RESPONSE OF MANDARIN CULTIVARS AND HYBRIDS TO CITRUS LEPROSIS VIRUS

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SUMMARY

Mandarins and their hybrids are considered resistant to citrus leprosis. However, there are no studies address-ing such resistance in the field under natural inoculation conditions. In this work we evaluated, through symptom analysis, the response to leprosis of 25 different geno-types of mandarins and hybrids from a ten-year-old or-chard with a long history of the disease. We observed higher resistance levels among Citrus reticulata Blanco va-rieties and some of their hybrids, such as the Murcott tangor [C. reticulatax C. sinensis (L.) Osbeck], which did not show any leprosis symptoms. C. deliciosaTenore and C. clementina hort. ex Tanaka accessions and hybrids, such as Lee tangelo [(C. clementina x (C. reticulata x C. paradisi Mac.)], were the most susceptible under natural conditions. However, even in those genotypes, most of the symptoms were observed in leaves and not in fruits, as often seen in sweet oranges (C. sinensis), considered the most susceptible species of citrus to leprosis. This suggests that even susceptible accessions of mandarins are likely to show some level of resistance when com-pared to sweet oranges.

Key words: Mandarin, citrus, CiLV, leprosis, resist-ance.

INTRODUCTION

Leprosis is considered the main viral disease for Brazilian citrus production due to the high costs of chem-ical control of its vector Brevipalpus phoenicis (Acari: Tenuipalpidae). Additionally, the global importance of citrus leprosis has recently increased with the north-bound spread of the virus to new areas in South and Cen-tral America (Rodrigues et al., 2003; Bastianel et al., 2006a). In all reports of the disease, sweet orange (Citrus sinensis) prevails as the most affected species.

Corresponding author: M. Bastianel Fax: +55. 1935461399

E-mail: mbastianel@centrodecitricultura.br

The common form of the virus is referred to as Cit-rus leprosis viCit-rus cytoplasmic type (CiLV-C) due to its cellular localization (Colariccio et al., 1995; Rodrigues et al., 2003). In contrast, a rare form of CiLV was first de-scribed in 1972 (CiLV-N) where virus particles accumu-late in the nucleus of infected cells (Kitajima et al., 1972; Rodrigues et al., 2003). The latter type has been reported only once from Panama (Dominguez et al., 2001) and from a few municipalities in Brazil (Bastianel et al., 2006a). Because of its morphology and presence either in the cytoplasm or in the nucleus of infected cells, CiLV-C has been considered a tentative member of the Rhabdoviridae family (Rodrigues et al., 2003). However, molecular studies have shown that the nu-clear and the cytoplasmic types of CiLV are completely different viruses (Freitas-Astúa et al., 2005) and that CiLV-C genome does not share sequence or organiza-tion similarity with rhabdoviruses (Locali-Fabris et al., 2006; Pascon et al., 2006). Hence, CiLV-C was pro-posed as the type member of a new plant virus genus named Cilevirus(Locali-Fabris et al., 2006).

Leprosis can cause typical chlorotic to necrotic circu-lar local lesions in citrus leaves, stems and fruits associ-ated with mite feeding sites, since the virus does not in-fect its hosts systemically (Rodrigues et al., 2003). Even though there is a common pattern observed among lep-rosis lesions, they can vary in size, shape, and colour de-pending on the host species, developmental stage of the plant, pathogen isolate and probably, environmental conditions (Locali et al., 2003; Bastianel et al., 2006a). Marques et al. (2007) analyzing leaves and twigs of sweet oranges affected by CiLV, noted histological changes in the phloem such as hyperplasia of the corti-cal and phloem parenchyma cells, causing obliteration of the phloem vessels; this could explain why the virus does not become systemic. The relationship between the virus and its vector is still unclear, but recent studies have suggested that, contrary to what has been pro-posed (Rodrigues et al., 2003), it can be a persistent cir-culative rather than propagative type of interaction (Nicolini et al., 2007; Freitas-Astúa et al., 2008).

Although plants of the genus Citrus are the only known natural hosts of CiLV (Rodrigues et al., 2003), the virus can be mite-transmitted to other plants usually

Journal of Plant Pathology (2008), 90(2), 307-312 Edizioni ETS Pisa, 2008 307

RESPONSE OF MANDARIN CULTIVARS AND HYBRIDS TO CITRUS LEPROSIS VIRUS

M. Bastianel1, J. Freitas-Astúa1,2, F. Nicolini1, N. Segatti1, V.M. Novelli1, V. Rodrigues1, C.L. Medina3 and M.A. Machado1

1 Centro APTA Citros Sylvio Moreira/IAC, C.P.04, Cordeirópolis, SP, Brazil 13490-970 2 Embrapa Mandioca e Fruticultura Tropical, s/n, Cruz das Almas, BA, Brazil 44380-000 3 Grupo de Consultores de Citros/GCONCI, C.P.144, Cordeirópolis, SP, Brazil 13490-000

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Mandarin and hybrids response to CiL

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Table 1.Citrus accessions evaluated for the presence of leprosis symptoms in different organs, disease incidence (%), and severity.

