Improvement of the specific detection of Xanthomonas phaseoli pv. manihotis based on the pthB gene

Modifications were made in the PCR conditions aiming to overcome the problem of non-amplification of the Xanthomonas phaseoli pv. manihotis (Xpm) fragment, using the primer pair XV / XK described in the literature. The objective of this study was to propose changes in the primers already described (XV / XK_MOD) and validate the use of these new primers in identifying Xpm. The validation procedure was carried out with the primer pair XV and XK_MOD, using different strains of Xpm, other plant pathogenic and endophytic bacteria genera and one isolate of X. phaseoli pv. passiflorae. As a control, additional reactions were conducted in multiplex with the universal primers for the 16S rRNA gene of the bacteria together with XV / XK and XV / XK_MOD. Using the forward primer (XV) described in the literature together with the modified reverse primer (XK_MOD), it was possible to achieve amplification from DNA extracted from in vitro cultures and from infected tissue, but no amplification was noticed for the primer pair described in the literature, confirming the effectiveness of the proposed modification.

Keywords:
Xpm; cassava bacterial blight; molecular diagnosis; resistance

Authorship

Rita de Cássia Cerqueira Melo

Universidade Federal do Recôncavo da Bahia, Av. Rui Barbosa, 710, 44380-000, Cruz das Almas, Bahia, Brazil.Universidade Federal do Recôncavo da BahiaBrazilCruz das Almas, Bahia, BrazilUniversidade Federal do Recôncavo da Bahia, Av. Rui Barbosa, 710, 44380-000, Cruz das Almas, Bahia, Brazil.

Carlos Augusto Dórea Bragança

Kátia Nogueira Pestana

Embrapa Mandioca e Fruticultura, Empresa Brasileira de Pesquisa Agropecuária, Cruz das Almas, Bahia, Brazil. Empresa Brasileira de Pesquisa AgropecuáriaBrazilCruz das Almas, Bahia, BrazilEmbrapa Mandioca e Fruticultura, Empresa Brasileira de Pesquisa Agropecuária, Cruz das Almas, Bahia, Brazil.

Harllen Sandro Alves da Silva

Cláudia Fortes Ferreira ^**Author for correspondence. E-mail: claudia.ferreira@embrapa.br

http://orcid.org/0000-0001-5476-2236

Saulo Alves Santos de Oliveira

*Author for correspondence. E-mail: claudia.ferreira@embrapa.br

SCIMAGO INSTITUTIONS RANKINGS

Figures | Tables

Figures (5)
Tables (1)

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Figure 1
Specificity test of the primers XV / XK_MOD, with bacteria isolates from different genera and species. (A):1: Xanthomonas phaeoli pv. manihotis (IBSBF 278); 2: X. axonopodis pv. passiflorae (CBEMF-8); 3: Xanthomonas campestris (CBEMF-5); 4: Ralstonia solanacearum (CBEMF-4); 5: Pseudomonas marginalis (CBEMF-3); 6: Pseudomonas syringae pv. maculicola (CBEMF-2); 7: Pectobacterium carotovorum subsp. carotovorum (CBEMF-7); 8: Pseudomonas viridiflava (CBEMF-1); 9: Enterobacter aerogenes (CBEMF-19); 10: Enterobacter cloacae (CBEMF-11); 11: Enterobacter asburiae (CBEMF-23); 12: Klebsiella oxytoca (CBEMF-105); 13: Kosakonia cowanii (CBEMF-112); 14: Klebsiella variicola (CBEMF-128); 15: Bacillus sp. (CBEMF-6); 16: control (without DNA). (B): Isolates of X. phaeoli pv. manihotis. 1: IBSBF 278; 2: IBSBF 1998; 3: IBSBF 291; 4: IBSBF 436, 5: CBEMF-9; and 6: control (without DNA). M: 200 bp Ladder (Invitrogen).

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Figure 2
PCR reaction in 1% agarose gel electrophoresis using DNA from the IBSBF 278 (Xanthomonas phaeoli pv. manihotis) isolate in different concentrations, in the following order: 100, 75, 50, 25, 1, and 0.25 ng with three replicates each (1, 2, and 3). A: Reaction with primers XV/XK_MOD; B: Reaction with primers XV/XK (Verdier et al., 1998Verdier, V., Mosquera, G., & Assigbétsé, K. (1998). Detection of the cassava bacterial blight pathogen, Xanthomonas axonopodis pv. manihotis, by polymerase chain reaction. Plant Disease, 82(1), 79-83, 1998.). M: 200 bp DNA ladder.

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Figure 3
PCR reaction in 1% agarose gel electrophoresis using DNA from the plants inoculated with IBSBF 278 (Xanthomonas phaeoli pv. manihotis) isolate. 1: BRS Tapioqueira (stem); 2: Cigana Preta (leaf); 3: Cigana Preta (stem); 4: Salangor (leaf); 5: BRS Poti Branca (leaf); 6: BRS Poti Branca (Stem); 7: negative control (plant material not infected with Xpm); 8: positive control (Xpm IBSBF 278 isolate). M: 200 bp DNA ladder.

