Detection and characterization of a Cucumber mosaic virus isolate infecting peperina , a species native to Argentina

Minthostachys mollis (Kunth.) Griseb., “peperina”, a member of the family Lamiaceae, is an aromatic species used in modern pharmacology and medicine. It is widely distributed in the Andes, from Venezuela and Colombia to Argentina. In Argentina, the main exploitation area of peperina includes the mountain area of the province of Córdoba, where the plant is indiscriminately harvested, leading to an irreversible loss of the germplasm. To preserve this plant as a native resource and regional source of incomes, the species has been domesticated. During this process, noticeable symptoms of yellow mosaic appeared. Biological, serological and molecular studies (RT-PCR, RFLP, cloning and sequencing) determined the presence of subgroup IA of Cucumber mosaic virus in domesticated peperina plants. The viral isolate studied is closely related to strain Y, which was reported from Japan. To our knowledge, this is the first report of a virus infecting peperina.


InTrODuCTIOn
Minthostachys mollis (Kunth.)Griseb., locally known as "peperina", is a member of the family Lamiaceae; it is a perennial, aromatic semi-shrub of about 0.3-2.0m in height.Due to its menthol content, its aroma resembles that of mint.It is widely used in infusions because of its stimulant, antispasmodic, antidiarrhoea, anticholera, antiemetic and digestive properties (Ordóñez et al., 2006).This aromatic plant is also used for liquor and mixed herb production.It has received increasing attention from modern pharmacology and medicine, as plant decoctions and extracted essential oils are tested for pharmacological effects (Ojeda et al., 2004;Ordóñez et al., 2006;Banchio et al., 2007).This species is widely distributed in the Andes, from Venezuela and Colombia to Argentina.In Argentina, it is present in the north-western region (provinces of Salta, Jujuy, Catamarca, Tucumán, and La Rioja) and in the central-western region (San Luis and Córdoba) (Schmidt-Lebuhn et al., 2008).In Córdoba, the plant grows between 700 and 1200 m a.s.l.In this area, the trade of these plants reaches its maximum levels; hence, it is indiscriminately gathered by collectors, who sometimes uproot whole plants without considering the phenological state or the ability of the species to recover.This practice has a strong impact on natural plant resources, modifies the environment, and leads to irrecoverable loss of germplasm through genetic erosion.For these reasons, research is carried out aiming at conservation, domestication and culture improvement of this native species.However, during the domestication process, loss of resistance genes takes place, as demonstrated for example in rice (Hiroaki & Takeshi, 2010).
Peperina plants were asymptomatic at their nat-ural habitat (Biderbost, unpublished data).However, when they were transferred to the experimental plots, conspicuous yellow mosaic symptoms, typical of virus infection, started to appear.
Cucumber mosaic virus (CMV), a member of the family Bromoviridae, and the type member of the genus Cucumovirus, is one of the most important viruses in the world, infecting more than 1200 species in over 100 families of mono and dicotyledonous plants, including vegetables, ornamentals, legumes and other important crops (Palukaitis & Garcia-Arenal, 2003).The virus is mechanically transmitted by sap and naturally spread by more than 80 aphid species in a non-persistent manner (García-Arenal et al., 2000).The virus genome consists of three positive-sense, single-stranded RNAs (RNA 1, RNA2 and RNA3) (Owen & Palukaitis, 1988).Many CMV strains have been described and classified into subgroups I and II on the basis of serology, nucleic acid hybridization, and restriction fragment length polymorphism (Owen & Palukaitis, 1988;Hu et al., 1995;Finetti Sialer et al., 1999;Szilassy et al., 1999).Subgroup I is further subdivided into IA and IB on the basis of gene sequence and phylogenetic analysis (Roossinck, 2002).The objective of the present work was to identify and characterize the possible viral agents infecting peperina in Argentina.

maTerIaLs anD meTHODs source of inoculum
Eight peperina plants showing conspicuous yellow mosaic symptoms (Figure 1) were collected from a field assay that included clones from different localities in the provinces of Córdoba (Villa Al-lende, Capilla del Monte, Tala Cañada and Cuesta Blanca), San Luis (Merlo), Tucumán (Tafí del Valle and Escaba), and Catamarca (Balcosna).The plants were transferred to pots and kept under greenhouse conditions for further analysis.

