Detection of two fungal biocontrol agents against root-knot nematodes by RAPD markers.
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.
Zhu
Ming Liang
ML
Yunnan Tobacco Science Research Institute, YNTC, 653100 Yuxi, Yunnan Province, P. R. China.
Mo
Ming He
MH
Xia
Zhen Yuan
ZY
Li
Yun Hua
YH
Yang
Shu Jun
SJ
Li
Tian Fei
TF
Zhang
Ke Qin
KQ
D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D009349:Nematode Infections / Q000517:prevention & control*; D010143:Paecilomyces / Q000235:genetics / Q000254:growth & development*; D010572:Pest Control, Biological / Q000379:methods*; D010935:Plant Diseases / Q000469:parasitology*; D018517:Plant Roots / Q000469:parasitology; D019105:Random Amplified Polymorphic DNA Technique; D017422:Sequence Analysis, DNA; D014026:Nicotiana
D004271:DNA, Fungal