First Report of Leaf Rust of Blueberry Caused by Thekopsora minima on Vaccinium corymbosum in the Western Cape, South Africa.

Southern highbush blueberry plants (Vaccinium corymbosum interspecific hybrids) showing rust-like symptoms were observed in July 2006 in Porterville in the Western Cape (WC), South Africa. Diseased plants were also found in Villiersdorp and George in the WC in 2007. In 2008, symptoms were observed in George, and in 2009, in all the previous reported areas. Cvs. Bluecrisp, Emerald, Jewel, Sharpblue, and Star were infected. Reddish-to-brown spots appeared on the adaxial surface of leaves and developed into yellow-to-orange erumpent uredinia with pulverulent urediniospores. Uredinia were hypophyllous, dome shaped, 113 to 750 µm wide, and occasionally coalescing. Urediniospores were broadly obovate, sometimes ellipsoidal or pyriform, with yellowish orange content, and measured 19 to 27 × 12 to 20 µm (average 24 × 15 µm, n = 30). Spore walls were echinulate, hyaline, 1 to 1.5 µm thick, and with obscure germ pores. No telia or teliospores were observed. Voucher specimens were lodged in the South African National Fungus Collection in Pretoria (PREM 60245). The isolate was initially identified as Thekopsora minima P. Syd. & Syd., based primarily on the absence of conspicuous ostiolar cells characteristic of Naohidemyces spp. (3). Genomic DNA was extracted from urediniospores. Approximately 1,400 bp were amplified spanning the 5.8S, ITS2, and 28S large subunit of the ribosomal DNA (1). The sequence (GU355675) shared 96% (907 of 942 bp; GenBank AF522180) and 94% (1,014 of 1,047 bp; GenBank DQ354563) similarities in the 28S portion, respectively, to those of Naohidemyces vaccinii (Wint.) Sato, Katsuya et Y. Hiratsuka and Pucciniastrum geoppertianum (Kuehn) Kleb, two of the three known rust species of blueberry (2). Although no sequences of T. minima were available for direct comparison, phylogenetic analyses of the 28S region strongly supported the South African blueberry rust as congeneric with T. guttata (J. Schröt.) P. Syd. & Syd. (GenBank AF426231) and T. symphyti (Bubák) Berndt (GenBank AF26230) (data not shown). Four 6-month-old cv. Sharpblue plants were inoculated with a suspension (approximate final concentration of 1 × 105 spores per ml) of fresh urediniospores in a water solution with 0.05% Tween 20. After incubation at 20°C for 48 h under continuous fluorescent lighting, the plants were grown in a glasshouse (18/25°C night/day temperatures). Identical uredinia and symptoms developed approximately 3 weeks after inoculation on the inoculated plants, but not on two control plants of cv. Sharpblue sprayed with distilled water and kept at the same conditions. The alternate host hemlock (Tsuga spp.) is not endemic to South Africa and not sold as an ornamental plant according to a large conifer nursery. Hosts of T. minima include Gaylussacia baccata, G. frondosa, Lyonia neziki, Menziesia pilosa, Rhododendron canadense, R. canescens, R. lutescens R. ponticum, R. prunifolium, R. viscosum, V. angustifolium var. laevifolium, V. corumbosum, and V. erythrocarpon (3). Visual inspection of possible hosts in the gardens in close proximity of Vaccinium production areas did not show any rust symptoms. To our knowledge, this is the first report of T. minima on blueberries outside of Asia and the United States (2). References: (1) M. C. Aime. Mycoscience 47:112, 2006. (2) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Botany and Mycology Laboratory. Online publication. USDA-ARS, 2009. (3) S. Sato et al. Trans. Mycol. Soc. Jpn. 34:47, 1993.

The toxicity of Senecio inaequidens DC.

This study was designed to confirm the toxicity of a plant implicated in an outbreak of poisoning of stock in Frankfort, Free State Province, South Africa. Cows died acutely after being introduced into a camp, where an abundant, green shrublet was noted to be heavily grazed. This plant was subsequently identified as Senecio inaequidens DC. (Asteraceae) by the South African National Biodiversity Institute (SANBI). Extraction and chemical analyses for pyrrolizidine alkaloids (PAs) in Senecio inaequidens revealed the presence of 4 different compounds, namely retrorsine and senecionine (known to be hepatotoxic) and 2 unidentified compounds. The average total PA (free base plus N-oxide) concentration in plant parts of S. inaequidens collected at Frankfort during the outbreak was 0.81%, compared with the total alkaloid content in the dried, milled S. inaequidens plant material, collected 7 weeks after the outbreak, of only 0.18%. Male Sprague-Dawley rats (n = 4), aged 8-9 weeks, were dosed per os. Each rat received a different dose of the crude Senecio inaequidens extract, ranging from 0.049 mg/g body weight (b.w.) to 0.25 mg/g b.w. No clinical signs were observed in the rat receiving the lowest dose. Rats receiving higher doses showed depression, an unsteady gait, pilo-erection and jaundice, which was particularly noticeable in the ears. Clinical chemistry evaluation revealed an increase in the activities of ALP (except Rat 4), AST and GGT in all animals. Total serum bilirubin, creatinine and urea concentrations were also elevated. All rats had low serum globulin concentrations with an A/G ratio above 1.2. Post mortem examination of the rats revealed marked hepatic lesions. Histopathologically, these changes were characterised by necrosis (variable in extent) of the centrilobular and midzonal hepatocytes (but sparing the portal hepatocytes), with extensive haemorrhage and congestion. Proliferation of the bile ducts, fibrosis and oedema were also present. Ultrastructural changes in affected rats were characterised by margination of chromatin, the presence of numerous autolysosomes in necrotic hepatocytes, intramitochondrial woolly inclusions and changes in the endoplasmic reticulum. A sheep, also dosed with the crude extract, failed to exhibit clinical signs, clinical chemistry aberrations or macroscopic lesions; however, examination of the liver of this sheep revealed histopathological and ultrastructural changes similar, though milder, to those displayed by the rats. Pyrrolizidine alkaloids were extracted from the liver and kidneys of the rats and the sheep. In the case of the sheep, retrorsine was also detected in the lungs, urine and bile.

