Biodiversity and Conservation 14: 841–848, 2005. DOI 10.1007/s10531-004-0652-9 Ó Springer 2005 Sources of resistance to downy mildew (Peronospora parasitica (Pers. ex Fr.) Fr.) in Sicilian germplasm of cauliflower and broccoli F. BRANCA1,*, K. BAHCEVANDZIEV2, V. PERTICONE1 and A. MONTEIRO3 1 Dipartimento di Orto-Floro-Arboricoltura e Tecnologie Agroalimentari (DOFATA), Università di Catania, Italy; 2Escola Superior Agrária, Polytechnical Institute of Coimbra, Portugal; 3Instituto Superior de Agronomia, Technical University of Lisbon, Lisboa, Portugal; *Author for correspondence (e-mail: fbranca@unict.it) Received 8 January 2003, accepted in revised form 25 November 2003 Key words: Broccoli, Cauliflower, Disease resistance, Peronospora parasitica, Sicilian landraces Abstract. Downy mildew (Peronospora parasitica (Pers. ex Fr.) Fr.) is a serious disease of brassicas in several countries. Seedlings are very susceptible to this pathogen and crops require frequent fungicide treatments to reach a good marketable yield. The use of resistant cultivars can be the most economical, reliable and environmental friendly method for managing this disease. In this work 32 Sicilian landraces and 16 commercial cultivars of cauliflower and broccoli (B. oleracea) were screened for downy mildew resistance at the cotyledon stage using one P. parasitica strain from Portugal and one from Sicily (Italy). Seven-day old seedlings were inoculated by deposing a droplet of a spore suspension on the cotyledons, incubated under controlled environment and scored 7 days later using a seven-class scale of interaction phenotype (IP), which took into consideration host response and pathogen sporulation. There were no differences in virulence between the two P. parasitica isolates. Accessions ranged from very susceptible to highly resistant to downy mildew showing a variable number of resistant individuals per accession. Forty accessions were very susceptible to downy mildew and are of no interest as sources of resistance, since most of the seedlings were scored in the most susceptible IP classes. Seven accessions had intermediate resistance and included individuals that expressed some degree of resistance. Accession Cv 90 (‘Cavolfiore Torino’) and Br 63 (‘Sparaceddu’) showed the majority of seedlings in the resistant IP classes and may constitute valuable sources of resistance to downy mildew to be used in breeding programs. Introduction The Mediterranean basin, and Sicily in particular, is the region of origin and diversification of broccoli (Brassica oleracea L. convar. italica) and cauliflower (Brassica oleracea L. convar. botrytis). Still many landraces are present and for this reason it could be of interest to look for sources of resistance to downy mildew in the Sicilian germplasm of Brassicaceae, which has already shown sources of resistance to other diseases (Gomez-Campo and Gustafsson 1991; Branca and Iapichino 1997; Catara et al. 2001). Downy mildew, caused by Peronospora parasitica (Pers. ex Fr.) Fr.), is a destructive pathogen of vegetable Brassica seedlings but may also cause damage on adult plants (Lucas et al. 1995). Cotyledons are very susceptible to infection and so 842 the disease represents a serious problem mainly in nurseries where intensive fungicide programs are required to produce healthy seedlings for transplanting (Verma and Thakur 1989). The disease is less destructive on adult plants but the infection on leafy and curded brassicas may cause a serious reduction in marketable yield (Michelmore et al. 1988). The opportunity for identifying and characterising genetically determined resistance, to be used as a component of an integrated disease management