Molecular and morphological characterisation of Scutellonema bradys from yam in Costa Rica and development of specific primers for its detection

Abstract

The yam nematode, Scutellonema bradys, which can cause dry rot disease of yam (Dioscorea spp.), was recorded for the first time from Costa Rica in four species of yam occurring in the Atlantic and north regions. Morphometric measurements from two populations from each region using ten female and 11 male characters corresponded with previous descriptions of this species. Canonical discriminant analysis of the female morphometric data separated the populations by region, whereas no separation by region was evident using the male data. Analysis of DNA sequences from the ITS region indicated that populations from Costa Rica were monophyletic with S. bradys from West Africa and clearly distinct from other Scutellonema species. No genetic separation by geographic region or Dioscorea species host was observed between Costa Rica populations. Species-specific primers were developed from the ITS region and supported the identity of 17 populations from 15 locations in Costa Rica as S. bradys: 14 populations from D. alata (greater or water yam) and one each from D. trifida (white yampee), D. cayenensis (yellow yam) and D. rotundata (white yam). Yam production in Costa Rica began in the Atlantic region, where the yam nematode was likely introduced from the Caribbean, progressively spreading to other locations through the use of infected vegetative planting material.

Morphological and molecular characterisation of Scutellonema species from yam (Dioscorea spp.) and a key to the species of the genus

“…Using only morphological-based identification, the margin of error for misidentifying as S. bradys the cohabiting Scutellonema species from the rhizosphere of yam is high. However, by combining molecular and morphological data, S. bradys appears to be a well defined monophyletic group with its morphological and morphometric characters aligning clearly with the available data (Sher, 1964;Van den Berg, 1973;Germani et al, 1985a;Humphreys-Pereira et al, 2014).…”

Characterisation of some Scutellonema species (Tylenchida: Hoplolaimidae) occurring in Botswana, South Africa, Costa Rica and the USA, with description of S. clavicaudatum sp. n. and a molecular phylogeny of the genus

“…In this study, the nematode was found in soil particles adhering to the surface of roots of Bermuda grass. It is important to note that in silico analysis of ITS sequences shows that specific primers recently developed for detection of S. bradys by Van den Berg et al (2013) and Humphreys-Pereira et al (2014) might not work for populations from the USA.…”

“…The newly obtained sequences for each gene (D2-D3 of 28S rRNA, ITS rRNA and the COI mtDNA) were aligned using ClustalX 1.83 (Thompson et al, 1997) with their corresponding published gene sequences (Chen et al, 2006;Subbotin et al, 2006Subbotin et al, , 2007Bae et al, 2009;Van den Berg et al, 2013;Humphreys-Pereira et al, 2014;Coyne et al, 2016;Tzortzakakis et al, 2016 and others). Outgroup taxa for each dataset were chosen based on previously published data (Subbotin et al, 2006).…”

Molecular diversity of Scutellonema bradys populations from Benin, based on ITS1 rDNA and COI mtDNA

“…The DNA of the samples for which sequences revealed the presence of S. bradys was used for PCR using the forward and reverse S. bradys -specific primers, SBVF1 (5′- CCTCTCCATGTGTCCCACTT-3′) and SBLR (5’TGCACAAGGCACACATCT-3′) developed by Humphreys-Pereira et al ( 2014 ). PCR was carried out in a volume of 25 μl comprising 2 μl of DNA with 23 μl of Master Mix prepared following the Fermentas protocol: 19.5 μl of water; 2.5 μl of 10X Pfu buffer with MgSO4; 0.5 μl of dNTPs; 0.15 μl of the forward and reverse primers as mentioned above; 0.2 μl of Pfu DNA polymerase.…”

“…Genetic diversity was reported also by Kolombia et al ( 2017 ) who studied the D2-D3 expansion segments of the 28 s rDNA and the COI sequence of the mtDNA of Scutellonema populations retrieved from yam fields in Nigeria and Ghana. Recently, S. bradys -specific primers have been developed and demonstrated to work with S. bradys populations from Costa Rica (Humphreys-Pereira et al 2014 ). Its applicability for identification of Benin’s populations is unknown.…”