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Specificity and sensibility of primer pair in the detection of Colletotrichum gossypii var. cephalosporioides in cotton seeds by PCR technique

Especificidade e sensibilidade de um par de primer na detecção de Colletotrichum gossypii var. cephalosporioides em sementes de algodão pela técnica de PCR

Abstract:

Cotton Ramulosis (Gossypium hirsutum) is an important disease affecting cotton plantations in Brazil, and its causal agent, Colletotrichum gossypiivar.cephalosporioides(Cgc), according to the Brazilian phytosanitary authority, was considered a regulated non quarantine pest. It makes this microorganism subject to standardization in seed certification programs. The current seed health testing for detecting that pathogen in seed samples does not provide reliable results for routine analysis. On this paper, attempts were made to design specific primers for detection of Cgc associated with cotton seed. Two primer sets were selected based on the analysis of a multiple alignment of gene’s sequence encoding the glyceraldehyde 3-phosphate dehydrogenase from Cgc, C. gossypii and reference strains of the C. gloeosporioides species complex. The conserved sites unique to Cgc strains were used to design specific fragment of 140 bp. The primer specificity was confirmed by using other fungi. The primers produced a detectable band of target DNA of Cgc in all inoculum potentials of the pathogen artificially inoculated by the water restriction technique. The developed primer pair represents, therefore, a reliable and rapid mean to diagnose the Ramulosis agent in cotton seed.

Index terms:
Colletotrichum gossypii; Colletotrichum gloeosporioides; Ramulosis; water restriction

Resumo:

A ramulose do algodão (Gossypium hirsutum), causada por Colletotrichum gossypii var. cephalosporioides (Cgc), é uma doença importante que afeta as plantações de algodão no Brasil. De acordo com as autoridades fitossanitárias brasileiras, esse organismo tem sido considerado uma praga quarentenária não regulamentada, o que faz com que ela seja objeto de padronização em programas de certificação de sementes. Neste trabalho, um par de primer foi selecionado com base na análise de um alinhamento múltiplo de sequências do gene que codifica a gliceraldeído-3-fosfato desidrogenase a partir de Cgc, C. gossypii e isolados de referência representantes de outras espécies do complexo C. gloeosporioides. Uma única região conservada de Cgc foi utilizada para desenhar um par de primer específico de 140 pb. A especificidade dos primers foi confirmada pela utilização de outros fungos isolados de semente algodão. Os primers produziram uma banda detectável de DNA de Cgc em todos os potenciais de inóculo artificialmente inoculados pela técnica de restrição hídrica. Os primers desenvolvidos representam, portanto, um meio confiável e rápido para diagnosticar Cgc em amostras de sementes de algodão.

Termos para indexação:
Colletotrichum gossypii; Colletotrichum gloeosporioides; ramulose; restrição hídrica

INTRODUCTION

Ramulosis is one of the most prominent diseases in cotton (Gossypium hirsutum) in Brazil, and it is caused by Colletotrichum gossypii var. cephalosporioides A. S. Costa. This organism belongs to the Colletotrichum gloeosporioides species complex, as well as Colletotrichum gossypii South. (Cg), which causes Anthracnose in cotton (Salustiano et al., 2014SALUSTIANO, M.E.; RONDON, M.N.; ABREU, L.M.; COSTA, S.S.; MACHADO, J.C.; PFENNING, L.H. The etiological agent of cotton Ramulosis represents a single phylogenetic lineage within the Colletotrichum gloeosporioides species complex. Tropical Plant Pathology , v.39, n.5, p.357-367, 2014. http://www.scielo.br/pdf/tpp/v39n5/v39n5a02.pdf
http://www.scielo.br/pdf/tpp/v39n5/v39n5...
).

These fungi (C. gossypii var. cephalosporioides and C. gossypii) belong to the Ascomycota phylum, having as main feature the production of conidial mass with orange color in acervuli and conidia morphologically similar (Bailey et al., 1996BAILEY, J.A; NASH, C.; MORGAN, L.W.; O’CONNELL, R.J.; TEBEEST, D.O. Molecular taxonomy of Colletotrichum species causing Anthracnose on the Malvaceae. Phytopathology, v.86, p.1076-1083, 1996. https://www.apsnet.org/publications/phytopathology/backissues/Documents/1996Articles/Phyto86n10_1076.PDF
https://www.apsnet.org/publications/phyt...
). Both pathogens are transmitted by seeds and cause damages in cotton plants (Silva-Mann et al., 2005SILVA-MANN, R.; VIEIRA, M.G.G.C.; MACHADO, J.C.; BERNARDINO-FILHO, J.R.; SALGADO, K.C.C.; STEVENS, M.R. AFLP markers differentiate Colletotrichum gossypii from C. gossypii var. cephalosporioides. Fitopatologia Brasileira , v.30, n.2, p.169-172, 2005. http://www.scielo.br/pdf/fb/v30n2/a11v30n2.pdf
http://www.scielo.br/pdf/fb/v30n2/a11v30...
; Mehta and Mehta, 2010MEHTA, Y.R.; MEHTA, A. Variabilidade genética entre isolados de Colletotrichum gossypii do algodoeiro. Summa Phytopathologica, v.36, n.1, p.40-44, 2010. http://www.scielo.br/pdf/sp/v36n1/07.pdf
http://www.scielo.br/pdf/sp/v36n1/07.pdf...
).

