Experimental and Applied Acarology 30: 203 213, 2003. 2003 Kluwer Academic Publishers. Printed in the Netherlands. Coffee ringspot virus vectored by Brevipalpus phoenicis (Acari: Tenuipalpidae) in coffee C.M. CHAGAS 1,, E.W. KITAJIMA 2 and J.C.V. RODRIGUES 3 1 Instituto Biológico, Avenida Conselheiro Rodrigues Alves 1252, 04014 São Paulo, SP, Brazil 2 Departamento de Entomologia, Fitopatologia e Zoologia Agrícola, Escola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo, Caixa Postal 9, 13418-900 Piracicaba, SP, Brazil 3 Entomology and Nematology Department, University of Florida, IFAS, Citrus Research and Education Center, 700 Experiment Station Road, Lake Alfred, FL 33850-2299, USA Abstract. Coffee ringspot is characterized by conspicuous ringspot symptoms on leaves, berries, and less frequently on twigs. It is caused by coffee ringspot virus (CoRSV), a short, bacilliform virus (40 nm 100 110 nm). The virus is not seed borne and is transmitted by Brevipalpus phoenicis (Geijskes). Transovarial transmission within the mite does not occur. CoRSV has been mechanically transmitted to Chenopodium amaranticolor Coste and Reynaud, C. quinoa Wildenow, Beta vulgaris L., and Alternanthera tenella Colla resulting in local lesions. Systemic infection within both C. amaranticolor and C. quinoa occurs. Virions are found in the nucleus or cytoplasm of infected cells, commonly associated with membranes. Occasionally, membrane bounded particles are found within the cisternae of the endoplasmic reticulum. A characteristic electron lucent, nuclear inclusion is commonly found in many infected cells. These cytopathic effects place CoRSV among the nuclear type of Brevipalpusborne viruses. The disease has been reported in several Brazilian states (São Paulo, Paraná, Minas Gerais, and Federal District) and recently found in Costa Rica. A similar disease is known in the Philippines, but no information exists about its relationship to CoRSV. Coffee ringspot had no economical significance until recently when a large scale infection was reported in Minas Gerais that resulted in yield loss. Key words: Brevipalpus phoenicis, Coffea sp., coffee ringspot virus, cytopathic effects, natural and experimental virus transmission Symptoms Coffee ringspot is a non-systemic coffee disease first reported in Brazil by Bitancourt (1938, 1939). He first suggested its viral nature. Affected coffee (Coffea arabica L.) plants show conspicuous local ringspot symptoms Author for correspondence (Tel.: +55-11-5087-1750; Fax: +55-11-5087-1796; E-mail: chagas@biologico.sp.gov.br)
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205 mainly on leaves and berries, and less commonly on twigs. On leaves, symptoms begin as small chlorotic spots which gradually increase in size, usually becoming ring-shaped, brownish, and sometimes with a necrotic center (Figure 1a). They may coalesce and cover larger foliar areas, sometimes along the midrib of secondary veins, acquiring a brown color as they age (Figure 1b). On senescent leaves, the ringspotted areas may stay green or brownish and surrounded by yellow areas (Figure 1c). Green fruits show whitish, rounded spots on the skin which turn into ringspots as the fruit mature. On mature berries the ringspots may be discolored and are frequently depressed (Figure 2a), or sometimes appear to be only superficial (Figure 2b). When heavily affected, coffee plants may drop both leaves and fruit. Apparently, symptoms do not differ between distinct coffee cultivars. As discussed below, this disease is considered of viral etiology and the agent is designated as coffee ringspot virus (CoRSV). Natural and Experimental Transmission Coffee ringspot was demonstrated to be transmitted by the false spider mite Brevipalpus phoenicis (Geijskes) (Acari: Tenuipalpidae). Under experimental conditions, transmission efficiency was about 24% using adult female mites which had access to infected leaves (Table 1). Transovarial transmission of CoRSV within the mite does not occur (Table 2) (Chagas, 1978). Such experiments were made with mites from a coffee plantation in Taubaté (São Paulo State, Brazil), in the same region where coffee ringspot was first reported by Bitancourt (1938, 1939). B. phoenicis also transmits citrus leprosis virus (CiLV). Another localized citrus disease in southeastern São Paulo State of uncertain etiology is referred to as citrus zonate chlorosis and is believed to be caused by B. phoenicis feeding or a virus. Attempts were made to transmit CoRSV from coffee to citrus and citrus leprosis or zonate chlorosis from citrus to coffee, but the results were negative (Rossetti et al., 1997). B. phoenicis colonizing coffee plants was also unable to transmit CiLV after exposure to citrus leprosis lesions (Chagas and Rossetti, 1982). These data suggest some Figure 1. Leaves of C. arabica L. with typical ringspot symptoms. Note chlorotic and necrotic ring spots (a); coalescent spots covering larger foliar areas along the midribs (b); and senescent leaf with rings surrounded by yellowish areas (c). Figure 2. Coffee berries with coffee ringspot symptoms. Berries from C. arabica cv. Mundo Novo with depressed ringspots (a) and from cv. Bourbom Amarelo with superficial ringspots (b).
