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Evaluation of tangerine hybrid resistance to Alternaria alternata
Scientia Horticulturae 123 (2009) 1–4
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Evaluation of tangerine hybrid resistance to Alternaria alternata Marcelo Claro de Souza a,*, Eduardo Sanches Stuchi b, Antonio de Goes a a b
Universidade Estadual Paulista, Departamento de Fitossanidade da FCAV, Jaboticabal, Rodovia Prof. Paulo Donato Castellane km 5, s/no, 14884-900 Jaboticabal, SP, Brazil Embrapa Mandioca e Fruticultura Tropical, EECB, CP 74, 14700-971 Bebedouro, SP, Brazil
A R T I C L E I N F O
A B S T R A C T
Article history: Received 13 February 2009 Received in revised form 7 July 2009 Accepted 10 July 2009
The Alternaria Brown Spot, caused by Alternaria alternata, is a major fungal disease in some kinds of tangerines, tangor, mandarins and pomelos. In Brazil as well as worldwide, A. alternata can cause necrosis in fruits, branches and leaves, causing substantial profit loss. In the present research, in laboratory conditions and in the field, we evaluated the resistance to the fungus, in leaves and fruits, for 22 varieties and hybrids of tangerines. To this end, we evaluated genotypes belonging to the Germplasm Bank of the Estac¸a˜o Experimental de Citricultura de Bebedouro. The resistant genetic materials (found in leaves and fruits) represented four varieties of clementines (Citrus clementina); six varieties of mandarins (two belonging to C. reticulata, two to C. tangerina, one to C. deliciosa and one to C. nobilis); one tangelo (C. tangerina C. paradisi); two mandarin hybrids (one resulting from crossing C. nobilis C. deliciosa and the other from crossing C. clementina C. reticulata); one tangor hybrid (C. clementina) and two satsuma hybrids (C. unshiu C. deliciosa). We also determined a relation between the inoculation of leaves and fruits. The resistance and susceptibility following inoculation in leaves and fruits supports a relationship between these organs and the physiological responses observed for the evaluated genotypes. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Citrus sp. Alternaria brown spot Varietal resistance
1. Introduction The first occurrence of the Alternaria Brown Spot (ABS) was in the mandarin ‘Emperor’, in Australia, 1903 (Pegg, 1966), and the causal agent was designated as Alternaria citri by Ellis and N. Pierce. In Florida, United States, ABS was found in 1974 to have infected the fruits and branches of Dancy tangerines (C. reticulata Blanco) (Whiteside, 1976). In 1989, ABS was reported in Israel (Solel, 1991); it has also been observed in South Africa (Schutte et al., 1992), Cuba (Herrera, 1992), Colombia (Castro Caicedo et al., 1994), Turkey (Canihos et al., 1997), Spain (Vicent et al., 2000), Italy (Bella et al., 2001), Argentina (Peres et al., 2003) and Peru (Marı´n et al., 2006). In Brazil, the initial symptoms of the disease were verified in Dancy tangerines (C. reticulata), in orchards located in Rio de Janeiro State (Goes et al., 2001). In subsequent years, the disease was reported in the States of Sao Paulo, Minas Gerais and Rio Grande do Sul (Spo´sito et al., 2003). Since then, this disease has proven to be a limiting factor in the production of tangerines and, especially, of the Murcott tangor, a hybrid resulting from the cross between C. sinensis (L.) Osbeck and C. reticulata Blanco. Two pathotypes of Alternaria alternata occur in citrus plants, defined by the production of host-specific toxin (HST) (Kohmoto
* Corresponding author. Tel.: +55 16 3209 2640; fax: +55 16 3209 2600. E-mail address: [email protected] (M.C. de Souza). 0304-4238/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2009.07.005
et al., 1979). One pathotype affecting tangerines is called ACT-toxin or ACTG-toxin, and it has been reported in Queensland, Australia (Pegg, 1966) and in Florida, in the USA (Whiteside, 1976). The other, referred to as ACR-toxin or ACRL-toxin, affects the ‘Rugoso’ lemon (C. jambhiri). The ACR pathotype, also reported in Florida by Whiteside (1976), is specific to the lemon tree, in particular the Rugoso and Rangpur lime (C. limonia) varieties. The ACT toxin, in addition to being expressed in tissues of Murcott tangor and in hybrids, is also expressed in young tissues of Minneola and ‘Orlando’ tangelos, and in mandarin hybrids like Sunburst, Nova, Robinson, Lee and Page (Nishimura and Kohmoto, 1983; Kohmoto et al., 1991; Solel, 1991; Peever et al., 2000). Symptoms of the disease appear in the form of spots with brown to black coloration and variable size, on leaves, branches and fruits. The symptoms appear up to 24 h after the start of the infection. However, the lesions usually continue to expand, and great areas of the foliar tissues can be destroyed by the action of the toxin (Kohmoto et al., 1993). According to Whiteside (1976), there is a relation between age and size of the fruits in the context of susceptibility. This finding was verified in the fruit of Dancy tangerines, in ‘Fortune’ mandarins (Vicent et al., 2004) and in Murcott tangor (Reis et al., 2006). Currently, fungicides represent the primary means of controlling ABS (Whiteside, 1979; Timmer and Zitko, 1997; Timmer et al., 2000). However, a great difficulty inherent in the control of ABS is the necessity of frequent spraying, rendering existing tissues susceptible and creating conditions conducive to pathogen