Colletotrichum taxonomy was subject of extensive discussion by the variability of species classified in this genus; so, there are difficulties in the identification and separation of these organisms. Traditionally, the identification of that genus’ members was based on some morphological characteristics, with emphasis on morphometry of conidia, colony color, mycelial growth rate and pathogenicity (Bailey et al., 1996BAILEY, J.A; NASH, C.; MORGAN, L.W.; O’CONNELL, R.J.; TEBEEST, D.O. Molecular taxonomy of Colletotrichum species causing Anthracnose on the Malvaceae. Phytopathology, v.86, p.1076-1083, 1996. https://www.apsnet.org/publications/phytopathology/backissues/Documents/1996Articles/Phyto86n10_1076.PDF
https://www.apsnet.org/publications/phyt...
; Tozze-Júnior et al., 2006TOZZE-JÚNIOR, H.J.; BUENO, C.R.N.C.; MASSOLA-JÚNIOR, N.S. Caracterização morfológica e molecular de isolados de Colletotrichum spp. de hortaliças solanáceas. Summa Phytopathologica , v.32, n.1, p.71-79, 2006. http://www.scielo.br/pdf/sp/v32n1/v32n1a11.pdf
http://www.scielo.br/pdf/sp/v32n1/v32n1a...
). Specifically for the Colletotrichum complex associated with cotton, it is not always possible to differ what are the pathogens involved in the symptomatology of Ramulosis and Anthracnose, as well as the different degrees of aggressiveness and symptoms (Carvalho et al., 2015CARVALHO, E.M.; FIGUEIRA, A.R.; MACHADO, J.C.; ARAÚJO, D.V.; MACHADO, C.F. Variability of seed-borne Colletotrichum strains in cotton based on its1 and its2 ribossomal genes analysis. Bioscience Journal , v.31, n.3, p.691-700, 2015. https://doi.org/10.14393/BJ-v31n3a2015-23126
https://doi.org/10.14393/BJ-v31n3a2015-2...
).

Within the seed pathology, the detection and differentiation between C. gossypii var. cephalosporioides and C. gossypii were carried out by using the “blotter” method, in which the assessment is based on mycelial growth habit of fungi developed in seeds after an incubation period (Tanaka et al., 1996TANAKA, M.A.S.; MENTEN, J.O.M.; MACHADO, J.C. Hábito de crescimento de Colletotrichum gossypii e C. gossypii var. cephalosporioides em sementes de algodoeiro. Bragantia, v.55, n.1, p.95-104, 1996. http://www.scielo.br/pdf/brag/v55n1/11.pdf
http://www.scielo.br/pdf/brag/v55n1/11.p...
). In this case, the high morphological similarities and isolate variability of these fungi make the results of such analysis questionable and not always consistent (Silva-Mann et al., 2002SILVA-MANN, R.; SALGADO, K.C.C.; VIEIRA, M.G.G.C.; MACHADO, J.C. Variabilidade genética de isolados do complexo Colletotrichum associados a sementes de algodoeiro, por meio de técnicas moleculares e inoculação em plantas. Fitopatologia Brasileira, v.27, n.1, p.27-32, 2002. http://www.scielo.br/pdf/fb/v27n1/8464.pdf
http://www.scielo.br/pdf/fb/v27n1/8464.p...
; Mehta and Mehta, 2010MEHTA, Y.R.; MEHTA, A. Variabilidade genética entre isolados de Colletotrichum gossypii do algodoeiro. Summa Phytopathologica, v.36, n.1, p.40-44, 2010. http://www.scielo.br/pdf/sp/v36n1/07.pdf
http://www.scielo.br/pdf/sp/v36n1/07.pdf...
), determining the need to develop more accurate and reliable methods for this task.

Accuracy in identification of C. gossypii var. cephalosporioides and C. gossypii is, thus, necessary and indispensable to diagnose and control the involved diseases, as well as demand for detection methods of these fungi in seed samples on laboratory routine activities (Carvalho et al., 2015CARVALHO, E.M.; FIGUEIRA, A.R.; MACHADO, J.C.; ARAÚJO, D.V.; MACHADO, C.F. Variability of seed-borne Colletotrichum strains in cotton based on its1 and its2 ribossomal genes analysis. Bioscience Journal , v.31, n.3, p.691-700, 2015. https://doi.org/10.14393/BJ-v31n3a2015-23126
https://doi.org/10.14393/BJ-v31n3a2015-2...
).