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Figure 4
1% agarose gel. PCR product from the detection test in different fragments of the stem. A: Hybrid 9624-09; B: BRS Caipira; C: Cigana Preta; D: BRS Tapioqueira; E: BRS Kiriris; F: BRS Poti Branca; G: Hybrid 98150-06; H: Solangor; I: BRS Verdinha; X: Control. 1= 1 cm in the base of the stem; 2 = 1 cm at the point of inoculation; 3 = 8 cm above the inoculation point; 4 = 16 cm above the inoculation point; 5 = Positive control (Xpm, isolate IBSBF 278); 6 = Negative control (Mix without DNA). M: 200 bp DNA ladder.

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Figure 5
PCR products from the sensitivity test with bacterial isolates from different genera and species. (A): Isolates of X. phaeoli pv. manihotis. 1: IBSBF 1994; 2: IBSBF 291; 3: 328; 4: IBSBF 436; 5: 726; 6-10: CBEMF-9 isolate (with DNA extraction conducted at different times); 11: IBSBF 278 and 12: Xanthomonas axonopodis pv. passiflorae (CBEMF-8), with primers 16S rRNA e XV / XK. (B): 1: IBSBF 1994; 2: IBSBF 291; 3: 328; 4: IBSBF 436; 5: 726; 6-10: CBEMF-9 isolate (with DNA extraction conducted at different times); 11: IBSBF 278 and 12: Xanthomonas axonopodis pv. passiflorae (CBEMF 8), with primers 16S rRNA and XV / XK_MOD. (C): 1: IBSBF 1994; 2: IBSBF 291; 3: 328; 4: IBSBF 436; 5: 726; 6-10: CBEMF-9 isolate (with DNA extraction conducted at different times); 11: IBSBF 278 and 12: Xanthomonas axonopodis pv. passiflorae (CBEMF 8), with primer 16S rRNA. M*: 100 bp DNA Ladder; M: 1 kb DNA ladder.

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Table 1
Bacteria species used in specificity tests of primers XV / XK_MOD.

Figure 1 Specificity test of the primers XV / XK_MOD, with bacteria isolates from different genera and species. (A):1: Xanthomonas phaeoli pv. manihotis (IBSBF 278); 2: X. axonopodis pv. passiflorae (CBEMF-8); 3: Xanthomonas campestris (CBEMF-5); 4: Ralstonia solanacearum (CBEMF-4); 5: Pseudomonas marginalis (CBEMF-3); 6: Pseudomonas syringae pv. maculicola (CBEMF-2); 7: Pectobacterium carotovorum subsp. carotovorum (CBEMF-7); 8: Pseudomonas viridiflava (CBEMF-1); 9: Enterobacter aerogenes (CBEMF-19); 10: Enterobacter cloacae (CBEMF-11); 11: Enterobacter asburiae (CBEMF-23); 12: Klebsiella oxytoca (CBEMF-105); 13: Kosakonia cowanii (CBEMF-112); 14: Klebsiella variicola (CBEMF-128); 15: Bacillus sp. (CBEMF-6); 16: control (without DNA). (B): Isolates of X. phaeoli pv. manihotis. 1: IBSBF 278; 2: IBSBF 1998; 3: IBSBF 291; 4: IBSBF 436, 5: CBEMF-9; and 6: control (without DNA). M: 200 bp Ladder (Invitrogen).

Figure 2 PCR reaction in 1% agarose gel electrophoresis using DNA from the IBSBF 278 (Xanthomonas phaeoli pv. manihotis) isolate in different concentrations, in the following order: 100, 75, 50, 25, 1, and 0.25 ng with three replicates each (1, 2, and 3). A: Reaction with primers XV/XK_MOD; B: Reaction with primers XV/XK (Verdier et al., 1998Verdier, V., Mosquera, G., & Assigbétsé, K. (1998). Detection of the cassava bacterial blight pathogen, Xanthomonas axonopodis pv. manihotis, by polymerase chain reaction. Plant Disease, 82(1), 79-83, 1998.). M: 200 bp DNA ladder.

Figure 3 PCR reaction in 1% agarose gel electrophoresis using DNA from the plants inoculated with IBSBF 278 (Xanthomonas phaeoli pv. manihotis) isolate. 1: BRS Tapioqueira (stem); 2: Cigana Preta (leaf); 3: Cigana Preta (stem); 4: Salangor (leaf); 5: BRS Poti Branca (leaf); 6: BRS Poti Branca (Stem); 7: negative control (plant material not infected with Xpm); 8: positive control (Xpm IBSBF 278 isolate). M: 200 bp DNA ladder.