serological characterization
The symptomatic peperina plants were screened for the presence of Cucumber mosaic virus (CMV), Alfalfa mosaic virus (AMV), Potato virus Y (PVY), Potato virus X (PVX), Tobacco mosaic virus (TMV), by PTA (Mowat & Dawson, 1987) and for Tomato ringspot virus (ToRSV), Tomato spotted wilt virus (TSWV) and Groundnut ringspot virus (GRSV) by DAS-ELISA (Clark & Adams, 1977).The reactions with 0.75 mg/ml p-nitrophenyl phosphate (ADGIA Inc., Elkhart, IN, USA) in 10% diethanolamine, pH 9.8, were quantified after 60 min at 410 nm (OD410) using a Dynatech MR 700 ELISA plate spectrophotometer (Chantilly, USA).Samples were considered positive when OD410 values were higher than the mean of the healthy controls plus three times the standard deviations (six healthy peperina controls per plate were used).Samples that tested positive with the polyclonal CMV antibody were subsequently analyzed with monoclonal CMV antisera (anti-CMV subgroups I and II ADGIA Inc, Elkhart, IN, USA).Tests were performed as described above, using commercial positive controls (ADGIA Inc., Elkhart, IN, USA).

molecular characterization. antigen-capture (aC) rT-PCr and rLFP
For molecular characterization of the CMV isolate, antigen capture followed by reverse transcription-polymerase chain reaction (AC-RT-PCR) was performed using Access RT-PCR System (Promega Corp. Madison, WI, USA) and primers 5´ CP (-) and 3´ CP (+) that amplify an 870 bp fragment, including the capsid protein (CP) gene, for all CMV isolates (Rizos et al., 1992).RT-PCR reaction volume was 50 µl and amplification conditions were as follows: 48 ºC for 45 min, 94 ºC for 4 min (40 cycles of 94 ºC for 30 s, 40 ºC for 30 s, 68 ºC for 1 min) and a final extension of 68 ºC for 7 min.Following PCR, 10 µl of the product was electrophoresed in a 1% agarose gel and stained with ethidium bromide.In order to determine the CMV subgroup by restriction fragment length polymorphism (RFLP), 10 µl of the PCR product were digested with MspI in a final volume of 20 µl (Rizos et al., 1992).Digestion was carried out at 37 ºC for 4 h in the buffers supplied by the manufacturer; products were electrophoresed in 1.5% agarose and stained as above.
The AC-RT-PCR products were cloned using the Topo TA Cloning ® Kit (Invitrogen Corp., San Diego, CA, USA) following the manufacturer's instructions.Three positive clones were purified using QIAprep Spin Miniprep kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions and further sequenced by the Unidad de Genómica, Instituto de Biotecnología-INTA (Argentina).
The nucleotide and deduced amino acid sequences were compared with those of other CMV isolates available in GenBank (www.ncbi.nlm.nih.gov).The accession number and assigned abbreviations of these isolates are listed in Table 1.Database searches were carried out using the Blastn algorithm (Altschul et al., 1990).Multiple sequence alignments of nucleotide and deduced amino acid sequences were performed with Clustal W (www. ebi.ac.uk/clustalw).Phylogenetic trees were generated with MEGA program version 4.0 (Kumar et al., 2004), using UPGMA method for nucleotide sequences and Neighbour-joining method for amino acids.The ER strain of Peanut stunt virus (acces- sion number U15730) was used as an out-group.Tree branches were bootstrapped with 2000 replications.Multiple alignments of the amino acid sequences of the coat protein gene were performed using LASERGENE (DNASTAR Inc.Madison, WI, USA program).

resuLTs
The virus was successfully transmitted by mechanical inoculation to all the inoculated plants.Chlorotic local lesions were observed on C. amaranticolor and severe systemic mosaic on N. rustica, N. occidentalis, N. glutinosa and V. unguiculata.Shoestring symptoms were observed on S. lycopersicum.On the other hand, original symptoms were reproduced in inoculated peperina plants.
All infected peperina plants reacted with the polyclonal CMV antiserum and with the monoclonal antiserum for CMV subgroup I, but not with subgroup II CMV antiserum.The strong reaction with subgroup I antiserum (Table 2), indicated its inclusion in this subgroup As all eight evaluated plants were positive with subgroup I antiserum, two of them were randomly selected for AC-RT-PCR.The amplification resulted of an approximately 900 bp fragment (Figure 2).After digestion of the amplified products with MspI and electrophoresis, the isolate gave two bands of 540 and 340 bp (Figure 3).This pattern allowed us to classify our local isolate as subgroup I according to Rizos et al. (1992).
The CP ORF length of the isolate was identical to that of previously described CMV strains, consisting of 657 bp, which therefore encoded a coat protein of 218 aa.
Sequence comparisons showed that the three sequenced clones shared 99.7-100% nucleotide sequence identity, indicating that they belong to the same isolate.The Minthostachys CMV isolate (CMV-Min) has the highest nucleotide sequence identity (95.2-98.0%)with those isolates belonging to Subgroup IA (Table 3).Comparisons of amino acid identity between the entire CMV-Min CP and other CMV strains revealed a homology of 81.6-83.9%for subgroup II members and of 97.7-99.1%    3).The highest homologies (99.1%) were found with strains Leg and Y, both of them reported for Japan.The multiple sequence alignment showed the local isolate differs from strain Y in that residue 17 of the capsid protein exhibits proline (P) as the remaining strains, whereas strain Y from Japan has leusine (L) at that site, and at residue 129 the Mint isolates has, as the rest of the strains, a proline while the Y has a serine (S) (Figure 4).It has been reported that substitution of this last amino acid affects symptom expression, inducing chlorosis instead of necrosis in infected tobacco plants (Mochizuki & Ohki, 2011).
The phylogenetic relationship between the nucleotides sequences of CMV-Min and those of other selected isolates is shown in Figure 5.The CMV-Min isolate clustered together with subgroup IA isolates and is closely related to CMV-Y, the strain reported for Japan.