Molecular techniques and DNA sequence analysis were important to distinguish and identify populations of organisms at different levels. Currently, the PCR technique is used for direct detection of fungi and other organisms in association with seeds (Lee et al., 2002LEE, H.K.; TEWARI, J.P.; TURKINGTON, T.K. Quantification of seedborne infection byRhinchosporium secalisin barley using competitive PCR.Plant Pathology, v.51, p.217-224, 2002.https://bsppjournals.onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-3059.2002.00685.x
https://bsppjournals.onlinelibrary.wiley...
; Munkvold, 2009MUNKVOLD, G.P. Seed pathology progress in academia and industry. Annual Review of Phytopathology , v.47, p.285-311, 2009. https://doi.org/10.1146/annurev-phyto-080508-081916
https://doi.org/10.1146/annurev-phyto-08...
; Barrocas et al., 2012BARROCAS, E.N.; MACHADO, J.C.; ALMEIDA, M.F.; BOTELHO, L.S.; VON PINHO, E.V.R. Sensibility of the PCR technique in the detection of Stenocarpella sp. associated to maize seeds. Revista Brasileira de Sementes, v.34, n.2, p.218-224, 2012. http://www.scielo.br/pdf/rbs/v34n2/05.pdf
http://www.scielo.br/pdf/rbs/v34n2/05.pd...
). This technology was successful in detecting, for example, Stenocarpella complex (S. maydis and S. macrospora) in maize (Romero and Wise, 2015ROMERO, M.P.; WISE, K.A. Development of molecular assays for detection of Stenocarpella maydis and Stenocarpella macrospora in corn. Plant Disease, v.99, p.761-769, 2015. http://apsjournals.apsnet.org/doi/pdf/10.1094/PDIS-09-14-0917-RE
http://apsjournals.apsnet.org/doi/pdf/10...
), Fusarium oxysporum f.sp. phaseoli in bean seeds (Sousa et al., 2015SOUSA, M.V.; MACHADO, J.C.; SIMMONS, H.E.; MUNKVOLD, G.P. Real-time quantitative PCR assays for the rapid detection and quantification of Fusarium oxysporum f. sp. phaseoli in Phaseolus vulgaris (common bean) seeds. Plant Pathology , v.64, p.478-488, 2015. https://bsppjournals.onlinelibrary.wiley.com/doi/epdf/10.1111/ppa.12257
https://bsppjournals.onlinelibrary.wiley...
), Sclerotinia sclerotiorum in soybean seeds (Botelho et al., 2015BOTELHO, L.S.; BARROCAS, E.N.; MACHADO, J.C.; MARTINS, R.S. Detection of Sclerotinia sclerotiorum in soybean seeds by conventional and quantitative PCR techniques. Journal of Seed Science, v.37, n.1, p.55-62, 2015. http://dx.doi.org/10.1590/2317-1545v37n1141460
http://dx.doi.org/10.1590/2317-1545v37n1...
) and Corynespora cassiicola in soybean seeds (Sousa et al., 2016SOUSA, M.V.; SIQUEIRA, C.S.; MACHADO, J.C. Conventional PCR for detection of Corynespora cassiicola in soybean seeds. Journal of Seed Science , v.38, n.2, p.85-91, 2016. http://www.scielo.br/pdf/jss/v38n2/2317-1545-jss-v38n2152049.pdf
http://www.scielo.br/pdf/jss/v38n2/2317-...
).

This study aimed to design specific primer pair to detect Colletotrichum gossypii var. cephalosporioides in cotton seeds and establish a protocol for safer and more sensitive sanitary analysis in the detection of this pathogen by PCR, ensuring to the cotton producers a safer quality control and providing better protection for agricultural production environments in the country.

MATERIAL AND METHODS

Isolates obtention: Colletotrichum gossypii var. cephalosporioides isolates and other fungi species were obtained from the mycological collection of the Mycology Laboratory and of the Seed Pathology Laboratory of the Universidade Federal de Lavras (UFLA), in Lavras, MG, Brazil (Table 1).

Table 1
Isolates of Colletotrichum gossypii var. cephalosporioides and others fungal species associated with cotton and others hosts used in the specificity test.

DNA extraction: genomic DNA was extracted from monosporic cultures of isolates grown on potato dextrose agar (PDA) for five days. The mycelium was scraped and homogenized in liquid nitrogen, and the extraction was performed using the Wizard®Genomic DNA purification kit (Promega, Madison, WI), according to the DNA extraction protocol recommended by the manufacturer. DNA concentrations were estimated using the NanoDrop 2000 instrument and visually in 1.2% agarose gel, by comparison of band intensity with a fragment size marker of 1 kb (Invitrogen).