206 Table 1. Transmission of coffee ringspot to healthy C. arabica L. seedlings individually infested by single females of B. phoenicis (Geijskes) a Infested seedlings of Mites from coffee ringspot-affected plantation Mites from coffee ringspot-free plantation C. arabica L. Colonized b Successful Transmission Colonized b Successful Transmission transmission c rate (%) transmission c rate (%) cv. Mundo Novo 26/33 9/33 27 26/33 0/33 0 cv. Bourbon Amarelo 25/33 7/33 21 27/33 0/33 0 Total 51/66 16/66 24 d 53/66 0/66 0 a From Chagas (1978). b Colonized seedlings/total number of infested seedlings. c Seedlings with symptoms/total number of infested seedlings. d Mean transmission rate considering total number of infested seedlings.
Table 2. Negative transmission of coffee ringspot through eggs of B. phoenicis (Geijskes) a Eggs from C. arabica L. cv. Mundo Novo Coffee seedlings individually infested with 1egg b 15 eggs b Colonized c Successful Transmission Colonized c Successful Transmission transmission d rate (%) transmission d rate (%) Ringspotted seedlings with 32/48 0/48 0 20/20 0/20 0 viruliferous mites Symptomless seedlings with 30/48 0/48 0 20/20 0/20 0 non-viruliferous mites Total 62/96 0/96 0 40/40 0/40 0 a From Chagas (1978). b Eggs from seedlings individually colonized with single viruliferous or non-viruliferous mites whose progeny were determined as B. phoenicis. c Colonized seedlings/total number of infested seedlings. d Seedlings with symptoms/total number of infested seedlings. 207
208 specificity between the virus, host plant, and mite populations. Mechanical inoculation of coffee tissues showing ringspots resulted in local lesions on Beta vulgaris L.,Chenopodium amaranticolor Coste and Reynaud, C. quinoa Wildenow (Chagas, 1978; Chagas et al., 1981; Vega and Costa, 1987), and Alternanthera tenella Colla (Carvalho and Figueira, 1998a). Carvalho and Figueira (1998b) described cases of systemic infection of mechanically inoculated C. amaranticolor and C. quinoa and attributed this to the presence of new virus strains. So far, no natural host for CoRSV other than coffee is known. Silberschmidt (1941) reported transmission of coffee ringspot to coffee 10 months after grafting. Also, in the Philippines, Reyes (1959) reported similar results. In both cases, the authors used donor plants directly from the field. Thus, the positive cases are likely due to the migration of viruliferous mite vectors, undetectable to the naked eye from donor to receptor plants. Although Valdez (1966) reported seed transmissibility of the ringspot disease in the Philippines, coffee ringspot in Brazil is not seed borne (Chagas, 1978). Etiology Based on transmission assays (mechanical and by B. phoenicis) and on morphological and cytopathic studies of CoRSV-infected host plants, it has been accepted that coffee ringspot indeed is a viral disease. CoRSV particles are short and bacilliform with 35 40 nm 100 110 nm (Figure 3). Transmission electron microscopy (TEM) of thin sections of infected leaves reveals that these particles are present only within the lesion areas either scattered in the nucleoplasm or in the cytoplasm. Such particles tend to occur associated with either a nuclear envelope or endoplasmic reticulum membranes, arranged perpendicularly as involved in a budding process which apparently seldom completes. Another remarkable feature is the presence of an electron lucent inclusion in many nuclei, referred to as viroplasm, in which rodshaped particles appear interspersed (Figure 3) (Kitajima and Costa, 1972; Chagas, 1980). Occasionally, membrane-bounded particles, generally measuring 60 80 nm 180 220 nm, have been seen within the cisternae of the endoplasmic reticulum (Figure 4). Similar alterations were also found in local lesions in mechanically inoculated C. amaranticolor and C. quinoa leaves (Chagas et al., 1981; Vega and Costa, 1987; Nogueira et al., 2000). These cytopathic effects are typical of the so-called nuclear type of Brevipalpus mite-borne viruses (Kitajima, 2001; Kitajima et al., 2003) represented by Orchid fleck virus (OFV) which may belong to the family Rhabdoviridae. Figueira et al. (1999) reported on the amplification of two DNA fragments
209 Figure 3. Electron micrograph of a mesophyll CoRSV-infected coffee cell showing a nucleus containing a lucent inclusion (I) interspersed with short bacilliform particles (V) and some particles associated with the inner nuclear membrane in budding process (arrow). using random primers in RT-PCR assays and claimed that these primers based on a cloned fragment were able to detect vrna in extracts from infected coffee.