Figure 4 1% agarose gel. PCR product from the detection test in different fragments of the stem. A: Hybrid 9624-09; B: BRS Caipira; C: Cigana Preta; D: BRS Tapioqueira; E: BRS Kiriris; F: BRS Poti Branca; G: Hybrid 98150-06; H: Solangor; I: BRS Verdinha; X: Control. 1= 1 cm in the base of the stem; 2 = 1 cm at the point of inoculation; 3 = 8 cm above the inoculation point; 4 = 16 cm above the inoculation point; 5 = Positive control (Xpm, isolate IBSBF 278); 6 = Negative control (Mix without DNA). M: 200 bp DNA ladder.

Figure 5 PCR products from the sensitivity test with bacterial isolates from different genera and species. (A): Isolates of X. phaeoli pv. manihotis. 1: IBSBF 1994; 2: IBSBF 291; 3: 328; 4: IBSBF 436; 5: 726; 6-10: CBEMF-9 isolate (with DNA extraction conducted at different times); 11: IBSBF 278 and 12: Xanthomonas axonopodis pv. passiflorae (CBEMF-8), with primers 16S rRNA e XV / XK. (B): 1: IBSBF 1994; 2: IBSBF 291; 3: 328; 4: IBSBF 436; 5: 726; 6-10: CBEMF-9 isolate (with DNA extraction conducted at different times); 11: IBSBF 278 and 12: Xanthomonas axonopodis pv. passiflorae (CBEMF 8), with primers 16S rRNA and XV / XK_MOD. (C): 1: IBSBF 1994; 2: IBSBF 291; 3: 328; 4: IBSBF 436; 5: 726; 6-10: CBEMF-9 isolate (with DNA extraction conducted at different times); 11: IBSBF 278 and 12: Xanthomonas axonopodis pv. passiflorae (CBEMF 8), with primer 16S rRNA. M*: 100 bp DNA Ladder; M: 1 kb DNA ladder.

Table 1 Bacteria species used in specificity tests of primers XV / XK_MOD.

Species	Strains	Origin	Crop
Pseudomonas viridiflava	CBEMF-1^a	Unknow	Chinese Cabbage
P. syringae pv. maculicola	CBEMF-2	Unknow	Cauliflower
Pseudomonas marginalis	CBEMF-3	Unknow	Lettuce
Ralstonia solanacearum	CBEMF-4	Unknow	Cucumber
Xanthomonas campestris	CBEMF-5	Unknow	Sweet Pepper
Bacillus sp.	CBEMF-6	Cruz das Almas - BA	Soil sample
Pectobacterium carotovorum subsp. carotovorum	CBEMF-7	Pernambuco (UFRPE)	Garlic
X. axonopodis pv. passiflora (Xap)	CBEMF-8	Pernambuco (UFRPE)	Passionfruit
X. phaseoli pv. manihotis	CBEMF-9	Brasília - DF	Cassava
Enterobacter aerogenes	CBEMF-19	Guanambi - BA	Cassava Endoph.^b
Enterobacter cloacae	CBEMF-11	Guanambi - BA	Cassava Endoph.^b
Enterobacter asburiae	CBEMF-23	Guanambi - BA	Cassava Endoph.^b
Klebisiela oxycota	CBEMF-105	Dourados- MS	Cassava Endoph.^b
Kosakonia cowanii	CBEMF-112	Laje - BA	Cassava Endoph.^b
Klebisiella variicola	CBEMF-128	Marechal C. Rondon - PR	Cassava Endoph.^b
X. phaseoli pv. manihotis (Xpm)	IBSBF 278	Unknow	Cassava
X. phaseoli pv. manihotis ^*	IBSBF 291	Unknow	Cassava
X. phaseoli pv. manihotis ^*	IBSBF 1994	Unknow	Cassava
X. phaseoli pv. manihotis ^*	IBSBF 436	Unknow	Cassava
X. phaseoli pv. manihotis ^*	IBSBF 328	Unknow	Cassava
X. phaseoli pv. manihotis ^*	IBSBF 726	Unknow	Cassava
X. phaseoli pv. manihotis	PR	Naviraí - PR	Cassava

CBEMF: Bacteria collection from the Phytopathology Laboratory at Embrapa Mandioca e Fruticultura; IBSBF: Collection of cultures of Phytobacteria from the Institutlo Biológico. bSpecies found as cassava endophyte associated with bacterial blight lesions Only DNA from the bacteria donated from the Instituto Biológico, SP. Abbreviation: BA: Bahia; MS: Mato Grosso do Sul; PR: Paraná; SP: São Paulo; UFRPE: Universidade Federal Rural de Pernambuco.

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Improvement of the specific detection of Xanthomonas phaseoli pv. manihotis based on the pthB gene

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[1] *Author for correspondence. E-mail: claudia.ferreira@embrapa.br