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ALTERNARIA SPECIES INFECTING BRASSICACEAE IN THE BRAZILIAN NEOTROPICS: GEOGRAPHICAL DISTRIBUTION, HOST RANGE AND SPECIFICITY A. Reis and L.S. Boiteux National Center for Vegetable Crops Research (CNPH), Embrapa Hortaliças, CP 0218, 70359-970 Brasilia-DF, Brazil

SUMMARY

Alternaria brassicae and A. brassicicola are the major leaf pathogens on Brassicaceae throughout the world. Here we report a spatio-temporal survey of naturally occurring isolates of these fungi in the Brazilian neotropics. Both fungi were able to induce similar symptoms in common hosts. The fungus A. brassicicola was more frequently isolated (187/322), being prevalent on the Brassica oleracea complex, whereas A. brassicae was prevalent on the B. rapa complex and weed species. Several new hosts and/or new geographical locations of occurrence in Brazil are reported. The presence of host-specificity in A. brassicae isolates was also investigated with cross-inoculation assays. A representative set of Brassicaceae accessions was inoculated with A. brassicae isolates obtained from distinct combinations of host and geographic origin. No host-specificity was observed for all isolates cross-infected all accessions. The present work is the most extensive survey of these fungal species conducted in the neotropics. Comprehensive information about these pathogens will have epidemiological implications, being useful for implementing effective disease management strategies. Key words: Brassicaceae, etiology, epidemiology, host range.

INTRODUCTION

The family Brassicaceae comprises a wide range of vegetable crops of economic, nutritional, and nutraceutical importance (D’Antuono et al., 2007). The most widely cultivated types are classified within the European (Brassica oleracea L.) group (haploid chromosome number n= 9), which includes: cabbage (B. oleracea L. var. capitata L.), kale or “couve-manteiga” (B. oleracea L. var. acephala DC.), perennial kale (B. oleracea L. var.

Corresponding author: A. Reis Fax: +55.61.35565744 E-mail: [email protected]

ramosa DC.), Brussels sprouts (B. oleracea L. var. gemmifera DC.), cauliflower (B. oleracea L. var. botrytis L.), broccoli and sprouting broccoli (B. oleracea L. var. italica Plenck), and kohlrabi or stem turnip (B. oleracea L. var. gongylodes L.) (Zeven et al., 1998; Dixon, 2007). B. rapa L. [Brassica campestris L. (haploid chromosome number n=10)] comprises the Oriental Brassicaceae group (Dixon, 2007). This species is divided in subgroups (former species): turnip (B. rapa L. var. rapa sensu auct mult), pak choi [B. rapa L. var. chinensis (Makino) Hanelt], Chinese cabbage [B. rapa L. var. pekinensis (Lour) Hanelt], caisin and choy sum [B. rapa L. var. parachinensis (Bailey) Tsen et Lee], as well as B. rapa L. var. narinosa, B. rapa L. var. japonica and B. rapa L. var. campestris (Dixon, 2007). Other important Brassicaceae species are garden (salad) rocket (Eruca sativa L.), radish (Raphanus sativus L.), oilseed rape (Raphanus sativus L. var. oleifera Metzg.) and Indian mustard or leaf mustard (Brassica juncea L.). Many species are weeds (Lorenzi, 2000), including wild mustard (Sinapis arvensis L.), weed turnip [Rapistrum rugosum (L.) All.], wild radish (Raphanus raphanistrum L.) and the model plant Arabidopsis thaliana (L.) Heynh. (Koornneef et al., 2004). Foliar diseases are one of the most important limiting factors for cultivation of Brassicaceae in tropical and sub-tropical areas. Fungal species of the genus Alternaria are among the most important causal agents of leaf spot diseases on members of the Brassicaceae family (Humpherson-Jones, 1992; Cucuzza et al., 1994; Verma and Saharan, 1994; Peruch et al., 2006). They cause rounded, well-defined, dark brown leaf spots with pale centre, which are usually delimited by a chlorotic halo (Humpherson-Jones, 1992). These pathogens also occur on many weed species, which may serve as inoculum reservoirs (Farr et al., 1989; Cucuzza et al., 1994; Maringoni, 1997). The two most prevalent fungi associated with dark leaf spot diseases of Brassicaceae around the world are Alternaria brassicae (Berk.) Sacc. ex Rape and Alternaria brassicicola (Schwein.) Wiltshire (Nakata and Takimoto, 1928; Rangel, 1945; Ellis, 1971; Degenhardt, 1982; Strandberg, 1992; Verma and Saharan, 1994). These fungi are causal agents of a complex foliar disease,

