Turfgrasses are among the most widely used ornamental plants in the world, serving important functions in soil stabilization and providing safe surfaces for recreational activities (Zeng et al., 2012a). Moreover, the quality of the turf in the sports areas, mainly football fields and golf courses, is crucial and any imperfection can have a huge impact (Oliveira et al., 2018). Of all turfgrass pests, nematodes are probably the least understood and most often overlooked. Due to this, nematode symptoms are often misdiagnosed because they appear similar to other factors such as localized soil conditions, fungal diseases, or insect attack. Ring nematodes can cause significant damage to grass if population densities are high enough. Feeding results in tiny lesions on the roots, and under high nematode pressure, roots can become discolored and stubby (Crow, 2005). Nematode damage usually appears as irregularly shaped declining areas in the lawn that may enlarge slowly over time. Grass will die under extreme nematode and environmental stress and often, as the grass thins out, spurge and other weeds may become prominent.
The plant parasitic nematode
In Winter 2016, yellow and declining patches were observed in the yards of a complex of houses in Caxias (Lisbon district), Portugal (Fig. 1). The instant roll-out lawn turf in the yard was a mixture provided by a private lawn and landscape company, with tall fescue as the predominant species (70%
To identify the problem, soil samples were collected after digging up to the depth of 10 to 15 cm along the margins of the chlorotic areas. Each sample consisted of 5 to 8 cores (30 mm diameter) sampled at roughly equal intervals following the patches across an area of 1000 m2 or less. Six composite soil samples were placed in polyethylene bags and immediately brought to laboratory for analysis. A 500 mL subsample was taken from each composite sample and processed to identify and count plant parasitic nematodes. Nematodes were extracted following the sieving and decanting technique (EPPO, 2013). The nematodes were collected from all samples and identified to genus level. At least 10 were placed in a drop of water on a glass slide and gently heat killed for morphological characterization using a brightfield light microscope (Olympus BX-51, Hamburg, Germany) and photographed with a digital camera (Olympus DP, Hamburg, Germany).
To confirm the morphological identification, DNA from selected female specimens was used for sequencing of the D2/D3 expansion segments of the 28S ribosomal RNA, following Subbotin et al. (2005). In total, 10 nematodes (juveniles and females) were handpicked and transferred individually to Eppendorf tubes with 10 µl of sterilized water, for DNA extraction, PCR amplification, and sequencing. Each nematode was frozen in liquid nitrogen and homogenized with a micro-pestle (Eppendorf, Hamburg, Germany). The homogenate was incubated at 56°C in lysis buffer and 100μg ml-1 proteinase K for 1 hr. After incubation, total genomic DNA extraction was performed using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. DNA quantity and purity were checked using a NanoDrop 2000 UV-V is Spectrophotometer (Thermo Fisher Scientific, Massachusetts, USA). PCR mixtures, using the primers D2A (5′-ACAAGTACCGTGGGGAAAGTTG-3′) and D3B (5′-TCGGAAGGAACCAGCTACTA-3′) (De Ley et al., 1999) and thermal cycling conditions were performed as described previously by Inácio et al. (2016). PCR products were cleaned using the QIAquick PCR Purification Kit (Qiagen, Hilden-Germany), according to the manufacturer’s instructions. Amplicons were sequenced at STABVida Sequencing Laboratory (Lisbon, Portugal) on a DNA analyzer ABI PRISM 3730xl (Applied Biosystems). Nucleotide sequences were edited and analyzed using BioEdit v7.2.0 (Hall, 2007). Sequencing of the one-single nematode PCR products resulted in sequences totally identical thus only one was included in this study. The resulting D2/D3 rDNA sequence was compared against a set of reference sequences of
A phylogenetic tree was estimated under maximum likelihood (ML) based on the model that best fit the data, which was identified as a Tamura 3-parameter model also integrated in MEGA 6. In total, 1,000 bootstrap replicates were performed to test the support of each node on the trees. One species of the genus
From the recovered nematodes (50-60 nematodes/100 ml soil), morphological characterization showed the affinity of specimens with
Comparison of the gross range of morphometrics recorded of the Portuguese population of
Character/ratio | Gross range (10♀♀) | As per Loof and De Grisse (1989) |
---|---|---|
L | 685 ± 20.7 (650–700) | 590 (560–670) |
a | 13.9 ± 0.9 (12.0–14.6) | 13.2 (12.0–15.0) |
b | 4.2 ± 0.1 (4.1–4.3) | 4.2 (4.0–4.3) |
c | 20.8 ± 1.3 (19.4–23.3) | 21.0 (19.0–23.4) |
%V | 92 ± 0.9 (91–93%) | 92 (91–93%) |
Stylet | 77.5 ± 2.1 (75.0–81.0) | 81.0 (77.5–85.0) |
R | 113 ± 1.9 (108–114) | 109 (104–116) |
Ran | 5–8 | 7–8 |
RVan | 0–4 | – |
RV | 7–8 | 8–9 |
Notes: R, total number of body annules; Ran, number of body annules between anus and tail tip; Rvan, number of body annules between vulva and anus; RV, number of body annules between vulva and tail tip.
One of the nucleotide sequences obtained in this study was deposited into the GenBank database (NCBI) under the accession number MG647831. Amplification of the D2/D3 rDNA loci resulted in a PCR product of 552 bp and the nucleotide sequence showed a similarity range between 88 and 98% with isolates of
The molecular phylogenetic status of samples as inferred from their D2/D3 sequences is presented in Figure 3. The phylogram reveals one clade, supported by a bootstrap value of 100%, that includes only two isolates of
The identification of