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survival. Therefore, given the conditions that prevail in many citrus plantations in Sao Paulo State, a large number of sprays are frequently required, sometimes exceeding 12 applications per cycle of cultivation. Even with this increased number of sprays, control of the pathogen is often incomplete. In view of the circumstances, the use of resistant varieties can be a highly variable alternative. This work sought to select varieties and hybrids of tangerines resistant to A. alternata infection, and to classify relative degrees of disease severity among leaves and fruits, in order to ultimately control the pathogen. 2. Materials and methods 2.1. Selection of the tested materials Two experiments were performed, both of which used materials belonging to the Germplasm Bank of the Estac¸a˜o Experimental de Citricultura de Bebedouro (EECB), Sao Paulo State, Brazil. Varieties/hybrids were selected based on data provided by Reis et al. (2007), as well as on the degree of potential agronomic value. This latter measure was determined by previous evaluations of palatability and phenotypic characteristics such as appearance, coloration and ease of peeling. 2.2. Isolation and production of A. alternata inoculum Symptomatic leaves of ‘Fortune’ tangor were obtained from plants at EECB and taken to the Laboratory of Phytopathology of the Department of Phytossanity of UNESP, in Jaboticabal, Sao Paulo, Brazil. For the isolation of the fungus, small pieces of the foliar tissue with symptoms of the disease were immersed in ethyl alcohol (50%, v/v) for 30 s. Samples were then immersed in a solution of 1% sodium hypochlorite in water (1:3, v/v) for 2 min. These fragments were rinsed in sterile distilled water and deposited in Petri dishes containing PDA (potato-dextrose-agar) culture medium. The dishes containing culture medium and fragments of tissues were incubated in BOD at 25 8C, under fluorescent light in photophase (12/12 h). After 7 days, we performed transfers to obtain pure colonies of the fungi. From pure cultures and with pathogenicity proven through preliminary inoculations in young Murcott tangor leaves, disks of 5 mm diameter containing culture medium and fungal colonies were transferred to test tubes containing PDA medium. After fungal growths were deposited in these tubes, we added sterile mineral oil to suspend fungal metabolism and then stored the test tubes in a cold chamber. At the same time, disks of culture medium and colonies of fungus were transferred to the Petri dishes containing PDA and incubated in BOD at 25 8C under fluorescent light (12/12 h photophase). To obtain the inoculum, 5 mL of sterile water was added to the surface of pure cultures; the surface was later scraped with a layer of glass to removal the mycelium. After this, the colonies were incubated at 20 8C for 3 days to facilitate the production of conidia. Then, we added 10 mL of sterile water containing Tween1 20 (0.02%, v/v) to the dishes, followed by superficial scraping of the colonies with soft brush bristles. The suspension obtained was filtered through double layers of sterile gauze. The suspension obtained was then calibrated to a concentration of 104 conidia mL 1. 2.3. Inoculation and evaluation of disease severity in fruits For evaluation, we used two or three plants from each variety/ hybrid of citrus with agronomic potential (Table 1). In February 2008, 20 fruits of around 20–40 mm in diameter, located in the
Table 1 Behavior of varieties and hybrids of Citrus spp. following natural and artificial inoculation in fruits with Alternaria alternata, causal agent of the Alternaria Brown Spot. Varieties/hybrids
Result of the inoculation Natural infection in fruits
Artificial infection in fruits
+
+
Clementines (C. clementina Hort. ex Tan.) Clementina 2 Kr Monreal Commune SRA-88 (B) Caffin SRA-385 (B) Bruno SRA-531 (B) Mandarin oranges (C. reticulata Blanco) Mand. Burgess SRA 412 (B) Fewtrell SRA-418 (B) Daisy Mandarim (C. deliciosa Ten.) Mand. Peau Lisse SRA 267 (B) (C. tangerina Hort. ex Tan.) Mand. Zanzibar SRA 442 (B) Mand. Beauty Of Glen Retreat SRA 261 (B) (C. nobilis Lour.) Mand. Rode king SRA 431 (B) Satsumas (C. unshiu Marc.) Satsuma CN (nucelar clone) Tangelos (C. tangerina C. paradisi Macf.) Nocatee SRA-452 (B) Mandarin oranges (hybrids) (C. nobilis C. deliciosa) Mand. Encore SRA 190 (B) (C. clementina C. reticulata) Fremont Tangors (hybrids) (C. temple Hort. ex. Y. Tan. C. tangerina Hort. ex Tan.) Temple Dancy Mandarin +
+
(C. clementina Hort. ex Tan. C. sinensis (L.) Osb.) Tangerina Clementina laranja Tarocco Satsumas (hybrids) (C. unshiu Marc. (C. reticulata Bla. C. sinensis Osb.)) Satsuma Murcott (34) Satsuma Murcott 4 (3) +
+
(C. unshiu Marc. C. sinensis Ten.) Satsuma Natal (30) Satsuma Natal (41) Satsuma Natal 3 (12) ( ) Absence of lesions, (+) one to two lesions, (++) three to six lesions, (+++) seven or more lesions. Botanical classification of the varieties and hybrids with the descriptions reported by Hodgson (1967), Ortiz Marcide (1985) and Ortiz Marcide (1986).