program, could be the most economical, reliable and environmental friendly solution to control it. There are various genes of resistance to downy mildew at cotyledon stage identified in B. oleracea for example in broccoli and cauliflower (Jensen et al. 1999; Natti et al. 1967; Silué et al. 1995; Thomas and Jourdain 1990). Leckie et al. (1996) identified several sources of resistance to P. parasitica in an European gene bank collection of horticultural brassicas. However little is known about the existence of resistance material in Italian broccoli and cauliflower germplasm. In this research a collection of Italian broccoli and cauliflower landraces and commercial varieties was screened for resistance to downy mildew with the objective of detecting resistant phenotypes. This work is part of a wider characterisation of this plant material. Materials and methods Thirty-two landraces and 16 commercial cultivars of cauliflower and broccoli (Table 1) were screened at the cotyledon stage for downy mildew resistance using the method described by Leckie et al. (1996). The accessions were tested in five different inoculation tests owing to space limitations. Each test consisted of two trays with 15 accessions
10 seedlings plus the susceptible control CrGC 3.1 (B. oleracea short cycle population, University of Wisconsin-Madison, USA). Forty seedlings were tested per accession with each accession included in two different inoculation tests, with 20 seedlings each time, to take into account eventual environmental influence between the tests. The accessions were grown in multipot trays filled with Levinston F2 compost (Fisons, UK) and covered with vermiculite in order to maintain high moisture for seeds. The trays were kept in a growth room at 20  1 8C, 70–80% RH and 16 h daylength. Seven-day-old seedlings were dual inoculated with the P. parasitica isolates P530 (from Italy) and P501 (from Portugal) by depositing two 10 ml droplets of a spore suspension on each cotyledon. One cotyledon per seedling was marked by puncturing with fine forceps to differentiate P530 from P501 strain. The inoculum was prepared by suspending in distilled water 25 cotyledons of maintenance plants (cabbage ‘Coração de Boi’) bearing 1–2-day-old sporophores. The spore suspension was agitated to dislodge the spores, filtered into centrifuge tubes, and centrifuged three times at 2000 RPM for 3 min. The supernatant was removed and the pellet re-suspended in a known volume of distilled water. Spore concentration was measured using a haemocytometer and adjusted to 4
105 conidia=ml. Seedlings were inoculated immediately to avoid spore germination. 843 Table 1. Identification and origin of the accessions used in the present study. Landraces Accessions Identification Name Origin County Br10 Br13 Br15 Br17 Br40 Br43 Br63 Br68 Cv14 Cv16 Cv19 Cv23 Cv24 Cv25 Cv27 Cv28 Cv31 Cv68 Cv72 Cv73 Cv75 Cv80 Cv81 Cv88 Cv89 Cv92 Cv93 Cv94 Cv95 Cv96 Cv98 Cv99 CrGC 3.1 Fruarola Natalisi Iannarino Sammartinaro Natalisi Natalino Sparaceddu Broccolo nero Natalino Viola Austina Natalino Marzolo Natalino Natalisa Purcella Frivarola Cavolfiore Settembrino Natalino Settembrino Natalino Natalino Violetto Violetto Timpurie di Bacau Natalino Sammartinaro Natalino Fevrarolo Sammartinaro Maiolino University of Wisconsin, - USA Modica Modica Modica Modica Aurnia Adrano P. Armerina Biancavilla Catania Lamezia P. Armerina Palermo Biancavilla Biancavilla Scicli Rosolini Rosolini Francavilla Catania Catania Acireale Biancavilla Biancavilla Francavilla Francavilla Romania Acireale Ragusa Ragusa Catania Adrano Adrano ISA – Portugal Commercial cultivars Name Company Br97 Br99 Cv26 Cv32 Cv33 Cv34 Cv40 Cv44 Cv45 Cv50 Cv52 