Detecção de Erwinia psidii via enriquecimento em extrato de folhas de goiabeira e imunodifusão radial dupla

Teixeira, Ana Cristina Oliveira; Ferreira, Maria Lurdes; Marques, Ana Maria Campos

doi:10.1590/s1982-56762008000300006

Cited by 6 publications

(8 citation statements)

References 16 publications

(12 reference statements)

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“…Although the anti‐ Ep antibody reacted positively with some E. psidii strains from P. guajava , lack of cross‐reaction with endophytic bacteria from that particular host plant must be verified before implementing it for guava disease diagnosis. This consideration is supported by the observation that an antibody raised against E. psidii in a different study showed cross‐reaction with strains that belong to other taxonomic groups when using bacterial enrichment from leaf tissue and the double immunodiffusion assay (Teixeira, Ferreira, & Marques, ).…”

Section: Discussionmentioning

confidence: 82%

An agglutination test for fast and specific detection of Erwinia psidii

et al. 2019

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Dieback and wilt, caused by Erwinia psidii (Ep), is one of the most important emergent diseases of Eucalyptus spp. in Brazil. Currently, pathogen detection relies on isolation of bacteria from infected plant tissue and either identification based on morphological, physiological and biochemical tests or DNA amplification using the polymerase chain reaction (PCR), which in many cases is laborious and cumbersome. Considering the need for a simpler and more rapid, yet reliable, method for detecting the pathogen, we obtained a polyclonal antibody (anti‐Ep) and developed an agglutination test for specific detection of E. psidii. The antiserum was produced against the E. psidii strain LPF534 and tested against 101 E. psidii isolates from Eucalyptus spp.; three E. psidii isolates from Psidium guajava; 23 Ralstonia solanacearum and 18 Xanthomonas axonopodis isolates pathogenic to Eucalyptus spp.; and seven endophytic isolates from Eucalyptus spp., three of which are phylogenetically related to the genus Erwinia. Results of direct ELISA indicated that a concentration as low as 3.5 µg/ml of the anti‐Ep antibody was able to detect the E. psidii antigen and that the antibody did not cross‐reacted with other bacteria pathogenic and non‐pathogenic to Eucalyptus spp. In the agglutination test, the anti‐Ep antibody showed positive reaction with all strains of E. psidii tested whereas cross‐reaction with none of the strains that belong to other taxonomic groups was observed. The agglutination test showed a detection limit of 105 colony‐forming units (CFU)/ml, and its specificity was the same as that obtained by PCR amplification using E. psidii‐specific primers. These results demonstrate that the agglutination test developed here is a useful tool for specific, fast and inexpensive detection of E. psidii although only operational on pure bacterial suspensions and not yet directly from infected tissues.

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Section: Discussionmentioning

confidence: 82%

An agglutination test for fast and specific detection of Erwinia psidii

et al. 2019

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“…For guava bacteriosis, a good strategy would be the use of BIO-PCR to test asymptomatic material, whereas conventional PCR would probably meet the requirements of sensitivity for symptomatic plants. The molecular detection methods described here, such as conventional PCR or BIO-PCR, and immunological methods described by Teixeira et al (2008), could be integrated in a reliable and specific protocol for certification of guava planting material. This would assuredly contribute to the reduction of disease dissemination into new areas in Brazil.…”

Section: Discussionmentioning

confidence: 99%

“…For IC-PCR, we used the polyclonal antibody As 14.1 produced against E. psidii type strain IBSBF 435 (Teixeira et al 2008). The antibody was diluted 1:200 in a coating buffer (1.59 g/L NaCO 3 ; 2.93 g/L NaHCO 3 ).…”

Section: Ic-pcrmentioning

confidence: 99%

“…Asymptomatic young plants will show symptoms approximately 2 years after their installation in the orchard, which is the time required for fructification (Gonzaga Neto et al 2001). Therefore, more sensitive and accurate methods for pathogen detection on asymptomatic guava plants are necessary (Marques et al 2007;Teixeira et al 2008).…”

Section: Introductionmentioning

confidence: 99%

“…Moreover, a method of inoculation using detached shoots was described, which proved to be useful to confirm pathogenicity and was less time-consuming than plant inoculations (Marques et al 2007). Bacterial detection using serology with a specific polyclonal antibody has been described as well (Teixeira et al 2008); however, due to its low sensitivity, an enrichment step in plant extracts was necessary to detect bacterial cells in small numbers. PCR, either conventional or real time, has been broadly employed for successful detection and diversity studies of plant pathogenic bacteria, including Pantoea stewartii (Wensing et al 2010), Erwinia amylovora (Powney et al 2011;Dreo et al 2012;Gehring and Geider 2012;Wensing et al 2012;Hannou et al 2013) and E. piriflorinigrans (Barbé et al 2014).…”

Section: Introductionmentioning

confidence: 99%

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PCR-based methods for detection of Erwinia psidii on guava

Silva

Torres

Oliveira

et al. 2015

Trop. plant pathol.

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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.

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Movement of the bacterial blight pathogen Erwinia psidii in guava varieties differing in susceptibility

Macagnan

Ferreira

2018

Trop. plant pathol.

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Erwinia psidii causes bacterial blight of guava, an important disease of this fruit crop in Brazil. Pathogen spread is associated with the movement of infected but asymptomatic propagating plant material. The objective of this study was to compare bacterial movement inside the host tissue and symptom development in two guava varieties commonly grown in Brazil. Guava plants of Pedro Sato and Kumagai varieties were inoculated with strain IBSBF435 by deposition of a bacterial suspension droplet followed by puncture at the axils of the first fully expanded pair of leaves. Disease severity was assessed in the upper third part of the plant and stem fragments were cut at intervals of 1 cm above and below the inoculation point. Samples were collected at 0.5, 1, 2, 4, 6, 8, 10 and 12 days after inoculation. Presence of viable cells of E. psidii at each sampling point was evaluated using Bio-PCR with species-specific primers after a 24 h-enrichment step. Bacterial movement was detected on both directions in both varieties, however a positive correlation between movement and disease severity was observed only for Pedro Sato. Kumagai plants remained symptomless despite the presence of E. psidii above and below the inoculation site.

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Detecção de Erwinia psidii via enriquecimento em extrato de folhas de goiabeira e imunodifusão radial dupla

Cited by 6 publications

References 16 publications

An agglutination test for fast and specific detection of Erwinia psidii

An agglutination test for fast and specific detection of Erwinia psidii

PCR-based methods for detection of Erwinia psidii on guava

Movement of the bacterial blight pathogen Erwinia psidii in guava varieties differing in susceptibility

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