Flow cytometry: a quick method to determine ploidy levels in honeybush (<Emphasis Type="Italic">Cyclopia</Emphasis> spp.)

Cyclopia (honeybush) species are widely-used in the production of a South African herbal tea and are endemic to the fynbos region of the Western and Eastern Cape Provinces. Honeybush is still one of the orphan agriculture crops and recent breeding efforts by researchers are hampered by the lack of basic genetic information e.g. basic chromosome numbers and ploidy levels. This study determined nuclear DNA content and ploidy level of various genotypes of three Cyclopia species using flow cytometry and cytological counting of chromosomes. Nuclei analysis of young leaves of C. genistoides, C. longifolia and C. subternata were done using a flow cytometer, while root tip squashes were carried out in order to correlate flow cytometry results. Flow cytometry analysis indicated differences in the nuclear DNA content among and within species whilst the DNA ploidy level only differed among species. Cyclopia genistoides had a higher DNA ploidy level (≥?10C) and DNA content (10.63 pg) than C. longifolia (6.09 pg) and C. subternata (5.99 pg), with no differences observed between the ploidy level of the latter two species (6C). The inferred ploidy level from nuclear DNA content by flow cytometry was consistent in all 30 genotypes of C. longifolia, and 24 of the 25 C. subternata and in only four of the 15 C. genistoides studied genotypes. These findings are important in breeding new cultivars with desired horticultural traits, thus improving the commercial characteristics for the sustainable production of honeybush.     

Influence of arbuscular mycorrhizal fungi,inoculum level and phosphorus placement on growth and phosphorus uptake of <Emphasis Type="Italic">Phyllanthus calycinus</Emphasis> under jarrah forest soil

Phosphorus uptake and biomass production may vary between arbuscular mycorrhizas (AM) species of contrasting life cycles and their modes of interaction with host plants. This research investigated differences among three AM fungi from different genera isolated from the jarrah forest with respect to their colonisation, biomass production and P uptake over time. The understorey plant Phyllanthus calycinus, has been shown to be extensively mycorrhizal. We examined differences in the capacity of fungi associated with this plant to access a P point source from increasing distances from the root. The methodology simulated “pockets” of P in forest soil. Phosphorus sources in root-exclusion mesh bags were inserted 2, 4 and 6 cm from plant roots restrained in separate mesh bags. Acaulospora laevis colonised plants to the greatest extent, producing higher plant biomass and P uptake compared to the other two fungi. Plant biomass and P uptake were enhanced where P sources were closest to the root, as expected, but there were differences among fungi in response to level of inoculum used for each fungus. The capacity of individual AM fungi to access a point source of P at different distances varied in a highly mycorrhizal native forest understorey plant in parallel with plant growth and P uptake.     

Clay addition to lime-amended biosolids overcomes water repellence and provides nitrogen supply in an acid sandy soil

The addition of clay to lime-amended biosolids has been proposed as a soil amendment (LaBC^®) for remediating acidic-sandy soil. We investigated whether the presence of clay in LaBC^® altered soil microbial processes over a 30-week period. Aerobic-incubation and CO₂ respiration assays were used to monitor water repellence and chemical and microbiological properties of amended soil. Dry LaBC^® was applied at equivalent wet weight of 50, 100 and 150 t ha^?1. In addition, dry components of LaBC^® (lime, clay, lime + clay (LAC) and lime + biosolids (LAB)) were applied separately at rates equivalent to their fractions within LaBC^®. Inclusion of clay in LaBC^® was effective in eliminating water repellence at all application rates. Inclusion of clay in LaBC^® decreased nitrogen (N) release from the biosolids (by 58 %), even at 50 t ha^?1, but only during the first 2 weeks of incubation. LaBC^® consistently decreased soil microbial respiration compared with LAB alone when applied at 150 t ha^?1 thereby protecting organic matter decomposition. There was no significant N release with lime and clay amendment alone or in combination (LAC) in the absence of the biosolids. There may be a complex interaction between clay, lime and organic matter, but each may have had a role in altering N release from biosolids at different times during the 30-week incubation. Addition of clay to LAB increased its effectiveness in remediating this water-repellent, acidic-sandy soil and prolonged the N release from the biosolids following soil amendment.     

