Molecular characterisation of 18 Pratylenchus species using rDNA Restriction Fragment Length Polymorphism

Abstract

The RFLP technique was used to establish a reliable diagnostic method for 18 Pratylenchus species: Pratylenchus agilis, P. bolivianus, P. brachyurus, P. coffeae, P. crenatus, P. fallax, P. goodeyi, P. loosi, P. mediterraneus, P. neglectus, P. penetrans, P. pratensis, P. pseudocoffeae, P. scribneri, P. subranjani, P. thornei, P. vulnus and P. zeae. The polymerase chain reaction (PCR) amplified the ITS regions from all species and populations examined and revealed large differences in length, ranging in size from approximately 900 to 1250 bp. The rDNA fragments were digested with five restriction enzymes (CfoI, DdeI, HindIII, HpaII, and PstI). All Pratylenchus species can be differentiated from each other by a combination of at least two enzymes. CfoI differentiated all nematode species with the exception of P. fallax, P. penetrans and P. pseudocoffeae. P. fallax was further separated by a DdeI restriction, and P. pseudocoffeae by a PstI digestion. Intraspecific RFLP were observed. Upon CfoI, DdeI, HindIII, or HpaII digestion, it was possible to separate the three P. coffeae populations studied from each other. La technique RFLP a été utilisée pour créer une méthode fiable de diagnostic pour 18 espèces de Pratylenchus: Pratylenchus agilis, P. bolivianus, P. brachyurus, P. coffeae, P. crenatus, P. fallax, P. goodeyi, P. loosi, P. mediterraneus, P. neglectus, P. penetrans, P. pratensis, P. pseudocoffeae, P. scribneri, P. subranjani, P. thornei, P. vulnus et P. zeae. La réaction de polymérisation en chaîne (PCR) a amplifié les régions de l’ITS pour toutes les espèces et populations étudiées et a mis en évidence de grandes différences dans la taille des gammes de longueur, de 900 à 1250 bp approximativement. Les fragments de rDNA ont été digérés à l’aide de cinq enzymes de restriction (CfoI, DdeI, HindIII, HpaII, and PstI). Toutes les espèces de Pratylenchus ont pu être différenciées les unes des autres par une combinaison d’au moins deux enzymes. CfoI a différencié toutes les espèces à l’exception de P. fallax, P. penetrans et P. pseudocoffeae. P. fallax a été ultérieurement séparé par une restriction DdeI, et P. pseudocoffeae par une digestion PstI. Des RFLP intraspécifiques ont été observés. Par les digestions CfoI, DdeI, HindIII, ou HpaII, il s’est révélé possible de séparer les unes des autres les trois populations étudiées de P. coffeae.

Morphological and molecular identification of cereal cyst nematodes from the eastern Mediterranean region of Turkey

“…The tubes were frozen at -80 °C for at least 10 min and then incubated at 65 °C for 1 h and 95 °C for 10 min consecutively in a thermocycler. After incubation, the tubes were centrifuged for 1 min at 14,000 rpm and kept at -20 °C until use (Waeyenberge et al, 2000).…”

Identification of Pratylenchus thornei, the cereal and legume root-lesion nematode, based on SCAR-PCR and satellite DNA

“…Although several DNA-based methods have been used to identify Pratylenchus species or populations (Al-Banna et al, 2004;Duncan et al, 1999;Ouri & Mizukubo, 1999;Pinochet, Cenis, Ferná ndez, Doucet, & Marull, 1994;Uehara et al, 1998;Waeyenberge, Ryss, Moens, Pinochet, & Vrain, 2000;), this is the first report of SCAR-based identification of a Pratylenchus species. The SCAR-PCR method described here has potential application to routine diagnostic purposes using DNA extracts from different life cycle stages, soil samples or infected plant Table 1 (except P. thornei isolates 1.4 and 1.5) was successfully hybridized with a DIG-11-dUTP labelled satDNA probe from P. thornei isolate 1.1.…”

Nematod taksonomisinde kullanılan moleküler markörler

“…Türkiye'den elde edilen 29 farklı kök-ur nematodu izolatı arasındaki farklılıklar ve benzerlikler AFLP yöntemi ile ortaya koyulmuştur (Devran et al, 2008). Önemli bitki paraziti nematod türlerini içeren Pratylenchus cinslerinin ayrımında da AFLP yöntemi sıklıkla ve yaygın olarak kullanılmıştır (Waeyenberge et al, 2000;Elbadri et al, 2002).…”