First Report of Diplodia intermedia Causing Canker and Dieback Diseases on Apple Trees in Canada.

A dieback of apple trees (Malus domestica (Suckow) Borkh.) associated with cankers was observed in commercial orchards in southwestern Ontario, Canada, in 2019. Fifteen 2 to 10-year-old symptomatic trees were collected from three orchards exhibiting up to 37% disease incidence. Small sections of diseased wood (1 cm long) were surface sterilized with 70% ethanol for 30 sec and 1% NaClO for 20 min, rinsed thrice in sterile water, placed on 2% PDA (Difco) amended with kanamycin (50 mg liter-1), and incubated at 22°C for 5 days in the dark (Ilyukhin et al. 2023). Fungal colonies that were consistently isolated were hyphal-tipped, transferred to individual PDA plates and incubated at 22°C for 7 days in the dark. Purified isolates with same characteristics were classed into morphotypes. One morphotype was initially white and turned dark olivaceous with dense aerial mycelium. Pycnidia were produced on pine needles on PDA (Fig. S2) after incubation at 22°C for 17 days in the dark. Conidia were brown, aseptate, ovoid, and measured 27.9 to 31.3 µm x 12.1 to 14.2 µm (mean ± S.D. of 15 conidia = 29.9 ± 0.9 µm × 13.2 ± 0.6 µm), the typical morphology of a Diplodia sp. (Phillips et al. 2012). Genomic DNA was extracted from a 7-day-old culture of a representative isolate M45-28, using the Plant/Fungi DNA Isolation Kit (Norgen Biotech, Canada). The internal transcribed spacer (ITS), translation elongation factor 1-α (EF1-α) and ß-tubulin gene regions were amplified and sequenced with primers ITS1/ITS4, EF1-728F/EF1-986R and Bt2a/Bt2b and deposited in GenBank with accession numbers MZ970605, MZ995430 and MZ995431, respectively. Based on the sequence, the fungus was identified as Diplodia intermedia A.J.L. Phillips et al. and matched isolates from different hosts and countries (ITS: 100%, MG220378; EF1-α: 100%, MG220385; ß-tubulin: 99.24%, MT592502). The maximum likelihood-based phylogenetic analysis of ITS, EF1-α and ß-tubulin concatenated sequences was performed using IQ-Tree 2.2.2.7 (Minh et al. 2020). M45-28 was clustered with high bootstrap support values with D. intermedia isolates from the Westerdijk Fungal Biodiversity Institute collection, including the ex-holotype (CBS 124462) (Fig. S1). A living culture of M45-28 was deposited in the Canadian Collection of Fungal Cultures (DAOMC 252253). Pathogenicity assay was conducted by inoculating mycelial plugs from a 7-day-old culture of M45-28 into wounds made on the trunk of 5 eight-month-old potted healthy 'Royal Gala' apple seedlings. Five control seedlings were inoculated with sterile plugs. Canker symptoms appeared 15 days after inoculation, spread around, up and down the main stem from the inoculation point, and by 7 weeks the upper portion of the seedling was dead (Fig. S2). Diplodia intermedia was re-isolated from all inoculated seedlings and species identity was confirmed by sequencing as described above, fulfilling Koch's postulates. Control seedlings remained symptomless and the fungus was not isolated from the wood. Diplodia intermedia was reported to cause cankers on apple in Uruguay (Delgado-Cerrone et al. 2016), wild apple (Malus sylvestris) in Portugal (Phillips et al. 2012), grapevines in France (Comont et al. 2016) and forest trees in Iran (Kazemzadeh Chakusary et al. 2019). To the best of our knowledge, this is the first report of D. intermedia causing canker and dieback diseases on apple trees in Canada. Further studies are required to better understand the epidemiology involved in the dynamic spread of the disease in order to recommend an adequate phytosanitary program for its control.

Effectiveness of mechanical traction in supine versus prone lying position for lumbosacral radiculopathy.

OBJECTIVES: To determine the effectiveness of mechanical traction in supine versus prone lying position for lumbosacral radiculopathy. METHODS: A quasi experimental trial was conducted from April to September 2020 among sixty patients of chronic lumbosacral radiculopathy at Ibn e Siena Hospital, Multan. Participants were divided into two groups. Group-A (Supine) participants received lumbar traction in supine lying along with conventional treatment. Group-B (Prone) underwent the same treatment except the lumbar traction being applied in prone lying position. Participants were evaluated twice: at pre- treatment (week 0) and at the post treatment (week 2). Oswestry Disability Index and Numeric Pain Rating Scale were used as outcome measure. Data was analyzed on SPSS 23. RESULTS: The mean (±S.D) age of the patients was 39±5.7 vs. 40±5.3 years in supine vs. prone group respectively. Mean ODI score was 25.2±6.13 vs. 26.0±6.26 at the start of treatment in supine vs. prone position respectively while it was 19.45±7.12 vs. 11.05±4.40 at end of treatment in supine vs. prone position respectively. Mean NPRS score was 7.73±1.23 vs. 7.67±0.96 at start of treatment in supine vs. prone position respectively while it was 4.63±0.89 vs. 3.13±0.90 at the end of treatment in supine vs. prone position respectively. CONCLUSION: Lumbar traction in prone lying position is more effective than lumbar traction in supine lying position for the treatment of chronic lumbosacral radiculopathy.

Cardiac glycoside-mediated turnover of Na, K-ATPases as a rational approach to reducing cell surface levels of the cellular prion protein.

It is widely anticipated that a reduction of brain levels of the cellular prion protein (PrPC) can prolong survival in a group of neurodegenerative diseases known as prion diseases. To date, efforts to decrease steady-state PrPC levels by targeting this protein directly with small molecule drug-like compounds have largely been unsuccessful. Recently, we reported Na,K-ATPases to reside in immediate proximity to PrPC in the brain, unlocking an opportunity for an indirect PrPC targeting approach that capitalizes on the availability of potent cardiac glycosides (CGs). Here, we report that exposure of human co-cultures of neurons and astrocytes to non-toxic nanomolar levels of CGs causes profound reductions in PrPC levels. The mechanism of action underpinning this outcome relies primarily on a subset of CGs engaging the ATP1A1 isoform, one of three α subunits of Na,K-ATPases expressed in brain cells. Upon CG docking to ATP1A1, the ligand receptor complex, and PrPC along with it, is internalized by the cell. Subsequently, PrPC is channeled to the lysosomal compartment where it is digested in a manner that can be rescued by silencing the cysteine protease cathepsin B. These data signify that the repurposing of CGs may be beneficial for the treatment of prion disorders.