New Source of Black Rot Disease Resistance in Brassica Oleracea and Genetic Analysis of Resistance

Euphytica (2016) 207:35–48 DOI 10.1007/s10681-015-1524-y

New source of black rot disease resistance in Brassica oleracea and genetic analysis of resistance

Partha Saha . Pritam Kalia . Munish Sharma . Dinesh Singh

Received: 1 May 2015 / Accepted: 22 July 2015 / Published online: 28 July 2015 Ó Springer Science+Business Media Dordrecht 2015

Abstract Black rot is the most widespread and Himjyoti x BR-207) and (Pusa Sharad x BR-207) were devastating disease in Brassica oleracea crops. The evaluated. Segregation ratios obtained were compared objective of this work was to identify new sources of by Chi square test at 5 % probability. The F1 resistance to races 1 and 4 of Xcc in 46 B. oleracea generations were resistant and segregation ratio of accessions and to understand genetics of resistance. resistant and susceptible plants indicated 3(R):1(S) in

Most of the accessions were susceptible to both the F2 and 1(R):2 (Seg):1(S) in F3 which suggested that races, except accessions BR-207, BR-1, BR-202-2 and resistance to Xcc race 1 in BR-207 is governed by a AL-15 of botrytis group to Xcc race 1. The partial single dominant gene. The information obtained in this resistant plants were observed in some accessions BR- study could be valuable for black rot resistance 202-2 (90 % plants) and AL-27 (10 % plants) of breeding and in planning a systematic breeding botrytis, DJ8012 (20 % plants) of capitata, 005426 programme to incorporate the black rot resistance (10 % plants) of italica. All the accessions were into the susceptible cultivars. susceptible to Xcc race 4 except one accession (DJ8012) in cabbage (capitata group) with mean Keywords Brassica oleracea Á Black rot Á Genetics disease score of 7.0 and 10 % plants were found partial resistant. The genetics of resistance in accession BR- 207 (botrytis group) was investigated by crossing this with susceptible accessions (Pusa Himjyoti and Pusa Introduction

Sharad). For the genetics study, F1,F2,F3 and backcrosses (BC1P1 and BC1P2) generations of (Pusa Brassica oleracea L. comprising crop like cauliﬂower, cabbage, broccoli, Brussels sprouts and kale which are P. Saha (&) Á P. Kalia widely grown vegetables in India and in other parts of Division of Vegetable Science, Indian Agriculture the world (Go´mez-Campo 1999). These crops are Research Institute, New Delhi 11002, India affected by several diseases, of which black rot caused e-mail: [email protected] by bacterium Xanthomonas campestris pv. campestris M. Sharma (Xcc) (Pammel) Dowson has been reported to be most Guru Angad Dev Veterinary and Animal Sciences devastating disease worldwide (Camargo et al. 1995; University, Ludhiana, Punjab 141004, India Taylor et al. 2002; Chen et al. 2003; Singh et al. 2011). The bacterium spreads through vascular tissue, clogg D. Singh Division of Plant Pathology, Indian Agriculture Research vessels and develops ‘V’-shaped chlorotic lesions Institute, New Delhi 11002, India (Fig. 1) (Tonguc and Grifﬁths 2004). Marginal leaf 123 36 Euphytica (2016) 207:35–48

