Detection of Meloidogyne incognita and Pochonia chlamydosporia by fluorogenic molecular probes*

Abstract

Fluorescent molecular probes were applied for detection of the plant parasitic nematode Meloidogyne incognita and the nematode-egg parasitic fungus Pochonia chlamydosporia var. chlamydosporia . A region in the M. incognita rDNA including ITS2 was selected for amplification and recognition with a real-time PCR assay, based on a combination of three specific motifs, each recognized by a specific fluorescent probe. Similarly, a Scorpion probe was designed for the RT-PCR quantification of P. c . chlamydosporia . For this purpose, the ITS-2 rDNA gene of the fungus was sequenced from a number of Italian isolates. A conserved region unique for P. c . chlamydosporia found in the ITS-2 rDNA gene was used. The probes allowed recognition of single juveniles of M. incognita and of the mycelium-or soil-extracted fungal DNA. The potentialities of the detection procedures are discussed.

Detection and biocontrol potential of Verticillium leptobactrum parasitizing Meloidogyne spp.

“…Molecular detection has the potential to discriminate between resident microbial populations and cells or propagules derived from micro-organisms intentionally released for biocontrol purposes (Ciancio et al 2005). When coupled to transcripts detection it can even yield informations not only about presence of a microorganism but also about its on-going biochemical activities.…”

“…This technology opens increasing opportunities to detect and study antagonistic fungi in their native environment, as well as to identify specific biochemical and metabolic pathways. By combining the sensitivity of conventional PCR with the generation of signals it is possible to detect in a short time specific targets, as shown by protocols developed for NF identification and quantification (Ciancio et al 2000(Ciancio et al , 2005Rosso et al 2007;Zhang et al 2006;Atkins et al 2009). …”

“…Efficient methodologies need to be optimized to study NFs ecology in soil and, in case of endophytic species, also in the root tissues (Atkins et al 2003a;Ciancio et al 2005;Rosso et al 2007;Lopez-Llorca et al 2008;Macia-Vicente et al 2009). The emergence of new DNA-based technologies involving the use of the polymerase chain reaction (PCR) allowed the development of techniques that are independent from the culture properties and/or density of target fungi (Van Elsas et al 2000).…”

Formulation of Pochonia chlamydosporia for Plant and Nematode Management

“…DNA-based technologies involving the use of the polymerase chain reaction (PCR) allow the development of detection techniques that are independent from the culture properties and/or density of target fungi. PCR-based detection assays have been described and applied to a wide range of fungi, including some P. chlamydosporia isolates (Hirsch et al 2001;Ciancio et al 2005;Atkins et al 2005Atkins et al , 2009Cordier et al 2007;Rosso et al 2007). To check for bacterial contaminants, PCR with universal 18S bacterial primers like 27F-1492R may be used.…”

A Review of Root Lesion Nematode: Identification and Plant Resistance

“…Sequence specific DNA probe combining hybridization was once used to detect and identify nematode [15][16][17]. However, this method required plenty of nematode to extract DNA and radioactivity examination equipment.…”