Development of specific primers for detection and identification of C. gossypii var. cephalosporioides: alignments generated from the sequences of the work of Salustiano et al. (2014)SALUSTIANO, M.E.; RONDON, M.N.; ABREU, L.M.; COSTA, S.S.; MACHADO, J.C.; PFENNING, L.H. The etiological agent of cotton Ramulosis represents a single phylogenetic lineage within the Colletotrichum gloeosporioides species complex. Tropical Plant Pathology , v.39, n.5, p.357-367, 2014. http://www.scielo.br/pdf/tpp/v39n5/v39n5a02.pdf
http://www.scielo.br/pdf/tpp/v39n5/v39n5...
, using ClustalW implemented by MEGA5 (Tamura et al., 2011TAMURA, K.; PETERSON, D.; PETERSON, N.; STECHER, G.; NEI, M.; KUMAR, S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance and maximum parsimony methods. Molecular Biology and Evolution, v.28, n.10, p.2731-2739, 2011. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3203626/pdf/msr121.pdf
https://www.ncbi.nlm.nih.gov/pmc/article...
), were obtained for the partial DNA of glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) of Cgc isolates and other species from the C. gloesporioides species complex. Unique sites in the sequences of the Ramulosis’ etiologic agent were identified and used to design species-specific primers. The primer sequences were compared using the BLAST program in order to verify its homology with sequences previously deposited in GenBank (https://www.ncbi.nlm.nih.gov/) (Table 2). The developed primer pair was analyzed for performance characteristics such as hairpin structure, potential self-dimer formation and stability of 3 termini, using OligoAnalyzer 3.1 integrated platform (https://www.idtdna.com/analyzer/Applications/OligoAnalyzer/). The primers’ synthesis was performed by Sigma-Aldrich Brazil LTD. The genomic material isolated from C. gossypii var. cephalosporioides was subjected to PCR analysis.

Table 2
GenBank accession numbers of Colletotrichum gossypii var. cephalosporioides and other species from the C. gloesporioides species complex used to obtain specific primer pair to Cgc.

Determining primer specificity: the specificity of the primer pair was tested by PCR amplification of genomic DNA of 28 Cgc’s isolates, ten isolates of Colletotrichum’s other species and 21 isolates of other fungal species, which were reported in cotton seed and other host (Table 1). PCR was performed using 25 µL mix for PCR OneTaq (BioLabs), containing 10 pmol of forward and reverse primers and DNA 10 ng. The DNA amplification was performed under the following cycle conditions: 94 °C for four minutes (initial denaturation), 94 °C for 45 seconds (denaturation), 65 °C for 45 seconds (annealing), 72 °C for one minute (extension), and 34 cycles of 72 °C for ten minutes (final extension). To separate PCR products, an aliquot of 10 µL was used on 1.2% agarose gel, stained with GelRed® (Biotium®, Hayward, 95 CA, USA). The PCR products were observed in UV transilluminator, L-Pix HE equipament (Loccus Biotechnology, Brazil). Before using the specific primers, a PCR reaction was performed using universal GDF primers GDF (5´- GCCGTCAACGACCCCTTCATTGA- 3’) and universal GDR primers GDR (5´- GGGTGGAGTCGTACTTGAGCATGT- 3’) (Templeton et al., 1992TEMPLETON, M.D.; RIKKERINK, E.H.A.; SOLON, S.L.; CROWHURT, R.N. Cloning and molecular characterization the glyceraldehyde-3- phosphate dehydrogenase-encoding gene and cDNA from the plant pathogenic fungus Glomerella cingulata. Gene, v.122, n.1, p.225-230, 1992. https://doi.org/10.1016/0378-1119(92)90055-T
https://doi.org/10.1016/0378-1119(92)900...
), with the genomic DNA of all species used in this study to test if the genomic DNA was adequate for PCR amplification. The experiments were repeated at least two times.

Sensivity evaluation of primers developed in seed samples: to evaluate the sensitivity of PCR reaction using primer pair, cotton seed with different infestation level inoculated with C. gossypii var. cephalosporioides was used, and a four-hundred-seed sample were prepared by mixing the artificially inoculated seeds with healthy seeds generating three infestation level (100%, 10% and 1%) per inoculum. For each infestation level of seeds, the test was performed in four replicates, and the experiment was repeated twice.

Seed inoculation: cotton seeds CV delta opal susceptible to the Ramulosis’ etiologic agent were disinfected in 70% alcohol for one minute, followed by 1% of sodium hypochlorite solution for two minutes, then washed four times with autoclaved distilled water. The sterilized seeds were arranged in trays where they remained for 24 hours at room temperature to complete drying. After drying, it was used physiological conditioning method or water restriction for seed inoculation (Machado et al., 2012MACHADO, J.C.; BARROCAS, E.N.; COSTA, L.N.; GUIMARÃES, R.M.; MACHADO, C. Uso da técnica de restrição hídrica ou condicionamento osmótico em patologia de sementes. Revisão Anual de Patologia de Plantas, v.20, p.37-63, 2012.; Barrocas et al., 2014BARROCAS, E.N.; MACHADO, J.C.; ALVES, M.C.; CORREA, C.L. Desempenho de sementes de algodão submetidas à deficiência hídrica e presença de Colletotrichum gossypii var. cephalosporioides. Bioscience Journal, v.30, n.2, p.421-428, 2014. http://www.seer.ufu.br/index.php/biosciencejournal/article/view/17993/13757
http://www.seer.ufu.br/index.php/bioscie...
).