DIsCussIOn
The need to preserve peperina as a natural resource of great regional importance led to its domestication.In the natural ecosystem, symptoms were not observed, but during the domestication, symptoms were developed.Serological and molecular characterization confirmed the presence of CMV infecting the species.Studies conducted worldwide have shown that the numerous CMV strains are classified into subgroups I and II (Rizos et al., 1992) on the basis of serology (Hu et al., 1995;Ilardi et al., 1995), nucleic acid hybridization (Owen & Palukaitis, 1988), gene sequencing (Owen et al., 1990;Hu et al., 1995;Szilassy et al., 1999) and RFLP (Rizos et al., 1992;Finetti Sialer et al., 1999).In turn, CMV subgroup I is further divided into IA and IB on the basis of sequences and phylogenetic relationships (Palukaitis & Zaitlin, 1997;Roossinck, 2002).Strains IA and II of this virus are distributed worldwide, whereas the strains of IB subgroup are mainly restricted to Asia (Roossinck, 2002;Koundal et al., 2011).Previous studies conducted in Argentina showed the presence of both subgroups.In 2003, CMV subgroup I was first mentioned infecting bean crops in northwestern Argentina (Rodríguez Pardina et al., 2003).It was further detected in the province of Córdoba, in greenhouse-grown plants of the ornamental Snapdragon (Antirrhinum majus L.) (Arneodo et al., 2005).However, this isolate was only serologically characterized by those researchers.Later, the presence of subgroup II in peanut crops was reported (de Breuil et al., 2005).
The present work is the first report of Cucumber mosaic virus infecting peperina in Argentina.The serological and molecular characterizations suggest that the isolate found in this aromatic plant be- longs to subgroup IA; the cluster analysis grouped the isolate with strain Y, which was first described in Japan (Nitta et al., 1988).However, the local isolate differs from strain Y in that residue 129 of the capsid protein exhibits proline (P) as the remaining strains, whereas strain Y from Japan has a muta-tion P x S (proline x serine) at that position.This fact could lead to differences in the expression of symptoms (Mochizuki & Ohki, 2011).
On the other hand it is necessary to consider that peperina can constitute a natural reservoir of CMV.

Figure 1 .
Figure 1.Peperina plants showing conspicuous yellow mosaic symptoms produced by Cucumber mosaic virus.

Figure 3
Figure 3 Electrophoretic analysis in 1.5% agarose of the amplified digested with MspI.Gels stained with ethidium bromide.M indicates 100 bp molecular weight marker.Lane 1 and 2: infected peperina samples.

Figure 4 .
Figure 4. Alignment of the deduced amino acid (aa) sequences of coat protein of the Minthostachys Cucumber mosaic virus isolate (CMV-Min) compared with other selected strains.Dots represent identical amino acids in all the isolates studied.

Figure 5 .
Figure 5. Phylogenetic trees based on the CP nucleotide (A) and amino acid (B) sequences of the Minthostachys Cucumber mosaic virus isolate (CMV-Min) and other selected CMV strains.The trees were constructed using UPGMA and neighbour joining methods with MEGA program version 4.0

Table 1 .
Cucumber mosaic virus strains used in comparative sequence analyses, with their corresponding subgroup and Gen-Bank accession numbers.

Table 2 .
OD410 values, of different peperina samples, when tested with polyclonal and monoclonal antisera

Table 3 .
Comparisons of percent nucleotide sequence and the deduced amino acids identities between Minthostachys Cucumber mosaic virus isolate (CMV-Min) and other CMV strains