Species Common name Number of plants

evaluated

Symptoms on: Leaf Branch Fruit

Percentage of

symptomatic plants Score (see text)

Vermelha mandarin 15 + + + 87.5 1.4 Cravo mandarin 16 + - + 56.2 1.3 Emperor mandarin 11 + - - 45.5 1 Harris mandarin 15 + - - 6.7 1 Muscia mandarin 15 + - - 6.7 1 Ponkan mandarin 15 + - - 6.7 1

África do Sul mandarin 15 - - - 0 0

De Wildt mandarin 15 - - - 0 0

Loose Jacket mandarin 13 - - - 0 0

Span Americana mandarin 15 - - - 0 0

Sunburst mandarin 14 - - - 0 0

Rosehaugh Nartjee mandarin 16 - - - 0 0

Citrus reticulata

Empress mandarin 15 - - - 0 0

Tardia da Sicilia mandarin 13 + - + 69.2 1.1

1144-12 mandarin 13 + - + 46.2 1.3

C. deliciosa

Mogi das Cruzes mandarin 14 + - + 42.9 1.2

Swzinkon mandarin 12 + - - 16.7 1

C. suhuiensis

Swzinkon x Swzinkon-Tizon hybrid 14 + - - 14.3 1

Clementine Caçula 3 mandarin 14 + - + 78.6 1.4

C. clementina x. C. reticulata Clementine Caçula 2 mandarin 12 + - + 50 1.5

Lee tangelo 15 + + + 73.3 1.2 Osceola tangelo 13 + - + 15.4 1.5 C. clementina x (C. reticulata x C. paradisi) Nova tangelo 16 - - - 0 0 Ortanique tangor 15 + - - 46.7 1.1 C. sinensis x C. reticulata Murcott tangor 16 - - - 0 0

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found in citrus orchards, such as weeds, windbreaks and hedgerows (Nunes, 2007). This may have conse-quences for the epidemiology of the disease.

Sweet oranges are considered the most susceptible citrus species and, even though there are differences in susceptibility amongst them, none of the commercial va-rieties are resistant (Rodrigues et al., 2000; Bastianel et al., 2006a). In Brazil leprosis is important in large areas cultivated with sweet orange, associated with the year-round presence of the vector and abundant source of inoculum in most orchards. In fact, approximately 80% of the citrus trees grown in São Paulo State, the main producer in Brazil, are sweet orange varieties, highly susceptible to CiLV. In 1995, more than 60% of the State’s citrus orchards had plants with leprosis symp-toms, which explains why chemical control of the vector is widely used (Salva and Massari, 1995).

Mandarins and some of their hybrids exhibit high levels of resistance to the disease (Rodrigues et al., 2003; Bastianel et al., 2006a), but there is very little if any in-formation on the behaviour of these genotypes under natural CiLV mite inoculation. This information is cru-cial for countries, like those of the Mediterranean basin, where mandarins are economically important and the mite is present.

MATERIALS AND METHODS

Plant material. The response to leprosis of twenty-five genotypes of mandarins and hybrids (Table 1) was studied under natural conditions of CiLV inoculation and based on the symptoms observed in the field, in a ten-year-old orchard with a long history of the disease, located in Mogi Mirim, São Paulo State, Brazil, a region of endemic leprosis. The experimental design was eight randomized blocks with two replicates for each geno-type, with 5 m spacing between rows and 6 m within rows. The area studied was surrounded by orchards of Cravo and Satsuma mandarins.

Disease assessment. Symptoms were evaluated every two months over a year period. Disease incidence was scored in the leaves, branches and fruits, and the per-centage of symptomatic plants was determined. Severity was scored according to an empirical scale from 0 (no lesions) to 5 (Rodrigues, 2000). In particular: 1 = few le-sions restricted to a particular region of the plant; 2 = lesions in more than one plant organ and/or distributed in more than one sector of the plant; 3 = plentiful le-sions throughout the plant; 4 = plentiful lele-sions throughout the plant accompanied by leaf and/or fruit drop; 5 = like 4 plus dieback of branches.

RESULTS AND DISCUSSION

From the 25 genotypes studied, 16 showed various

levels of leprosis symptoms, which remained unaltered throughout the six assessments (Table 1). Although symptoms were similar to those observed in sweet or-anges (Fig. 1), they occurred mainly in the leaves and rarely in branches and fruits.

High disease variability was observed particularly within varieties of C. reticulata, which correlates with the high genetic variability within the group (Machado et al.,1996). The two varieties showing highest leprosis incidence and severity were Vermelha and Cravo man-darins (Table 1), both possible hybrids of C. reticulata with C. deliciosa(Machado et al.,1996; Coletta Filho et al., 1998). While Vermelha mandarin is not an imptant variety in Brazil, Cravo is grown in commercial or-chards and, reportedly, often shows leprosis symptoms on leaves and branches (Salvo Filho et al., 1997), which corroborates our data. Interestingly, other C. reticulata accessions, considered true varieties, showed low lepro-sis or remained symptomless throughout (Table 1).

