Sensibilidade do método de obtenção das células bacterianas e da técnica de PCR para detecção de Curtobacterium flaccumfaciens pv. flaccumfaciens em sementes de feijão

Abstract

RESUMOAtu a lmente , existe a necessid a de de se desenvolv er métodos sen sí ve is, b a r a t os, r ep ro du z ív ei s e r á p id os pa ra a d e te cç ã o d e fitobactérias em sementes. O objetivo deste tra balho foi otimiza r um método de obtenção das células bacterianas e a técnica de PCR p a ra d e t ec ç ã o de C u rt o b a c t e r i u m f l a c c u m fa c i e n s p v. flaccumfaciens (Cff), em sementes de feijão. Foi utilizado o primer CffFOR2-REV4 , desenhado a partir do fra gmento amplificado via PC R b a se a do na se qü ê nci a r epet iti va (Re p-P CR) . Ava l ia r a m-se ta mbém qu a tro métodos de prepa ra çã o dos extra tos de sementes d e fe i jã o pa r a a o bt e n çã o cé l u la s de C ff : 1 ) e x tr a t o b r u t o d e se me n t e s; 2 ) e x t r a to c o nc e n t ra Essa s du a s técnica s, a lém de detecta r a ba ctéria , a presenta m a lta sensibilida de, detecta ndo a té 1 semente inocu la da artificialmente a rti ficia lm ente c om Cff em 9 9 9 seme ntes sa di a s. Ana lisa ra m -se dezessete lotes comercia is de sementes de feijã o pelo método de co nc en tra çã o de ex tra to p or ce nt rifu g a ç ã o, send o qu e e m do ze detectou-se a bactéria Cff, observado pela presença de bandas com 3 0 6 p b. Porta nto, foi possível otimiza r u m métod o de obt ençã o da s célula s bacterianas e técnica de PCR pa ra detecção de Cff em sem e n te s de fei j ã o, q u e sej a sen sí ve l , r e p ro d u zí v e l e de fá c i l execução, que poderá ser utilizado rotineira mente em labora tórios de a ná lises de sementes.Keywords: Detection of bacteria inoculation, water restriction. ABSTRACTCu rrently, there is a need to develop sensitive methods, inexpensive, reproducible and rapid detection of phytobacteria seeds. The objective of this study was to optimize a method for obtaining ba cteria l cells a nd the PCR technique to detect Cu rto b a c te riu m flaccumfaciens pv. flaccumfaciens (Cff), in bean seeds using PCR. It was used one pairs of primers CffFOR2-REV4, designed from an PCR amplified fragment of the conserved repetitive sequence (Rep-PCR). The primer pair CffFOR2-REV4 was selected in this study, because demonstrated higher reproducibility and efficacy in the detection of Cff strains in bean seeds. Four methods were also evaluated in the seed extra ct prepa ra tion for PCR: 1 ) rou gh seed extra ct; 2) millipore membrane filter (0 .2 2Mu dia meter) concentrated seed extract and subsequently ressuspended in water; 3) centrifuge concentrated seed

Protocol for detecting Curtobacterium flaccumfaciens pv. flaccumfaciens in the leaves of infected bean plants (Phaseolus vulgaris L.)

“…The following three protocols were used to extract bacterial DNA from infected plant leaves: the method proposed by Doyle and Doyle (1987), the alkaline extraction method developed by Xin, Velten, Oliver, and Burke (2003), and a method proposed by Deuner, Souza, Zacaroni, Figueira, and Camera (2012), which we optimized and will thereafter denote as the protocol for detecting Curtobacterium flaccumfaciens pv. flaccumfaciens in the leaves of infected bean plants (Phaseolus vulgaris L.).…”

“…The first two protocols were used to isolate bacterial DNA from plant tissues to detect bacterial cells (Moreira, Noschang, Neiva, Carvalho, & Vicente, 2010;Trindade, Marques, Lopes, & Ferreira, 2007). The protocol of Deuner et al (2012) was developed for Cff cell extraction from infected seeds, and we modified it for bacterial cell extraction from the leaf tissues of bean plants as follows. We modified the extraction method by reducing the amount of water used, replacing seeds with leaf tissues (obtained from the veins and petioles), and using the CffFOR2 and CffREV4 primers specific for Cff.…”

A Powerful LAMP Weapon against the Threat of the Quarantine Plant Pathogen Curtobacterium flaccumfaciens pv. flaccumfaciens

“…Some semi-selective culture media and immunological tests have also been reported for Cff, but they generally have low sensitivity and specificity, and are time-consuming [for a recent review, 4]. Currently, among the PCR-based assays developed so far for Cff [15,16], the primer pair Cff FOR2/Cff REV4 has been demonstrated to be highly effective and sensitive for the detection of Cff in contaminated bean seeds [16][17][18], and in/on alternative host plants and weeds [19][20][21][22].…”