Fig. 1 Black rot disease symptom in Brassica oleracea chlorosis, necrosis and darkening of leaf veins and which is generally recommended to farmers has not vascular tissue within the stem are main characteristic provided a satisfactory and durable solution; in symptoms. The disease has a wide geographical addition, it is costly and environment pollutant (Mason distribution and is destructive to these vegetables et al. 2000). Therefore, development of resistant causing yield (50–65 %) and quality loss due to varieties and/or hybrids is the best approach (Pico cultivar susceptibility (Williams 1980; Ignatov et al. et al. 1996; Lapidot et al. 1997). The utilization of host 1999; Kashyap and Dhiman 2010; Dhar and Singh resistance has been recognized as one of the most 2014). This is a seed borne bacterial disease. Besides economic and effective strategies. Numerous studies these, the disease spreads by rain drops and the have been carried out to identify new sources of pathogen survives in the crop debris, seeds, and weeds resistance to black rot, but most of them are not race for longer periods (Tonguc and Griffiths 2004). The specific resulting in difficulties to incorporate black rot pathogen remains in the vascular system of the resistant gene in desirable genetic background. Many infected plants until favourable environment for related crops of Brassica species have also been found bacterial growth take place (Lema et al. 2012). Since to exhibit resistance to Xcc (Taylor et al. 2002; Tonguc the first recognition of Xcc races by Kamoun et al. and Griffiths 2004). Presently, the control of this (1992), several studies analyzed black rot resistance in disease is complicated, and very few sources of relation to race types (Ignatov et al. 2000; Vicente resistance are available in B. oleracea groups. Specific et al. 2000; Taylor et al. 2002). Initially, Vicente et al. resistance to races 1 and/or 4 has been previously (2001) identified six races (1–6), and later on, Fargier reported in different Brassica species (Ribeiro and and Manceau (2007) added three additional races Dias 1997; Tonguc and Griffiths 2004; Griffiths et al. (7–9). Races 1 and 4 have been found to be the most 2009; Vicente et al. 2002; Singh et al. 2011). The R1 virulent, widespread and accounting for more than gene conferring resistance to race 1 is present in the B 90 % of black rot disease around the world (Vicente genome of Brassica carinata (BBCC), Brassica et al. 2001). juncea (AABB) and Brassica nigra (BB), while the It is very difficult to control this disease and it can R4 gene conferring resistance to race 4 is present in the only be achieved by the use of disease free seeds and A genome of Brassica rapa (AA), Brassica napus adoption of proper cultural practices, and the elimi- (AACC) and B. juncea (AABB) (Vicente et al. 2002; nation of other potential inoculum sources (infected Taylor et al. 2002). In B. oleracea (CC) with the C crop debris and cruciferous weeds). Chemical control, genome, specific resistance to races 1 and 4 has been

