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., 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).

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

Species	CML1	Other code2	Geographic origine3	Host	Specific Primer4
C. gossypii var. cephalosporioides		LAPS 22	Maracaju, MT	Gossypium hirsutum	+
C. gossypii var. cephalosporioides		LAPS 23	Maracaju, MT	Gossypium hirsutum	+
C. gossypii var. cephalosporioides		LAPS 24	Tangará da Serra, MT	Gossypium hirsutum	+
C. gossypii var. cephalosporioides		LAPS 32	Primavera do Leste, MT	Gossypium hirsutum	+
C. gossypii var. cephalosporioides	2371	LAPS 259	Santa Helena de Goiás, GO	Gossypium hirsutum	+
C. gossypii var. cephalosporioides	2372	LAPS 260	Primavera do Leste, MT	Gossypium hirsutum	+
C. gossypii var. cephalosporioides	2373	LAPS 261	Pedra Preta, MT	Gossypium hirsutum	+
C. gossypii var. cephalosporioides	2374	LAPS 263	Primavera do Leste, MT	Gossypium hirsutum	+
C. gossypii var. cephalosporioides	2375	LAPS 264	Mineiros, GO	Gossypium hirsutum	+
C. gossypii var. cephalosporioides	2376	LAPS 265	Campo Verde, MT	Gossypium hirsutum	+
C. gossypii var. cephalosporioides	2377	LAPS 266	Primavera do Leste, MT	Gossypium hirsutum	+
C. gossypii var. cephalosporioides	2378	LAPS 267	Mineiros, GO	Gossypium hirsutum	+
C. gossypii var. cephalosporioides	2379	LAPS 268	Mineiros, GO	Gossypium hirsutum	+
C. gossypii var. cephalosporioides	2380	LAPS 269	Nova São Joaquim, MT	Gossypium hirsutum	+
C. gossypii var. cephalosporioides	2381	LAPS 270	Nova São Joaquim, MT	Gossypium hirsutum	+
C. gossypii var. cephalosporioides	2382	LAPS 271	Pedra Preta, MT	Gossypium hirsutum	+
C. gossypii var. cephalosporioides	2383	LAPS 272	Itiquira, MT	Gossypium hirsutum	+
C. gossypii var. cephalosporioides	2384	LAPS 273	Itiquira, MT	Gossypium hirsutum	+
C. gossypii var. cephalosporioides	2386	LAPS 275	Itiquira, MT	Gossypium hirsutum	+
C. gossypii var. cephalosporioides	2387	LAPS 276	Itiquira, MT	Gossypium hirsutum	+
C. gossypii var. cephalosporioides		LAPS 277	Primavera do Leste, MT	Gossypium hirsutum	+
C. gossypii var. cephalosporioides		LAPS 392	Primavera do Leste, MT	Gossypium hirsutum	+
C. gossypii var. cephalosporioides		LAPS 393	Primavera do Leste, MT	Gossypium hirsutum	+
C. gossypii var. cephalosporioides		LAPS 396	Primavera do Leste, MT	Gossypium hirsutum	+
C. gossypii var. cephalosporioides		LAPS 397	Primavera do Leste, MT	Gossypium hirsutum	+
C. gossypii var. cephalosporioides		LAPS 398	Primavera do Leste, MT	Gossypium hirsutum	+
C. gossypii var. cephalosporioides		LAPS 400	Primavera do Leste, MT	Gossypium hirsutum	+
C. gossypii var. cephalosporioides		CGCUber	Uberlândia, MG	Gossypium hirsutum	+
Colletotrichum gossypii	2327	CG3LEM	Luís Eduardo Magalhães, BA	Gossypium hirsutum	-
Colletotrichum siamense sensu lato	2884	CCJ73	Campo Grande, PB	Anacardium occidentale	-
Colletotrichum tropicale	2888	CCJ105	Fortaleza, CE	Anacardium occidentale	-
Colletotrichum asianum	2893	CCJ204	São Luís, MA	Anacardium occidentale	-
Colletotrichum theobromicola	2931	MT68	Pacajus, CE	Anacardium occidentale	-
Colletotrichum truncatum		LAPS133	Rio Verde, GO	Phaseolus vulgaris	-
Colletotrichum gloeosporioides		CAA115/1	Acari, MG	Annona reticulata	-
Colletotrichum fructicola		CAA137	Acari, MG	Annona crassiflora	-
Colletotrichum karstii		CAA81	Umbuzeiro, MG	Annona crassiflora	-
Colletotrichum gigasporum	3316	LabioMMi3311	Brazil	Piper aducum	-
Aspergillus flavus	2708		Montividiu, GO	Solo (Gossypium hirsutum)	-
Aspergillus clavatus	2734		Ibiá, MG	Seed (Glycine max)	-
Aspergillus chevalieri	2737		Lavras, MG	Seed (Phaseolus vulgaris)	-
Bipolaris sorokiniana	3315	LabioMMi285	São Carlos, SP	Piper aducum	-
Curvularia sp.		CTC15	Pará		-
Alternaria alternata	3314	LabioMMi06	Brazil		-
Diaphorte sp.		LAPS559	São Paulo, SP	Glycine max	-
Phoma tarda	716		Campanha, MG	Coffea arabica	-
Phoma exígua	940		Coromandel, MG	Coffea arabica	-
Penicillium citrunum	3310	LabioMMi249	Teresina, PI		-
Penicillium terrigenum	1226		Montividiu, GO	Gossypium hirsutum	-
Fusarium oxysporum f. sp. vasinfectum	1119		Mato Grosso	Gossypium hirsutum	-
Ascochyta sp.	361		Lavras, MG	Baccharis sp.	-
Phomopsis sp.		FEL89	Brazil		-
Clonostachys roseum		CSO36	Brazil		-
Cercospora sp.		LAPS255	Campo Verde, MT	Glycine max	-
Fusarium paranaense	1830		Brazil	Glycine max	-
Didymella sp.	193		Machado, MG	Coffea arabica	-
Macrophomina sp.		MA01	Primavera do Leste, MT	Glycine max	-
Corynespora cassiicola		LAPS467	São Paulo, SP	Glycine max	-
Sclerotinia sp.		LAPS242	Uberlândia, MG	Glycine max	-

