ABSTRACT
The stem and bulb nematode Ditylenchus dipsaci is a destructive nematode pest on many crops and is internationally quarantined in many countries, whereas Ditylenchus weischeri, only known to infect a weed plant (Cirsium arvense), is an unregulated nematode species with no known economic importance. In this study, we used comparative genomics to identify multiple gene regions and developed novel real-time PCR assays for the detection of D. dipsaci and D. weischeri. We sequenced the genomes of two mixed-stage nematode populations of D. dipsaci and two mixed-stage nematode populations of D. weischeri. The assembled genomes of D. dipsaci were 228.2 Mb and 239.5 Mb, and the genomes of D. weischeri were 177.0 Mb and 196.3 Mb. Depending on the species, 21,403-27,365 gene models were predicted. Using orthologous group analysis, single-copy and species-specific genes were identified. Primers and probes were designed targeting two species-specific genes in each species. The assays detected as low as 12 pg of DNA from the target species, or as few as five nematodes, with a Cq of 31 cycles or less. Our study provides genome data for two additional D. dipsaci isolates and two D. weischeri isolates, and four new and validated molecular assays to be used for rapid detection and identification of the two species.
ABSTRACT
The root-lesion nematode of the genus Pratylenchus Filipjev (1936) has a worldwide distribution and cause severe production constraints on numerous important crops. In 2013-14, during a survey of the apple nurseries and orchards in center of Tunisia (Kairouan, Zaghouan, Monastir and Kasserine), 70 different roots and soil samples were collected. The populations of root-lesion nematode were identified on the basis of their morphological and morphometric characters, and by molecular methods. Microscopic observation of females and males demonstrated the occurrence of Pratylenchusd vulnus on apple trees. The ribosomal DNA D2-D3 expansion segments of the 28S rRNA and of the Pratylenchus populations were PCR amplified and sequenced. The sequences were compared with those of Pratylenchus species in the GenBank database with high similarity (99%). This comparison reconfirmed the morphological identifications. Phylogenetic studies placed those populations with P. vulnus. This is the first report of P. vulnus infecting apple in Tunisia.The root-lesion nematode of the genus Pratylenchus Filipjev (1936) has a worldwide distribution and cause severe production constraints on numerous important crops. In 2013-14, during a survey of the apple nurseries and orchards in center of Tunisia (Kairouan, Zaghouan, Monastir and Kasserine), 70 different roots and soil samples were collected. The populations of root-lesion nematode were identified on the basis of their morphological and morphometric characters, and by molecular methods. Microscopic observation of females and males demonstrated the occurrence of Pratylenchusd vulnus on apple trees. The ribosomal DNA D2-D3 expansion segments of the 28S rRNA and of the Pratylenchus populations were PCR amplified and sequenced. The sequences were compared with those of Pratylenchus species in the GenBank database with high similarity (99%). This comparison reconfirmed the morphological identifications. Phylogenetic studies placed those populations with P. vulnus. This is the first report of P. vulnus infecting apple in Tunisia.
ABSTRACT
The genus Ditylenchus has been divided into 2 groups: the Ditylenchus triformis-group, and the Ditylenchus dipsaci-group based on morphological and biological characters. A total of 18 populations belong to 5 species of Ditylenchus was studied: Ditylenchus africanus, Ditylenchus destructor, Ditylenchus myceliophagus and dipsaci, Ditylenchus weischeri, the first 3 belong to the Ditylenchus triformis-group, the last 2 the Ditylenchus dipsaci-group. The species of Ditylenchus triformis-group were cultured on fungi, while the species from Ditylenchus dispaci-group cultured on excised roots of plant hosts in petri dish. DNA sequences of regions of the nuclear ribosomal first internal transcribed spacer (ITS1) and the small subunit 18S were PCR amplified, sequenced and the phylogenetic analyses also including the sequences of the closely related species from the GenBank. The randomly amplified polymorphisms of genomic DNA (RAPD) were also generated. Two clusters or clades corresponding to the 2 groups were consistently observed with significant statistical support from the 3 datasets. The phylogenetic analysis also revealed that the genus is paraphyletic, separating the 2 groups by species of Anguina and Subanguina.