middle of the plant as well as distributed across all four quarters, were marked and inoculated with a suspension of A. alternata. The inoculation was performed at dusk with a manual atomizer (De Vilbs type), with a flow that ensured sufficient coverage (Canihos et al., 1999, with modifications). After the inoculation, all fruits were bagged in clear plastic bags previously sprayed inside with water to create a moist chamber. On the next day, in the morning
M.C. de Souza et al. / Scientia Horticulturae 123 (2009) 1–4
and at dusk, the bags were again sprayed with water in order to ensure a chamber that was continuously moist until the morning of the next day, when the bags were removed. Fruits from the control group were submitted to the same treatments, but without pathogen inoculation. The evaluations were performed from the fourth day after the inoculation for seven subsequent days. The following scoring Table 2 Behavior of varieties and hybrids of Citrus spp. following natural and artificial inoculation in leaves with Alternaria alternata, causal agent of the Alternaria Brown Spot. Varieties/hybrids
Result of the inoculation Natural infection in leaves
Artificial infection in detached leaves
+
+++
Clementines (C. clementina Hort. ex Tan.) Clementina 2 Kr Monreala Commune SRA-88 (B)a Caffin SRA-385 (B)a Bruno SRA-531 (B)a Mandarin oranges (C. reticulata Blanco) Mand. Burgess SRA 412 (B)a Fewtrell SRA-418 (B)a Daisy Mandarim (C. deliciosa Ten.) Mand. Peau Lisse SRA 267 (B)a (C. tangerina Hort. ex Tan.) Mand. Zanzibar SRA 442 (B)a Mand. Beauty Of Glen Retreat SRA 261 (B)a (C. nobilis Lour.) Mand. Rode king SRA 431 (B)a Satsumas (C. unshiu Marc.) Satsuma CN (nucelar clone)
+
Tangelos (C. tangerina C. paradisi Macf.) Nocatee SRA-452 (B)a Mandarin oranges (hybrids) (C. nobilis C. deliciosa) Mand. Encore SRA 190 (B)a (C. clementina C. reticulata) Fremont Tangors (hybrids) (C. temple Hort. ex. Y. Tan. C. tangerina Hort. ex Tan.) Temple Dancy Mandarin +
+++
(C. clementina Hort. ex Tan. C. sinensis (L.) Osb.) Tangerina Clementina laranja Tarocco Satsumas (hybrids) (C. unshiu Marc. (C. reticulata Bla. C. sinensis Osb.)) Satsuma Murcott (34) Satsuma Murcott 4 (3) + (C. unshiu Marc. C. sinensis Ten.) Satsuma Natal (30) Satsuma Natal (41) Satsuma Natal 3 (12)
+ +++
+
( ) Absence of lesions, (+) one to two lesions, (++) three to six lesions, (+++) seven or more lesions. Botanical classification of the varieties and hybrids with the descriptions reported by Hodgson (1967), Ortiz Marcide (1985) and Ortiz Marcide (1986). a Test of susceptibility in detached leaf performed by Reis et al. (2007).
3
system was used: zero (absence of symptoms), 1 (presence of one to two lesions per fruit), 2 (presence of three to six lesions) and 3 (seven or more lesions). 2.4. Inoculation and evaluation of disease severity in detached leaves For this evaluation, we used the Citrus spp. varieties/hybrids listed in Table 2. Using pruning scissors, we collected young branches 20–30 days old. The branches were stored in Styrofoam boxes and brought to the Laboratory of Phytopathology at UNESP, in Jaboticabal City, Sao Paulo State, Brazil, a few hours after the collection. From these branches, we collected leaves 20–40 mm in length, intact and of uniform maturity. Petioles were surrounded by wads of cotton, subjected to humidification and deposited in Petri dishes (15 cm 2 cm). For the inoculation, we used criteria similar to those described above. Dishes were isolated and maintained in a horizontal plane, on a laboratory bench. For each variety/hybrid, experiments were performed in triplicate (20 leaves in total). The same procedure was used for the control leaves, but using only water spray (Canihos et al., 1999, with modifications). After the inoculation, the Petri dishes were maintained at environmental temperature (24– 26 8C), under a photophase of 12/12 h. The evaluation was performed 48 h after the inoculation, following the same procedures used for the evaluation of fruits. 3. Results and discussion The responses to natural infection, exhibited following artificial inoculation with A. alternata in fruits, were similar in the Daisy mandarin; the Temple tangor mandarin Dancy hybrid (C. temple Hort. ex. Y. Tan. C. tangerina Hort. ex Tan.); and the satsuma Murcott 4 tangor hybrid (3) (C. unshiu Marc. (C. reticulata Bla. C. sinensis Osb.)). These varieties all displayed low susceptibility (Table 1). The other varieties and hybrids evaluated did not display symptoms and were therefore considered resistant to artificial inoculation as well as to natural infections. Through evaluations in detached leaves, we verified high susceptibility in the ‘Daisy’ mandarin (C. reticulata Blanco); the Temple tangor Dancy mandarin hybrid (C. temple Hort. ex. Y. Tan. C. tangerina Hort. ex Tan.); and the satsuma Murcott 4 tangor hybrid (3) (C. unshiu Marc. (C. reticulata Bla. C. sinensis Osb.)). We verified low susceptibility in Satsuma CN (nuclear clone) (C. unshiu Marc.); the satsuma Murcott tangor hybrid (34) (C. unshiu Marc. (C. reticulata Bla. C. sinensis Osb.)); and in the satsuma Natal hybrid (41) (C. unshiu Marc. C. deliciosa