Cv61 Calabrese tardivo Mariner Napolet. Natalino Di Jesi Violetto di Sicilia Violetto di Sicilia Artemis F1 Precoce di Riesi Palla di neve precocis Romanesco Natalino Violetto di Sicilia Tarocke F1 SAIS Peto seed La Rosa Royal Sluis Royal Sluis Ingegnoli Asgrow Ingegnoli Ingegnoli Ingegnoli Ingegnoli Vilmorin 844 Table 1. (continued) Commercial cultivars Cv64 Cv65 Cv66 Cv90 Accessions Identification Name Origin County Star F1 Cristina F1 Snow ball Cavolfiore Torino Peto seed Peto seed Peto seed Clause Semen After inoculation the trays were placed inside a sealed plastic bag (RH ¼ 100%) and incubated in a growth chamber in the dark for 16–20 h at 16  0.5 8C. Then the plastic bags were removed and the trays moved to the growth room. Six days later the trays were returned to the dew chamber for 24 h to induce sporulation (16  0.5 8C, 100% RH). Subsequently the interaction phenotype (IP) was evaluated using the discrete seven-class scale proposed by Leckie et al. (1996). The IP classes took into account the host response and pathogen sporulation: NN (0) ¼ no symptoms; HN (1) ¼ small necrotic fleck, no sporulation; FN (3) ¼ necrotic flecks, no sporulation; SS (5) ¼ necrotic lesions, sometimes with accompanying chlorosis, a few conidiophores confined to the point of inoculation; CS (7) ¼ necrotic lesions, sometimes with accompanying chlorosis, concentrated (CS-C) or dispersed (CS-D) sporulation in both chlorotic and necrotic areas; HS (9) ¼ necrosis and some chlorosis may be evident, uniformly heavy sporulation over abaxial surface of leaf. The numerical score of each seedling IP was used to calculate a disease index (DI) for each accession consisting of the weighted mean of the disease scores (Williams 1987). Results and discussion The screening for resistance to P. parasitica showed a significant effect of genotype (accession) on the level of the resistance (Table 2). There were no significant differences between the mean DI of isolates P501 and P530 confirming that the two isolates had similar aggressiveness (Table 2). There was no significant effect of the different inoculation tests on the mean DI, which confirms that the different trials had no influence on the expression of resistance. The significant accession by inoculation–test interaction and accession by isolate interaction (Table 2) could have been due to the existence of a few escape seedlings (individuals scored in class NN) that occurred in different tests for accessions Br 99, Cv 19, Br 43, Br 10 and Br 63 (data not shown). There was high variability in the level of resistance among individuals within some accessions ranging from very susceptible to resistant individuals. The disease index (DI) was a good indicator of the resistance of each accession and allowed the distribution of the accessions into four groups of resistance (Table 3). Group 1, with DI between 8.3 and 7.7, includes five accessions with the highest number of susceptible individuals and the susceptible control. Accession DIs in this 845 Table 2. Mean accession disease index in relation to the P. parasitica isolate and to the inoculation test. Mean Inoculation test Isolate I II P530 P501 6.5 6.6 6.6 6.5 FAccessions(Ac) ¼ 32.86*; FInoculation test(It) ¼ 2.71 ns; Fisolate (Is) ¼ 3.03 ns; FAcxIt ¼ 10.00*; FAcxIs ¼ 1.68*; FItxIs ¼ 0.01 ns; FacxItxIs ¼ 1.23 ns. *Significant at p ¼ 0.05, ns – not significant (p ¼ 0.05). Table 3. Accession disease index and percentage of plants interaction–phenotype class. Accession DI Interaction–phenotype class NN HN FN SS CS-C CS-D HS CrGC 3.1 Br97 Cv52 Br17 Cv33 8.3 8.0 7.9 7.7 7.7 6.3 5.5 0.0 4.5 1.