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which may have both pathogens in the same field, infecting a single host and/or host tissue at the same time (Verma and Saharan, 1994; Maringoni, 1997). In Brazil, A. brassicae and A. brassicicola have been reported to be associated with leaf spots of many Brassicaceae (Maringoni, 1997; Mendes et al., 1998). However, the information about natural host range, relative prevalence, and geographical distribution of Alternaria species on Brassicaceae in neotropical areas (including Brazil) is still scarce. In addition, some reports of either A. brassicae or A. brassicicola are unreliable as they do not provide a precise pathogen identification and/or the fulfilment of the Koch’s postulates. There are also doubtful records as, for example, that of A. brassicae in host plants belonging to families other than Brassicaceae (Mendes et al., 1998). In this context, the major objective of the present work was to conduct an extensive geographical and temporal survey in neotropical Brazil to assess the relative importance of A. brassicae and A. brassicicola as leaf spots agents of Brassicaceae species, their distribution pattern and natural host range. An additional study was also carried out to investigate potential patterns of host adaptation (specificity) of A. brassicae isolates.

Journal of Plant Pathology (2010), 92 (3), 661-668 MATERIALS AND METHODS

Alternaria isolates obtained from Brassicaceae species. Samples displaying leaf spots were obtained from distinct Brassicaceae species in several growing regions of Brazil ranging from latitude 4o 10’ 01’’ S to latitude 31o 46’ 19’’ S (Fig. 1). The field survey was conducted from 2003 to 2008. Leaf samples displaying typical necrotic, concentric spots with or without chlorotic halo (Fig. 2 and 3), were kept in moist chambers for 24 to 48 h. Individual conidia (four to five) were collected from the lesions with a sterilized metal loop and transferred to four to five different points in Petri plates containing water agar medium. The development of mycelia from these conidia was observed after 2-3 days. Hyphal tips from each isolate were transferred under a stereomicroscope to Petri plates containing 10% V8 juice agar medium. Fungal cultures were grown in culture tubes on V8 medium supplemented with penicillin (50 mg/l) and rifampicin (30 mg/l) and long-term conserved in distilled water (Castellani, 1939) and mineral oil (Buell and Weston, 1947). Pathogen identification. The morphological and

Fig. 1. Distribution of the Brazilian regions where the isolates of Alternaria brassicicola (Abcc) and A. brassicae (Abce) were sampled. Abbreviations of the Brazilian States: RS = Rio Grande do Sul, SC = Santa Catarina, PR = Paraná, SP = São Paulo, RJ = Rio de Janeiro, MG = Minas Gerais, ES = Espírito Santo, MS = Mato Grosso do Sul, MT = Mato Grosso, GO = Goiás, DF = Distrito Federal, BA = Bahia, SE = Sergipe, AL = Alagoas, PE = Pernambuco, PB = Paraíba, RN = Rio Grande do Norte, CE = Ceará, PI = Piauí, MA = Maranhão, TO = Tocantins, PA = Pará, AP = Amapá, AM = Amazonas, RR = Roraima, AC = Acre, and RO = Rondônia.

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Fig. 2. Symptoms caused by Alternaria brassicae on Chinese cabbage (A), radish (B), pak-choy (C), and oilseed rape (D).

