First report of Pratylenchus penetrans (Nematoda: Pratylenchidae) associated with amaryllis (Hippeastrum x hybrid), in Portugal

Abstract

The world flower business is a highly dynamic industry and in Portugal yields around EUR 500 million a year (INE 2013). Root lesion nematodes (RLNs), Pratylenchus spp., are considered the third most important group of plant parasitic nematodes (PPNs) worldwide due to their large distribution, wide host range, and impact on economically important crops (Castillo and Vovlas 2007). The most recent report of RLNs parasitizing amaryllis (Hippeastrum spp.) was the amaryllis lesion nematode, Pratylenchus hippeastri, in Florida (U.S.A.) (Gozel et al. 2007). In 2016, soil and root samples of amaryllis hybrid Hippeastrum × hybridum cv. Benfica were collected from potted plants produced in greenhouses in Montijo (Setúbal Peninsula, Central Portugal), 50 km from Lisbon. Nematodes were extracted from soil samples according to the protocol PM 7/119 (1) (EPPO 2013) and identified to genus level based on morphometrical traits of 10 females and 11 males. DNA extraction was conducted from 10 nematodes separately, followed by PCR amplification and sequencing of the D2/D3 expansion segment of the 28S rRNA and 18S rRNA genes using the primers D2A and D3B (Nadler et al. 1999) and 1813F and 2646R (Holterman et al. 2006). The nucleotide sequences were deposited as MN756841 and MN756509 at the NCBI. RLNs were detected in all five samples in a concentration of around 100 nematodes/100 cm3 of soil. Some primary roots of affected plants showed small, elliptical, reddish-brown lesions and poor growth. More severely affected plants presented stunted, chlorotic foliage and had the cortical lesions enlarged to the point that rotting was severe and many roots were easily detached from the bulb; in some cases, there was not emergence from bulbs. The female body was moderately slender, almost straight when killed by gentle heat, lateral field with four straight lines, body annules distinct, lip with three annules, stylet knobs round, median bulb round, spermatheca round and filled with sperm, tail tapering to rounded terminus, sometimes with two striae. Body length was 558.99 ± 48.50 μm, body width 22.45 ± 2.02 μm, stylet length 18.52 ± 1.02 μm, and tail length 32.91 ± 7.50 μm. Values of a, c, and c′ ratios were 25.95 ± 2.53, 17.62 ± 3.45, and 38.49 ± 4.42; V (%) value was 80.92 ± 1.69. Males were smaller than females with body length 511.37 ± 29.90 μm, stylet length 17.55 ± 1.04 μm, and spicule length 18.53 ± 1.17 μm. No other PPN was found in the suspensions extracted from the soil samples. BLAST hits showed that partial sequence of D2/D3 rRNA loci (540 bp) was 96.2% identical (e-value 0.0) to Pratylenchus penetrans isolate MU2 (KP161612) and voucher T143 (KY828357). The partial sequence of 18S rRNA (650 bp) was 100% identical to P. penetrans isolate ILVO-Pp (MH983023) and 99.81% to P. penetrans Praty-A (AB661627). In the corresponding phylogenetic trees, both P. penetrans sequences clustered together with other species from the penetrans group (P. fallax, P. convallariae, and P. oleae) with good support (Janssen et al. 2017). Nevertheless, although all species within this group share many morphological characteristics (Subbotin et al. 2008), our specimens presented higher similarity, both morphological and molecular, with P. penetrans. So far, this RLN has been found associated mainly with potato fields in Portugal (Abrantes et al. 1987; Esteves et al. 2015); thus, to our knowledge, this is the first report of P. penetrans parasitizing amaryllis, in Portugal.