Garlic viruses: characterization, importance and management

Vilma C. Conci

Instituto de Patologia Vegetal (IPAVE) de INTA CIAP y Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) - E-mail: conci.vilma@inta.gob.ar

Garlic viruses are widely distributed across all the producing areas worldwide, causing serious losses. To date, several viral entities have been detected which do not kill the plant but cause systemic and chronic infection. Due to the exclusive agamic propagation of garlic, viruses accumulate in bulbs or cloves, therefore persisting from generation to generation. As a consequence, all plants have become infected by a virus mixture, forming a complex. Leaf striping is frequently observed, which varies from shades of green to bright yellow, depending on the garlic cultivar, the season in the crop cycle and the different combinations of the viruses present.

A study was conducted in Argentina comparing virus-free White type garlic plants with plants infected with the viral complex naturally affecting garlic. A field assay was performed to evaluate yield of healthy garlic plants for five years. The results showed that the plants were reinfected with viruses in the first crop cycle. However, from the first year an increase in garlic bulb weight of healthy garlic plants was detected with respect to those infected with the virus complex. Such increase range was 66-216% in year 1, 46-127% in year 2, 27-62% in year 3, 33-44% in year 4, and 49% bulb weight increase in the last year, without statistical difference from the previous year (Conci et al., 2003).

In this species, viruses form mixed infections that include aphid-borne viruses of the genera Potyvirus and Carlavirus, and others transmitted by mites of the genus Allexivirus.

Within Potyvirus, Onion yellow dwarf virus (OYDV) and Leek yellow stripe virus (LYSV) were reported (Bos, 1982, Van Dijk, 1993 a, Tsuneyoshi et al., 1998, Fajardo et al., 2001)

OYDV is one of the most frequently found viruses in garlic crops. In Argentina, it was first detected in onion (Docampo and Muñoz, 1974) and later in garlic (Conci and Nome, 1988). The presence of OYDV in onion is not frequent probably because low susceptible cultivars are used and because this virus cannot be transmitted by seeds. By contrast, in garlic the virus was detected in all the producing regions and in all cultivars, with incidence values ranging between 33 and 100%. Virus-free plants and plants infected with the isolated OYDV were compared in repeated assays during two crop cycles. The results showed that bulb weight of healthy plants was between 44 and 69% higher than that of infected plants. No differences between plants infected with the viral complex and those infected with OYDV isolates were detected, suggesting that this virus is the main one causing yield losses in the garlic virus complex (Conci, 1997).

The comparison of the nucleotide (nt) and amino acid (aa) sequences of the capsid protein (CP) gene of garlic and onion viruses showed more than 80% of nt and aa identity. Hence, the viruses detected in both plants were considered to be the same species, according to the criteria of the International Committee on Taxonomy of Viruses (ICTV) for Potyvirus species demarcation. However, the virus detected in garlic was efficiently transmitted to garlic, but not to onion. In turn, the OYDV isolate of garlic was successfully transmitted to onion but not to garlic. According to this result, along with some molecular differences found, the researchers considered the existence of a strain infecting onion and another one infecting garlic (Dijk, 1993 a, Kobayashi et al., 1996, Conci et al., 1999).

In Argentina, the complete genomic sequences of two OYDV isolates from onion have been recently obtained. The sequences were compared with other complete sequences obtained from garlic virus by Chen et al. (2003). The comparison of the complete sequences of the viruses in the genomic regions P1, HC-Pro, P3, CI, VPg and NIa-Pro, and of the cleavage sites of the genomes of the garlic and onion viruses revealed lower nt identity values than those suggested as necessary to be considered a single species by the ICTV taxonomic classification criteria and by Adams et al., (2005). These results, as well as the differences in the host mentioned above, suggest that the garlic and onion viruses detected may be different species (Celli et al., 2013).

LYSV was reported in leek in 1940 and later detected in garlic in several countries (Delecolle and Lot, 1981, Walkey, 1990, Van Dijk, 1993 a, Barg et al., 1994, Bos, 1981). In Argentina, LYSV was detected in garlic, leek, “Castaño” garlic (A. sativum var. Ophioscorodom) and great-headed garlic (A. ampeloprasum var. ampeloprasum) using serological and molecular tests. In addition, the vector capacity of seven aphid species was demonstrated: Myzus persicae, Rhopalosiphum padi, R. maidis, Schizaphis graminum, Aphis nerii, A. gossyppii, Hyperomyzus carduellinus, and Uroleucon sonchi (Lunello et al., 2002, 2005).

LYSV was detected in all garlic producing regions of Argentina (Conci et al., 2002). Comparative yield assays between plants infected with the LYSV isolate and virus-free plants, which were repeated during two crop cycles, showed that the virus caused a decrease of up to 36% in bulb weight with respect to healthy plants (Lunello et al., 2007).