Then, the seeds were artificially inoculated with the C. gossypii var. cephalosporioides strain CML2374 that growed in petri dishes with fifteen cm diameter containing PDA medium, modified by the addition of manitol adjusted with water potential of -1.0 MPa, as SPPM Software (computer program that relates solute potencial to solution composition). A sequence of data was generated over temperature, concentration, or potential ranges by specifying an initial value (Michel and Radcliffe, 1995MICHEL, B.E.; RADCLIFFE, D.A. Computer program relating solute potencial to solution composition for five solutes. Agronomy Journal, v.87, p.131-136, 1995.), remaining seven days in BOD at 25 °C with a photoperiod of twelve hours. The seeds were placed in a single layer on the fungus colony, where they remained for 24 and 48 hours, being removed and placed in sterilized trays and dried in a laminar flow chamber for 24 hours. As controls, seeds were used without the fungus and with incubation in substrate with water restriction.

DNA extraction of seed samples: the inoculated seed samples were macerated in mill (IKA® A11 analytical basic mill) with liquid nitrogen to obtain a thin powder. Samples with 0.04 g of this powder were placed in 1.5 mL microtubes in four replicates. The extraction was carried out with the use of Wizard®Genomic DNA purification kit (Promega, Madison, WI), according to the DNA extraction protocol recommended by the manufacturer. The PCR reaction and the cycle conditions were the same described for the specificity of the primer pair.

RESULTS AND DISCUSSION

Colletotrichum gossypii var. cephalosporioides specific primers designed from the GAPDH gene had the following sequences: CGC1F (5’- CAG ACT ACA AGG CCA ACG C- 3’) and CGC1R (5’- GAG TCG TAC TTG AGC ATG TAG- 3’). This primer pair amplifies a fragment of 140bp. This primers’ pair specifically amplified DNA of only its respective target, Cgc, in all reactions (Figure 1A). The primers did not cross-react with DNA of any other Colletotrichum species or other fungal species tested (Figure 1B and Table 1).

Figure 1
Specificity test of conventional PCR with primer pair CGC1F/ CGC1R.

The sensitivity of the primers’ pair may be considered high due to their capacity of detecting the pathogen in seed samples with minimal incidence of 1%, which was the limit used in this study. In the controls, there was no amplification of the genomic DNA from the causative agent of cotton Ramulosis (Figure 2).

Figure 2
Sensitivity test of conventional PCR with primer pair CGC1F/ CGC1R in the detection of Colletotrichum gossypii var. cephalosporioides in samples of cotton seeds with different infection levels.

A PCR-based diagnostic assay using specific primers derived from the gene encoding the glyceraldehyde 3-phosphate dehydrogenase was developed for the Ramulosis’ causal agent from cotton, C. gossypii var. cephalosporioides. Furthermore, the primers were able to detect the pathogen in artificially infested cotton seeds.

The PCR products obtained from the seeds showed characteristic bands, as observed in the pathogen’s DNA amplification in pure cultures. Thus, it was evident that the primer pair was effective in detecting the Ramulosis’ etiological agent in artificially infested cotton seeds, indicating no false positive result for contamination. These primer pair allowed the amplification of the genomic DNA samples from the C. gossypii var. cephalosporioides tested, being effective in detection of fungal incidences from 1 to 100% at different inoculum potential tested.

In a study conducted by Guimarães et al. (2017GUIMARÃES, M.R.F.; SIQUEIRA, C.S.; MACHADO, J.C.; FRANÇA, S.K.S.; GUIMARÃES, G.C. Evaluation of inoculum potential of pathogens in seeds: relation to physiological quality and DNA quantification by qPCR. Journal of Seed Science , v.39, n.3, p.224-233, 2017. http://www.scielo.br/pdf/jss/v39n3/2317-1545-jss-39-03-00224.pdf
http://www.scielo.br/pdf/jss/v39n3/2317-...
), the pair of primers designed and described was used to quantify C. gossypii var. cephalosporioides in artificially inoculated cotton seeds by cPCR and qPCR techniques. The results showed that the primers used were reliable. Primers showed linearity in the standard curve generated by qPCR technique at each dilution level of Cgc DNA extracted from pure culture. The quantification of the inoculum potential by qPCR was 1.44 pg/ μL DNA at P24, which increases to 6.89 pg/ μL at P48 and 24.5 pg/ μL at P96. The authors concluded that there was proportionality between fungal DNA, inoculum potential, effects on germination and seed vigor.