morphometrical characteristics of conidia were recorded from colonies grown on 5% V8 agar medium, which induced a more profuse sporulation than the 10% V8 medium. The length and the width of 50 conidia were mesured from one randomly chosen isolate of each crop/geographic origin combination. These measurements as well as other morphological characteristics of the colonies and conidia, including the presence (or absence) of conidial chains, number of conidia per chain, colour and number of septa of mature conidia, presence and length of the rostrum (beak) of the conidia were compared with Alternaria descriptions of Ellis (1971) and Simons (1995, 2007). Pathogenicity and cross-inoculation assays with A. brassicae isolates. Two experiments were carried out to test the pathogenicity and potential host speciation of A. brassicae isolates. In pathogenicity tests, one isolate from each host and place of origin was used to inoculate its original host under greenhouse conditions. Thirty four A. brassicae isolates were used in this test. In the second experiment, one A. brassicae isolate from each host was inoculated on a set of seedlings of the host

plants found in the survey. Stem radish, wild radish, and wild mustard were not included due to the lack of seeds. The experimental design of both assays was completely randomized factorial arrangement 10 x 7 x 3 (10 isolates, seven hosts, and three replications). Each replication was constituted by one pot with two plants. Both experiments were conducted twice. The following cultivars were employed in these assays: ‘Pe-Tsai’ (Chinese cabbage), ‘Rúcula Cultivada’ (salad rocket), ‘Flórida Lisa’ (leaf mustard), ‘Chouyou F1’ (pak choi), ‘Minoware’ (turnip), ‘Crinson Gigante’ (radish) and one wild accession of oilseed rape. Plants were inoculated with a conidial suspension (104 conidia/ml) until leaf runoff. Conidia were produced in Petri plates containing 20 ml of 5% V8 medium (12 h of darkness and 12 h of black light). The negative control was one pot of each cultivar sprayed with distilled sterile water. The presence/absence of leaf spot symptoms was evaluated 10 days after inoculation. The presence of necrotic leaf lesions typically induced by Alternaria species was recorded and the diameter of five lesions on the lowest leaf of each plant was measured.

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Pathogenicity of A. brassicicola isolates. In the pathogenicity test a single A. brassicicola isolate from each host and place of origin was inoculated on its own host. Inoculation and evaluation were essentially as described in the A. brassicae experiment. This experiment was also carried out twice. Cross-inoculation assays with the A. brassicicola isolates were not done.

RESULTS

A. brassicae and A. brassicicola inducd quite similar symptoms in inoculated plants, which hampered direct pathogen identification under both greenhouse and field conditions. Both Alternaria species were successfully re-isolated from leaf spots on inoculated plants and Koch’s postulates were fulfilled for both Alternaria species on all hosts. A. brassicae colonies were initially white, turning

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brown within 5 to 7 days from plating on growth media. At this time, darker spots developed on the culture plate which contained a large quantity of conidiophores and conidia typical of Alternaria. In general, conidia were solitary but some rare chains of 2-3 conidia were also observed. Conidia were pale brown in colour and exhibited 8 to 16 transversal septa and 0 to 7 longitudinal septa. The beak (rostrum) length ranged from 26 to 162 µm, whereas conidial length and width ranged from 68 to 310 µm and 18 to 28 µm, respectively. A. brassicicola colonies were dark brown to black. Conidiophores ranged from 45 to 66 µm in length, while conidia were pale to light brown and showed 1 to 9 transversal septa and 1 to 4 longitudinal septa. The apical cells were very short and brighter than the conidial body. Conidia length and weight ranged from 27 to 142 µm and 7 to 22 µm, respectively. Conidia were most of the time in long chains of up to 23 elements, sometimes branched, arising through small pores on the conidiophores.

Fig. 3. Symptoms caused by Alternaria brassicicola on wild mustard (A), oilseed rape (B), radish (C), and Chinese cabbage (D).

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Two to five Alternaria isolates were obtained from each geographical site, totalling 322 from cultivated and wild Brassicaceae species. Of these, 187 were A. brassicicola and 135 A. brassicae, giving an overall prevalence of A. brassicicola (58%) over A. brassicae (42%). Of the

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230 isolates collected from the B. oleracea complex 170 (73.9%) were A. brassicicola and only 60 (26.1%) were classified as A. brassicae. On the other hand, A. brassicae was more frequent (66.5%) than A. brassicicola (33.5%). on the other cultivated and weed species.

