INTRODUCTION

Achira (Canna edulis Ker.) belongs to the Cannaceae (^{Tanaka, 2004}) family; it is of South American origin and is grown in Latin America, Caribbean, Australia and Asia (^{Corpoica, 2001}; ^{Rodríguez et al. 2003}), mainly to extract starch from its rhizomes, which is used in food preparation such as bread, cakes and pasta. In Colombia, these activities are important for rural economy of several departments and farmers generally use local planting materials (^{Caicedo et al. 2003}; ^{Torres, 2004}; ^{Corpoica, 2001}).

In Taiwan, Vietnam and southern China, the species is sown between 2,000 and 3,000m. a.s.l. (^{Kasemwong et al. 2003}; ^{Roca and Manrique, 2005}) and in Colombia, the species is grown in areas with altitudes below 2,700m (^{Ariza León et al. 2013}), it is estimated that the achira starch demand per year in the country is 1,681.65t. The main producers are the departments of Nariño with 502ha, Huila 81ha and Cauca 24ha (^{Agronet, 2016}). The plantations are established with genotypes local of regional origin, selected by farmers (^{Caicedo et al. 2003}).

Among the limiting factors for crop production are phytosanitary problems, and particularly diseases associated with viruses, since they significantly reduce yields reaching losses of 70% or more, and are hard to manage (^{Hull, 2014}).

Worldwide, several species of viruses affecting achira have been reported for years, Bean yellow mosaic virus, Potyvirus (BYMV) (^{Castillo et al. 1956}), Tomato aspermy virus, Cucumovirus (TAV) (^{Hollings et al. 1971}), Cucumber mosaic virus, Cucumovirus (CMV) (^{Lockhart, 1988}), Canna yellow mottle virus, Badnavirus (CYMV) (^{Yamashita et al. 1985}), Canna yellow streak virus, Potyvirus (CaYSV) (^{Monger et al. 2007}), Tomato spotted wilt virus, Tospovirus (TSWV) (^{Moini et al. 2000}) and Sugarcane mosaic virus, Potyvirus (SCMV) (^{Tang et al. 2016}).

In Colombia, infections caused by CMV have been registered in the Canna edulis species (^{Castaño et al. 1994}; ^{Castaño et al. 1995}; ^{Reichel et al. 1995}) and Banana streak virus, Badnavirus (BSV) (^{Reichel et al. 1996}). Mixed infections of filamentous particles similar in their morphology to species of the genus Potexvirus (^{Reichel, 2007}) and bacilliform particles similar to species of the genus Badnavirus have also been observed in achira plants with viral symptoms, from the department of Nariño (Reichel, data sin publish).

In the department of Nariño, achira is grown in the municipalities of La Cruz, San Pablo, El Tablón, Colón, Belén and Albán (Consolidado Agropecuario - Nariño, 2015). At present, there is no detailed knowledge of the virus species present in the crops, nor have been applied the diagnostic methods based on serological or molecular techniques that are available worldwide for these pathogens. In addition, their means of transmission and other biological characteristics of epidemiological interest are not known.

This situation motivated the development of the current research between the University of Nariño, Agrosavia and CIAT, aiming to establish solutions in the diagnosis and detection of viruses, through the standardization of serological and molecular protocols that serve as a starting point for such diseases management.

MATERIALS AND METHODS

Localization. This research was carried out at the University of Nariño (Pasto - Nariño) and at the CIAT International Center for Tropical Agriculture (Palmira - Valle del Cauca), under laboratory conditions.

Plant material. Plant samples were taken in La Cruz municipality, which is the main achira production area in Nariño and in an Achira germoplasm collection of AGROSAVIA (Obonuco-Pasto), with genotypes from the departments of Cundinamarca, Nariño and Huila.

Samples were taken from symptomatic and asymptomatic plants, based on symptoms reported for pathologies associated with viruses in achira (^{Castaño et al. 1994}; ^{Castaño et al. 1995}; ^{Reichel et al. 1995}; ^{Rajakaruna et al. 2014}; ^{Verchot & Rajakaruna, 2013}). The sampling included the recollection of foliar tissues, which were labeled, wrapped in paper towels and placed in plastic bags for transport to the laboratories. The plant material was kept in a refrigerator at 4°C until processing.

Evaluation and visual description of viral symptoms. The monitoring and visual description of 10 achira plants was carried out for six weeks, with photographic record. The symptoms evolution was evaluated in each plant, from the first leaf outbreak, following recommendations of other authors (^{Armitage et al. 1990}; ^{Hunger & Sherwood, 1985}; ^{Lockhart, 1988}; ^{Monger et al. 2007}; ^{Pappu & Druffel, 2008}; ^{Zhang et al. 2011}).

Virus detection by molecular tests

DNA extraction. Leaf samples from symptomatic achira plants were used, following the method of ^{Prince et al. (1993)}, taking 0.4g of tissue in each case, which was frozen and macerated in liquid nitrogen using a sterile mortar and pestle. After the final wash in ethanol, the extracted nucleic acid was suspended in 30 to 50uL of Tris-EDTA buffer solution (10 mM Tris-HCl, pH 8.0, and 1mM EDTA, pH 8.0) and stored at 4ºC until use.

