PCR-based methods for detection of Erwinia psidii on guava

Abstract

Erwinia psidii causes bacterial blight of guava (Psidium guajava), one of the most important diseases of this crop in Brazil. Control measures are not effective, and dissemination often occurs through contaminated but asymptomatic propagating plant material. Considering the need for a reliable and sensitive method for detecting the pathogen in asymptomatic plant material, E. psidii-specific PCR primers were designed from a 355-bp fragment of the recombinase A gene (recA) amplified from E. psidii type strain. Primer pair Ep2L/2R only amplified DNA from E. psidii and its detection limit was 10 −5 ng/μL of purified DNA and 10 CFU (colony forming units) of bacterial cell suspension/mL. Three methods, conventional PCR, IC-PCR, and BIO-PCR were evaluated with the selected primers for their potential to detect E. psidii on guava leaves. BIO-PCR and conventional PCR were more sensitive and less time-consuming than IC-PCR. The detection limits on extracts of macerated guava leaves spiked with bacterial suspensions at different concentrations were 10 and 10 3 CFU/mL for BIO-PCR and conventional PCR, respectively. The PCR method here described could be useful for developing a protocol for early detection of this pathogen in asymptomatic guava plants.

Molecular detection of Erwinia psidii in guava plants under greenhouse and field conditions

“…Those four apparently healthy shoots probably harbor a bacterial population which was not large enough to induce symptoms (CHOUDHARY & SCHMIDT-DANNERT, 2010), or that modifies the environment to improve its habitat without causing disease, and at the same time suppressing or evading plant defenses (BEATTIE & LINDOW, 1999). However, this small population was within the detection limit of our BIO-PCR assay with primers Ep2L/2R which is 10CFU ml -1 or 20CFU g -1 of leaf tissue according to our previous research (SILVA et al, 2015). The other 93.3% of the samples were PCR negative but may contain a population below the detection limit of the primers.…”

“…Plants were covered with plastic bags to provide high humidity for 72 hours. Samples were collected from inoculated plants, regularly, at 0, 5, 10 and 15 days after inoculation, and submitted to conventional PCR and BIO-PCR using E. psidii-specific primers, Ep2L (3' CCA AAA AGC TTG GTG TGG ATA 5') and Ep2R (3' AAA TTG GTG ACT CGC ACA TG 5') and the protocols described by SILVA et al (2015). Plants were daily inspected for symptom development and disease progress.…”

“…Aliquots of 100µL were transferred to solid 523 culture medium, in triplicate Petri dishes, and incubated at 29ºC for 24 hours. Small but typical colonies of E. psidii can be observed after this period and there are no differences in the amplification results employing 24 or 48h of growth (SILVA et al, 2015). Plates were then washed with 2mL of distilled sterile water, and 1mL of this suspension was diluted to 10 -1 .…”

“…Plates were then washed with 2mL of distilled sterile water, and 1mL of this suspension was diluted to 10 -1 . PCR reactions were carried out by using E. psidii specific primers Ep2L/ Ep2R, which amplifies a ~200bp fragment of the rec A gene (SILVA et al, 2015). Amplification reactions were performed in 25µL, containing 0.1mM of dNTPs, PCR reaction buffer (10mM Tris HCl, 50mM KCl), 1.25mM MgCl 2 , 0.5µM of each primer, 0.5U of Taq DNA polymerase and 3µL of the diluted plate washings.…”

“…Considering the need for validated methods to detect E. psidii in asymptomatic plant material, recently it was designed a specific PCR protocol for E. psidii (SILVA et al, 2015). The objective of the present research was to show the application of this method in detecting the pathogen in inoculated guava plants by conventional PCR and BIO-PCR over time, and to validate BIO-PCR as a method suitable for inspection of guava orchards, under natural infection conditions.…”