Degenerate primers were designed and a one-step IC-RT-PCR protocol was developed to differentiate LYSV from OYDV in single and mixed infections in several Allium spp. The detection of these potyviruses (OYDV and LYSV) was also implemented via Real Time PCR (Lunello et al., 2004, 2005).

The first record of Carlavirus infecting garlic in Argentina was serologically related to Carnation latent virus (CLV) (Conci et al., 1992). Further studies reported that garlic virus is different from CLV and proposed the name Garlic common latent virus (GarCLV) (Van Dijk, 1993 b; Barg et al., 1994). The results of comparison of the genomic sequence of the viral CP gene with the reported GarCLV sequences showed higher identity percentages than those established by ICTV to be considered the same virus (Nieto et al., 2004). The virus was detected in all the producing regions in Argentina, with incidence percentages that ranged between 7 and 22%.

Another Carlavirus detected in garlic is Shallot latent virus (SLV). In Argentina, it was first reported in shallot (Conci, 1992) and later in garlic (Torrico et al., 2010). The gene encoding the virus CP was cloned and sequenced; identity percentage ranges were 77-92.2% nt and 90-99% aa, which were higher than values determined by ICTV to consider the virus as belonging to this species.

Allexivirus are transmitted by eriophyid mites (Aceria tulipae) and their host range is restricted to Alliaceae. Results of the comparison of genomic sequences allowed the identification of Garlic virus A, B, C, D, E, y X (Sumi et al., 1993, Tsuneyoshi and Sumi, 1996; Chen et al., 2001, 2004; Song et al., 1998). In Argentina Garlic virus A, C and D (GarV-A, C and D) were detected in garlic plants from different producing regions.

GarV-A was detected in Argentina in 1997. A fragment of the virus genome was cloned and sequenced, including the CP gene of GarV-A (accession number X98991), which showed 91% identity with the GarV-A detected in Japan (Helguera et al., 1997). Unlike other garlic viruses, GarV-A was not detected in all the cultivars analyzed, and the infection percentages were variable (Lunello et al., 2000).

GarV-C was isolated by mechanical transmissions and characterized using biological and molecular techniques. The virus CP gene was cloned and sequenced; identity with sequences published in GeneBank ranged between 95.8 and 97.3% aa, with these values being higher than those determined by ICTV for species demarcation (Cafrune et al., 2006 a).

The isolation of GarV-A and GarV-C allowed us to study the effect of each of them on garlic yield. Results of an assay comparing yield in two garlic cultivars for two years showed that the isolate GarV-C does not cause important damage. Decreases in bulb weight of less than 15% were detected. GarV-A was the isolate that most affected yield, causing reductions of between 32 and 14% in bulb weight (Cafrune et al., 2006 b).

However, after the fourth growth cycle, non-significant differences in garlic yield were detected among treatments infected with the viral complex, GarV-C and GarV-A, whereas yield for the virus-free garlic plant was 22% higher than that of the viral complex treatment. Garlic yield decreased more rapidly in plants previously infected with at least one Allexivirus and then infected with other naturally occurring viruses than in the plants that were virus-free at the beginning (Perotto et al., 2010).

The presence of GarV-D was also detected in the virus mixture infecting garlic in Argentina using serological and molecular techniques. A comparison between the CP virus gene sequences with those corresponding to GarV-D published in GenBank showed 91.9% of identity.

Production of virus-free garlic

All the plants are infected by a mixture of viruses in the field; hence, the only way to obtain healthy plants is by meristem culture (Conci and Nome, 2001, Conci et al., 2005). This process, however, is not completely efficient. Therefore, once the plants are obtained in vitro, thorough analyses are necessary to detect if the viruses have been eliminated. In addition, when plants are maintained in vitro, the viruses are present at very low concentrations, and highly sensitive diagnosis methods are required. At IPAVE the analyses are conducted using immunosorbent electron microscopy plus decoration (ISEM-D) techniques, which have proven to be the most reliable in terms of their sensitivity and the wide range of viruses that can be detected. To detect all the possible viruses affecting garlic, even those whose identity still remains unclear, the antisera employed are those obtained from OYDV, LYSV, GarCLV, SLV, GarV-A, and GarV-C, as well as an antiserum obtained from garlic plants naturally infected with the viral complex. In addition, the use of RT-PCR with specific primers for the viruses identified in the country has been implemented.

Ex-vitro plant multiplication stage is conducted in anti-aphid cages and then in the fields isolated from Alliaceae until they obtain enough bulbs to start a commercial production. In the field, plants are tested using DAS-ELISA for different viruses.

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