123 Euphytica (2016) 207:35–48 37 never reported. However, race-nonspecific resistance (cauliflower) are contradictory. The polygenic domi- has been identified in this species (Williams 1972; nance of inheritance was reported by Sharma et al. Sharma et al. 1995; Camargo et al. 1995; Taylor et al. (1972; Tewari et al. (1987); Thakur et al. (2003); Tonu 2002; Griesbach et al. 2003). In cabbage, Bain (1952) et al. (2013) whereas, single dominant gene was initially found resistance in the cv. Huguenot and in reported by Jamwal and Sharma (1986). Pandey et al. the Japanese cv. Early Fuji and made selections from (1995) reported that resistance to black rot in cauli- them. Hunter et al. (1987) reported resistance in the flower was governed by non-additive genes. In India, introduction from China PI 436606 (Heh Yeh da Ping available varieties/hybrids of B. oleracea crops in the Tou). This genotype together with Early Fuji has been market are susceptible to black rot disease. The extensively used in cabbage breeding resulting in farmers waste huge amount of money in managing cabbage lines like NY 4002 and Badger Inbred-16 this disease in standing crop. Most of the above utilized in the development of several resistant described resistant sources in B. oleracea are not cabbage cultivars. Jensen et al. (2005) found field commercially cultivated due to other undesirable resistance to black rot in hybrid cultivars T-689 F1, characters or these are not being utilized in developing Gianty F1, No. 9690 F1, N 66 F1 and SWR-02 F1. varieties or hybrids. There is urgent need to identify Griesbach et al. (2003) reported strong resistance in new resistant breeding lines of B. oleracea against Xcc cabbage inbred line AU 4518 and some degree of isolate and to develop resistant cultivars for wider resistance in cv. Green Challenger, Utopia, Shinzan adaptability with good quality attributes. The devel- No. 2, Natsutae, Rotan and Tenacity against mixture oped lines/varieties/hybrids can be grown in other of isolates containing races 1 and 4. Resistant source countries as breeding materials for the improvement of such as Pusa Snowball K-1 (late group cauliflower B. oleracea crops. Besides breeding strategy needs to variety) was reported by Gill (1993) under temperate be formulated using identified resistance sources to conditions. Sharma et al. (1995) reported B. oleracea develop black rot resistant varieties and/or hybrids. In var. botrytis line EC-162587 line as highly resistant this experiment, the new source of resistance and the while RSK-1301 and MRS-1 as moderately resistant genetics of resistance in B. oleracea, this will be of to black rot disease. immense use in the development of resistant varieties The progress has been limited in the development and/or hybrids against this bacterial pathogen. There- of resistant varieties and/or hybrids and elusive due fore, the present study was framed to identify new inefficient phenotyping of resistant plants and unac- black rot resistance source (s) and know the genetics of ceptable co-segregating attributes. Breeders are there- resistance in B. oleracea. fore still seeking resistant varieties with good quality and yield attributes, which requires new sources of resistance, known genetics of resistance and better Materials and methods strategies for gene transfer. Genetic resistance was proposed by Williams (1980) as an efficient approach Experimental site against this disease, along with the utilization of healthy plant material and disease-free seeds. The The experiments were carried out at research farm of contradictory report on genetics of resistance and Division of Vegetable Science, Indian agricultural involvement of resistant genes in different B. oleracea Research Institute, New Delhi, India located at an background has been reported, and these resulted in elevation of about 228 m above MSL, 20°400 north lack of development of durable resistant varieties or latitude and 77°130 east longitude. The climate is of hybrids. Kaur et al. (2009) reported single dominant the sub-tropical type. The average temperatures are gene in January King of cabbage. Vicente et al. (2002) between 20 and 32 °C. The soil is loamy sand, black reported resistance to race 3 in DH line derived from and rich in organic matter. cv. Bohmerwaldkohl is controlled by single dominant gene. Ignatov et al. (1998) attributed race specific Plant material resistance (possibly race 3) to a single dominant locus. Literature on sources of resistance and the genetics of Forty-six B. oleracea accessions (Table 1) including resistance to black rot in B. oleracea var. botrytis 19 B. oleracea var. botrytis (two are commercial 123 38 Euphytica (2016) 207:35–48

Table 1 Descriptions of the accessions of Brassica oleracea S. No. Species Crops Genotypes/ Source Distinguished features breeding lines

1 Brassica oleracea var. botrytis Cauliflower BR-1 IARI, New Delhi Curd are creamish, loose and small sized 2 Brassica oleracea var. botrytis Cauliflower BR-207 IARI, New Delhi Curd are white, compact and small sized 3 Brassica oleracea var. botrytis Cauliflower AL-15 IARI, New Delhi Curd are creamish, compact and small sized 4 Brassica oleracea var. botrytis Cauliflower BR-202-2 IARI, New Delhi Curds are creamish, loose and small sized 5 Brassica oleracea var. botrytis Cauliflower DPCA-2 IARI, New Delhi Curds are white, compact and large sized 6 Brassica oleracea var. botrytis Cauliflower Pusa IARI, New Delhi Curds are white, compact and Himjyoti medium sized 7 Brassica oleracea var. botrytis Cauliflower Pusa Sharad IARI, New Delhi Curds are creamish, compact and large sized 8 Brassica oleracea var. botrytis Cauliflower BR-36 IARI, New Delhi Curds are creamish, loose and small sized 9 Brassica oleracea var. botrytis Cauliflower DB-1 IARI, New Delhi Curds are creamish, loose and medium sized 10 Brassica oleracea var. botrytis Cauliflower DB-2 IARI, New Delhi Curds are creamish, loose and medium sized 11 Brassica oleracea var. botrytis Cauliflower AL-3 IARI, New Delhi Curds are creamish, loose and small sized 12 Brassica oleracea var. botrytis Cauliflower AL-10 IARI, New Delhi Curds are creamish, loose and small sized 13 Brassica oleracea var. botrytis Cauliflower AL-23 IARI, New Delhi Curds are creamish, loose and small sized 14 Brassica oleracea var. botrytis Cauliflower AL-26 IARI, New Delhi Curds are creamish, loose and small sized 15 Brassica oleracea var. botrytis Cauliflower AL-27 IARI, New Delhi Curds are creamish, loose and small sized 16 Brassica oleracea var. botrytis Cauliflower CC-3 IARI, New Delhi Curds are creamish, loose and medium sized 17 Brassica oleracea var. botrytis Cauliflower CCm IARI, New Delhi Curds are white, compact and medium sized 18 Brassica oleracea var. botrytis Cauliflower SR-2 IARI, New Delhi Curds are creamish, loose, and small sized 19 Brassica oleracea var. botrytis Cauliflower HR-6-5-1 IARI, New Delhi Curds are white, loose and medium sized 20 Brassica oleracea var. gemmifera Brussels sprout DJ4763 HRIGRU Sprouts are small and compact 21 Brassica oleracea var. capitata Cabbage DJ8012 HRIGRU Head are compact and medium sized 22 Brassica oleracea var. capitata Cabbage Pusa Agethi IARI, New Delhi Heads are compact and medium sized 23 Brassica oleracea var. capitata Cabbage Golden Acre Denmark Heads are small to medium sized green, round, solid with few outer leaves 24 Brassica oleracea var. italica Cavolo broccolo 005426 HRIGRU, UK Heads are small and green aprilotu calabrase

