Detection of Two Fungal Biocontrol Agents against Root-knot Nematodes by RAPD Markers

Abstract

The strain ZK7 of Pochonia chlamydosporia var. chlamydosporia and IPC of Paecilomyces lilacinus are highly effective in the biological control against root-knot nematodes infecting tobacco. When applied, they require a specific monitoring method to evaluate the colonization and dispersal in soil. In this work, the randomly amplified polymorphic DNA (RAPD) technique was used to differentiate between the two individual strains and 95 other isolates, including isolates of the same species and common soil fungi. This approach allowed the selection of specific fragments of 1.2 kb (Vc1200) and 2.0 kb (Vc2000) specific for ZK7, 1.4 kb (P1400) and 0.85 kb (P850) specific for IPC, using the random Primers OPL-02, OPD-05, OPD-05 and OPC-11, respectively. These fragments were cloned, sequenced, and used to design sequence-characterized amplification region (SCAR) primers specific for the two strains. In classical polymerase chain reaction (PCR), with serial dilution of ZK7 and IPC pure culture DNAs template, the detection limits of these oligonucleotide SCAR-PCR primers were found to be 10, 1000, 500, 100 pg, respectively. In the dot blotting, digoxigenin (DIG)-labeled amplicons from these four primers specifically recognized the corresponding fragments in the DNAs template of these two strains. The detection limit of these amplicons were 0.2, 0.2, 0.5, 0.5 mug, respectively.

Fungal chitinases and their biological role in the antagonism onto nematode eggs. A review

“…In addition, molecular methods reveal identification and taxonomic affinities, and genetic diversity among strains, and provide tools for the monitoring of biopesticides in the environment (to track the movements of strain and to test survival and the genetic stability over successive generations of propagation) (Bidochka et al 1999;Gunasekera et al 2000;Hirsch et al 2001;Mauchline et al 2002;Punja and Utkhede 2003;Atkins and Clark 2004;Atkins et al 2005;Inglis et al 2005;Zhu et al 2006). Furthermore, molecular techniques are being used in microbial ecology to understand the soil ecosystem and to achieve an integrated management of soil microbial populations (Atkins and Clark 2004;Barea et al 2005).…”

“…Interestingly, chitin is present only in the eggshell of nematodes, and for this reason it is the target of possible control strategies (Spindler et al 1990, Veronico et al 2001Khan et al 2004;Morton et al 2004;De Jin et al 2005). Biological control and other management methods are becoming more and more important because the use of methyl bromide as a soil fumigant (for the control of nematodes, soil borne diseases, and weeds in agriculture and horticulture) was banned in 2001 due to its adverse environmental effects (Zhu et al 2006). Substantial economic losses can be caused by root-parasite nematodes.…”