Detecção molecular de Colletotrichum lindemuthianum em amostras de sementes de feijão

Colletotrichum lindemuthianum é o agente causal da antracnose do feijoeiro, tendo a semente como uma forma de disseminação. O uso de sementes de feijão livres do patógeno é uma das estratégias de manejo, e o uso de técnicas de detecção precisas e rápidas torna-se indispensável para a análise sanitária prévia de sementes. Neste estudo, as técnicas de PCR convencional (cPCR) e PCR em tempo real (qPCR) foram utilizadas para a detecção e quantificação de C. lindemuthianum em amostras de sementes de feijão. Para isso, sementes foram inoculadas por meio de sua exposição às colônias do fungo por tempos variados, 0, 36, 72, 108, e 144 horas, cada período correspondendo a um potencial de inóculo, P0, P36, P72, P108 e P144. As sementes inoculadas foram misturadas com sementes sadias, obtendo-se incidências de 0,25% 0,50%, 1%, 10% e 100% de sementes para cada potencial de inóculo em amostras de 400 sementes. Tanto a cPRC como a qPCR foram eficazes na detecção do fungo. A partir das amostras de sementes com 10% de incidência e com P36, foi possível detectar o fungo por cPCR. Pela técnica de qPCR o patógeno foi detectado em amostras a partir da incidência de 0,25% no menor potencial de inóculo P36.

Termos para indexação:
Phaseolus vulgaris; antracnose; PCR

Autoria

Stélio Jorge Castro Gadaga

Departamento de Fitopatologia, Universidade Federal de Lavras, 37200-000 - Lavras, MG, Brasil.Universidade Federal de LavrasBrazilLavras, MG, BrazilDepartamento de Fitopatologia, Universidade Federal de Lavras, 37200-000 - Lavras, MG, Brasil.

Carolina da Silva Siqueira ^**Corresponding author <kerolpet@gmail.com>

José da Cruz Machado

*Corresponding author <kerolpet@gmail.com>

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Figuras | Tabelas

Figuras (3)
Tabelas (5)

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Figure 1
Specificity of the pair of primers CD1/ CD2 in detecting Colletotrichum lindemunthianum by conventional PCR. M: 1 kb marker (Qiagen GelPilot®); Columns 1-3: Sclerotinia sclerotiorum; 4-6: Fusarium oxysporum f. sp. phaseoli; 7-9: Rhizoctonia solani; 10-12: negative control; and 13-15: positive control.

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Figure 2
Limit of detection of the pair of primers CD1/ CD2 for Colletotrichum lindemuthianum, by the conventional PCR technique. M: 1 Kb marker (Qiagen GelPilot^®). Columns 1-2: 200 ng/µL; 3-4: 20 ng/µL; 5-6: 2 ng/µL; 7-8: 0.2 ng/µL; 9-10: 0.02 ng/µL; 11-12: 0.002 ng/µL; 13-14: 0.0002 ng/µL; 15-16: 0.00002 ng/µL; and 17: negative control.

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Figure 3
Real-time PCR for the detection and quantification of Colletotrichum lindemuthianum. A - Amplification curve with serial dilutions of the target DNA, from 200 ng/μL to 0.002 ng/μL, and negative control (NC). B - Standard curve; Threshold cycle (Ct), graphically represented as a function of the logarithm values of dilution and fluorescence emitted during the reaction.

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Table 1
Incidence levels obtained from mixing healthy bean seeds (cultivar Pérola) with seeds inoculated with the isolate LV238 of Colletotrichum lindemuthianum race 65, for different seed-fungus contact times, corresponding to the inoculum potentials P0 - 0 h, P36 - 36 h, P72 - 72 h, P108 - 108 h, and P144 - 144 h.

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Table 2
Primer sequences used in the conventional PCR (cPCR) and real-time PCR (qPCR) reactions for detecting Colletotrichum lindemuthianum in bean seeds artificially inoculated.

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Table 3
Amplification of the genomic DNA of Colletotrichum lindemuthianum by conventional PCR (cPCR), using the pair of primers CD1/ CD2, in samples of 400 bean seeds with different levels of incidence and treated with different inoculum potentials P0 - 0 h, P36 - 36 h, P72 - 72 h, P108 - 108 h, and P144 - 144 h.

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Table 4
Quantification of Colletotrichum lindemuthianum (ng/μL*1000), by real-time PCR, in bean samples with different incidence levels of inoculated seeds (0.25%, 0.5%, 1%, 10%, 100%), treated with different seed-fungus contact time, corresponding to the inoculum potentials P0 - 0 h, P36 - 36 h, P72 - 72 h, P108 - 108 h, and P144 - 144 h.

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Table 5
Concentration of Colletotrichum lindemuthianum DNA (ng/μL*100), quantified by real-time PCR, in bean samples (whole seeds, tegument, and embryo) treated with different seed-fungus contact time, corresponding to the inoculum potentials P0 - 0 h, P36 - 36 h, P72 - 72 h, P108 - 108 h, and P144 - 144 h.

Figure 1 Specificity of the pair of primers CD1/ CD2 in detecting Colletotrichum lindemunthianum by conventional PCR. M: 1 kb marker (Qiagen GelPilot®); Columns 1-3: Sclerotinia sclerotiorum; 4-6: Fusarium oxysporum f. sp. phaseoli; 7-9: Rhizoctonia solani; 10-12: negative control; and 13-15: positive control.

Figure 2 Limit of detection of the pair of primers CD1/ CD2 for Colletotrichum lindemuthianum, by the conventional PCR technique. M: 1 Kb marker (Qiagen GelPilot^®). Columns 1-2: 200 ng/µL; 3-4: 20 ng/µL; 5-6: 2 ng/µL; 7-8: 0.2 ng/µL; 9-10: 0.02 ng/µL; 11-12: 0.002 ng/µL; 13-14: 0.0002 ng/µL; 15-16: 0.00002 ng/µL; and 17: negative control.

