Pi uptake shifts from an epidermal to a hyphal pathway in ectomycorrhizal plants (Scleroderma sp.), similar to arbuscular mycorrhizal symbioses, whereas A. occidentalis benefits its host indirectly. The enhanced rhizosphere carboxylates are linked to growth and nutritional benefits in the novel symbiosis. • This work is a starting point for detailed mechanistic studies on other basidiomycete-woody plant relationships, where a continuum between heterotrophic rhizosphere fungi and plant beneficial symbioses is likely to exist.

The reduced mycorrhizal colonisation (rmc) mutation of tomato disrupts five gene sequences including the CYCLOPS/IPD3 homologue.

Arbuscular mycorrhizal (AM) symbiosis in vascular plant roots is an ancient mutualistic interaction that evolved with land plants. More recently evolved root mutualisms have recruited components of the AM signalling pathway as identified with molecular approaches in model legume research. Earlier we reported that the reduced mycorrhizal colonisation (rmc) mutation of tomato mapped to chromosome 8. Here we report additional functional characterisation of the rmc mutation using genotype grafts and proteomic and transcriptomic analyses. Our results led to identification of the precise genome location of the Rmc locus from which we identified the mutation by sequencing. The rmc phenotype results from a deletion that disrupts five predicted gene sequences, one of which has close sequence match to the CYCLOPS/IPD3 gene identified in legumes as an essential intracellular regulator of both AM and rhizobial symbioses. Identification of two other genes not located at the rmc locus but with altered expression in the rmc genotype is also described. Possible roles of the other four disrupted genes in the deleted region are discussed. Our results support the identification of CYCLOPS/IPD3 in legumes and rice as a key gene required for AM symbiosis. The extensive characterisation of rmc in comparison with its 'parent' 76R, which has a normal mycorrhizal phenotype, has validated these lines as an important comparative model for glasshouse and field studies of AM and non-mycorrhizal plants with respect to plant competition and microbial interactions with vascular plant roots.