123 Euphytica (2016) 207:35–48 39

Table 1 continued S. No. Species Crops Genotypes/ Source Distinguished features breeding lines

25 Brassica oleracea var. italica Broccoli precocisima 005311 HRIGRU, UK Heads are small and loose calabrase 26 Brassica oleracea var. italica Heading broccoli 006702 HRIGRU, UK heads are small (charteuse) 27 Brassica oleracea var. italica Broccoli green 010709 HRIGRU, UK Heads are small and green heading broccoli 28 Brassica oleracea var. italica Green heading 010711 HRIGRU, UK Heads are small, loose broccoli and green 29 Brassica oleracea var. italica Calabrase ramoso 010784A HRIGRU, UK Heads are small and green calabrase broccolo 30 Brassica oleracea var. italica Calabrase broccolo 005585A HRIGRU, UK Heads are medium and Innorota/gennorota yellow 31 Brassica oleracea var. italica (Broccolo) Ramoso di 010771A HRIGRU, UK Heads are small and green calabria calabrase 32 Brassica oleracea var. italica Ramoso calabrase 004701 Heads are small (calabrase) 33 Brassica oleracea var. italica Calabrase 003198A HRIGRU, UK Heads are small and green 34 Brassica oleracea var. italica Calabrase verde 005404A HRIGRU, UK Heads are small, loose and calabrase green 35 Brassica oleracea var. italica Calabrase verde 004706A HRIGRU, UK Heads are small calabrase 36 Brassica oleracea var. italica Clabrase (ramoso 004711A HRI, GRI, UK Heads are small and green calabrase verde) 37 Brassica oleracea var. italica Calabrase (bravo) 008663A HRIGRU, UK Heads are small and green 38 Brassica oleracea var. italica (Ramoso calabrase 003207 HRIGRU, UK Heads are small and green Calabrase 39 Brassica oleracea var. italica Calabrase cavolo 005388A HRIGRU, UK No head formation broccoli invernale type B 40 Brassica oleracea var. italica Calabrase cavolo 005403A HRIGRU, UK Heads are small and loose broccolo 41 Brassica oleracea var. italica Clabrase (Ramoso 004704A HRI, GRI, UK Heads are small calabrase verde) 42 Brassica oleracea var. italica Calabrase cavolo 004740A HRIGRU, UK Heads are very small broccolo di sarno and green 43 Brassica oleracea var. italica Calabrase verde 004703A HRIGRU, UK Heads are small, green calabrase and loose 44 Brassica oleracea var. italica Cavolo broccolo 005425 HRIGRU, UK Heads are small and compact natalino calabrase 45 Brassica oleracea var. italica Calabrase paciﬁca 008636 Heads are small and loose 46 Brassica oleracea var. italica Calabrase ramoso 005281A HRIGRU, UK Heads are small calabrase precoce variety and 17 are improved breeding lines and oleracea var. italica landraces collected from HRI- belongs to subtropical heat tolerant type), one B. GRU, UK. All the accessions were screened at the oleracea var. gemmifera collected from HRIGRU, research farm of Division of Vegetable Science during UK, three B. oleracea var. capitata (two commercial 2009–2010 and 2010–2011, separately. The seeds of varieties and one improved breeding line), 23 B. all the lines were sown in raised nursery beds in the

