First report of the root lesion nematode, pratylenchus bolivianus, on aspalathus linearis in South Africa

Pratylenchus bolivianus Corbett, 1983 (Nematoda: Pratylenchidae) is an obligate plant-parasitic nematode that was originally described from soil around the roots of oats and potatoes in the Bolivian Andes, at an altitude of about 3,500 m above sea level (Corbett 1983). More recently an amphimictic population of this species was reported and characterized from sword ferns in Florida (Troccoli et al. 2016). Aspalathus linearis (Burm. f.) Dahlgren, commonly called rooibos, is a unique herbal plant that is endemic to the Fynbos biome of South Africa. The plant is the only source of aspalathin, which is a unique phenolic compound that possesses exceptional health benefits that include antiaging properties and the ability to reduce the risk of type 2 diabetes and associated cardiovascular diseases (South African Rooibos Council 2016). Currently, 36,000 ha of rooibos are grown, yielding about 14,000 metric tons annually. Increased demand, owing to a large export market of the fermented and green rooibos tea in Europe and the United States, has led to increased acreage of rooibos in the Cyderberg and Sandveld areas of the Western Cape province of South Africa. In December 2016, an amphimictic population of P. bolivianus was extracted from soil samples obtained from a rooibos orchard (32°10′12.5″S, 18°49′03.6″E) at an altitude of 375 m above sea level during a field survey of rooibos monocultures in the Cyderberg area of the Western Cape. A high population of P. bolivianus (800/250 cm3 of soil) was found in the soil samples that were taken from the root zone of the tea plants. A modified Baermann tray method was used for the extraction of nematodes from soil samples. The morphological and morphometric characters of the nematodes are similar to those obtained from the amphimictic population reported from Florida (Troccoli et al. 2016). Both male and female nematodes were observed. They are straight or slightly curved with tails that are asymmetrically tapered and possess the characteristic thickened cuticles consisting of about 16 to 19 annuli. Stylet knobs are rounded, median bulb is ovate, and pharyngeal gland lobe overlaps the intestine dorsally and ventrally. Vulva is located posteriorly, and the ovary is monoprodelphic. The spermatheca are not very conspicuous, and the postuterine sac ranges between 22 and 28 µm. Male reproductive features are composed of an outstretched testis, paired spicule, and a curved gubernaculum. Tails are conoid and enveloped with a bursa. Polymerase chain reaction (PCR) was used to verify the identification of the species. DNA was extracted from individual nematodes using a modified method (Nguyen 2007). PCR amplification of the D2-D3 expansion segments of 28S rRNA gene was done using KAPA2 Robust Hotstart ReadyMix (KAPA Biosystems). The primers used were D2A 5′-ACA AGT ACC GTG AGG GAA AGT TG-3′ (forward) and D3B 5′-TCG GAA GGA ACC AGC TAC TA-3′ (reverse). The sequence generated from the PCR product (MG871467) corresponded 99% with the Troccoli et al. (2016) sequence (KU198956, KU198957) in the GenBank database. Further studies were conducted to determine the host status of rooibos to P. bolivianus under glasshouse conditions. The nematode was cultured in vitro on carrot discs (Fourie et al. 2003) to produce inoculum for a pot experiment. Three rooibos seedlings were inoculated with 150 nematodes per plant. After a period of 40 days after inoculation, approximately 480 nematodes/5 g of root were recovered from each rooibos seedling. To our knowledge, this is the first report of P. bolivianus in Africa and the first report on rooibos.