Table 1. List of 50 isolates of Alternaria brassicae recorded on new hosts and/or new geographical locations in Brazil. Isolate code

Host

665

Host species

EH-0416 Chinese cabbage Brassica campestris var. pekinensis EH-0418 Pack-choi Brassica campestris var. chinensis EH-0523 Chinese cabbage Brassica campestris var. pekinensis EH-0569 Chinese cabbage Brassica campestris var. pekinensis EH-0570 Chinese cabbage Brassica campestris var. pekinensis EH-0571 Pack-choi Brassica campestris var. chinensis EH-0828 Salad rocket Eruca sativa EH-0829 Chinese cabbage Brassica campestris var. pekinensis EH-0834 Turnip Brassica rapa EH-0876 Leaf mustard Brassica juncea EH-0877 Leaf mustard Brassica juncea EH-0905 Chinese cabbage Brassica campestris var. pekinensis EH-0911 Oilseed rape Raphanus sativus var. oleifera EH-0998 Pack-choi Brassica campestris var. chinensis EH-1005 Chinese cabbage Brassica campestris var. pekinensis EH-1025 Chinese cabbage Brassica campestris var. pekinensis EH-1042 Wild mustard Sinapis arvensis EH-1043 Wild radish Raphanus raphanistrum EH-1081 Salad rocket Eruca sativa EH-1083 Chinese cabbage Brassica campestris var. pekinensis EH-1084 Radish Raphanus sativus EH-1139 Chinese cabbage Brassica campestris var. pekinensis EH-1161 Chinese cabbage Brassica campestris var. pekinensis EH-1194 Wild radish R. raphanistrum EH-1198 Wild radish R. raphanistrum EH-1269 Wild radish R. raphanistrum EH-1297 Turnip Brassica rapa EH-1326 Chinese cabbage Brassica campestris var. pekinensis EH-1353 Stem radish B. olaracea var. gongylodes EH-1388 Oilseed rape Raphanus sativus var. oleifera EH-1396 Chinese cabbage Brassica campestris var. pekinensis EH-1424 Leaf mustard Brassica juncea EH-1425 Leaf mustard Brassica juncea EH-1450 Leaf mustard Brassica juncea EH-1451 Leaf mustard Brassica juncea EH-1497 Salad rocket Eruca sativa EH-1519 Leaf mustard Brassica juncea EH-1520 Leaf mustard Brassica juncea EH-1545 Leaf mustard Brassica juncea EH-1546 Leaf mustard Brassica juncea EH-1606 Wild radish R. raphanistrum EH-1609 Wild mustard S. arvensis EH-1611 Chinese cabbage Brassica campestris var. pekinensis EH-1621 Radish R. sativus EH-1750 Oilseed rape Raphanus sativus var. oleifera EH-1762 Chinese cabbage Brassica campestris var. pekinensis EH-1763 Oilseed rape Raphanus sativus L. var. oleifera EH-1775 Chinese cabbage Brassica campestris var. pekinensis EH-1778 Wild radish R. raphanistrum EH-1779 Oilseed rape Raphanus sativus var. oleifera * For abbreviations of the Brazilian States see Figure 1

Geographic area*

Month and year of isolation

Brazlândia – DF Brazlândia – DF Ceilândia – DF Chã-Grande – PE Chã-Grande – PE Chã-Grande – PE Vargem-Bonita – DF Vargem Bonita – DF Vargem-Bonita – DF Pelotas – RS Pelotas – RS Guapiara – SP S. Francisco de Paula – RS Brazlândia – DF Paranaguá – PR Planaltina – DF Vargem-Bonita – DF Vargem-Bonita – DF Vargem-Bonita – DF Vargem Bonita – DF Vargem Bonita – DF Camocim de São Félix–PE São José dos Pinhais – PR Araucária – PR Araucária – PR Águas Claras – DF Brazlândia – DF Guaraciaba do Norte – CE Guaraciaba do Norte – CE Carandaí – MG Igarapé – MG São Marcos – MG Carandaí – MG Elias Fausto – SP Elias Fausto – SP Biguaçu – SC Muniz Freire – ES Muniz Freire – ES Rancho Queimado – SC Rancho Queimado – SC Nova Friburgo – RJ Nova Friburgo – RJ Nova Friburgo – RJ Nova Friburgo – RJ Reserva – PR Antônio Carlos – SC Antônio Carlos – SC Nova Friburgo – RJ Nova Friburgo – RJ Nova Friburgo – RJ