123 40 Euphytica (2016) 207:35–48 mid of September and 1 month old seedlings having Accessions with mean disease scores of 1–3 were 50 plants of each accession were transplanted in the considered resistant, 3.1–6 were partially resistant, field in three replications in randomized block desigh and 6.1–9 were considered susceptible. In the next (RBD). The seedlings were planted with a planting year 2010–2011, the accessions which showed resis- distance of 60 cm between rows and 45 cm within tant or partial resistant were tested again with a row. Recommended cultivation practices was fol- minimum of 50 plants per accession to confirm lowed for raising the crop included the application of previous results. The resistant and susceptible other- 20 tonnes of farmyard manure; 60 kg N: 75 kg P: wise desirable genotypes were selected and integrated 60 kg K per hectare at the time of field preparation into crossing programs to develop generations and to prior to transplanting the seedlings. Another dose of study the genetics of black rot resistance. 30 kg N was applied along with first earthing up about a month after transplanting, which was further Development of generations to study the genetics supplemented by a similar dose at the time of curd of resistance initiation with a second earthing up. The temperature conditions during the experimental period were as the From the tested accessions, the resistant B. oleracea maximum average temperature ranged from 24.4 to var. botrytis accession BR-207, and highly susceptible 30.3 °C (day temperature), while the minimum varied B. oleracea var. botrytis accessions Pusa Sharad and between 5.5 and 12.2 °C (night temperature). Pusa Himjyoti were selected and plants were bud pollinated for selfing. The two commercial varieties Bacterial strains and preparation of inoculum Pusa Himjyoti and Pusa Sharad developed good curd and disease resistance assay but were very susceptible to Xcc race 1. The resistant line BR-207 develop small sized curd but resistant to Xanthomonas campestris pv. campestris (Pammel) black rot disease. Crosses were attempted to develop

Dowson (Xcc) was isolated (isolate number Xcc-C 1 F1 generations (Pusa Himjyoti 9 BR-207; Pusa Shar- and C-25) from infected leaves of Brassica crops in ad 9 BR-207). The F1S were advanced to F2 gener- 2006 and race 1 and 4 type strain typed. Bacterial ation, and BC1P1 (the F1 backcrossed to susceptible cultures were grown in nutrient sucrose broth medium, parent), and BC1P2 (the F1 backcrossed to resistant adjusted to 0.1 optical densities at 600 nm, diluted to parent) in both the cross combinations. The resulting 8 contain 1 9 10 CFU/ml. Forty-eight hours old cul- F2 plants were selfed to obtain F3 progenies and all the ture of Xcc race 1 and 4 was inoculated on 45 days old F2 seeds were harvested individually and that were plants (15 days after transplanting of crops). Ten used to determine the genotype of F2 plants by plants per accession were inoculated by clipping the analyzing the segregation of resistance in F3. secondary veins at the margins with small scissors dipped in the bacterial suspension. Inoculations with Experimental design and challenge inoculation the two strains were made onto four leaves per plant, of parents and segregating generations two with each strain. Ten points of inoculation were made per leaf. The total number of inoculated points Seeds of parental lines together with F1,F2 (generated and number of points showing disease symptoms were by self-pollination of the F1), and backcross genera- recorded and the percentage of infected points were tions consisting of 10 plants of each of parent, 10 calculated. For each inoculated leaf, disease scores plants of F1, 40 plants of F2, and 30 plants of each were taken at 21 days post inoculation. The severity of BC1P1 and BC1P2 of the two crosses were planted in symptoms were assessed on a six point scale of 0–9 RBD with three replications having one row of each of based on the relative lesion size as 0 = no symptoms, parents and F1 generation, 5 rows of F2 generation 1 = small necrosis or chlorosis surrounding the with 10 plants in each row in each replication (Gomez infection point, 3 = typical small V-shaped lesion and Gomez 1984). In the next year the F3 plants were with black veins, 5 = typical lesion half way to the studied using 220 plants of each F3 families. Cultiva- middle vein, 7 = typical lesion progressing to the tion practices and inoculation technique were fol- middle vein, and 9 = lesion reaching the middle vein lowed as described earlier. Infector row of Pusa (Vicente et al. 2002). Himjyoti and Pusa Sharad were planted after each of 123 Euphytica (2016) 207:35–48 41