For other pathosystems, the sensitivity in detecting seeds’ pathogens is variable. For example, in a study conducted by Barrocas et al. (2012BARROCAS, E.N.; MACHADO, J.C.; ALMEIDA, M.F.; BOTELHO, L.S.; VON PINHO, E.V.R. Sensibility of the PCR technique in the detection of Stenocarpella sp. associated to maize seeds. Revista Brasileira de Sementes, v.34, n.2, p.218-224, 2012. http://www.scielo.br/pdf/rbs/v34n2/05.pdf
http://www.scielo.br/pdf/rbs/v34n2/05.pd...
), Sternocarpella was detected in maize seeds infected with minimal incidence of 2% in the studied samples. In a study conducted by Sousa et al. (2015SOUSA, M.V.; MACHADO, J.C.; SIMMONS, H.E.; MUNKVOLD, G.P. Real-time quantitative PCR assays for the rapid detection and quantification of Fusarium oxysporum f. sp. phaseoli in Phaseolus vulgaris (common bean) seeds. Plant Pathology , v.64, p.478-488, 2015. https://bsppjournals.onlinelibrary.wiley.com/doi/epdf/10.1111/ppa.12257
https://bsppjournals.onlinelibrary.wiley...
), Fusarium oxysporum f. sp. phaseoli fungus was detected in lower levels of infection, and 0.25% incidence in beans seeds. One possibility of increasing the PCR sensitivity is prior incubation in favorable conditions for the development of fungi in seeds. Other example, cPCR and qPCR techniques were effective in detecting Colletotrichum lindemuthianum in beans seeds. It was possible using cPCR to detect the fungus in seed samples with 10% of incidence and with 0.25% incidence by qPCR technique (Gadaga et al., 2018GADAGA, S.J.C.; SIQUEIRA, C.S.; MACHADO, J.C. Molecular detection of Colletotrichum lindemuthianum in bean seed samples. Journal of Seed Science , v.40, n.4, p.370-377, 2018. http://dx.doi.org/10.1590/2317-1545v40n4192761
http://dx.doi.org/10.1590/2317-1545v40n4...
).

CONCLUSIONS

The results of this study, which complement previous work done by the pathologist group involved in this project in order to detect the causal agent of cotton Ramulosis in seed samples, meet a long-year demand from seed producers in Brazil. This technology enables a sanitary quality control of cotton seeds with greater accuracy and speed, making health analysis of seeds, which is viable and extremely important for the cotton producers.

It is also important to point out that, in practical terms, the health test protocol for the detection of C. gossypii var. cephalosporioides in cotton seed samples for quality certification programs can be made by implementing a health test by two methods, a molecular and a biological. In this case, samples would be initially subjected to PCR and subsequently applying the blotter test, as it was done by the current Rules for Seed Testing (Brasil, 2009aBRASIL. Ministério da Agricultura, Pecuária e Abastecimento. Manual de análise sanitária de sementes. Ministério da Agricultura, Pecuária e Abastecimento. Secretaria de Defesa Agropecuária. Brasília: MAPA/ACS, 2009a. 200p. http://www.agricultura.gov.br/assuntos/insumos-agropecuarios/insumos-agricolas/sementes-e-mudas/publicacoes-sementes-e-mudas/manual-de-analise-sanitaria-de-sementes/view
http://www.agricultura.gov.br/assuntos/i...
, bBRASIL. Ministério da Agricultura, Pecuária e Abastecimento. Regras para análise de sementes. Ministério da Agricultura, Pecuária e Abastecimento. Secretaria de Defesa Agropecuária. Brasília: MAPA/ACS, 2009b. 395p. http:// www.agricultura.gov.br/arq_editor/file/2946_regras_analise__sementes.pdf
http:// www.agricultura.gov.br/arq_edito...
) for samples that had positive results in molecular testing. It is understood that combining these two methods makes the diagnosis of Ramulosis’ agent in cotton seed samples safer and feasible from an operational point of view on health routine analytical laboratories.

ACKNOWLEDGEMENTS

The authors thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for providing the scholarship for the first author. To the CNPq, the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) for funding and supporting for research.