210 Figure 4. Electron micrograph of a mesophyll CoRSV-infected cell depicting complete, membrane bounded bacilliform particles in the endoplasmic reticulum (arrows).
211 Geographical Distribution of the Disease and its Vector in Coffee Plantations Brevipalpus phoenicis has been known to infest coffee plantations in the main coffee growing Brazilian states of São Paulo, Paraná, and Minas Gerais, but the effects of damage to the plants was not reported (Flechtmann, 1981). Under favorable laboratory conditions, coffee seedlings infested with viruliferous or non-viruliferous mites usually show higher mite concentrations on the lower leaf surfaces. These infested leaves frequently wither and drop prematurely (Chagas, unpublished data). After the first report in the state of São Paulo (Bitancourt, 1938; 1939), coffee ringspot has been found in coffee plantations in other states: Federal District (Branquinho et al., 1988), Minas Gerais (Figueira et al., 1995; Juliatti et al., 1995), and Paraná (Rodrigues and Nogueira, 2001). In the Federal District and Paraná State, coffee ringspot was identified by symptomatology associated with the presence of viral particles. In two distinct regions of Minas Gerais State, B. phoenicis was identified in heavily infested coffee trees in association with the disease. In Costa Rica, a recent survey showed the occurrence of coffee ringspot disease with the typical viral particles in association with B. phoenicis infestations (Rodrigues et al., 2002). There have been reports of ringspot-like disease in the Philippines (Reyes, 1959) but confirmation of its relationship with CoRSV is not available. Economic Impact and Control Since the first report in 1938, coffee ringspot has been considered a minor disease without economic significance. However, in recent years there have been reports of epidemics of the disease especially in two regions in the State of Minas Gerais (Triângulo Mineiro and Alto Parnaíba). Both have resulted in intense defoliation and fruit drop resulting in yield losses (Figueira et al., 1995; Juliatti et al., 1995). In the Triangulo Mineiro region, the epidemic has disappeared and according to Juliatti (2002, personal communication) this is due to the growers not using harvesting devices that produce strong air current that further distribute mites and infested leaves across greater distances. In the region of Alto Parnaíba, the losses have persisted (Figueira et al., 1998). Knowledge of the epidemiology of CoRSV is scarce, including information about the biology and phenology of the Brevipalpus mites in coffee orchards, possible alternate hosts for both virus and vector, as well as possible sources of genetic resistance. Effective control measures are also lacking although acaricide applications provide temporary control. However, acaricide
212 and application costs are very high and if adequate coverage is not provided then poor control and potential acaricide resistance by B. phoenicis populations can become additional problems. The elimination of natural predacious mites has been reported by Rossetti et al. (1997). All mites involved in the natural or experimental transmission of coffee ringspot virus were identified as B. phoenicis. Ten and twenty-five individual Brevipalpus specimens were identified in 1973 by Prof D.M. Tuttle (Arizona University, USA) and by Prof C.H.W. Flechtmann (São Paulo University, Brazil), respectively. Another 130 specimens were identified by C.M. Chagas (Instituto Biológico, São Paulo, Brazil) in 1973 and 1974. Additional B. phoenicis were identified by Prof Paulo Rebelles Reis (Universidade Federal de Lavras, MG, Brazil) and by Prof Fabio Maximiano (Universidade Federal de Uberlândia, MG, Brazil) in 1995. Acknowledgements The authors thank Drs Kenneth S. Derrick and Carl C. Childers for critical review of this manuscript. Publication of the color plate was supported by Prof M.W. Sabelis (University of Amsterdam, Institute for Biodiversity and Ecosystem Dynamics, Section Population Biology). References Bitancourt, A.A. 1938. A mancha anular, uma nova doença do cafeeiro. O Biológico 4: 404-405. Bitancourt, A.A. 1939. Lesões nas frutas da mancha anular do cafeeiro. O Biológico 4: 53 54. Branquinho, W.G., Cupertino, F.P., Takatsu, A., Kitajima, E.W. and Boiteux, L.S. 1988. Doenças que afetam plantios de café e de mandioca no Distrito Federal. Fitopatol. Bras. 13: 140 (abstract). Carvalho, C.M. and Figueira, A.R. 1998a. Alternanthera tenella as a potential wild host for coffee ringspot virus. Virus Res. Rev. 3(1): 146 (abstract). Carvalho, C.M. and Figueira, A.R. 1998b. Isolados atuais da mancha anular do cafeeiro invadem sisemicamente hospdeiras experimentais. Fitopatol. Bras. 23: 314 (abstract). Chagas, C.M. 1978. Mancha anular do cafeeiro: transmissibilidade, identificação do vetor e aspectos anatomo-patológicas da espécie Coffea arabica L. afetada pela moléstia. PhD Thesis, São Paulo, Universidade de São Paulo, 132 pp. Chagas, C.M. 1980. Morphology and intracellular behaviour of coffee ringspot virus (CRV) in tissues of coffee (Coffea arabica L.). Phytopathol. Z. 99: 301 309. Chagas, C.M. and Rossetti, V. 1982. Novos aspectos sobre a transmissibilidade da leprose dos citros. Fitopatol. Bras. 7: 536 (abstract). Chagas, C.M., July, J.R. and Alba, A.P.C. 1981. Mechanical transmission and structural features of coffee ringspot virus (CRV). Phytopathol. Z. 102: 100 106.