10 rows to ensure an ample availability of inoculum score was 7.0. In broccoli (italica group), the mean for the spread of the disease. disease score ranged from 4.3 (010784A) to 9.0 (008636, 005425, 004703A, 005403A, 008663A, Statistical analysis 005404A 004701, 005585A and 010711). In this group 10 and 40 % plants were found partial resistant For screening of B. oleracea accessions, comparisons in accessions 005426 and 003207, respectively. of means were performed for mean disease score by Among the various groups, the botrytis group, with a using Fisher’s least significant difference (LSD) at the mean disease score of 7.28, was significantly different 0.05 level of probability (Steel et al. 1997). To study from capitata (8.33) and gemmifera (8.20) and italica the genetics of resistance to black rot, Chi square (v2) (8.11) groups (Table 3). In cauliflower (botrytis was used to test the goodness of fit of the observed group), 5 accessions were found to have both resistant ratio of resistant and susceptible plants in F2, back- and partial resistant plants, whereas, one accession in cross generations and F3 as suggested by Panse and capitata group and two accessions in italica group Sukhatme (1967). Analysis of variance was performed were partial resistant. The selfed progeny of the by using SAS 9.4 software package available at Indian resistant plants of three accessions BR-207 (100 Agricultural Statistics Research Institute, New Delhi, resistant plant), BR-202-2 (10 % resistant and 90 % India. partial resistant) and AL-15 (80 % resistant plant and 20 % partial resistant) were further evaluated by inoculating with Xcc race 1. All the plants of BR-207, Results BR-202 showed resistant reaction, while plants of AL- 27 showed variable mean disease score. Screening of B. oleracea accessions resistance to Xcc race 1 and 4 Resistance to Xcc race 4