Direct PCR. Total DNA was used for direct PCR amplification using CaYMV-specific primers: CaYMV-3 and CaYMV-4, which produce a 565bp fragment (^{Momol et al. 2004}). PCR was carried out using a denaturation cycle at 95°C for 5 min and 35 cycles of amplification (95°C for 60s, 60°C for 45s, 72°C for 60s), with a final extension at 72°C for 7 min. A mixture of 12.5μL of go taq master mix 2X Promega, 0.5μL of each primer, 9.5μL of water and 2μL of each DNA sample was prepared for a final volume of 25μL.

RNA extraction and RT-PCR. RNA extraction and cDNA synthesis were developed at CIAT (Palmira, Valle del Cauca). The RT-PCR technique was used according to the protocol described by ^{Carvajal et al. (2014)}. Between 2 and 4μg of RNA and 200 to 400ng of random primers were denatured at 65°C for 5 min, followed by one minute on ice. The first cDNA chain was synthesized at 37°C for 1.5 hours after adding 5x first Strand buffer, 0.1 M DTT, 10mM dNTPs and 200 U of MMLV reverse transcriptase (invitrogen) to a final volume of 15μL. The quality of the cDNA was checked with a PCR of internal controls.

Once the cDNA was obtained, PCR tests were performed with specific primers for: BYMV (BYMV-F and BYMV-R) (^{Skelton et al. 2007}), CaYSV (Canna-F1 and Canna-R2) (^{Monger et al. 2007}), SCMV (S400-551 and S400-910) (^{Smith et al. 1994}) and CMV and TAV (CPTALL-3 and CPTALL-5) (^{Choi et al. 1999}), which produce fragments of 382, 695, 359 and 938 and 966bp, respectively. Primers and the positive control for SCMV were provided by Cenicaña.

In the PCR amplification tests, a mixture of 12.5μL of go taq master mix 2X Promega, 0.5μL of each primer, 9.5μL of water and 2μL of each cDNA sample was prepared for a final volume of 25μL. For the detection of BYMV, CaYSV and SCMV, 35 cycles were performed (95°C for 60s, 50°C for 45s, and 72°C for 60s), with a final extension of 72°C for 7 min. For Cucumovirus the same program was used, but using a temperature of the hybridization phase of 40°C for 60s.

Sequencing. The PCR amplification products were cleaned using the DNA precipitation method with polyethylene glycol and ethanol (^{Ziv et al. 2007}) and sequenced in Macrogen. The obtained sequences were compared by maximum mating and higher percentages of homology with those of reference in the GenBank, using the BLAST tool of the National Biotechnology Information Center (http://www.ncbi.nlm.nih.gov/BLAST) (^{Altschul et al. 1990}).

Serological tests. Leaf tissue samples from asymptomatic and symptomatic achira plants were also subjected to serological tests of DAS-ELISA, with commercial antibodies (AGDIA) for the Potyvirus group, to verify the presence of viruses, following the methodology described by ^{Clark & Adams (1977)} and recommendations from other authors such as ^{Reichel et al. (1997)}.

Electron Microscopy. It was carried out at the University of Antioquia. The resin immersion method was used to process foliar tissue from very young leaves, from symptomatic achira plants. Fixation was made in 2.5% glutaraldehyde and washed in saline buffer, postfixed in 1% osmium tetra-oxide, washed with buffer and propylene oxide. Then it was dehydrated in an ascending gradient of ethanol from 10% to 100%, to then imbibe it and include it in epoxy resin. Polymerization was carried out at 70°C, and ultrafine cuts at 60nm. Subsequently, the sections were contrasted with uranyl acetate and lead citrate and were observed under a Tecnai G2 F20 transmission electron microscope.

RESULTS AND DISCUSSION

Evaluation and visual description of viral symptoms. In the department of Nariño, the achira crops have a high incidence of virus, the evaluated plants showed a wide range of foliar symptoms, which start as a yellowing, mottled mosaics, and advance to the leaf distortion; especially on the edges, chlorotic streaking, vein clearing, leaf necrosis and sometimes dwarfism, as the most outstanding (Figure 1).

Figure 1 a.-b. symptoms with veins deformation and chlorosis; c.-d. chlorosis and vein necrosis together with leaf rolling; e.-f. chlorotic bands on leaves; g.-h. symptoms of chlorotic veins and streaks; i.-k. symptoms of mosaics with different tonalities (yellow, white, green, orange and purple), set to deformation of the edge of the leaves; l.-n. symptoms of streaks and chlorotic spots; o.-p. symptoms of chlorotic speckling on the final third of the leaf. 