Ten.). The other varieties and hybrids did not display the symptoms of ABS. Moreover, by evaluating the form of natural infection in leaves, we verified low susceptibility in the ‘Daisy’ mandarin (C. reticulata Blanco); the Temple tangor Dancy mandarin hybrid (C. temple Hort. ex. Y. Tan. C. tangerina Hort. ex Tan.); and the satsuma Murcott 4 tangor hybrid (3) (C. unshiu Marc. (C. reticulata Blanco C. sinensis Osb.)). The other varieties were highly resistant, with no symptoms in the leaves evaluated (Table 2). Several research efforts have sought to test resistance and susceptibility to phytotoxins such as ACT and ACR in citrus (Kohmoto et al., 1979; Solel and Kimchi, 1997; Peever et al., 2000). The ACT toxin produced by A. alternata is specific for a particular set of hosts (HST) (Kohmoto et al., 1991; Peever et al., 1999), comprising tangerines and hybrids (Pegg, 1966). In the present research, susceptibility to ACT toxin was verified in crosses derived from C. reticulata, C. temple, C. tangerina, C. unshiu, C. sinensis and C. deliciosa (Reis et al., 2007), as well as in some cultivars of C. deliciosa and C. temple. Pegg (1966) has already reported the sensitivity of C. tangerina to A. alternata and Reis et al. (2007) performed studies to determine the behavior of varieties of tangerines, satsumas,
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clementines and tangors in relation to the resistance of A. alternata. In this study 79 varieties/hybrids were evaluated, among these some materials resistant to the fungus were identified. Some exhibited notable agronomic potential. Among the mandarins derived from C. reticulata, only the ‘Daisy’ variety of mandarin showed susceptibility to fruit or leaf inoculation and to natural infection. Similar results were observed in the Temple tangor Daisy mandarin hybrid (C. temple Hort. ex. Y. Tan. C. tangerina Hort. ex Tan.) and in the satsuma Murcott 4 hybrid (3) (C. unshiu Marc. (C. reticulata Bla. C. sinensis Osb.)). In all three cases, we observed that the infections in detached leaves were more severe than in attached leaves or fruit. Similar results were found following inoculation of Satsuma CN (nuclear clone) (C. unshiu Marc.; satsuma Murcott hybrid (34) (C. unshiu Marc. (C. reticulata Bla. C. sinensis Osb.)); and satsuma Natal (41) (C. unshiu Marc. C. deliciosa Ten.). Two resistant hybrids were obtained from the cross between satsuma Natal (C. unshiu Marc. C. sinensis Ten.); these did not display symptoms. Another hybrid resulting from the satsuma Natal (41) cross presented weak infection in detached leaves. This finding requires further investigation, since detached leaves show physiological changes that can reflect the level of resistance, which may render the plant more susceptible to A. alternata or other opportunistic microorganisms. Resistance in hybrids with a C. sinensis parent was also observed by Vicent et al. (2004) on Ellendalle and Ortanique, originated from the breed between C. sisnesis C. reticulata, and submitted to three inoculations of isolated A. alternata, AF 2999-1, AF 3499-1 and AF 5199-2. In general terms, when theoretically similar progenitors are utilized there is the possibility that the hybrids behavior, pertaining to A. alternata, be fenotopically similar. However, in this case study variations in their response levels when exposed to this fungus inoculation were verified. This differentiated behavior, although it was known of from literature (Vicent et al., 2004; Chagas et al., 2007), the reasons of its occurrence remain unknown since the total environment conditions and methodologies, including the inoculation method, were equally favorable and followed the concepts adopted by various researchers (Canihos et al., 1999; Reis et al., 2006, 2007). Many factors can contribute to such variations, for example, the ones pertaining to the intrinsic host–pathogen relationship, including an improved specialization of the pathogen. In this context, Kohmoto et al. (1991) verified that King tangerines and its’ hybrids Encore and Kara-Kara were resistant to Aac originated from Florida. According to these authors, in the Kara-Kara hybrid case, this resistance was also observed with the inoculation of isolated Aac, originated from Australia. However, this hybrid along with Wilking and King tangerines showed to be susceptible when inoculated with isolated Aac originated from Israel (Solel and Kimchi, 1997). With this in sight, you cannot rule out a possible genetic effect of the very varieties and hybrids tested since the plants are predominantly cross-pollinating and maintained and/or cultivated in common or neighboring areas. A consistent relationship was found between the level of susceptibility in leaves (as evaluated following either natural infection or artificial inoculation) and the severity and susceptibility of fruit, suggesting a new methodology for the diagnosis, identification and selection of varieties and/or hybrids of Citrus spp. resistant to A. alternata. References Bella, P., Guarino, C.R., Catara, A., 2001. Severe infections of Alternaria spp. on a mandarin hybrid. Journal of Plant Pathology 83, 231.