01/2003 01/2003 02/2003 03/2003 03/2003 03/2003 01/2004 09/2003 09/2003 10/2003 10/2003 02/2004 04/2004 07/2004 08/2004 09/2004 10/2004 10/2004 12/2004 10/2004 10/2004 03/2005 04/2005 04/2005 04/2005 07/2005 08/2005 08/2005 08/2005 08/2005 08/2005 08/2005 08/2005 08/2005 08/2005 01/2006 02/2006 02/2006 03/2006 03/2006 07/2006 07/2006 07/2006 07/2006 03/2008 10/2008 10/2008 11/2008 11/2008 11/2008

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Mustard, pak-choi, and turnip were prevalent hosts of A. brassicae, which was also more frequent than A. brassicicola on Chinese cabbage, since of the 52 isolates from this host 44 (84.6%) were A. brassicae and only 8 (15.4%). were identified as A. brassicicola. As to the geographic distribution, A. brassicae was limited to areas with mild subtropical climate (southern and south-eastern regions) and to highland tropical areas [e.g. Distrito Federal (DF) and Goiás (GO)] and also to high elevation micro-regions (sierras and mountains) near to the seashore [e.g. Ceará (CE) and Pernambuco (PE)]. By contrast, A. brassicicola was prevalent in warm areas of the central-west, north-east, and northern regions. Both species had almost the same frequency only in the south-east (Fig. 1). A. brassicicola was the most cosmopolitan of the two species, being present in all Brazilian states sampled, except for the warm and humid equatorial areas in the Amazonas (AM) State in the north. Cross-inoculation assays indicated an apparent lack of host-specificity for A. brassicae isolates. All those that were tested were able to cause some level of disease on all inoculated plants regardless the host they came from. Differential levels of aggressiveness of the isolates from any given plant host was not observed. However, distinct degrees of rate-reducing resistance (Nelson, 1978) to A. brassicae were observed in accessions of radish and oilseed rape, which was characterized by the presence of lesions with smaller diameters (range from 3 to 7 mm, average 4 mm). On the contrary, leaf mustard, pak-choi and Chinese cabbage accessions displayed leaf spots with the largest diameters (range from 9 to 17 mm, average 11 mm).

Journal of Plant Pathology (2010), 92 (3), 661-668 DISCUSSION

We report, for the first time, the occurrence of A. brassicae and A. brassicicola causing leaf spots on different combinations of host/geographical location in Brazil. These new records are listed in the Table 1 (A. brassicae) and Table 2 (A. brassicicola). The present work is therefore the most extensive survey of A. brassicae and A. brassicicola conducted in the neotropics. Koch’s postulates were fulfilled for each combination of Alternaria and host species many of which are reported for the first time from Brazil. All morphometric characteristics of the isolates were in agreement with those described for both A. brassicicola and A. brassicae by Ellis (1971) and Simmons (1995, 2007). Although long conidial chains observed in all A. brassicicola isolates are also a characteristic of A. alternata, a fungus that colonizes decaying leaf tissue of many plant species (Ellis, 1971), nevertheless A. brassicicola can redily be distinguishable from A. alternata due to the presence of a brighter apical cell and the lack of a conspicuous beak (rostrum) in most conidia. In addition, all isolates from Brassicaceae displaying long conidial chains, when inoculated on its original host, were able to induce typical lesions in actively growing leaf tissue, which A. alternata isolates are not usually able to do (Ellis, 1971). Both pathogens were found as complexes in some crops. However, in our survey A. brassicae was observed with high frequency on Chinese cabbage, mustard, pakchoi and turnip, whereas A. brassicicola was predominant on the B. oleracea complex. This may be explained by a possible preference of A. brassicae for host species of the Oriental group. Moreover, B. oleracea species are cultivated all over Brazil, comprising regions of tropical and subtropical (milder) climates, which proved to be