The inoculated plants initiated symptoms as translu- Analyses of variance showed significant differences in cent tissue near the cut portion of leaf after 10 days of mean disease score among the accessions under study. inoculation. Characteristic symptoms as yellowing of The mean disease score among the 46 accessions and the leaves were observed on next day. Inoculation with within each corresponding group are presented in Xcc race 1 and 4, the disease is easily recognized by Tables 2 and 3. In cauliflower (botrytis group) all the the presence of ‘V’-shaped chlorotic areas extending accessions had mean disease score of 9, except Al-15 inward from the margin of a leaf, and by black veins in (8.6) and DB-2 (8.0). In cabbage (capitata group), one the infected area. accession (DJ8012) had mean disease score of 7.0 and 10 % plants were found partial resistant. In broccoli Resistance to Xcc race 1 (italica group), the mean disease score ranges from 7 to 9 within the accessions. The accession 008663A had Significant difference (P B 0.05) was observed for mean disease score of 7.0 which was lowest in the disease score among 46 B. oleracea accessions in italica group and 10 % plants were found partial analysis of variance (Table 2). In cauliflower (botrytis resistant in this accession. The mean disease score of group) the minimum mean disease score (1.2) was gemmifera group was highest (9.0) as compared to found for accession BR-207 followed by Al-15 (1.9). other groups, though all the groups are susceptible The maximum (9.0) was found for HR-6-5-1, Cc-3, based on their mean disease score (Table 3). In all the Al-23, AL-10, DB-2, Pusa Sharad, Pusa Himjyoti. In four groups no plants were found to be resistant to race this group there was considerable variability for 4 except accession DJ8012 (capitata group) having disease score among the accessions ranging from 10 % partial resistant plant. score 1-9. One accession of Brussels sprout (gemmifera group) had mean disease score of 8.2. In Resistance to race 1 and 4 cabbage (capitata group), mean disease score ranged from 7 to 9. In this group 20 % plants was found In all the groups only one accession (DJ8012) in partial resistant in DJ8012, though the mean disease cabbage (capitata group) showed plants with partial 123 42 123 Table 2 Percentage of resistant, partial resistant, susceptible and very susceptible plants and mean and range of disease score for different accession in four Brassica oleracea groups following inoculations with races 1 and 4 of Xanthomonas campestris pv. campestris Genotypes/ Race 1 Race 4 breeding lines/ a a accessions number Plants (%) Disease score Plants (%) Disease score Resistant Partial Susceptible Very Rangeb Mean Resistant Partial Susceptible Very Rangeb Mean resistant susceptible resistant susceptible

Cauliﬂower (botytris group) 1 BR-1 0 100 0 0 3–5 3.9 0 0 0 100 99.0 9.09 2 BR-207 100 0 0 0 1–2 1.2 0 0 0 100 99.0 9.09 3 AL-15 80 20 0 0 1–3 1.9 0 0 10 90 7–98.6 8.67–9 4 BR-202-2 10 90 0 0 3–5 4.2 0 0 0 100 99.0 9.09 5 DPCA-2 0 0 10 90 7.9 8.2 0 0 0 100 99.0 9.09 6 Pusa Himjyoti 0 0 0 100 9 9.0 0 0 0 100 99.0 9.09 7 Pusa Sharad 0 0 0 100 9 9.0 0 0 0 100 99.0 9.09 8 BR-36 0 0 40 60 5–9 7.6 0 0 0 100 99.0 9.09 9 DB-1 0 0 0 100 7–9 8.4 0 0 40 60 7–98.0 8.07–9 10 DB-2 0 0 0 100 9 9.0 0 0 0 100 99.0 9.09 11 AL-3 0 0 30 70 7–9 8.4 0 0 0 100 99.0 9.09 12 AL-10 0 0 0 100 9 9.0 0 0 0 100 99.0 9.09 13 AL-23 0 0 0 100 9 9.0 0 0 0 100 99.0 9.09 14 AL-26 0 0 40 60 5–9 7.8 0 0 0 100 99.0 9.09 15 AL-27 0 10 30 60 5–9 7.6 0 0 0 100 99.0 9.09 16 CC-3 0 0 0 100 9 9.0 0 0 0 100 99.0 9.09 17 CCm 0 0 0 100 7–9 8.4 0 0 0 100 99.0 9.09 18 SR-2 0 0 20 80 5–9 7.8 0 0 0 100 99.0 9.09 19 HR-6-5-1 0 0 0 100 9 9.0 0 0 0 100 99.0 9.09 Brussels sprout (gemmifera group) uhtc 21)207:35–48 (2016) Euphytica 20 DJ4763 0 0 0 100 7–9 8.2 0 0 0 100 99.0 9.09 Cabbage (capitata) group 21 DJ8012 0 20 30 50 5–9 7.0 0 10 80 10 5–97.0 7.05–9 22 Pusa Agethi 0 0 0 100 9 9.0 0 0 0 100 99.0 9.09 23 Golden Acre 0 0 0 100 9 9.0 0 0 0 100 99.0 9.09 Table 2 continued 207:35–48 (2016) Euphytica Genotypes/ Race 1 Race 4 breeding lines/ a a accessions number Plants (%) Disease score Plants (%) Disease score Resistant Partial Susceptible Very Rangeb Mean Resistant Partial Susceptible Very Rangeb Mean resistant susceptible resistant susceptible

