The commercial varieties of poinsettia are the bestselling pot plants worldwide (^{Canul et al., 2012}). In 2018, the value of poinsettia production in the United States of America reached more than 148 million dollars (^{USDA, 2019}). In the same year, 19 million plants were produced in Mexico, valued at 718 million Mexican pesos (^{SADER, 2019}). Poinsettia cropping is threatened by pests and diseases, and those caused by viruses are the most important (^{Bertaccini et al., 1996}). Poinsettia varieties are hosts of Poinsettia latent virus (PnLV) (^{aus dem Siepen et al., 2005}), Euphorbia leaf curl virus (EuLCV) (^{Ma et al., 2004}) and Poinsettia mosaic virus (PnMV), of which Poinsettia mosaic virus (PnMV) is the most important and widely distributed poinsettia viral pathogen in the world (^{Clarke et al., 2006};^{Okano et al., 2010}).

Commercial poinsettia varieties infected with PnMV usually show leaf mosaic symptoms and mottling, as well as foliar deformation that includes the bracts, but some plants can be asymptomatic (^{Fulton and Fulton, 1980}; ^{Lebas et al., 2007}). When infections are severe, the PnMV interferes with the process of bract pigmentation, poinsettias’ main attractive feature, and puts the production of the crop at risk (^{Brunt et al., 1996}). In Mexico, PnMV infects wild poinsettia plants, backyard plants, and the ‘Freedom’ and ‘Red Prestige’ commercial varieties (^{Jacobo et al., 2015}; ^{Ocampo et al., 2013}). However, the presence of PnMV has not been detected on other varieties cultivated in the country. One of the limitations in the biological study of PnMV is the low success rate of the mechanical transmission of the virus to differential species (0-10%) (^{Guy et al., 1985}). Based on the above, the objectives of this research were to identify the PnMV in 20 commercial poinsettia varieties and evaluate two buffer solutions in the mechanical transmission of PnMV to differential species. The hypotheses formulated were that the poinsettia varieties commercialized in Mexico are infected with PnMV and that at least one buffer solution is adequate for transmitting PnMV by mechanical means to differential species.

In December 2016-2018, asymptomatic plants (A) and plants with putative virus symptoms (S) of 20 commercial poinsettia varieties (100% pigmented) were obtained from a greenhouse located in Texcoco, State of Mexico. The advantages of obtaining materials in the winter season are the total differentiation of bracts and the presence of developed leaves on the plants, which makes it easier to observe the viral symptoms. The plant material (rooted cuttings) came from the flower companies Floraplant^® and Vivero Internacional de México^® that import plant material mainly from the United States.

The total number of pot plants that were obtained of each variety depended on the availability of the plant material. The varieties and the number of plants each year were: 2016: ‘Silverstar Marble’ (three plants, A), ‘Silverstar Red’ (two plants, A), ‘Sparkling Punch’ (one plant, S), ‘Ice Punch’ (three plants, A), ‘Marblestar’ (two plants, A), ‘Cortez Electric Fire’ (one plant, A), ‘Carousel Dark Red’ (one plant, A) and ‘Primero White’ (six plants, A); 2017: ‘Cortez Red’ (two plants, S), ‘Enduring Marble’ (two plants, S), ‘Freedom Pink’ (two plants, A), ‘Monet Early’ (two plants, A), ‘Polar Bear’ (two plants, S), ‘Premier Pink’ (two plants, A), ‘Viking Cinnamon’ (two plants, A), ‘Winter Rose Early Red’ (two plants, S) and ‘Winter Rose White’ (two plants, A); 2018: ‘Amaris Hot Pink’ (11 plants, S), ‘Primero Red Glitter’ (six plants, S) and ‘Orange Spice’ (six plants, A). The plants were established in the Floriculture greenhouse of the Plant Breeding Department of Chapingo Autonomous University (UACh, its acronym in Spanish), kept at 22 °C temperature during the day and 15 °C at night, with a photoperiod of 11-12 h, and agronomically managed as indicated by ^{Cabrera et al. (2006}). Samples of young leaves, bracts, and stems of all the varieties were taken and kept in refrigeration at -20 °C until they were analyzed.