Canihos, Y., Erkilic, A., Timmer, L.W., 1997. First reporter of Alternaria Brown spot of Minneola tangelo in Turkey. Plant Disease 81, 1214. Canihos, Y., Peever, T.L., Timmer, L.W., 1999. Temperature, leaf wetness, and isolate effects on infection of Minneola tangelo leaves by Alternaria sp. Plant Disease 83, 429–433. Castro Caicedo, B.L., Leguizamon, J.E., Lopez, J.A., 1994. La mancha foliar de los cı´tricos em la zona cafetera. Avances Te´cnicos Cenicafe´ 198, 1–8. Chagas, E.A., Cazetta, J.O., Lemos, E.G.M., Pasqual, M., Goes, A., Ramos, J.D., PIO, R., Barbosa, W., Mendonc¸a, V., Ambrosio, L.A., 2007. Identificac¸a˜o de hı´bridos de citros resistentes a` mancha-marrom-de-alterna´ria por meio de fAFLP e testes de patogenicidade. Pesquisa Agropecua´ria Brasileira 42, 975–983. Goes, A., Montes de Oca, A.G., Reis, R.F., 2001. Ocurrencia de la mancha de Alternaria em mandarina ‘‘Dancy’’ en el estado de Rio de Janeiro. Fitopatologia Brasileira 26 (Suppl.), 386. Herrera, L., 1992. 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Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Evaluation of tangerine hybrid resistance to Alternaria alternata Marcelo Claro de Souza a,*, Eduardo Sanches Stuchi b, Antonio de Goes a a b
Universidade Estadual Paulista, Departamento de Fitossanidade da FCAV, Jaboticabal, Rodovia Prof. Paulo Donato Castellane km 5, s/no, 14884-900 Jaboticabal, SP, Brazil Embrapa Mandioca e Fruticultura Tropical, EECB, CP 74, 14700-971 Bebedouro, SP, Brazil
A R T I C L E I N F O
A B S T R A C T
Article history: Received 13 February 2009 Received in revised form 7 July 2009 Accepted 10 July 2009
The Alternaria Brown Spot, caused by Alternaria alternata, is a major fungal disease in some kinds of tangerines, tangor, mandarins and pomelos. In Brazil as well as worldwide, A. alternata can cause necrosis in fruits, branches and leaves, causing substantial profit loss. In the present research, in laboratory conditions and in the field, we evaluated the resistance to the fungus, in leaves and fruits, for 22 varieties and hybrids of tangerines. To this end, we evaluated genotypes belonging to the Germplasm Bank of the Estac¸a˜o Experimental de Citricultura de Bebedouro. The resistant genetic materials (found in leaves and fruits) represented four varieties of clementines (Citrus clementina); six varieties of mandarins (two belonging to C. reticulata, two to C. tangerina, one to C. deliciosa and one to C. nobilis); one tangelo (C. tangerina C. paradisi); two mandarin hybrids (one resulting from crossing C. nobilis C. deliciosa and the other from crossing C. clementina C. reticulata); one tangor hybrid (C. clementina) and two satsuma hybrids (C. unshiu C. deliciosa). We also determined a relation between the inoculation of leaves and fruits. The resistance and susceptibility following inoculation in leaves and fruits supports a relationship between these organs and the physiological responses observed for the evaluated genotypes. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Citrus sp. Alternaria brown spot Varietal resistance
1. Introduction The first occurrence of the Alternaria Brown Spot (ABS) was in the mandarin ‘Emperor’, in Australia, 1903 (Pegg, 1966), and the causal agent was designated as Alternaria citri by Ellis and N. Pierce. In Florida, United States, ABS was found in 1974 to have infected the fruits and branches of Dancy tangerines (C. reticulata Blanco) (Whiteside, 1976). In 1989, ABS was reported in Israel (Solel, 1991); it has also been observed in South Africa (Schutte et al., 1992), Cuba (Herrera, 1992), Colombia (Castro Caicedo et al., 1994), Turkey (Canihos et al., 1997), Spain (Vicent et al., 2000), Italy (Bella et al., 2001), Argentina (Peres et al., 2003) and Peru (Marı´n et al., 2006). In Brazil, the initial symptoms of the disease were verified in Dancy tangerines (C. reticulata), in orchards located in Rio de Janeiro State (Goes et al., 2001). In subsequent years, the disease was reported in the States of Sao Paulo, Minas Gerais and Rio Grande do Sul (Spo´sito et al., 2003). Since then, this disease has proven to be a limiting factor in the production of tangerines and, especially, of the Murcott tangor, a hybrid resulting from the cross between C. sinensis (L.) Osbeck and C. reticulata Blanco. Two pathotypes of Alternaria alternata occur in citrus plants, defined by the production of host-specific toxin (HST) (Kohmoto
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et al., 1979). One pathotype affecting tangerines is called ACT-toxin or ACTG-toxin, and it has been reported in Queensland, Australia (Pegg, 1966) and in Florida, in the USA (Whiteside, 1976). The other, referred to as ACR-toxin or ACRL-toxin, affects the ‘Rugoso’ lemon (C. jambhiri). The ACR pathotype, also reported in Florida by Whiteside (1976), is specific to the lemon tree, in particular the Rugoso and Rangpur lime (C. limonia) varieties. The ACT toxin, in addition to being expressed in tissues of Murcott tangor and in hybrids, is also expressed in young tissues of Minneola and ‘Orlando’ tangelos, and in mandarin hybrids like Sunburst, Nova, Robinson, Lee and Page (Nishimura and Kohmoto, 1983; Kohmoto et al., 1991; Solel, 1991; Peever et al., 2000). Symptoms of the disease appear in the form of spots with brown to black coloration and variable size, on leaves, branches and fruits. The symptoms appear up to 24 h after the start of the infection. However, the lesions usually continue to expand, and great areas of the foliar tissues can be destroyed by the action of the toxin (Kohmoto et al., 1993). According to Whiteside (1976), there is a relation between age and size of the fruits in the context of susceptibility. This finding was verified in the fruit of Dancy tangerines, in ‘Fortune’ mandarins (Vicent et al., 2004) and in Murcott tangor (Reis et al., 2006). Currently, fungicides represent the primary means of controlling ABS (Whiteside, 1979; Timmer and Zitko, 1997; Timmer et al., 2000). However, a great difficulty inherent in the control of ABS is the necessity of frequent spraying, rendering existing tissues susceptible and creating conditions conducive to pathogen