Table 2. List of 17 isolates of Alternaria brassicicola recorded on new hosts and/or new geographical locations in Brazil. Isolate Host Host species code EH-0159 Chinese cabbage Brassica campestris var. pekinensis EH-0197 Chinese cabbage Brassica campestris var. pekinensis EH-0704 Chinese cabbage Brassica campestris var. pekinensis EH-906 Oilseed rape Raphanus sativus var. oleifera EH-1007 Chinese cabbage Brassica campestris var. pekinensis EH-1160 Chinese cabbage Brassica campestris var. pekinensis EH-1198 Wild radish Raphanus raphanistrum EH-1355 Stem turnip Brassica olaracea var. gongylodes EH-1416 Leaf mustard Brassica juncea EH-1492 Salad rocket Eruca sativa EH-1498 Turnip Brassica rapa EH-1606 Wild mustard Sinapis arvensis EH-1633 Chinese cabbage Brassica campestris var. pekinensis EH-1770 Radish Raphanus sativus EH-1774 Oilseed rape R. sativus var. oleifera EH-1777 Wild radish R. raphanistrum * For abbreviations of the Brazilian States see Figure 1.

Geographic area* Mutuns – PE Chã Grande – PE Brazlândia – DF São Francisco de Paula- RS Paranaguá – PR São José dos Pinhais – PR Araucária – PR Guaraciaba do Norte – CE Carandaí – MG Biguaçu – SC Vargem Bonita – DF Nova Friburgo – RJ Guaraí – TO Nova Friburgo – RJ Nova Friburgo – RJ Nova Friburgo – RJ

Month and year of isolation 02/2003 02/2003 06/2003 04/2004 08/2004 04/2005 06/2005 08/2005 08/2005 01/2006 01/2006 07/2006 08/2006 11/2008 11/2008 11/2008

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the prevalent ecological niche of A. brassicae. A. brassicae was prevalent on Chinese cabbage, which could be once more explained by the preference of this fungus for hosts of the Oriental Brassicaceae group combined with the fact that Chinese cabbage is grown only in subtropical and highland areas, where cooler temperatures predominate throughout the year. According to our survey, which corroborates previous studies (Maringoni, 1997; Mendes et al., 1998), A. brassicae is a natural pathogen of at least eight host species in Brazil. However, some past reports are doubtful because Koch’s postulates were not fulfilled. In addition, our study confirmed that A. brassicae is a very common pathogen of Brassicaceae in our country as in other regions of the world (Nakata and Takimoto, 1928; Degenhardt et al., 1982; Farr et al., 1989; Hampherson-Jones, 1992; Verma and Saharan, 1994; Mendes et al. 1998). Alternaria brassicicola appered to be the most cosmopolitan of the two species, our observations being in agreement with the notion that A. brassicae is better adapted to mild climates and to host plants outside the B. oleracea complex (Strandberg, 1992; Verma and Saharan, 1994; Peruch et al., 2006). The B. oleracea complex is cultivated in all regions of Brazil contributing for the prevalence of A. brassicicola in the country. In the North, where average temperatures and humidity are very high, only species of the B. oleracea complex are grown, which may be the main reason for the lack of A. brassicae on diseased plants sampled in this region. We have detected A. brassicae and A. brassicicola for the first time on different combinations of host and geographical locations of several Brazilian States. Therefore, these plant pathogens are able to infect a broad range of hosts under distinct environmental conditions. This set of alternative host species provides field inoculum all year round, perpetuating the presence of both fungal species in a range of environmental conditions. The identification of new alternative hosts of A. brassicae and A. brassicicola is a very important piece of information that has epidemiological implications and a bearing on the implementation of more effective disease management strategies in Brassicaceae-producing areas of the country. Weeds such as S. arvensis, wild mustard and oilseed rape, can also serve as reservoirs of both pathogens, providing initial inoculum for infection of economically important hosts. This situation may cause difficulties for the management of both pathogens in areas where these alternative hosts occur endemically.

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Received January 19, 2010 Accepted April 19, 2010

Journal of Plant Pathology (2010), 92 (3), 661-668 Zeven A.C., Dehmer K.J., Gladis T., Hammer K., Lux H., 1998. Are the duplicates of perennial kale (Brassica oleracea L. var. ramosa DC.) true duplicates as determined by RAPD analysis? Genetic Resources and Crop Evolution 45: 105-111.