REFERENCES

  • BRASIL. Ministério da Agricultura, Pecuária e Abastecimento. Manual de análise sanitária de sementes Ministério da Agricultura, Pecuária e Abastecimento. Secretaria de Defesa Agropecuária. Brasília: MAPA/ACS, 2009a. 200p. http://www.agricultura.gov.br/assuntos/insumos-agropecuarios/insumos-agricolas/sementes-e-mudas/publicacoes-sementes-e-mudas/manual-de-analise-sanitaria-de-sementes/view
    » http://www.agricultura.gov.br/assuntos/insumos-agropecuarios/insumos-agricolas/sementes-e-mudas/publicacoes-sementes-e-mudas/manual-de-analise-sanitaria-de-sementes/view
  • BRASIL. Ministério da Agricultura, Pecuária e Abastecimento. Regras para análise de sementes Ministério da Agricultura, Pecuária e Abastecimento. Secretaria de Defesa Agropecuária. Brasília: MAPA/ACS, 2009b. 395p. http:// www.agricultura.gov.br/arq_editor/file/2946_regras_analise__sementes.pdf
    » http:// www.agricultura.gov.br/arq_editor/file/2946_regras_analise__sementes.pdf
  • BAILEY, J.A; NASH, C.; MORGAN, L.W.; O’CONNELL, R.J.; TEBEEST, D.O. Molecular taxonomy of Colletotrichum species causing Anthracnose on the Malvaceae. Phytopathology, v.86, p.1076-1083, 1996. https://www.apsnet.org/publications/phytopathology/backissues/Documents/1996Articles/Phyto86n10_1076.PDF
    » https://www.apsnet.org/publications/phytopathology/backissues/Documents/1996Articles/Phyto86n10_1076.PDF
  • BARROCAS, E.N.; MACHADO, J.C.; ALMEIDA, M.F.; BOTELHO, L.S.; VON PINHO, E.V.R. Sensibility of the PCR technique in the detection of Stenocarpella sp. associated to maize seeds. Revista Brasileira de Sementes, v.34, n.2, p.218-224, 2012. http://www.scielo.br/pdf/rbs/v34n2/05.pdf
    » http://www.scielo.br/pdf/rbs/v34n2/05.pdf
  • BARROCAS, E.N.; MACHADO, J.C.; ALVES, M.C.; CORREA, C.L. Desempenho de sementes de algodão submetidas à deficiência hídrica e presença de Colletotrichum gossypii var. cephalosporioides Bioscience Journal, v.30, n.2, p.421-428, 2014. http://www.seer.ufu.br/index.php/biosciencejournal/article/view/17993/13757
    » http://www.seer.ufu.br/index.php/biosciencejournal/article/view/17993/13757
  • BOTELHO, L.S.; BARROCAS, E.N.; MACHADO, J.C.; MARTINS, R.S. Detection of Sclerotinia sclerotiorum in soybean seeds by conventional and quantitative PCR techniques. Journal of Seed Science, v.37, n.1, p.55-62, 2015. http://dx.doi.org/10.1590/2317-1545v37n1141460
    » http://dx.doi.org/10.1590/2317-1545v37n1141460
  • CARVALHO, E.M.; FIGUEIRA, A.R.; MACHADO, J.C.; ARAÚJO, D.V.; MACHADO, C.F. Variability of seed-borne Colletotrichum strains in cotton based on its1 and its2 ribossomal genes analysis. Bioscience Journal , v.31, n.3, p.691-700, 2015. https://doi.org/10.14393/BJ-v31n3a2015-23126
    » https://doi.org/10.14393/BJ-v31n3a2015-23126
  • GADAGA, S.J.C.; SIQUEIRA, C.S.; MACHADO, J.C. Molecular detection of Colletotrichum lindemuthianum in bean seed samples. Journal of Seed Science , v.40, n.4, p.370-377, 2018. http://dx.doi.org/10.1590/2317-1545v40n4192761
    » http://dx.doi.org/10.1590/2317-1545v40n4192761
  • GUIMARÃES, M.R.F.; SIQUEIRA, C.S.; MACHADO, J.C.; FRANÇA, S.K.S.; GUIMARÃES, G.C. Evaluation of inoculum potential of pathogens in seeds: relation to physiological quality and DNA quantification by qPCR. Journal of Seed Science , v.39, n.3, p.224-233, 2017. http://www.scielo.br/pdf/jss/v39n3/2317-1545-jss-39-03-00224.pdf
    » http://www.scielo.br/pdf/jss/v39n3/2317-1545-jss-39-03-00224.pdf
  • LEE, H.K.; TEWARI, J.P.; TURKINGTON, T.K. Quantification of seedborne infection byRhinchosporium secalisin barley using competitive PCR.Plant Pathology, v.51, p.217-224, 2002.https://bsppjournals.onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-3059.2002.00685.x
    » https://bsppjournals.onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-3059.2002.00685.x
  • MACHADO, J.C.; BARROCAS, E.N.; COSTA, L.N.; GUIMARÃES, R.M.; MACHADO, C. Uso da técnica de restrição hídrica ou condicionamento osmótico em patologia de sementes. Revisão Anual de Patologia de Plantas, v.20, p.37-63, 2012.
  • MEHTA, Y.R.; MEHTA, A. Variabilidade genética entre isolados de Colletotrichum gossypii do algodoeiro. Summa Phytopathologica, v.36, n.1, p.40-44, 2010. http://www.scielo.br/pdf/sp/v36n1/07.pdf
    » http://www.scielo.br/pdf/sp/v36n1/07.pdf