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survival. Therefore, given the conditions that prevail in many citrus plantations in Sao Paulo State, a large number of sprays are frequently required, sometimes exceeding 12 applications per cycle of cultivation. Even with this increased number of sprays, control of the pathogen is often incomplete. In view of the circumstances, the use of resistant varieties can be a highly variable alternative. This work sought to select varieties and hybrids of tangerines resistant to A. alternata infection, and to classify relative degrees of disease severity among leaves and fruits, in order to ultimately control the pathogen. 2. Materials and methods 2.1. Selection of the tested materials Two experiments were performed, both of which used materials belonging to the Germplasm Bank of the Estac¸a˜o Experimental de Citricultura de Bebedouro (EECB), Sao Paulo State, Brazil. Varieties/hybrids were selected based on data provided by Reis et al. (2007), as well as on the degree of potential agronomic value. This latter measure was determined by previous evaluations of palatability and phenotypic characteristics such as appearance, coloration and ease of peeling. 2.2. Isolation and production of A. alternata inoculum Symptomatic leaves of ‘Fortune’ tangor were obtained from plants at EECB and taken to the Laboratory of Phytopathology of the Department of Phytossanity of UNESP, in Jaboticabal, Sao Paulo, Brazil. For the isolation of the fungus, small pieces of the foliar tissue with symptoms of the disease were immersed in ethyl alcohol (50%, v/v) for 30 s. Samples were then immersed in a solution of 1% sodium hypochlorite in water (1:3, v/v) for 2 min. These fragments were rinsed in sterile distilled water and deposited in Petri dishes containing PDA (potato-dextrose-agar) culture medium. The dishes containing culture medium and fragments of tissues were incubated in BOD at 25 8C, under fluorescent light in photophase (12/12 h). After 7 days, we performed transfers to obtain pure colonies of the fungi. From pure cultures and with pathogenicity proven through preliminary inoculations in young Murcott tangor leaves, disks of 5 mm diameter containing culture medium and fungal colonies were transferred to test tubes containing PDA medium. After fungal growths were deposited in these tubes, we added sterile mineral oil to suspend fungal metabolism and then stored the test tubes in a cold chamber. At the same time, disks of culture medium and colonies of fungus were transferred to the Petri dishes containing PDA and incubated in BOD at 25 8C under fluorescent light (12/12 h photophase). To obtain the inoculum, 5 mL of sterile water was added to the surface of pure cultures; the surface was later scraped with a layer of glass to removal the mycelium. After this, the colonies were incubated at 20 8C for 3 days to facilitate the production of conidia. Then, we added 10 mL of sterile water containing Tween1 20 (0.02%, v/v) to the dishes, followed by superficial scraping of the colonies with soft brush bristles. The suspension obtained was filtered through double layers of sterile gauze. The suspension obtained was then calibrated to a concentration of 104 conidia mL 1. 2.3. Inoculation and evaluation of disease severity in fruits For evaluation, we used two or three plants from each variety/ hybrid of citrus with agronomic potential (Table 1). In February 2008, 20 fruits of around 20–40 mm in diameter, located in the
Table 1 Behavior of varieties and hybrids of Citrus spp. following natural and artificial inoculation in fruits with Alternaria alternata, causal agent of the Alternaria Brown Spot. Varieties/hybrids
Result of the inoculation Natural infection in fruits
Artificial infection in fruits
+
+
Clementines (C. clementina Hort. ex Tan.) Clementina 2 Kr Monreal Commune SRA-88 (B) Caffin SRA-385 (B) Bruno SRA-531 (B) Mandarin oranges (C. reticulata Blanco) Mand. Burgess SRA 412 (B) Fewtrell SRA-418 (B) Daisy Mandarim (C. deliciosa Ten.) Mand. Peau Lisse SRA 267 (B) (C. tangerina Hort. ex Tan.) Mand. Zanzibar SRA 442 (B) Mand. Beauty Of Glen Retreat SRA 261 (B) (C. nobilis Lour.) Mand. Rode king SRA 431 (B) Satsumas (C. unshiu Marc.) Satsuma CN (nucelar clone) Tangelos (C. tangerina C. paradisi Macf.) Nocatee SRA-452 (B) Mandarin oranges (hybrids) (C. nobilis C. deliciosa) Mand. Encore SRA 190 (B) (C. clementina C. reticulata) Fremont Tangors (hybrids) (C. temple Hort. ex. Y. Tan. C. tangerina Hort. ex Tan.) Temple Dancy Mandarin +
+
(C. clementina Hort. ex Tan. C. sinensis (L.) Osb.) Tangerina Clementina laranja Tarocco Satsumas (hybrids) (C. unshiu Marc. (C. reticulata Bla. C. sinensis Osb.)) Satsuma Murcott (34) Satsuma Murcott 4 (3) +
+
(C. unshiu Marc. C. sinensis Ten.) Satsuma Natal (30) Satsuma Natal (41) Satsuma Natal 3 (12) ( ) Absence of lesions, (+) one to two lesions, (++) three to six lesions, (+++) seven or more lesions. Botanical classification of the varieties and hybrids with the descriptions reported by Hodgson (1967), Ortiz Marcide (1985) and Ortiz Marcide (1986).