Farmers call the disease as "yellow streak" because of its predominant symptom, which they correlate to a decrease in the yield of starch. Cannas economically speaking can be separated in two main groups, those used as source of food, i.e. C. edulis and those used for ornamental purposes, i.e. C. indica and related hybrids. Because ornamental and edible cannas can have different leaf color and can also have variegations in their leaves, the expression of viral symptoms on them are somewhat different depending on the variety and could lead to confusion when observing samples in the field (^{Rajakaruna et al. 2014}). In this case, the plantations studied were planted with two different varieties (green or black), none of those are known to have variegations in their leaves, suggesting that the observed symptoms are due to physiological responses possibly caused by the presence of virus. Similar symptoms were reported in China in achira plantations affected by SCMV (^{Tang et al. 2016}). Also, similar symptoms description due to Potyvirus infections in canna have been reported by several authors (^{Alexandre et al. 2017}; ^{Zakubanskiy et al. 2017}; ^{Kumari et al. 2016}; ^{Monger et al. 2007}). Other virus species like a Tomato aspermy virus, Cucumovirus (TAV) (^{Hollings et al. 1971}), Cucumber mosaic virus, Cucumovirus (CMV) (^{Lockhart, 1988}), Canna yellow mottle virus, Badnavirus (CYMV) (^{Yamashita et al. 1985}), because similar symptoms, but none were detected in this study.

Virus detection by molecular tests

Direct PCR. None of the collected samples amplified by direct PCR. Thus, there was no presence of Canna yellow mottle virus (CaYMV) Badnavirus (Caulimoviridae family) using specific primers. In exploration studies in Brazil, ^{Alexandre et al. (2017)} reported that Badnavirus amplifications were not obtained in a Canna species, with symptoms of chlorotic mosaic, but subsequent studies using RT-PCR showed the presence of a Potyvirus in the same samples.

RT-PCR. Negative results were obtained for the TAV, CMV, BYMV and CaYSV viruses, but positive results were obtained for the Sugarcane mosaic virus (SCMV) Potyvirus, in (20/30) symptomatic samples examined, corresponding to 66%. One of the gels is shown in figure 2.

Figure 2 1% of agarose gel, Lanes 1 to 5 and 6 to 10 cDNA samples from achira plant tissue, M: molecular weight marker of 100 bp (Thermoscientific), +: positive control, SCMV cDNA donated by CENICAÑA, -: Negative control, reaction mix without cDNA. 

Molecular diagnostic tests revealed that a high proportion of plants were infected with SCMV, pointing to an association with the "yellow stripped" disease in the department of Nariño. In China, ^{Tang et al. (2016)} similarly detected SCMV infecting Canna spp. While the similarity of symptoms of the “yellow stripped” disease might be confused with those of caused by CaYSV, (^{Rajakaruna et al. 2014}), there is a phylogenetic relationship between these two viruses as shown by ^{Chauhan et al. (2015)}, whom showed that CaYSV (Potyvirus), a virus that affects achira, belongs to the subgroup of SCMV.

Sequencing. Sanger sequencing of the sugarcane mosaic virus (SCMV) RT-PCR amplification products were performed and the sequences compared for maximum identity and coverage against the available sequences deposited at the Gene Bank of the National Center for Biotechnology Information (NCBI), using the BLAST tool of the National Biotechnology Information Center. The results indicated the resemblance of the amplified sequences to the id. AF006734.1, the partial coding sequence of the sugar cane mosaic virus coat protein, isolate Brisbane, of Australian origin. The matching fragment had a length of 286bp with no gaps and a homology of 98% to the consensus sequence of the experimental samples of (Nariño) Colombia.

Serological tests. Serological tests of DAS-ELISA were performed with commercial serological kits (AGDIA) for the genus Potyvirus in which no positive results were obtained. Negative and positive control for the assay were satisfactory (data not shown). The results suggest that perhaps a low titer of viral particles precluded the assay from recognizing the pathogen and reinforce the notion that molecular assays have higher sensitivity than serological tests. Nonetheless, in the available literature about the Agdia Poty test, there is no information about the sensitivity of the assay to different strains of SCMV. The same reported (^{Alexandre et al. 2017}), in serological studies with polyclonal antibodies to Potyvirus in Canna.

Electron Microscopy. The tissues examination under observation in transmission electron microscope did not confirm the presence of viral particles and inclusion bodies (Figure 3). This situation is not indicative of the absence of virus because its detection requires a correct selection of samples. On the other hand, the concentration of virus in plant tissues can be low, which makes it difficult to detect it for electron microscopy.

Figure 3 Electron micrographs of achira leaf tissue of the green variety. White arrow grana thylakoid and red arrow Chloroplast stroma. 

Different viruses have been reported that are infecting the Canna spp., in countries such as the United Kingdom, Belgium, Holland, France, Italy, Austria, Israel, Kenya, Iran, India, China, Thailand, Japan, the United States and Brazil (^{Zakubanskiy et al. 2017}).

In conclusion, this is the first report of the Sugarcane Mosaic Virus, Potyvirus (SCMV) infecting achira crops (Canna edulis ker.) in Nariño (Colombia), through the use of molecular detection techniques. The epidemiological implications on the crop are serious, because its propagation is vegetative. It is necessary to initiate studies related to vectors and cleaning of planting material.