The serological tests were conducted at the National Research Laboratory and Agri-Food and Forestry Service, in December 2019. The diagnosis was made using the DAS-ELISA method with polyclonal antibodies to detect PnMV; the polyclonal antibodies were purchased from Agdia^® (catalog number SRA 90700/0500). The analysis of each poinsettia variety was conducted in duplicate (two samples per plant of each variety), according to the manufacturer’s protocol. The samples consisted of 0.5 g of young leaves which were individually macerated in sterile mortars using liquid nitrogen. The samples placed on sensitized plates with the enzymatic conjugate were incubated at 4 °C for 12 h. The ‘Red Prestige’ commercial variety, provided by the UACh’s Agricultural Virology Laboratory, was used as the positive control, and a wild poinsettia plant collected in Tehuilotepec, Guerrero, as the negative control.

The absorbance values were measured at 405 nm using a Varioskan Flash Thermo Scientific^® microplate reader 60 min after incubation. According to ^{Ruiz et al. (2009}), samples with values higher than three times the means of the negative control were considered positive.

The molecular tests were conducted at the Agricultural Virology Laboratory of UACh’s Department of Agricultural Parasitology. The extraction of total RNA was carried out using the PureLink™ Plant RNA Reagent (catalog number 12322012) from the Thermo Fisher Scientific^® company, according to the manufacturer’s protocol. From each poinsettia variety, samples of 0.1 g of plant tissue from young leaves, stems and/or bracts were taken and individually macerated with liquid nitrogen in sterile mortars, and then each macerate was placed in a microcentrifuge tube (1.5 mL) to which 500 µL of lysis solution were added. The samples were centrifuged at 12,000 x g for 2 min at 4 °C, the supernatant was recovered and mixed with 100 µL of 5 M sodium chloride and 300 µL of chloroform, and centrifuged for 10 min; the superficial part was recovered, and 1.5 volumes of isopropanol were added (relative to what was recovered) and incubated at -4 °C for 12 h. Then, the samples were centrifuged again for 10 min; RNA cleanup was performed with 70% ethanol; and, finally, the RNA tablet was diluted in water free of nucleases (Promega^®) and stored at -20 °C.

The concentration of the total RNA was determined in a NanoDrop ThermoFisher Scientific^® spectrophotometer, while RNA integrity was verified in 1% p/v agarose gel electrophoresis (120V/60 min) and visualized in a Quantum Studio^® photodocumenter. To determine RNA viability, one fragment of the 18S ribosomal gene was amplified using the primers 18S-F (5’-ACGGATCGCACGGCCTTCGTG-3’) and 18S-R (5’-ACCAGACTTGCCCTCCAATGG-3’), which amplify a 300 pb fragment, according to the RT-PCR conditions indicated by ^{Zamboni et al. (2008}).

To detect PnMV on the poinsettia varieties, a fragment of the virus capsid protein was amplified with the specific primers PnMV-F (5´-GTGCCAGCCGCCGTTCTTCT-3´) and PnMV-R (5´-GAGCCGGCGACTCCAT CCA-3´), which amplify a 700 pb fragment. This procedure was carried out under the RT-PCR conditions indicated by ^{Ocampo et al. (2013}). The RT-PCR amplified fragments were analyzed using 2% p/v agarose gel electrophoresis (120V/60 min). The primers used in the molecular tests (18S-F, 18S-R, PnMV-F and PnMV-R) were synthetized at the UNAM’s Institute of Biology in Cuernavaca, Morelos. The ‘Red Prestige’ commercial variety was used as the positive control., and one plant of Nicotiana clevelandii as the negative control; foliar tissue was used for the analysis in both cases.