middle of the plant as well as distributed across all four quarters, were marked and inoculated with a suspension of A. alternata. The inoculation was performed at dusk with a manual atomizer (De Vilbs type), with a flow that ensured sufficient coverage (Canihos et al., 1999, with modifications). After the inoculation, all fruits were bagged in clear plastic bags previously sprayed inside with water to create a moist chamber. On the next day, in the morning
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and at dusk, the bags were again sprayed with water in order to ensure a chamber that was continuously moist until the morning of the next day, when the bags were removed. Fruits from the control group were submitted to the same treatments, but without pathogen inoculation. The evaluations were performed from the fourth day after the inoculation for seven subsequent days. The following scoring Table 2 Behavior of varieties and hybrids of Citrus spp. following natural and artificial inoculation in leaves with Alternaria alternata, causal agent of the Alternaria Brown Spot. Varieties/hybrids
Result of the inoculation Natural infection in leaves
Artificial infection in detached leaves
+
+++
Clementines (C. clementina Hort. ex Tan.) Clementina 2 Kr Monreala Commune SRA-88 (B)a Caffin SRA-385 (B)a Bruno SRA-531 (B)a Mandarin oranges (C. reticulata Blanco) Mand. Burgess SRA 412 (B)a Fewtrell SRA-418 (B)a Daisy Mandarim (C. deliciosa Ten.) Mand. Peau Lisse SRA 267 (B)a (C. tangerina Hort. ex Tan.) Mand. Zanzibar SRA 442 (B)a Mand. Beauty Of Glen Retreat SRA 261 (B)a (C. nobilis Lour.) Mand. Rode king SRA 431 (B)a Satsumas (C. unshiu Marc.) Satsuma CN (nucelar clone)
+
Tangelos (C. tangerina C. paradisi Macf.) Nocatee SRA-452 (B)a Mandarin oranges (hybrids) (C. nobilis C. deliciosa) Mand. Encore SRA 190 (B)a (C. clementina C. reticulata) Fremont Tangors (hybrids) (C. temple Hort. ex. Y. Tan. C. tangerina Hort. ex Tan.) Temple Dancy Mandarin +
+++
(C. clementina Hort. ex Tan. C. sinensis (L.) Osb.) Tangerina Clementina laranja Tarocco Satsumas (hybrids) (C. unshiu Marc. (C. reticulata Bla. C. sinensis Osb.)) Satsuma Murcott (34) Satsuma Murcott 4 (3) + (C. unshiu Marc. C. sinensis Ten.) Satsuma Natal (30) Satsuma Natal (41) Satsuma Natal 3 (12)
+ +++
+
( ) Absence of lesions, (+) one to two lesions, (++) three to six lesions, (+++) seven or more lesions. Botanical classification of the varieties and hybrids with the descriptions reported by Hodgson (1967), Ortiz Marcide (1985) and Ortiz Marcide (1986). a Test of susceptibility in detached leaf performed by Reis et al. (2007).
3
system was used: zero (absence of symptoms), 1 (presence of one to two lesions per fruit), 2 (presence of three to six lesions) and 3 (seven or more lesions). 2.4. Inoculation and evaluation of disease severity in detached leaves For this evaluation, we used the Citrus spp. varieties/hybrids listed in Table 2. Using pruning scissors, we collected young branches 20–30 days old. The branches were stored in Styrofoam boxes and brought to the Laboratory of Phytopathology at UNESP, in Jaboticabal City, Sao Paulo State, Brazil, a few hours after the collection. From these branches, we collected leaves 20–40 mm in length, intact and of uniform maturity. Petioles were surrounded by wads of cotton, subjected to humidification and deposited in Petri dishes (15 cm 2 cm). For the inoculation, we used criteria similar to those described above. Dishes were isolated and maintained in a horizontal plane, on a laboratory bench. For each variety/hybrid, experiments were performed in triplicate (20 leaves in total). The same procedure was used for the control leaves, but using only water spray (Canihos et al., 1999, with modifications). After the inoculation, the Petri dishes were maintained at environmental temperature (24– 26 8C), under a photophase of 12/12 h. The evaluation was performed 48 h after the inoculation, following the same procedures used for the evaluation of fruits. 3. Results and discussion The responses to natural infection, exhibited following artificial inoculation with A. alternata in fruits, were similar in the Daisy mandarin; the Temple tangor mandarin Dancy hybrid (C. temple Hort. ex. Y. Tan. C. tangerina Hort. ex Tan.); and the satsuma Murcott 4 tangor hybrid (3) (C. unshiu Marc. (C. reticulata Bla. C. sinensis Osb.)). These varieties all displayed low susceptibility (Table 1). The other varieties and hybrids evaluated did not display symptoms and were therefore considered resistant to artificial inoculation as well as to natural infections. Through evaluations in detached leaves, we verified high susceptibility in the ‘Daisy’ mandarin (C. reticulata Blanco); the Temple tangor Dancy mandarin hybrid (C. temple Hort. ex. Y. Tan. C. tangerina Hort. ex Tan.); and the satsuma Murcott 4 tangor hybrid (3) (C. unshiu Marc. (C. reticulata Bla. C. sinensis Osb.)). We verified low susceptibility in Satsuma CN (nuclear clone) (C. unshiu Marc.); the satsuma Murcott tangor hybrid (34) (C. unshiu Marc. (C. reticulata Bla. C. sinensis Osb.)); and in the satsuma Natal hybrid (41) (C. unshiu Marc. C. deliciosa Ten.). The other varieties and