  • MICHEL, B.E.; RADCLIFFE, D.A. Computer program relating solute potencial to solution composition for five solutes. Agronomy Journal, v.87, p.131-136, 1995.
  • MUNKVOLD, G.P. Seed pathology progress in academia and industry. Annual Review of Phytopathology , v.47, p.285-311, 2009. https://doi.org/10.1146/annurev-phyto-080508-081916
    » https://doi.org/10.1146/annurev-phyto-080508-081916
  • ROMERO, M.P.; WISE, K.A. Development of molecular assays for detection of Stenocarpella maydis and Stenocarpella macrospora in corn. Plant Disease, v.99, p.761-769, 2015. http://apsjournals.apsnet.org/doi/pdf/10.1094/PDIS-09-14-0917-RE
    » http://apsjournals.apsnet.org/doi/pdf/10.1094/PDIS-09-14-0917-RE
  • SALUSTIANO, M.E.; RONDON, M.N.; ABREU, L.M.; COSTA, S.S.; MACHADO, J.C.; PFENNING, L.H. The etiological agent of cotton Ramulosis represents a single phylogenetic lineage within the Colletotrichum gloeosporioides species complex. Tropical Plant Pathology , v.39, n.5, p.357-367, 2014. http://www.scielo.br/pdf/tpp/v39n5/v39n5a02.pdf
    » http://www.scielo.br/pdf/tpp/v39n5/v39n5a02.pdf
  • SILVA-MANN, R.; SALGADO, K.C.C.; VIEIRA, M.G.G.C.; MACHADO, J.C. Variabilidade genética de isolados do complexo Colletotrichum associados a sementes de algodoeiro, por meio de técnicas moleculares e inoculação em plantas. Fitopatologia Brasileira, v.27, n.1, p.27-32, 2002. http://www.scielo.br/pdf/fb/v27n1/8464.pdf
    » http://www.scielo.br/pdf/fb/v27n1/8464.pdf
  • SILVA-MANN, R.; VIEIRA, M.G.G.C.; MACHADO, J.C.; BERNARDINO-FILHO, J.R.; SALGADO, K.C.C.; STEVENS, M.R. AFLP markers differentiate Colletotrichum gossypii from C. gossypii var. cephalosporioides Fitopatologia Brasileira , v.30, n.2, p.169-172, 2005. http://www.scielo.br/pdf/fb/v30n2/a11v30n2.pdf
    » http://www.scielo.br/pdf/fb/v30n2/a11v30n2.pdf
  • SOUSA, M.V.; SIQUEIRA, C.S.; MACHADO, J.C. Conventional PCR for detection of Corynespora cassiicola in soybean seeds. Journal of Seed Science , v.38, n.2, p.85-91, 2016. http://www.scielo.br/pdf/jss/v38n2/2317-1545-jss-v38n2152049.pdf
    » http://www.scielo.br/pdf/jss/v38n2/2317-1545-jss-v38n2152049.pdf
  • SOUSA, M.V.; MACHADO, J.C.; SIMMONS, H.E.; MUNKVOLD, G.P. Real-time quantitative PCR assays for the rapid detection and quantification of Fusarium oxysporum f. sp. phaseoli in Phaseolus vulgaris (common bean) seeds. Plant Pathology , v.64, p.478-488, 2015. https://bsppjournals.onlinelibrary.wiley.com/doi/epdf/10.1111/ppa.12257
    » https://bsppjournals.onlinelibrary.wiley.com/doi/epdf/10.1111/ppa.12257
  • TAMURA, K.; PETERSON, D.; PETERSON, N.; STECHER, G.; NEI, M.; KUMAR, S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance and maximum parsimony methods. Molecular Biology and Evolution, v.28, n.10, p.2731-2739, 2011. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3203626/pdf/msr121.pdf
    » https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3203626/pdf/msr121.pdf
  • TANAKA, M.A.S.; MENTEN, J.O.M.; MACHADO, J.C. Hábito de crescimento de Colletotrichum gossypii e C. gossypii var. cephalosporioides em sementes de algodoeiro. Bragantia, v.55, n.1, p.95-104, 1996. http://www.scielo.br/pdf/brag/v55n1/11.pdf
    » http://www.scielo.br/pdf/brag/v55n1/11.pdf
  • TEMPLETON, M.D.; RIKKERINK, E.H.A.; SOLON, S.L.; CROWHURT, R.N. Cloning and molecular characterization the glyceraldehyde-3- phosphate dehydrogenase-encoding gene and cDNA from the plant pathogenic fungus Glomerella cingulata Gene, v.122, n.1, p.225-230, 1992. https://doi.org/10.1016/0378-1119(92)90055-T
    » https://doi.org/10.1016/0378-1119(92)90055-T
  • TOZZE-JÚNIOR, H.J.; BUENO, C.R.N.C.; MASSOLA-JÚNIOR, N.S. Caracterização morfológica e molecular de isolados de Colletotrichum spp. de hortaliças solanáceas. Summa Phytopathologica , v.32, n.1, p.71-79, 2006. http://www.scielo.br/pdf/sp/v32n1/v32n1a11.pdf
    » http://www.scielo.br/pdf/sp/v32n1/v32n1a11.pdf

Publication Dates

  • Publication in this collection
    03 Apr 2020
  • Date of issue
    2020

History

  • Received
    03 Oct 2019
  • Accepted
    05 Feb 2020
ABRATES - Associação Brasileira de Tecnologia de Sementes Av. Juscelino Kubitschek, 1400 - 3° Andar, sala 31 - Centro,, CEP 86020-000 Londrina/PR - Londrina - PR - Brazil
E-mail: jss@abrates.org.br