The RT-PCR fragments were sent to Macrogen, in Korea, for sequencing, and then compared to those recorded for PnMV in the GenBank using BLAST (Basic Local Alignment Search Tool). On the other hand, PnMV was mechanically inoculated in differential plants of Nicotiana benthamiana, N. glutinosa, N. clevelandii and Chenopodium amaranticolor 60 days after plant emergence. The inoculum was obtained from the PnMV positive commercial varieties that were analyzed using RT-PCR, except ‘Amaris Hot Pink’, and for this reason, there were 12 inoculum sources. Two buffer solutions were used: Phosphates buffer + DIECA (diethyldithiocarbamic acid) with pH 8.6, and Na phosphates (mono and dibasic) with pH 7.8, in order to increase the success rate of the virus transmission due to its low transmission rate in differential species (^{Chung et al., 2004}; ^{Guy et al., 1985}). To prepare the viral solutions and inoculate differential plants, the methodology described by ^{Jacobo et al. (2015}) was used. Young leaves of each inoculum source were individually macerated in a sterile mortar to which the corresponding buffer in a 1/10 p/v ratio was added. Twenty-four plants per differential species were inoculated: two plants per inoculum source (one for each buffer solution). Two plants were established as negative controls in each species and inoculated only with each of the buffer solutions. Then 4-5 young leaves per differential plant that had been previously sprayed with 600 mesh carborundum, were rubbed with the viral solution or buffer solution (negative controls) using a sterile cotton swab.

The inoculated materials were kept under observation in the Floriculture greenhouse of UACh’s Plant Breeding Department. These materials were not fertilized. Sixty days after inoculation (dai), all the materials were molecularly analyzed to determine the transmission of PnMV (according to the previously described methodology used to extract RNA and RT-PCR from the commercial poinsettia varieties).

The commercial poinsettia varieties with no symptoms when they were bought had no apparent changes during the time they were kept in the greenhouse and accounted for 65% of the evaluated population. According to ^{Lebas et al. (2007}), PnMV sometimes does not induce symptoms in infected plants, which would explain the “healthy appearance” of these varieties. 35% of the poinsettia population showed putative virus symptoms from the time when they were bought and while kept in the greenhouse (approximately 1 year). The symptoms were mosaic, mottling, chlorosis, and foliar deformation. ^{Carballo et al. (2001}) and ^{Chung et al. (2004}) mentioned that these symptoms are characteristic of poinsettia varieties infected with PnMV. The ‘Enduring Marble’ variety showed rib yellowing, a secondary symptom that ^{Bertaccini et al. (1996}) associated with PnMV infection. The bracts of variety ‘Primero Red Glitter’ were severely deformed and not totally pigmented, and according to ^{Brunt et al. (1996}), these symptoms are associated with PnMV infection (Figure 1).

The absorbance values (DAS-ELISA) of all the commercial varieties were higher than those of the detection limit (DL). The average absorbance values were between 0.908 and 1.837. ‘Monet Early’ had the highest absorbance values, while ‘Sparkling Punch’ had lower values. The results of the serological analysis indicated that 100% of the plants were infected with the PnMV virus.

The amplification of the fragment of the 18S ribosomal gene with the RNA obtained from the commercial varieties ensured RNA viability in the molecular tests. PnMV was identified using the RT-PCR technique with specific primers. The presence of the virus was confirmed in 13 varieties: symptomatic: ‘Sparkling Punch’, ‘Amaris Hot Pink’, ‘Polar Bear’, ‘Primero Red Glitter’ and ‘Winter Rose Early Red’; and asymptomatic: ‘Silverstar Marble’, ‘Silverstar Red’, ‘Ice Punch’, ‘Marblestar’, ‘Cortez Electric Fire’, ‘Carousel Dark Red’, ‘Primero White’ and ‘Monet Early (Table 1), by amplifying the expected fragment. PnMV was molecularly identified in 65% of the evaluated varieties; the virus was identified in different plant tissues in order to reduce the risk of obtaining false positives due to the lack of viral load in certain tissues. PnMV was identified through serological and molecular tests in both symptomatic and asymptomatic plants, and the results showed that the virus may be present in poinsettia plants without inducing symptoms, as mentioned by ^{Lebas et al. (2007}), which is a latent source of pathogenic inoculum that puts the plant’s health at risk during crop production.

Figure 1. Putative virus symptoms in commercial poinsettia varieties. A-E) ‘Primero Red Glitter’; A-C): foliar blade deformation and chlorotic mosaic; D): deformed bract (in differentiation process); E): cluster of severely deformed bracts; F) ‘Winter Rose Early Red’, mosaic; G): ‘Cortez Red’, deformation and mosaic; H): ‘Enduring Marble’, chlorosis and rib yellowing; I and J) ‘Polar Bear’; I): chlorosis: J): mottling; K) ‘Sparkling Punch’, chlorotic spots; L): ‘Primero White’ asymptomatic leaf. 