hybrids did not display the symptoms of ABS. Moreover, by evaluating the form of natural infection in leaves, we verified low susceptibility in the ‘Daisy’ mandarin (C. reticulata Blanco); the Temple tangor Dancy mandarin hybrid (C. temple Hort. ex. Y. Tan. C. tangerina Hort. ex Tan.); and the satsuma Murcott 4 tangor hybrid (3) (C. unshiu Marc. (C. reticulata Blanco C. sinensis Osb.)). The other varieties were highly resistant, with no symptoms in the leaves evaluated (Table 2). Several research efforts have sought to test resistance and susceptibility to phytotoxins such as ACT and ACR in citrus (Kohmoto et al., 1979; Solel and Kimchi, 1997; Peever et al., 2000). The ACT toxin produced by A. alternata is specific for a particular set of hosts (HST) (Kohmoto et al., 1991; Peever et al., 1999), comprising tangerines and hybrids (Pegg, 1966). In the present research, susceptibility to ACT toxin was verified in crosses derived from C. reticulata, C. temple, C. tangerina, C. unshiu, C. sinensis and C. deliciosa (Reis et al., 2007), as well as in some cultivars of C. deliciosa and C. temple. Pegg (1966) has already reported the sensitivity of C. tangerina to A. alternata and Reis et al. (2007) performed studies to determine the behavior of varieties of tangerines, satsumas,
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clementines and tangors in relation to the resistance of A. alternata. In this study 79 varieties/hybrids were evaluated, among these some materials resistant to the fungus were identified. Some exhibited notable agronomic potential. Among the mandarins derived from C. reticulata, only the ‘Daisy’ variety of mandarin showed susceptibility to fruit or leaf inoculation and to natural infection. Similar results were observed in the Temple tangor Daisy mandarin hybrid (C. temple Hort. ex. Y. Tan. C. tangerina Hort. ex Tan.) and in the satsuma Murcott 4 hybrid (3) (C. unshiu Marc. (C. reticulata Bla. C. sinensis Osb.)). In all three cases, we observed that the infections in detached leaves were more severe than in attached leaves or fruit. Similar results were found following inoculation of Satsuma CN (nuclear clone) (C. unshiu Marc.; satsuma Murcott hybrid (34) (C. unshiu Marc. (C. reticulata Bla. C. sinensis Osb.)); and satsuma Natal (41) (C. unshiu Marc. C. deliciosa Ten.). Two resistant hybrids were obtained from the cross between satsuma Natal (C. unshiu Marc. C. sinensis Ten.); these did not display symptoms. Another hybrid resulting from the satsuma Natal (41) cross presented weak infection in detached leaves. This finding requires further investigation, since detached leaves show physiological changes that can reflect the level of resistance, which may render the plant more susceptible to A. alternata or other opportunistic microorganisms. Resistance in hybrids with a C. sinensis parent was also observed by Vicent et al. (2004) on Ellendalle and Ortanique, originated from the breed between C. sisnesis C. reticulata, and submitted to three inoculations of isolated A. alternata, AF 2999-1, AF 3499-1 and AF 5199-2. In general terms, when theoretically similar progenitors are utilized there is the possibility that the hybrids behavior, pertaining to A. alternata, be fenotopically similar. However, in this case study variations in their response levels when exposed to this fungus inoculation were verified. This differentiated behavior, although it was known of from literature (Vicent et al., 2004; Chagas et al., 2007), the reasons of its occurrence remain unknown since the total environment conditions and methodologies, including the inoculation method, were equally favorable and followed the concepts adopted by various researchers (Canihos et al., 1999; Reis et al., 2006, 2007). Many factors can contribute to such variations, for example, the ones pertaining to the intrinsic host–pathogen relationship, including an improved specialization of the pathogen. In this context, Kohmoto et al. (1991) verified that King tangerines and its’ hybrids Encore and Kara-Kara were resistant to Aac originated from Florida. According to these authors, in the Kara-Kara hybrid case, this resistance was also observed with the inoculation of isolated Aac, originated from Australia. However, this hybrid along with Wilking and King tangerines showed to be susceptible when inoculated with isolated Aac originated from Israel (Solel and Kimchi, 1997). With this in sight, you cannot rule out a possible genetic effect of the very varieties and hybrids tested since the plants are predominantly cross-pollinating and maintained and/or cultivated in common or neighboring areas. A consistent relationship was found between the level of susceptibility in leaves (as evaluated following either natural infection or artificial inoculation) and the severity and susceptibility of fruit, suggesting a new methodology for the diagnosis, identification and selection of varieties and/or hybrids of Citrus spp. resistant to A. alternata. References Bella, P., Guarino, C.R., Catara, A., 2001. Severe infections of Alternaria spp. on a mandarin hybrid. Journal of Plant Pathology 83, 231.
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