Partial sequencing of a putative Alstroemeria necrotic streak orthotospovirus isolate detected on lettuce in Colombia 1

Lettuce is one of the most cultivated leafy salad vegetables worldwide. In Colombia, the principal producing region is the Department of Cundinamarca, with Madrid being the second largest producer, with a yield of 13,514 ton ha -1 , in 2019 (Agronet 2021). This crop is affected by several pathogens, being viruses, after fungi, the most limiting biotic agents, causing devastating losses (Raid 2004, Lebeda et al. 2014). Among the main viruses affecting lettuce crops so far reported are the Lettuce mosaic ABSTRACT


PALAVRAS-CHAVE:
The symptoms associated with TSWV and INSV in lettuce crops are indistinguishable from one another, consisting of brown necrotic spots forming an extended necrotic area, leaf chlorosis, leaf distortion and plant stunting (Koike et al. 2008, Moreno & Fereres 2012, Beris et al. 2020).
All types of lettuce (iceberg, romaine, greenleaf, redleaf, butter) are susceptible to TSWV and INSV, and both viruses are not seedborne, i.e., they infect several crops, weed species and are spread by thrips.
Alstroemeria necrotic streak orthotospovirus (ANSV) is endemic in Colombia, being found naturally infecting ornamental alstroemeria (Alstroemeria sp.) crops (Hassani-Mehraban et al. 2010).Furthermore, other surveys have identified natural infections in solanaceous crops, including tomato (Solanum lycopersicum), bell pepper (Capsicum annuum) and lulo (Solanum quitoense), in Colombia (Olaya et al. 2017, Gallo et al. 2018, Gallo et al. 2019).Recently, symptoms similar to those of orthotospovirus infection have been reported in commercial lettuce crops in the municipality of Madrid, Cundinamarca.Thus, to establish the causal agent thereof, the present study aimed to identify the viral species associated with those symptoms, in order to update the lettuce phytosanitary status in this region.
Random sampling was used to evaluate seven romaine lettuce commercial crops, with samples collected from 30 points in each crop, of which 6 plants per point were evaluated, during the first crop cycle, in 2017.The disease incidence was 30 % in the municipality of Madrid, Department of Cundinamarca, Colombia, with foliar symptoms characterized by brown necrotic spots forming an extended necrotic area, chlorosis, leaf distortion and plant stunting (Figure 1).
The collected plant material underwent serological and molecular testing.The serological tests were performed according to the decision scheme for some of the orthotospoviruses reported in diagnosis protocols for regulated pests (EPPO 2004), avoiding indicator plants (Figure 2).Enzymelinked immunosorbent assays (DAS-ELISA) for Orthotospovirus (Agdia Inc., Elkhart, IN) and lateral flow ImmunoStrip™ assays (Agdia Inc.) were carried out for TSWV and INSV using sap obtained directly from collected symptomatic lettuce plants.
RNA extraction was performed from symptomatic lettuce plants using the Thermo Scientific GeneJET™ kit, according to the manufacturing instructions.The first-strand cDNA synthesis was done using a universal degenerate primer for the coding region of the orthotospovirus nucleocapsid protein (N) gene (Uga & Tsuda 2005), in a volume of 20 µL.The reaction mixture was composed of 0.1 μM final concentration of universal primer   1), with 2 μL of cDNA, 1X buffer (Invitrogen™), 1.5 mM of MgCl 2 (Invitrogen™), 0.2 mM dNTPs (Thermo Fisher Scientific™), 0.2 μM of each primer and 1.25 U Taq Pol (Invitrogen™).It was carried out following this thermal profile: 95 ºC for 5 min, followed by 35 cycles at 95 ºC for 30 s, 55 ºC for 40 s and 72 ºC for 30 s, and a final step of 6 min at 72 ºC.Alstroemeria lyophilized material infected with TSWV was used as a positive control.The PCR products were analyzed in 1.5 % agarose gel electrophoresis and sequenced, and the BioEdit software (version 7.1.9)was used for the analysis, whose results were compared with the sequences available in the GenBank, in which the consensus sequences generated in this study were deposited.
