Distribution and diversity of root-lession nematode (Pratylenchus spp.) associated with Miscanthus × giganteus and Panicum virgatum used for biofuels, and species identification in a multiplex polymerase chain reaction

Abstract

The distribution of Pratylenchus spp. from bioenergy field plots in six states (Illinois, Iowa, South Dakota, Kentucky, Tennessee, Georgia) of the USA were surveyed. The species were identified based on morphology and morphometrics and further characterised based on fragment sequences of the 28S rRNA of the D2-D3 region. The region revealed variations in sequencing information that supports the morphological identification. In this work, six Pratylenchus spp. were detected: Pratylenchus brachyurus, P. crenatus, P. hexincisus, P. neglectus, P. penetrans and P. scribneri. Pratylenchus scribneri, P. crenatus, and P. penetrans were distributed most widely, with detection of 34, 29 and 15%, respectively. Pratylenchus hexincisus, P. brachyurus and P. neglectus were distributed sporadically, with detection rates of 10.0, 2.6 and 2.0%, respectively. A one-step multiplex PCR was developed for the simultaneous detection of P. scribneri, P. crenatus and P. penetrans. Sequence data from this research and NCBI were used to generate different primer sets that are species-specific. We have therefore designed three sets of primers that discriminate P. scribneri, P. crenatus and P. penetrans in multiplex PCR. All the tested primers have shown specificity and have no cross-reaction with the nontarget species. When used in a uniplex, duplex and triplex PCR, the selected three primers gave a unique electrophoretic DNA banding pattern characterised by a single DNA fragment for P. scribneri (ca 750), P. crenatus (ca 690), and P. penetrans (ca 520). The method could be used for routine diagnostic programmes.

Phytotechnology with Biomass Production

“…are known as very dangerous plant pathogens responsible for lesion disease that rank third place in the world list of the most economically important species (Jones & Fosu-Nyarko, 2014). This finding for Ukraine and Poland was in agreement with results obtained earlier by Mekete et al (2011a) in the US.…”

Characterization of Pratylenchus crenatus and P. neglectus (Nematoda: Pratylenchidae) associated with wheat crop

“…Sequence analysis of D2/D3 expansion segments of P. crenatus (accession numbers KM580543, KM580544, KM580545) among the three Czech populations also did not reveal any variation. The sequences of D2/D3 of P. crenatus differs from published sequences EU130852, EU130853 , HM921215 (Mekete et al, 2011) by 0.45 to 1.36 % and these six sequences (KM580543-KM580545, EU130852, EU130853, HM921215) varied by 0 -1.82 %. Sequences of 18S gene of P. neglectus among fi ve populations (accession numbers KM580538-KM580542) from the Czech Republic differ by 0 -0.14 %.…”

“…In recent years it has become a standard to identify species on the basis of morphological and molecular data with more emphasis on molecular work. Therefore in this work species specifi c PCR was performed to confi rm the morphological identifi cation of the both species using published primers (Al-Banna et al,2004;Mekete et al, 2011). Moreover 18S and 28S partial genes of ribosomal DNA have been analyzed and compared.…”

“…Morphological methods can be time-consuming and impractical when rapid results are required. The use of molecular diagnostic tools like PCR with species specifi c primers (Al-Banna et al, 2004, Mekete et. al., 2011 and sequencing of diagnostic rDNA regions in combination with phylogenetic analyses contributes to overcome such a problem.…”

Genetic diversity of Bursaphelenchus cocophilus in South America

“…based on this molecular marker. In addition to the ITS-1/2 region, the D2-D3 region of rDNA LSU has also been studied for plant-parasitic nematode taxonomy and species identification (Kaplan et al, 2000;de la Peña et al, 2007;Ye et al, 2007;Múnera Uribe et al, 2010;Mekete et al, 2011;Oliveira et al, 2011). Based on the LSU region, we could clearly distinguish Brazilian and Colombian populations.…”