213 Figueira, A.R., Reis, P.R., Carvalho, V.I. and Pinto, A.C.S. 1995. Vírus da mancha anular do cafeeiro tem causado prejuízos relevantes. Fitopatol. Bras. 20: 299 (abstract). Figueira, A.R., Paseto, L.A. and Carvalho, C.M. 1998. A disseminação do vírus da mancha anular do cafeeiro em Minas Gerais tem aumentado acima das expectativas. Fitopatol. Bras. 23: 317 (abstract). Figueira, A.R., Carvalho, C.M., Boari, A.J. and Nogueira, N.L. 1999. Studies on the Brazilian coffee ringspot virus using RT-PCR with random primers and electron microscopy. Virus Res. Rev. 4(1): 152 (abstract). Flechtmann, C.H.W. 1981. Ácaros de Importância Agrícola. Livraría Nobel S.A., São Paulo. Juliatti, F.C., Báo, S.N., Araújo, A.C.G., Kitajima, E.W., Neves, J.B. and Peixoto, J.R. 1995. Mancha anular do cafeeiro: etiologia viral e danos em lavouras da região de Araguarí. Fitopatol. Bras. 20: 337 (abstract). Kitajima, E.W. 2001. Brevipalpus-mite transmitted plant viruses. In: Proc. 11th Congr. Mediterranean Union of Phytopathology and 3rd Congress of the Sociedade Portuguesa de Fitopatologia, Oeiras, Portugal, 16 20 September 2001. Kitajima, E.W. and Costa, A.S. 1972. Partículas baciliformes associadas à mancha anular do cafeeiro. Ciência e Cultura 24: 542 545. Kitajima, E.W., Chagas, C.M. and Rodrigues, J.C.V. 2003. Brevipalpus-transmitted plant virus and virus-like diseases: reports of some recent cases and cytopathology. Exp. Appl. Acarol. 30: 135 160. Nogueira, N.L., Figueira, A.R., Mort, A.E. and Boari, A.J. 2000. Comprovação da infecção sistêmica do Chenopodium quinoa por vírus da mancha anular do cafeeiro (CoRSV) através de microscopia eletrônica. Fitopatol. Bras. 25: 446 (abstract). Reyes, T.T. 1959. Ringspot of coffee in the Philippines. FAO Plant Bull. 8: 11 12. Rodrigues, J.C.V. and Nogueira, N.L. 2001. Ocorrência da mancha anular do cafeeiro no Estado do Paraná e sobrevivência do inóculo à geada. Fitopatol. Bras. 26: 513 (abstract). Rodrigues, J.C.V., Rodriguez, C.M., Moreira, L., Villalobos, W., Rivera, C. and Childers, C.C. 2002. Occurrence of coffee ringspot virus, a Brevipalpus mite-borne virus in coffee in Costa Rica, Central America. Rodrigues, J.C.V., Kitajima, E.W., Childers, C.C. and Chagas, C.M. 2003. Citrus leprosis virus vectored by Brevipalpus phoenicis (Acari: Tenuipalpidae) in citrus in Brazil. Exp. Appl. Acarol. 30: 161 179. Rossetti, V., Colariccio, A., Chagas, C.M., Sato, M.E. and Raga, A. 1997. Leprose dos citros. Bol. Téc. Inst. Biol. 6: 5 27. Silberschmidt, K. 1941. A transmissão experimental da mancha anular do cafeeiro. O Biológico 7: 93 99. Valdez, R.B. 1966. The current status of the ring spot disease of coffee in the Philippines. Philippine Agric. Vol. L: 267 275. Vega, J. and Costa, A.S. 1987. Alterações intracelulares induzidas pelo vírus da mancha anular do cafeeiro em lesões locais de Chenopodium. Summa Phytopathol. 13: 28 (abstract).