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.3 0.0 8.8 7.3 6.3 25.0 4.4 2.5 61.4 16.1 16.2 39.3 42.4 25.0 72.1 57.5 51.8 45.0 Cv65 Br68 Cv34 Cv40 Cv89 Cv94 Cv45 Cv81 Cv16 Cv25 Cv99 Br15 Cv27 Cv24 Cv72 Cv64 Br99 Cv95 Cv73 Cv66 Cv23 Cv88 Cv75 Cv68 Cv32 Cv44 Br13 Cv31 7.6 7.5 7.5 7.5 7.5 7.4 7.4 7.3 7.3 7.2 7.2 7.2 7.2 7.1 7.0 7.0 7.0 6.9 6.9 6.9 6.7 6.6 6.6 6.6 6.6 6.6 6.6 6.3 2.5 2.5 7.1 5.3 1.3 0.0 3.8 0.0 0.0 1.3 1.3 0.0 1.3 2.5 2.6 0.0 18.0 2.5 1.3 0.0 0.0 1.3 18.2 0.0 0.0 0.0 6.3 2.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.3 0.0 0.0 1.3 4.3 1.3 0.0 0.0 0.0 0.0 1.3 0.0 0.0 0.0 1.3 0.0 0.0 0.0 0.0 1.3 0.0 0.0 2.5 0.0 0.0 0.0 1.3 0.0 0.0 1.2 0.0 0.0 1.6 0.0 2.5 2.6 5.0 2.5 0.0 5.0 0.0 1.4 0.0 0.0 1.3 2.5 9.0 7.4 6.5 1.3 1.3 2.5 6.4 1.2 3.8 0.0 0.0 0.0 8.8 3.8 8.6 2.6 0.0 6.7 3.8 1.3 3.8 2.5 6.5 3.6 11.2 3.6 7.5 13.8 9.6 5.0 5.1 17.4 21.3 9.1 2.5 8.8 3.7 21.4 15.0 21.3 21.3 12.4 11.4 35.4 3.8 7.7 8.6 1.3 23.7 18.8 5.1 35.5 25 5.7 29.4 49.7 41.3 11.2 34.7 32.5 22.4 22.5 36.2 57.4 67.2 27.6 57.4 51.2 38.6 57.4 35.8 25.1 71.2 54.3 68.8 10.3 46.2 55.0 62.8 52.6 52.4 26.5 57.6 13.0 13.0 40.0 41.9 46.3 50.0 58.8 49.6 31.3 24.0 47.2 26.3 26.3 30.0 23.8 38.3 34.3 20.0 26.1 13.8 66.6 22.5 17.4 25.6 6.9 8.8 46.0 4.2 21.0 27.1 28.8 9.3 846 Table 3. (continued) Accession DI Interaction–phenotype class NN HN FN SS CS-C CS-D HS Cv80 Cv96 Cv98 Cv19 Br40 Cv14 Cv26 6.3 6.3 6.2 6.2 6.2 6.1 6.1 0.0 5.0 5.0 14.9 1.3 21.3 3.8 1.3 2.5 2.5 0.0 0.0 0.0 0.0 5.8 0.0 1.3 0.0 6.3 1.6 1.3 11.7 0.0 2.5 7.6 11.2 0.0 21.2 27.9 46.2 56.2 23.2 33.8 5.3 23.7 48.3 35.0 17.5 25.3 43.6 39.3 46.2 5.0 11.3 15.0 29.0 3.8 32.5 3.8 Cv92 Cv93 Cv50 Cv61 Br43 Cv28 Br10 5.9 5.9 5.9 5.7 5.4 5.0 4.5 1.6 2.5 13.4 2.5 20.7 1.3 13.3 0.0 3.8 7.5 2.5 3.3 2.5 3.3 9.9 0.0 1.3 1.3 8.3 18.8 3.3 21.9 13.2 2.5 33.8 1.7 44.8 8.3 34.7 46.1 21.9 32.4 9.9 18.8 16.7 24.4 31.8 23.3 25.0 14.1 13.8 25.1 7.5 2.6 30.1 2.5 42.0 0.0 30.0 Cv90 Br63 LSD (p¼0.05) LSD (p¼0.01) 3.0 2.1 0.6 0.7 0.0 5.0 – – 46.3 42.5 – – 30.0 51.2 – – 6.3 1.3 – – 1.3 0.0 – – 13.6 0.0 – – 2.5 0.0 – – group did not significantly differ from the susceptible control (CrGG 3.1); HS was the predominant IP class (Table 3). Group 2, with DI between 7.6 and 6.1, includes 35 accessions, which were less susceptible than CrGC 3.1 but did not present any interest as sources of resistance because almost all seedlings were scored in the most susceptible classes CS and HS. Group 3, with DI between 5.9 and 4.5, includes seven accessions with genotypes expressing some degree of resistance to downy mildew. For instance seedlings in accession Cv 93 were not fully compatible with the pathogen because 78% of them were CS, 13% SS and only 3% HS. Most individuals in accessions Cv 92 and Cv 61 fell into SS and CS classes (Table 3). The accessions of Group 3 could not be regarded as useful sources of resistance because most of the seedlings were in the sporulating classes SS and CS. However, the existence of a reasonable number of individuals in classes SS and sometimes FN shows that they were not fully compatible with the pathogen and therefore may contain some sources of resistance (Table 3). Finally Group 4 includes the accessions Cv 90 and Br 63 with a DI of 3.0 and 2.1 respectively, confirming their resistance to downy mildew. Cv 90 showed the majority of the seedlings in classes HN and FN. Cv 90 was slightly more susceptible to stain P501 than to P530, as expressed by a lower percentage of seedlings in class FN and a higher percentage in classes SS, CS and HS (data not shown). Broccoli landrace ‘Sparaceddu’ (Br 63) was the most resistant accession tested. Almost all the seedlings fell in non-sporulating classes (Table 3). The IP with isolate P530 was 847 predominantly class FN and the IP with isolate P501 class