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Introduction
Garlic (Allium sativumL.) is propagated vegetatively. This procedure allows for the accumulation of pathogens (especially viruses), which are perpetuated by bulbs from one production cycle to the next. Typically, garlic is infected by several viruses belonging to theAllexivirus, Carlavirus, andPotyvirusgenera (Barg et al., 1994;Sumi et al., 1993), which significantly reduce crop yield.
Potyvirusspecies are the most common and damage more garlic when plants are mix-infected with other genera of viruses (Salomon, 2002). They are represented by theOnion yellow dwarf virus (OYDV) andLeek yellow stripe virus (LYSV) species (Chen et al., 2001;Maeso et al., 1997). As regards theCarlavirus genus, the most common species found is theGarlic common latent virus(GarCLV) (Fajardo et al., 2001), andShallot latent virus (SLV) was recently reported in Brazil (Mituti et al., 2011).
Species in theAllexivirus genus include theGarlic mite-borne filamentous virus(GarMbFV),Garlic virus A(GarV-A),Garlic virus B (GarV-B),Garlic virus C (GarV-C),Garlic virus D(GarV-D),Garlic virus E (GarV-E),Garlic virus X (GarV-X), andShallot virus X(ShVX) (Kanyuka et al., 1992;Sumi et al., 1993;Yamashita et al., 1996). Species reported in Brazil include the GarMbFV, GarV-A, GarV-B, GarV-C, GarV-D, and GarV-X (Melo Filho et al., 2004;Oliveira et al., 2013).
The mid-western and southeastern regions of Brazil are currently the most important areas which produce noble garlic in Brazil, and no prior surveys in commercial fields indicating the prevalence of viruses in these areas exist. Thus, the goal of the authors in this study was to evaluate the occurrence of viruses belonging to theAllexivirus,Carlavirus, andPotyvirusgenera.
Materials and Methods
Sample collection
520 samples of garlic (both from symptomatic and asymptomatic plants) presenting mosaic and chlorotic streaking were collected in commercial fields approximately 70 days after planting in four Brazilian states and seven municipalities, including the following: Goiás (Campo Alegre 9° 46' 46'' latitude S, 36° 21’ 1” longitude W, and Ipameri 17° 43’ 29” latitude S, 48° 9’ 35” longitude W) , Minas Gerais (Santa Juliana 19º 18’ 32” latitude S, 47º 31’27”, longitude W and São Gotardo 19° 18' 27'' latitude S, 46° 3’ 22'' longitude W), Paraná (Bandeirantes 23º 06’ 36” latitude S, 50º 22’ 0” longitude W, Piraquara 25º 26’ 30” latitude S, 49º 03' 48” longitude W, and Guarapuava 25º 23’ 43” latitude S, 51º 27’ 29” longitude W), and São Paulo (São Manuel 22º 43’ 52” latitude S, 48º 34’ 14” longitude W). Collections were made from May 2007−Oct 2011, for a total of 34 collections. Samples collected in Guarapuava were cultivated in a greenhouse, where samples that were free of the garlic virus were propagated.
Genera and species-specific primer development and analysis
Multiple alignments of the main species described in garlic around the world were done using the MEGA 4.0 (Tamura et al., 2007) program. Universal and specific primers were synthesized to detect genera and different species and to partially amplify the region of coat protein (CP) except for LYSV, of which the primers amplified the partial region of the P1 gene (Table 1).
Table 1 – Primer sequences used to detect various viruses in garlic.