The DNA sequences of PnMV obtained from 11 of the commercial varieties had 95-98% similarity with the sequence of a virus isolate reported in Japan with accession number AB550788.1. According to ^{Jacobo et al. (2015}), the DNA sequences obtained from the virus in the ‘Freedom’ and ‘Red Prestige’ varieties were aligned with this sequence with 92% similarity. ‘Marblestar’ had 97% similarity with the DNA sequence of the virus reported in Germany with accession number AJ271595.1. The amplicon obtained from ‘Amaris Hot Pink’ was not sent for sequencing.

The N. benthamiana plants that were inoculated with PnMV (phosphates + DIECA solution) had warts, systemic chlorosis, foliar deformation and chlorotic spots on the leaves (Figure 2); these symptoms are in agreement with those described by ^{Lebas et al. (2007}), who inoculated plants of this species with the virus and observed warts and systemic chlorosis on the leaves. The plants that were inoculated with PnMV using a Na phosphates buffer (mono and dibasic) had no symptoms, possibly due to the incompatibility of the buffer and the virus. According to ^{Guy (1985}), PnMV has a 0-10% success rate in the mechanical transmission of the virus.

In the particular case of N. glutinosa, N. clevelandii and C. amaranticolor, no differences were observed in the expressed symptoms compared to the use of the buffer solutions. N. glutinosa showed light mosaic symptoms that were attributed to virus transmission. The N. clevelandii plants had chlorosis symptoms on the leaves, possibly in response to viral infection. The inoculated plants of C. amaranticolor had no symptoms (Figure 2).

All the differential plants that were inoculated with PnMV were analyzed using RT-PCR, and the results obtained were negative. The differential plants of N. benthamiana, N. glutinosa and N. clevelandii are reported as PnMV hosts, while C. amaranticolor is not susceptible to infection by this pathogen (^{Brunt et al., 1996}). Despite the negative results of the molecular tests, the symptoms observed on N. benthamiana can be attributed to PnMV infection, since according to Brunt et al. (1996), ^{Floeistad and Blystad (1999)} and ^{Lebas et al. (2007}), this species has been mechanically inoculated and the virus re-isolated; the authors also point out that this species is adequate for diagnosing the pathogen, because the plants that are inoculated with the virus are systemically infected.

The negative results of the RT-PCR tests for PnMV in the differentiated plants can be attributed to the presence of inhibitors, as was observed on the commercial poinsettia varieties, where using serological tests, positive results were obtained (validated by negative and positive controls) that could not be molecularly confirmed. ^{Lebas et al. (2007}) mentioned that the probable causes could be the presence of inhibitors and latex, which make it difficult to extract RNA, and suggest using RT-PCR to identify the virus and characterize isolates, and the DAS-ELISA tests to detect the pathogen.

Figure 2. Symptoms observed on differentiated plants inoculated with PnMV. A-E): N. benthamiana; A): warts; B): systemic chlorosis; C): deformation; D): chlorotic spots; E): Control (-), mechanical damage; F-I): N. glutinosa; F): deformation; G): white spots; H): slight mosaic; I): Control (-), mechanical damage; J-L): N. clevelandii; J): chlorosis; K): mechanical damage; L): Control (-), healthy leaf; M-O): C. amaranticolor; M and N): mechanical damage; O): Control (-), mechanical damage. 

Poinsettia mosaic virus was detected on 100% of the commercial poinsettia varieties using the DAS-ELISA serological technique, and on 65% using RT-PCR molecular tests. 35% of the varieties showed characteristic symptoms of viral infection, while 65% showed no symptoms. PnMV was identified in both symptomatic and asymptomatic plants. The origin of the plant material did not influence the results. The use of the buffer solution prepared with phosphates + DIECA, pH 8.6, to mechanically inoculate PnMV in Nicotiana benthamiana plants contributed to the development of putative virus infection symptoms but this was not molecularly confirmed.