Potential Inoculum Sources of Phaeomoniella chlamydospora in South African Grapevine Nurseries.

Petri disease of grapevine is primarily caused by Phaeomoniella chlamydospora. This pathogen affects mostly young grapevines, but is also implicated in esca disease of older grapevines. Little is known about the disease cycle of this fungus. Infected propagation material was identified as a major means of dissemination of the pathogen. Recently, the pathogen was also detected from soil in South Africa and airborne conidia have been found in vineyards. The aim of this study was to use a molecular detection technique to test different samples collected from nurseries in South Africa at different nursery stages for the presence of P. chlamydospora. A one-tube nested-PCR technique was optimised for detecting P. chlamydospora in DNA extracted from soil, water, callusing medium and grapevine wood. The one-tube nested-PCR was sensitive enough to detect as little as 1 fg of P. chlamydospora genomic DNA from water and 10 fg from wood, callusing medium and soil. PCR analyses of the different nursery samples revealed the presence of several putative 360 bp P. chlamydospora specific bands. Subsequent sequence analyses and/or restriction enzyme digestions of all 360 bp PCR bands confirmed that all bands were P. chlamydospora specific, except for five bands obtained from callusing media and one from water. Phaeomoniella chlamydospora was positively detected in 25% of rootstock cane sections collected from mother blocks, 42% of rootstock cuttings and 16% of scion cuttings collected during grafting, 40% of water samples collected after pre-storage hydration, 67% of water samples collected during grafting, 8% of callusing medium samples and 17% of soil samples collected from mother blocks. These media can therefore be considered as possible inoculum sources of the pathogen during the nursery stages.

Neurotoxicity in calves induced by the plant, Nierembergia hippomanica Miers var. violacea Millán in South Africa.

The plant Nierembergia hippomanica var. violacea has been incriminated in field outbreaks of neurotoxicity in calves in the Free State Province. Hepatotoxicity and electrocardiogram (ECG) deviations were induced in a sheep dosed with 5 g/kg dried plant material on four consecutive days. A calf dosed with 2.5 g/kg dried plant material, on two consecutive days, did not show overt clinical changes. Voluntary ingestion of approximately 30 g/kg fresh flowering plants by a second calf resulted in nervous signs characterized by chewing motions, protrusion of the tongue, dysphagia, hypermetria, ataxia, paresis and lateral recumbency. Salivation, dehydration and cardiac irregularities completed the clinical picture. Clinical chemistry changes revealed muscle damage and increased serum urea and creatinine concentrations indicative of kidney involvement. This is the first confirmed outbreak of Nierembergia hippomanica var. violacea intoxication of stock in South Africa.

Krimpsiekte in a sheep following a single dose of Tylecodon ventricosus (Burm. f.) Toelken and the isolation of tyledoside D from this plant species.

Tylecodon ventricosus induced severe respiratory distress in two penned sheep without any electrocardiographic abnormalities being recorded. Based on the results it appears as if T. ventricosus predominantly induces the neuromuscular syndrome referred to as krimpsiekte. A single, relatively large intraruminal dose of 10.0 g/kg induced krimpsiekte in one sheep. Treatment with 5.0 g/kg activated charcoal on two consecutive days did not prevent the development of krimpsiekte. A bufadienolide, tyledoside D, was isolated from semi-dried plant material.

Vermeersiekte caused by Geigeria burkei Harv. subsp. burkei var. hirtella merxm. in the northern province of South Africa.

The 1st field outbreak of vermeersiekte induced by Geigeria burkei Harv. subsp. burkei var. hirtella Merxm, is reported. It is also the first recorded outbreak of this disease in the arid sweet bushveld of the Northern Province of South Africa. The toxicosis was experimentally reproduced in a sheep following daily intraruminal administration of 2.5-5.0 g/kg dried, milled plant material for 18 consecutive days. Neither the sheep in the field outbreak nor the ewe in the experiment exhibited any signs of regurgitation of rumen contents (vermeersiekte). All developed only the stiff or paretic/paralytic forms of the disease. Serum activities of CK and GGT were slightly raised in clinically affected sheep (n = 11) during the field outbreak, and serum activities of AST, GLDH, GGT, LDH and CK increased in the ewe dosed with the plant material. Analysis of dried, milled Geigeria plant material confirms that this species is moderately nutritious.

Regional chromosome mapping of human collagen genes alpha 2(I) and alpha 1(I) (COLIA2 and COLIA1).

For the assignment of the genes for the pro-alpha 2(I) (COLIA2) and the pro-alpha 1(I) (COLIA1) collagens, cDNA and genomic DNA probes were used in in situ hybridization experiments on human prometaphase chromosomes. An improved staining method is reported for the simultaneous identification of chromosomes and the autoradiographic grains after the hybridization procedures. With this procedure more cells with higher resolution could be used for the assignment of genes by in situ hybridization. Statistical analysis of the grains located on respectively 660 and 302 metaphases using pro-alpha 2(I) and pro alpha 1(I) DNA probes, confirmed the assignment of these genes to human chromosomes 7 and 17. Analysis of the grain distribution on prometaphase chromosomes showed that the location of the pro-alpha 2(I) collagen gene is in the region 7q21.3-22.1. The location of the pro-alpha 1(I) collagen gene was found to be in band 17q21.31-2205.