| Genus or species | Sequence | Reference |
|---|---|---|
| Carlavirus | 5’-GGNTKKGAAWCTGGGAGDCC-3’ | Designed for this work |
| 5’-CATKTMATTCCAAACAACNGGYGC-3’ | ||
| Potyvirus | 5'-GAT TTA GGT GAC ACT ATA GT16-3' | Gibbs et al., 1997 |
| 5'-ATG GTT TGG TGY ATY GAR AAT-3' | Mota et al., 2004 | |
| Allexivirus | 5’ CTACCACAATGGTTCCTC 3’ | Oliveira et al., 2013 |
| 5’ GATTTCTTTAACGCAGTG 3’ | ||
| OYDV | 5’ CRCCARTTCTGGATAAYGC 3’ | Designed for this work |
| 5’ CTCCGTGTCCTCATCCG 3’ | ||
| LYSV | 5’ CTTCMTCRCASTCATGKTCC 3’ | Designed for this work |
| 5’ AATCTCAACACAACTTATRC 3’ | Yoshida et al., 2011 | |
| SLV | 5’-CTTTTGGTTCACTTTAGG-3’ | Mituti et al., 2011 |
| 5’-GCACGCAATAGTCTACGG-3’ | ||
| GarCLV | 5’-GGSTTTGARACTGGGAGGCC-3’ | Designed for this work |
| 5’-CATKTMATTCCAAACAACNGGYGC-3’ | ||
| GarV-A | 5’-CCCAAGCTTACTGGAAGGGTGAATTAGAT-3’ | Melo Filho et al., 2004 |
| 5’-CCCAAGCTTAGGATATTAAAGTCTTGAGG-3’ | ||
| GarV-B | 5’ GCAGAATAARCCCCCYTC 3’ | Oliveira et al., 2013 |
| 5’ RAAGGGTTTATTCTGTTG 3’ | ||
| GarV-C | 5’-CCCAAGCTTCATCTACAACAACAAAGGCG-3’ | Melo Filho et al., 2004 |
| 5’-CCCAAGCTTATAAGGGTGCATGATTGTGG-3’ | ||
| GarV-D | 5’-CCAAGCTTAAGCAAGTGAAGAGTGTAAG-3’ | Melo Filho et al., 2004 |
| 5’-CCAAGCTTTTTGGAAGAGGAGGTTGAGA-3’ |
OYDV=Onion yellow dwarf virus; LYSV=Leek yellow stripe virus ; SLV=Shallot latent virus; GarCLV=Garlic commom latent virus; GarV-A=Garlic virus A; GarV-B=Garlic virus B; GarV-C=Garlic virus C; GarV-D =Garlic virus D.
Total RNA was extracted (Bertheau et al., 1998) and used in a one-step reverse transcription polymerase chain reaction (RT-PCR) assay. A total volume of 25 µL, 12.5 µL of 2X PCR master mix, 1 µM of each primer, one unit of AMV (Avian Mieloblastosis virus) reverse transcriptase, 2.5 µL of total RNA, and nuclease-free water were used. The reaction consisted of the following: 30 min at 42 °C; 1 min at 95 °C; 40 cycles of 94 °C for 40 s, 50 °C for 60 s, and 72 °C for 60 s; and a final extension for 10 min at 72 °C. The same reaction cycle was used to detect the species of theCarlavirusgenus, GarCLV, OYDV, and LYSV. For the other primers, the reaction cycle was performed in accordance with the bibliographic references cited inTable 1. Amplicons of five samples of each genus and species were sequenced to evaluate the efficiency of primers.
Results and Discussion
Potyvirus was the most common genus identified in 306 samples (81 %), of which 155 (41 %) were infected only by potyviruses (Table 2). Mixed infections between OYDV and LYSV were found in 61 samples (16 %). 94 samples (25 %) had potyviruses and allexiviruses; 23 (6 %) were infected with potyviruses plus carlaviruses, and 34 (9 %) were infected with potyviruses, allexiviruses, and carlaviruses, indicating that approximately half of the collected samples had mixed infections of viruses belonging to the three different genera tested.
Table 2 – Detection of genera and species ofAllexivirus,Carlavirus andPotyvirusby the reverse transcription polymerase chain reaction (RT-PCR), using universal and specific primers.