Figure 3 Real-time PCR for the detection and quantification of Colletotrichum lindemuthianum. A - Amplification curve with serial dilutions of the target DNA, from 200 ng/μL to 0.002 ng/μL, and negative control (NC). B - Standard curve; Threshold cycle (Ct), graphically represented as a function of the logarithm values of dilution and fluorescence emitted during the reaction.

Table 1 Incidence levels obtained from mixing healthy bean seeds (cultivar Pérola) with seeds inoculated with the isolate LV238 of Colletotrichum lindemuthianum race 65, for different seed-fungus contact times, corresponding to the inoculum potentials P0 - 0 h, P36 - 36 h, P72 - 72 h, P108 - 108 h, and P144 - 144 h.

Inoculum potential*	Incidence levels (%)	Nº of infected seeds	Nº of healthy seeds	Total of seeds
P36	0.25	1	399	400
	0.5	2	398	400
	1	4	396	400
	10	40	360	400
	100	400	0	400
P72	0.25	1	399	400
	0.5	2	398	400
	1	4	396	400
	10	40	360	400
	100	400	0	400
P108	0.25	1	399	400
	0.5	2	398	400
	1	4	396	400
	10	40	360	400
	100	400	0	400
P144	0.25	1	399	400
	0.5	2	398	400
	1	4	396	400
	10	40	360	400
	100	400	0	400

*For each inoculum potential, all incidence levels were tested separately

Table 2 Primer sequences used in the conventional PCR (cPCR) and real-time PCR (qPCR) reactions for detecting Colletotrichum lindemuthianum in bean seeds artificially inoculated.

Technique	Primers	Sequence	Reference
cPCR	CD1	5’-ACC TGG ACA CAT AAG TCA AAG-3’	Wang et al. (2008WANG, W.; TANG, J.H.; WANG, Y.C. Molecular detection of Colletotrichum lindemuthianum by duplex PCR. J. Phytopathology, v.156, p.431-437, 2008. http://onlinelibrary.wiley.com/doi/10.1111/j.1439-0434.2007.01386.x/epdf http://onlinelibrary.wiley.com/doi/10.11... )
cPCR	CD2	5’-CAA CAA TGC CAG TAT CAG AG-3’
qPCR	ClF432	5’-GGA GCC TCC TTT GCG TAG TAAC-3’	Chen et al. (2013CHEN, Y.Y.; CONNER, R. L.; GILLARD, C.L.; McLAREND, D.L.; BOLANDE, G.J.; BALASUBRAMANIANF, P.M.; STASOLLAG, C.; ZHOUH, Q.X.; HWANGH, S.F.; CHANGH, K.F.; BABCOCK, C. A quantitative real-time PCR assay for detection of Colletotrichum lindemuthianum in navy bean seeds. Plant Pathology, v.62, p.900-907, 2013. http://onlinelibrary.wiley.com/doi/10.1111/j.1365-3059.2012.02692.x/epdf http://onlinelibrary.wiley.com/doi/10.11... )
qPCR	ClR533	5’-ACC TGA TCC GAG GTC AAC CTT GTT-3’

Table 3 Amplification of the genomic DNA of Colletotrichum lindemuthianum by conventional PCR (cPCR), using the pair of primers CD1/ CD2, in samples of 400 bean seeds with different levels of incidence and treated with different inoculum potentials P0 - 0 h, P36 - 36 h, P72 - 72 h, P108 - 108 h, and P144 - 144 h.

Inoculum potential*	Incidence of seeds inoculated with Colletotrichum lindemuthianum (%)	cPCR detection in seeds (positive results/ replications)
Negative control	-	0/3
Positive control	+	3/3
P36	0.25	0/3
	0.5	0/3
	1	0/3
	10	3/3
	100	3/3
P72	0.25	3/3
	0.5	3/3
	1	3/3
	10	3/3
	100	3/3
P108	0.25	3/3
	0.5	3/3
	1	3/3
	10	3/3
	100	3/3
P144	0.25	3/3
	0.5	3/3
	1	3/3
	10	3/3
	100	3/3

*Regarding the different seed-fungus contact time.

Table 4 Quantification of Colletotrichum lindemuthianum (ng/μL*1000), by real-time PCR, in bean samples with different incidence levels of inoculated seeds (0.25%, 0.5%, 1%, 10%, 100%), treated with different seed-fungus contact time, corresponding to the inoculum potentials P0 - 0 h, P36 - 36 h, P72 - 72 h, P108 - 108 h, and P144 - 144 h.

Incidence levels * (%)	Quantification of Colletotrichum lindemuthianum (ng/μL)
Incidence levels * (%)	P36	P72	P108	P144
0.25	0.161	3.24	5.25	8.71
0.5	0.354	6.22	11.44	19.1
1	0.443	12.33	19.6	27.4
10	14.9	41.8	99.3	131
100	120	204	443	842

*Incidence levels in lots of 400 seeds.

Table 5 Concentration of Colletotrichum lindemuthianum DNA (ng/μL*100), quantified by real-time PCR, in bean samples (whole seeds, tegument, and embryo) treated with different seed-fungus contact time, corresponding to the inoculum potentials P0 - 0 h, P36 - 36 h, P72 - 72 h, P108 - 108 h, and P144 - 144 h.

Structure	P36	P72	P108	P144
Whole seed	0.987	5.835	48.35	60.3
Tegument	0.513	0.792	6.497	20.433
Embryo	0.021	0.017	0.081	0.145

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Detecção molecular de Colletotrichum lindemuthianum em amostras de sementes de feijão

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[1] *Corresponding author <kerolpet@gmail.com>