The visualized symptoms (Figure 1) corresponded to those associated with both the TSWV and INSV species (Kuo et al. 2014), which were reported in Europe (Moreno & Fereres 2012), Asia (Al-Saleh et al. 2014), Brazil (Pavan et al. 2008) and the United States (Abad et al. 2005).However, the differentiation between both viruses in the field is difficult, due to the ressemblance of symptoms produced by these two species (Kuo et al. 2014).In this way, the identification of such viral species in this study was carried out following the decision scheme for orthotospovirus detection (EPPO 2004), which was modified, avoiding indicator plants (Figure 2).The symptomatic samples were positive for the Orthotospovirus genus by the DAS-ELISA test and showed a positive reaction to TSWV and negative to INSV by the ImmunoStrip™ test, thus suggesting that TSWV is the causal agent of this disease.A cross reaction of TSWV antiserum with other orthotospovirus species, such as Groundnut ringspot orthotospovirus, Tomato chlorotic spot orthotospovirus and ANSV, was reported by Margaria & Rosa 2015, Hassani-Mehraban et al. 2016, Agdia 2017and Olaya et al. 2017, based on the highly conserved capsid protein used for these tests (Gallitelli 2004).For this reason, we used molecular tests to verify the viral species.
The RT-PCR for nucleocapsid N gene detection was performed using the primers TSWV-709/TOS-R15 (Uga & Tsuda 2005) and Pr-dTS-f/ Pr-dTS-r (Liu-Yuan et al. 2017), and the sequence analysis confirmed that the disease is caused by orthotospoviruses.To determine the viral species, a product of 709 pb was obtained from the symptomatic lettuce samples (accession number MK085115) and the positive control (Alstroemeria infected with TSWV, accession number MK08511), using the primer combination TSWV-709 (specific for TSWV)/TOS-R15 (universal for Orthotospovirus) (Figure 3; Table 1).The sequence analysis of the lettuce samples showed nucleotide identity higher than 99 % for the ANSV genome segment S and the N gene (MK275264.1,MK275263.1,GQ478668.1,KX833218.1),and 78.44 % of nucleotide identity for the TSWV nucleocapsid protein N gene (AJ296600.1).Meanwhile, the Alstroemeria control infected with TSWV (MK085117) showed nucleotide identity higher than 99 % for the TSWV genome segment S and the N gene (KC261967.1,HQ830187.1).Additionally, there was not an amplification product using the primers INSV-589 (specific for INSV)/TOS-R15 (universal for Orthotospovirus), confirming the absence of INSV in the samples.According to these results, in order to confirm the presence of ANSV, the pair of primers Pr-dTS-f/ Pr-dTS-r (Table 1) was used, which identified six orthotospovirus species.As a result, a 460 bp product (accession number MK085116.1)(Figure 4) was obtained, with nucleotide identity of more than 99 % for the ANSV genome segment S and the N gene (MK275264.1,GQ478668.1,KX833218.1).
According to the molecular analysis, all sequences showed nucleotide identity higher than 99 % for ANSV, discounting the presence of the TSWV identified by the ImmunoStrip™ test.Therefore, ANSV is another orthotospovirus present in lettuce which had not yet been reported for this crop in Colombia, as this species had been previously detected only for solanaceous like tomato, lulo and pepper (Olaya et al. 2017, Gallo et al. 2018, Gallo et al. 2019), what supports the extensive host range previously proposed for this virus (Hassani-Mehraban et al. 2016).It would be interesting to sequence the complete genome of ANSV infecting lettuce to establish whether it belongs to the American clade, as the ANSV infecting solanaceous plants, and evaluate in different regions such viral presence in lettuce crops and thrips vectors.However, specific primers have been recently designed from the genome sequencing of ANSV from Colombian solanaceous samples, which could as well be used to confirm the presence of ANSV in other vegetables by RT-PCR and RT-qPCR (Gallo et al. 2018, Gallo et al. 2019).
In conclusion, an accurate orthotospovirus diagnosis between Tomato spotted wilt orthotospovirus and Alstroemeria necrotic streak orthotospovirus requires the use of molecular RT-PCR test with different sets of specific primers followed by sequence analysis, because of cross reactions when immunological tests are used.Specific molecular tests detected the presence of Alstroemeria necrotic streak orthotospovirus in lettuce, and could be used in other crops and regions to update the phytosanitary status of this viral species in Colombia.

Figure 1 .
Figure 1.Symptoms in lettuce plants (brown necrotic spots forming an extended necrotic area, chlorosis, leaf distortion and plant stunting), in the municipality of Madrid, Cundinamarca, Colombia.

Table 1 .
PCR with universal and specific primers to detect the nucleocapsid (N) gene of Orhtotospovirus.