| Genus (no. positive samples) | No. of species | No. of samples (%) |
|---|---|---|
| Potyvirus only (155) | One species | 94 (25 %) |
| Two species | 61 (16 %) | |
| Carlavirus only (15) | One species | 13 (3 %) |
| Two species | 2 (1 %) | |
| Allexivirus only (53) | One species | 33 (9 %) |
| Two species | 13 (3 %) | |
| More than three species | 7 (2 %) | |
| Potyvirus + Carlavirus (23) | Two species | 11 (3 %) |
| More than three species | 12 (3 %) | |
| Potyvirus + Allexivirus (94) | Two species | 54 (14 %) |
| More than three species | 40 (11 %) | |
| Carlavirus + Allexivirus (6) | Two species | 6 (2 %) |
| Potyvirus + Allexivirus+ Carlavirus (34) | Three species | 4 (1 %) |
| Four species | 9 (2 %) | |
| Five species | 16 (4 %) | |
| Six species | 5 (1 %) | |
| Total of positive samples | 380 | |
LYSV and OYDV were found with the same frequency in noble garlic from Brazil. Our results differ from previous studies byFayad-Andre et al. (2011), which showed LYSV to be predominant compared to OYDV. In this study, only noble garlic was analyzed, whileFayad-Andre et al. (2011) collected garlic from different production systems. Noble garlic has the higher commercial price compared to tropical garlic, which produces lower quality and smaller bulbs over a shorter cycle (Filgueira, 2007).
Followed by potyviruses, allexiviruses were found with high incidence, being detected in 187 samples collected (49 %). GarV-D and GarV-A can be considered the predominant species, with 109 (29 %) and 107 (28 %) positive samples, respectively. 41 samples (11 %) were positive for GarV-C, while GarV-B (the rarest) was found only in 18 samples (5 %).
The transmission of allexiviruses can easily occur because the mite is a pest commonly found in bulbs, which facilitates the transmission of viruses (Cafrune et al., 2006). Our results also differed from those obtained byFayad-Andre et al. (2011), because GarV-C was seen to be the prevalent species in all production systems, while GarV-D was limited to the Cerrado region.
Carlaviruses were detected in 78 samples (21 %), indicating relatively low prevalence, with GarCLV being the predominant species. SLV was detected only in 11 samples (3 %). In Brazil, the occurrence of carlaviruses is low, with GarCLV being the prevalent species. This genus can cause crop losses which are somewhat limited, but it can cause significant yield losses when plants are co-infected with potyviruses as a result of synergistic effects (Takaichi et al., 1998).
Potyviruses, allexiviruses, and carlaviruses were found in most regions, except for the state of Goiás, where GarV-B, GarV-C, and GarV-D were not detected. Also, SLV was not found in Paraná (Table 3). 141 samples were negative for the three genera. Most of them were found in Paraná (34 %), where garlic seed free of viruses is maintained in greenhouses, and Minas Gerais (27 %), where the same seed is multiplied in fields (Table 3).
Table 3 – Occurrence of species of Potyvirus (Leek yellow stripe virus -LYSV andOnion yellow dwarf virus -OYDV), Carlavirus (Garlic common latent virus -GarCLV andShallot latent virus -SLV) and Allexivirus (Garlic virus A -GarV-A;Garlic virus B -GarV-B;Garlic virus C -GarV-C andGarlic virus D -GarV-D) in Brazil, analyzed from April 2007 to Oct 2011.
| Genera | Species | Collection Site (number of collected samples) | |||
|---|---|---|---|---|---|
| Minas Gerais (228 samples) | Goiás (49 samples) | Paraná (149 samples) | São Paulo (94 samples) | ||
| Potyvirus | LYSV | 98 (43 %) | 21 (43 %) | 42 (28 %) | 47 (50 %) |
| OYDV | 91 (40 %) | 24 (49 %) | 47 (32 %) | 49 (52 %) | |
| Carlavirus | GarCLV | 33 (14 %) | 5 (10 %) | 3 (2 %) | 37 (39 %) |
| SLV | 5 (2 %) | 1 (2 %) | 0 | 5 (5 %) | |
| Allexivirus | GarV-A | 45 (20 %) | 2 (4 %) | 20 (13 %) | 40 (43 %) |
| GarV-B | 3 (1 %) | 0 | 7 (5 %) | 8 (9 %) | |
| GarV-C | 9 (4 %) | 0 | 5 (3 %) | 27 (29 %) | |
| GarV-D | 40 (2 %) | 0 | 39 (26 %) | 30 (32 %) | |
| Virus Free | - | 62 (27 %) | 9 (18 %) | 50 (34 %) | 19 (20 %) |
Infected bulbs seem to be common in commercial fields, and the occurrence of two or more viruses of different taxonomic groups is also common. There was no correlation between the occurrence of species with producing regions of the country, probably as a result of the exchange of garlic seed in the southern, southeastern, and mid-western regions of Brazil.