HN. This does not express a clear differential reaction between the isolates and the two resistant accessions, but only a difference in the host response of Br 63. The necrotic fleck under the point of inoculation was much larger after inoculation with strain P530 (class FN) than with P501 (class HN). These tests showed that Sicilian Brassica germplasm expresses high variability for resistance producing two interesting and horticulturally valuable sources of resistance to downy mildew, one in cauliflower and another in broccoli. Accession Cv 90 is a white-curded cauliflower cultivar and its resistance may be transferred to other cultivars by classic breeding methods. Br 63 is a Sicilian landrace of sprouting broccoli, grown in the western areas of Sicily (Branca and Iapichino 1997), and it showed a surprisingly uniform resistance to both P. parasitica isolates. The high and uniform level of resistance within the accession suggests that this landrace could have been previously selected for downy mildew resistance by the growers. Further research is still required to determine the mechanism and the inheritance of the resistance. Conclusions The data showed a great diversity in the Brassica accessions tested. Some of these accessions proved to be of interest for the widespread gene pool for this genus in the Mediterranean basin. The high variability for resistance to P. parasitica both within and among the accessions makes it reasonable to consider some of the accessions tested as potentially useful sources of resistance. The evaluation method used in this research permits a rapid identification of the best sources of resistance to downy mildew among this large group of accessions, which could be utilised to improve widely grown Brassica crops for different production systems, but in particular, sustainable agrosystems. The source of resistance to P. parasitica expressed by accession Br 63 is probably derived from the broad Brassica gene pool present in Sicily, which is likely shared across cultivated and wild Brassica species. The widespread resistance to P. parasitica detected by the present research confirms the great potential of Sicilian landraces of Brassica species as sources of disease resistance reported by Gomez-Campo and Gustafsson (1991), and Branca and Iapichino (1997). Sicilian Brassica germplasm is worth being preserved in situ or ex situ and regularly evaluated owing to its potential for the genetic improvement of commercial cultivars. References Branca F. and Iapichino G. 1997. Some wild and cultivated Brassicaceae exploited in Sicily as vegetable. Plant Genetic Resource Newsletter 110: 22–28. Catara V., Branca F. and Grasso S. 2001. Response of local cultivars of Sicilia violet cauliflower to Xanthomonas campestris pv. Campestris. Proceedings of the 5th Congress of the European Foundation for Plant Phatology, pp. 460–463. 848 Gomez-Campo C. and Gustafsson M. 1991. Germplasm of wild n ¼ 9 Mediterranean species of Brassica. Botany Chronicle 10: 429–434. 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New sources of resistance in cauliflower (Brassica oleracea var. botrytis) to crucifer downy mildew caused by Peronospora parasitica. J. Phytopathology, 143: 659–661 Thomas C.E. and Jourdain E.L. 1990. Evaluation of broccoli and cauliflower germplasm for resistance to race 2 of Peronospora parasitica. HortScience, 25: 1429–1431. Verma T.S. and Thakur P.C. 1989. Comparative field resistance of cabbage collection to downy mildew at seedling stage. Indian Journal of Plant Protection, 17: 79–80. Williams P.H. 1987. GrGC Resource Book. Department of Plant Pathology, University of Wisconsin, Madison, WI, USA. View publication stats