Detecting and monitoring endophytic colonization by Pseudomonas putida BP25 in black pepper (Piper nigrum L.) using quantitative real-time PCR

Abstract

A quantitative real-time PCR assay was developed to quantify Pseudomonas putida BP25, an antagonistic endophyte against a broad range of pathogens in black pepper such as Phytophthora capsici, Colletotrichum gloeosporioides, Rhizoctonia solani, Gibberella moniliformis, Athelia rolfsii and a plant parasitic nematode, Radopholus similis. The real-time PCR primers were designed based on the16S rRNA sequences of P. putida strains and specificity of the primers was confirmed. The detection limit of the assay was found to be 1 pg. The assay detected and quantified the bacterial colonization in the roots at weekly intervals after inoculation. The P. putida DNA was quantified to be 0.4 ng in roots corresponding to 5.4 log10 CFU g-1 at 7th and 14th day after inoculation (DAI). A decline in endophyte population was observed during 21st and 28th DAI and the DNA concentration ranged from 3.7-4.6 pg corresponding to 3.4-3.5 log10 CFU g-1 of root. No amplification could be obtained in stem and leaf samples. The newly developed real-time PCR could be useful for detection, quantification and monitoring of endophytic P. putida BP25 in different plant tissues.