• ¹CML = mycological collection of the Plant Pathology Department, Universidade Federal de Lavras, Lavras, MG, Brazil. ²LAPS = mycological collection of the Seed Pathology Laboratory, Universidade Federal de Lavras, Lavras, MG, Brazil. ²LaBioMMi = Microorganisms Micromolecular Biochemistry Laboratory, Chemistry Department, Universidade Federal de São Carlos, São Carlos, SP, Brazil. ³States of Brazil: BA = Bahia; CE = Ceará; GO = Goiás; MG = Minas Gerais; MA = Maranhão; MT = Mato Grosso; PB = Paraíba; PI = Piauí; SP = São Paulo. ⁴Specific primer; (+) PCR amplification; (-) no PCR amplification.

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-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.

Species	CML1	Other code2	Host	Origin3	GenBank number
C. gossypii var. cephalosporioides	2373	LAPS 261	Gossypium hirsutum	Pedra Preta, MT	JX847009
C. gossypii var. cephalosporioides	2379	LAPS 268	Gossypium hirsutum	Mineiros, GO	JX847010
C. gossypii var. cephalosporioides	2384	LAPS 273	Gossypium hirsutum	Itiquira, MT	JX847011
C. gossypii var. cephalosporioides	2388	IAC 13350	Gossypium hirsutum	Piracicaba, SP	JX847012
C. gossypii var. cephalosporioides	2389	IAC 12405	Gossypium hirsutum	Ituverava, SP	JX847013
Colletotrichum gossypii	2324	IAC 1025	Gossypium hirsutum	Campinas, SP	JX847014
Colletotrichum gossypii	2325	CG 1 LEM	Gossypium hirsutum	Luis Eduardo Magalhães, BA	JX847015
Colletotrichum gossypii	2327	CG 3 LEM	Gossypium hirsutum	Luis Eduardo Magalhães, BA	JX847016
C. kahawae subsp. kahawae		ICMP 17905	Coffea arabica	Kenya	JX010012
Colletotrichum gloeosporioides		IMI 356878	Citrus sinensis	Italy	JX010056
Colletotrichum fructicola		ICMP 18581	Coffea arabica	Thailand	JX010033
Colletotrichum siamense		ICMP 18578	Coffea arabica	Thailand	JX009924
Colletotrichum asianum		ICMP 18580	Coffea arabica	Thailand	JX010053
Colletotrichum theobromicola		ICMP 17958	Stylosanthes guianensis	Australia	JX009948
Colletotrichum boninense		CBS 112115	Leucospermum sp.	Australia	JQ005247

• ¹CML = mycological collection of the Plant Pathology Department, Universidade Federal de Lavras, Lavras, MG, Brazil. ²LAPS = mycological collection of the Seed Pathology Laboratory, Universidade Federal de Lavras, Lavras, MG, Brazil. IAC = Campinas Agronomic Institute, Campinas, SP, Brazil. ICMP = International Collection of Microorganisms from Plants, New Zeland. CBS = Centralalbureau voor Schimmelcultures, Utrecht, The Netherlands. ³States of Brazil: BA = Bahia; GO = Goiás; MT = Mato Grosso; SP = São Paulo.

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.

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., 2012; 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).

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. (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 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., 2018).

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.

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.

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Publication Dates

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

History

• Received
  03 Oct 2019
• Accepted
  05 Feb 2020