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

: Cotton Ramulosis ( Gossypium hirsutum ) is an important disease affecting cotton plantations in Brazil, and its causal agent, Colletotrichum gossypii var. 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. 25 µL PCR OneTaq 10 pmol forward and reverse primers and DNA 10 ng. 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). separate PCR an aliquot 10 µL on with PCR transilluminator, L-Pix Before the specific primers, a PCR reaction performed using universal GDF primers GDF (5´- and universal GDR primers GDR with genomic of all species used in to test if genomic for PCR The repeated at least two times.


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., 2014).
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., 1996). Both pathogens are transmitted by seeds and cause damages in cotton plants (Silva-Mann et al., 2005;Mehta and Mehta, 2010).
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., 1996;Tozze-Júnior et al., 2006). 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., 2015).
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., 1996). 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., 2002;Mehta and Mehta, 2010), 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., 2015).
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., 2002;Munkvold, 2009;Barrocas et al., 2012). This technology was successful in detecting, for example, Stenocarpella complex (S. maydis and S. macrospora) in maize (Romero and Wise, 2015), Fusarium oxysporum f.sp. phaseoli in bean seeds (Sousa et al., 2015), Sclerotinia sclerotiorum in soybean seeds (Botelho et al., 2015) and Corynespora cassiicola in soybean seeds (Sousa et al., 2016).
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).
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), using ClustalW implemented by MEGA5 (Tamura et al., 2011), 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-Journal of Seed Science, v.42, e202042012, 2020 Aldrich Brazil LTD. The genomic material isolated from C. gossypii var. cephalosporioides was subjected to PCR analysis.
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., 1992), 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.  Journal of Seed Science, v.42, e202042012, 2020 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 Barrocas et al., 2014).
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, 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).
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).
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. (2017), 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. (2012), Sternocarpella was detected in maize seeds infected with minimal incidence of 2% in the studied samples. In a study conducted by Sousa et al. (2015), Fusarium oxysporum f. sp. phaseoli fungus was detected  Lane M: 50kb marker; lanes 1 and 2 -Cgc -CML2384 and 2374 isolates; lanes 3 to 6: 1% infection with seeds inoculated for 24 hours; lanes 7 to 10: 10% infection with seeds inoculated for 24 hours; lanes 11 to 14: 1% infection with seeds inoculated for 48 hours; lanes 15 to 18: 10% infection with seeds inoculated for 48 hours; lanes 19 to 22: control without fungus; lanes 23 to 26: 100% seeds infected with inoculation of 24 hours; lanes 27 to 30: 100% seeds infected with inoculation of 48 hours.

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, 2009a, b) 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.