Overall, C. deliciosa(Mediterranean mandarin) vari-eties were more susceptible than those of C. reticulata. Machado et al. (1996), using molecular markers, ob-served complete genetic similarity in approximately 20 accessions of Mediterranean mandarins, suggesting very low genetic variability and, possibly, widespread suscep-tibility to CiLV within the group. The Swzinkon man-darin (C. suhuiensis) and hybrids were also susceptible to leprosis, as were most C. clementina hybrids (Table 1). Interestingly, the two C. clementina x C. reticulata hybrids evaluated (Clementine Caçula 2 and Clemen-tine Caçula 3) have a Cravo mandarin parent (Pio et al., 2000), and may have inherited their susceptibility from that genotype.

Amongst tangelos [C. clementinax (C. reticulata x C. paradisi)], Lee exhibited the highest susceptibility, while Osceola showed few symptomatic plants and Nova re-mained symptomless throughout (Table 1).

Differential response to CiLV was also observed beetwen Ortanique and Temple tangors (C. sinensisx C. reticulata). In the field, Ortanique showed high disease incidence, similar to that observed by Freitas-Astúa et al.(unpublished data) under greenhouse conditions for Temple. Ortanique (and Temple) have likely inherited their susceptibility from the sweet orange parent, but Murcott tangor, which remained symptomless through-out the evaluation, may have inherited the resistance trait from its C. reticulata parent (Bastianel et al., 2006b). The resistance (or tolerance) of Murcott to lep-rosis is known from previous studies that have ascer-tained by biological (Boaretto et al., 1994; Rodrigues, 1995) and molecular (Bastianel et al., 2004; Nicolini et al., 2008) methods that Murcott has low CiLV titres.

It was not possible to detect the presence of the virus by RT-PCR in any of the field samples collected in this study, despite of the endemic character of the disease in neighbouring sweet orange plantings. This is probably

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due to low level of target RNA in the mandarin grove, due to the natural resistance observed in these species and their hybrids.

Since Murcott is one of the main citrus genotypes grown in Brazil for fresh fruit consumption and exhibits resistance to leprosis and citrus variegated chlorosis (CVC, caused by Xylella fastidiosa), one of the other main citrus diseases in the country, it has been widely used in breeding programs (Oliveira et al., 2002). In fact, all information available on the genetic inheritance of re-sistance to leprosis has been obtained by Bastianel et al. (2006b) using Murcott tangor. These authors assessed the incidence and severity of leprosis in a population result-ing from a cross between Murcott (highly resistant) and Pêra sweet orange (highly susceptible) infested with

vir-uliferous B. phoenicis, concluding that inheritance of re-sistance to leprosis is probably controlled by few genes present in Murcott (Bastianel et al., 2006b).

Differential behaviour towards leprosis amongst cit-rus varieties, even among the susceptible ones, has been reported by some authors (Roessing and Salibe, 1967; Rodrigues et al., 2000, 2003) and may be a reaction ei-ther to the virus or to the vector (Rodrigues, 2000). However, in our study mites were observed on all geno-types assessed, indicating that the variation in symptoms was due to differential responses to CiLV.

It is known that lemons [Citrus limon (L.) Osbeck], limes [C. aurantifolia (Christmann) Swingle], mandarins (various species), grapefruit [C. paradisi (McFad.) Hooker] and some tangors (hybrids between sweet or-Fig. 1.Typical local lesions induced by leprosis (cytoplasmic type) in the leaves of (A) Lee tangelo (A), Osceola tangelo (B), Sicilia orange (C), Vermelha orange (D), Clementine Caçula 2 (E), Cravo mandarins (F), Clementine Caçula 2 fruits (G) and Mogi das Cruzes mandarin (H).

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anges and mandarins) show various levels of resistance to CiLV (Roessing and Salibe, 1967; Rodrigues et al., 2003; Bastianel et al., 2006a, 2006b). However, few studies have been made with the objective to compare resistance amongst these genotypes. Our results were based on the assessment of leprosis symptoms in a com-mercial orchard containing several varieties and hybrids of different species of mandarins, located in a region where CiLV is endemic. This combination of factors, uncommon to find under natural conditions, provided an unique opportunity to study the behaviour of these genotypes against the disease. Broadening our experi-ence on the response of mandarin cultivars and hybrids to leprosis under natural inoculation conditions is rele-vant for a better understanding of the epidemiology of the disease and its management.

ACKNOWLEDGEMENTS

We would like to thank the owners of the Esmeralda farm (Mogi Mirim, São Paulo, Brazil), where the work was done. Financial support was from the Fund for Cit-rus Plant Protection (FundecitCit-rus), Brazil and FAPESP (Proc. 04/10511-3). N. Segatti had a scholarship from Fundecitrus.

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Received January 23, 2008 Accepted March 19, 2008

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