Bursaphelenchus hofmanni Braasch, 1998 associated with peat growth substrate in hops nurseries in the Czech Republic

Abstract

To date, seven Bursaphelenchus species have been re- ported in surveys of the Czech Republic (Cˇ ermák et al., 2013). However, the occurrence of some species, such as Bursaphelenchus hofmanni Braasch, 1998, is limited to single detections in imported coniferous wood (un- publ. data in Braasch, 2001). During a survey to deter- mine pathogenic agents on hops (Humulus lupulus L.) conducted by State Phytosanitary Administration (CZ) in hops fields and nurseries in 2012, B. hofmanni was found in a mixture of peat and soil in a hops seedling nurs- ery in Žatec (Bohemia, Czech Republic). The occurrence of Bursaphelenchus species in non-woody plants or sub- strates has been previously reported for species of the fungivorus group, such as B. gonzalezi Loof, 1964, B. hunti Giblin & Kaya, 1983 and B. fungivorus Franklin & Hooper, 1962. Species belonging to the hofmanni group are often associated with wood products such as packag- ing material (Gu et al., 2006) and, to our knowledge, this is the first time that this species has been found associated with peat substrate and soil. Nematodes were isolated from 60 g of peat substrate (3 parts peat to 1 part soil) associated with hops seedlings, and extracted using the Baermann funnel technique. Specimens belonging to Bursaphelenchus were killed and fixed in hot 4% formalin and transferred to pure glycerin according to De Grisse (1969). Two mature females and two males were used to establish a culture maintained in Botryotinia fuckeliana, growing on 5% malt extract agar (MEA). Nematode identification was confirmed by both mor- phological and molecular analyses (sequencing of the ITS, 18S and 28S rDNA loci). DNA was extracted as fol- lows: single specimens were collected into 20 μl of ex- traction buffer (10 mM Tris-HCl, pH 8.8; 1 mM EDTA; 1% Triton X-100 (v/v); 100 μg ml−1 Proteinase K) in a 1.5 ml Eppendorf tube. Each sample was ground using a micropestle and incubated at 55°C for 1 h and subse- quently at 95°C for 10 min. The mixture was used as a DNA template for PCR. The SSU, LSU and ITS regions of rDNA were amplified using a Phusion High Fidelity DNA polymerase (having 3′ → 5′ exonuclease activity) (NEB). GoTaq DNA polymerase (Promega) was added to a terminal 10 min/72°C elongation step to create an A- overhang for subsequent TA cloning.