Microscopy Observations of Habitable Space in Biochar for Colonization by Fungal Hyphae From Soil

Biochar is a potential micro-environment for soil microorganisms but evidence to support this suggestion is limited. We explored imaging techniques to visualize and quantify fungal colonization of habitable spaces in a biochar made from a woody feedstock. In addition to characterization of the biochar, it was necessary to optimize preparation and observation methodologies for examining fungal colonization of the biochar. Biochar surfaces and pores were investigated using several microscopy techniques. Biochar particles were compared in soilless media and after deposition in soil. Scanning electron microscopy （SEM） observations and characterization of the biochar demonstrated structural heterogeneity within and among biochar particles. Fungal colonization in and on biochar particles was observed using light, fluorescence and electron microscopy. Fluorescent brightener RR 2200 was more effective than Calcofluor White as a hyphal stain. Biochar retrieved from soil and observed using fluorescence microscopy exhibited distinct hyphal networks on external biochar surfaces. The extent of hyphal colonization of biochar incubated in soil was much less than for biochar artificially inoculated with fungi in a soilless medium. The location of fungal hyphae was more clearly visible using SEM than with fluorescence microscopy. Observations of biochar particles colonized by hyphae from soil posed a range of difficulties including obstruction by the presence of soil particles on biochar surfaces and inside pores. Extensive hyphal colonization of the surface of the biochar in the soilless medium contrasted with limited hyphal colonization of pores within the biochar. Both visualization and quantification of hyphal colonization of surfaces and pores of biochar were restricted by two-dimensional imaging associated with uneven biochar surfaces and variable biochar pore structure. There was very little colonization ofbiochar from hyphae in the agricultural soil used in this study.     

Pyoderma caused by Pseudomonas aeruginosa infection in dogs: 20 cases

In this report we describe the historical, clinical, histopathological and microbiological features, as well as treatments and clinical outcome, of pyoderma where Pseudomonas aeruginosa alone was isolated on bacterial culture from lesional skin. Twenty dogs were included in this retrospective study. Seven dogs without prior history of systemic or skin disease presented with acute deep pseudomonal pyoderma characterized by a sudden onset of dorsal truncal pain. Skin lesions in these dogs consisted of erythematous papules, haemorrhagic bullae, ulcers and haemorrhagic crusts confined to the dorsum. An excellent clinical response was achieved with 3-4 weeks of treatment with oral fluoroquinolones. Thirteen dogs with a more gradual onset of skin lesions associated with pseudomonal pyoderma had a history of prior skin, ear or systemic disease and had previously been treated with antibiotics and/or immunomodulatory drugs. Skin lesions in these dogs were variable and similar to those described for superficial and deep staphylococcal pyoderma. In this group, one dog was euthanized prior to commencement of treatment, two dogs were lost to follow up, and 9 had resolution of lesions following treatment with topical silver sulfadiazine (one dog), fluoroquinolones (six dogs) or cephalexin (two dogs) administered orally for 3 to 12 weeks. Rod-shaped bacteria were not always detected on cytology. Histopathology of dogs with deep pseudomonal pyoderma was characterized by severe perforating suppurative folliculitis and furunculosis.     

Barbiturate ingestion in three adult captive tigers (Panthera tigris) and concomitant fatal botulism of one

Zoo animals, including tigers, have been reported to suffer from barbiturate intoxication, with pentabarbitone being most commonly recorded. Clinical signs range from mild ataxia to general anaesthesia with recovery over hours to days with several factors affecting hepatic barbiturate metabolism and tissue partitioning. Botulism is an often fatal intoxication in man, animals, birds and certain fish. The occurrence in carnivores is uncommon to rare, with only 2 reports found of botulism in felids. This report relates to 3 adult captive cohabiting tigers that simultaneously developed signs of abdominal discomfort, progressive ataxia, recumbency and comatose sleep resembling stage 2 anaesthesia, alternating with periods of distracted wakefulness and ataxic movements. These signs occurred 4 days after being fed the carcass of a horse that had ostensibly died of colic and not been euthanased. The male tiger that was the dominant animal in the feeding hierarchy was worst affected and had to be given intravenous fluids. The female that was lowest in hierarchy was unaffected. After 48-72 hours of treatment at the Onderstepoort Veterinary Academic Hospital the females could eat and made an uneventful recovery. The male tiger showed partial recovery but died during the night a few hours after drinking water on his return to the owner. Necropsy revealed severe oesophageal dilation and impaction with decaying grass; some of this material and water were present in the pharynx and trachea, and had been aspirated causing acute widespread bronchopneumonia. Colon content tested negative for common pesticides but, together with liver, tested positive for barbiturate. Serum taken on the day of admission had tested negative for barbiturate and the residual serum from the 3 animals later tested negative for botulinum toxin. Colon and oesophageal content from the male at necropsy were positive for Clostridium botulinum toxin type C by the mouse bioassay neutralisation test, confirming that this male had had concomitant barbiturate toxicity and botulism, and had succumbed to aspiration bronchopneumonia secondary to pharyngeal, laryngeal and oesophageal paralysis and oesophageal