First Report of the Hop Cyst Nematode Heterodera humuli from Common Nettle Urtica dioica in Turkey

Abstract

Cyst nematodes (Heterodera spp.) are one of the most important groups of plant parasitic nematodes in many countries throughout the world (Subbotin et al. 2010). The hop cyst nematode Heterodera humuli Filipjev. 1934, the most common plant parasitic nematode found on hops (Humulus lupulus L.), was reported from Russia, Australia, the United States, Canada, New Zealand, South Africa, and several European countries (England, Belgium, Germany, Italy, Spain, Greece, and the Czech Republic). However, it has not been found in Turkey. The host range of this nematode was noted to be predominantly in the Urticaceae family (Sen 1968) and includes hops and Urtica dioica L. (common nettle); both plants are widespread in Turkey. Hops have been commercially cultivated in Bilecik city (Marmara Region) since 1965, and common nettle is used by people as an herbal medicine and food. In 2017, we randomly surveyed a naturally occurring common nettle growing area in Ordu province (N 40°58′36.55″, E 37°0.59′51.23″) located in the Black Sea Region of Turkey. Cyst-forming nematodes were found on the roots of nettle. Cysts and infective second-stage juveniles (IJ2s) were collected from the infested roots and soil by modified flotation (Jenkins 1964) and sieving methods (Byrd et al. 1966) and identified by morphometric and molecular studies. Morphometrics (mean ± SD) of J2 (n = 15): body length (L) 407.5 ± 28.5 µm; stylet length 23.6 ± 1.4 µm; maximum body width 20.1 ± 1.1 µm; excretory pore (Exp) 94.4 ± 10.3 µm; body width at anus (ABW) 12.5 ± 0.4 µm; tail length 52.8 ± 3.7 µm; hyaline tail length 28.6 ± 3.0 µm. Cysts were lemon shaped, and their coloring darkened with age. Morphometrics of cysts (n = 15): length (without neck) 509.4 ± 48.9 µm; width 390.1 ± 45.6 µm; neck length 93.2 ± 14.1 µm. The vulval cone was bifenestrate without bullae. Semifenestrae were circular. Morphometrics of vulval cone (n = 10): fenestral length = 54.3 ± 3.2 µm; fenestral width 26.9 ± 5.5 µm; vulval slit = 38.3 ± 4.7 µm; underbridge length = 41.13 ± 8.6 µm; semifenestral length 22.6 ± 2.5 µm. Morphometrics of eggs (n = 10): length 93.3 ± 3.7 µm; width 38.7 ± 1.4 µm. Morphometrics of male (n = 1): body length 914 µm; maximum body width 28.86 µm; stylet length 27.2 µm; spicule 33.64 µm; gubernaculum 7.81 µm. These measurements were highly similar with those for H. humuli as described by Sen (1968). Genomic DNA was extracted from individual juveniles (n = 5). The ribosomal region spanning the internal transcribed spacer (ITS) 1, 5.8S gene, and ITS2 was amplified with forward primer TW81 (5′-GTTTCCGTAGGTGAACCTGC-3′) and reverse primer AB28 (5′-ATATGCTTAAGTTCAGCGGGT-3′). A 1,100-bp fragment was obtained, and the sequence was compared with those in GenBank using BLAST. Results revealed a sequence similarity of 100% with H. humuli (e.g., GenBank accession nos. AY347926.1 and AF498384.1) with 100% query cover. Morphological and molecular identification demonstrated that the population of cyst nematodes from Turkey was H. humuli. Reproduction on nettle was demonstrated by inoculating 500 eggs per plant onto 5-week-old nettle grown in pots of sterilized sand at 23°C. Noninoculated plants were used as controls. Sixty days after inoculation, cysts were harvested from the plants, and extraction produced approximately 1,000 eggs. To our knowledge, this detection represents a new record of H. humuli in Turkey. The nematode may be expected to reduce the quality of the nettle plant, but regional impacts on yield still need to be investigated.