Broccoli (italica) group 24 005426 0 10 40 50 5–9 6.5 0 0 50 50 7–97.8 7.87–9 25 005311 0 0 0 100 7–9 8.6 0 0 0 100 99.0 9.09 26 006702 0 0 0 100 9 9.0 0 0 0 100 99.0 9.09 27 010709 0 0 20 80 7–9 8.2 0 0 0 100 99.0 9.09 28 010711 0 0 0 100 9 9.0 0 0 0 100 99.0 9.09 29 010784A 0 0 100 0 5–7 4.3 0 0 0 100 99.0 9.09 30 005585A 0 0 0 100 9 9.0 0 0 0 100 99.0 9.09 31 010771A 0 0 10 90 7–9 8.8 0 0 0 100 99.0 9.09 32 004701 0 0 0 100 9 9.0 0 0 0 100 99.0 9.09 33 003198A 0 0 30 70 5–9 7.4 0 0 0 100 99.0 9.09 34 005404A 0 0 0 100 9 9.0 0 0 0 100 99.0 9.09 35 004706A 0 0 50 50 5–9 7.4 0 0 0 100 99.0 9.09 36 004711A 0 0 30 70 7–9 7.8 0 0 0 100 99.0 9.09 37 008663A 0 0 0 100 9 9.0 0 10 80 10 5–77.0 7.05–7 38 003207 0 40 10 50 3–7 5.8 0 0 0 100 99.0 9.09 39 005388A 0 0 50 50 7–9 7.4 0 0 0 100 99.0 9.09 40 005403A 0 0 0 100 9 9.0 0 0 0 100 99.0 9.09 41 004704A 0 0 40 60 7–9 8.0 0 0 0 100 99.0 9.09 42 004740A 0 0 60 40 7–9 7.8 0 0 0 100 99.0 9.09 43 004703A 0 0 0 100 9 9.0 0 0 0 100 99.0 9.09 44 005425 0 0 0 100 9 9.0 0 0 0 100 99.0 9.09 46 008636 0 0 0 100 9 9.0 0 0 0 100 99.0 9.09 46 005281A 0 0 20 80 7–9 8.6 0 0 0 100 99.0 9.09 LSD (0.05) 1.12 0.16 a Disease score based on a rating scale from 1 to 9 where 1 = plants without lesions and 9 = severely diseased plants, where 1–3 = resistant, 4–6 = intermediate or partially

123 resistant and 7–9 = susceptible b Minimum and maximum disease score 43 44 Euphytica (2016) 207:35–48

Table 3 Frequency and distribution of resistant palants to two races of Xanthomonas campestris pv. campestris among a subset of 46 accessions of Brassica oleracea Crops Total no No. of resistant accessions Mean Disease score (1–9) Race 1 Race 4 Race 1 Race 4

Cauliﬂower (Brassica oleracea var. botrytis) 19 5a 0 7.28 8.92 Cabbage (Brassica oleracea var. capitata)31b 0 8.33 8.33 Brussels sprout (Brassica oleracea var. gemmifera) 1 0 0 8.2 9.0 Broccoli (Brassica oleracea var. italica)232b 0 8.11 8.86 LSD (0.05) 0.12 0.14 a Accessions with both resistant and partial resistant plants b Accessions with partial resistant plants; LSD least signiﬁcant difference

resistance to races 1 and 4. The plant number 4 in this with very high confidence (P = 0.67). The F2 gener- accession showed partial resistance to both the races. ation derived from the cross of Pusa Sharad (S) 9 BR- 207 (R) segregated into 88 resistant and 32 susceptible Genetics of resistance to black rot disease plants, with a good fit to the 3:1 ratio expected for a single gene inheritance (v2 = 0.18, P = 0.53). Back- To investigate the genetics of resistance, the reaction cross generations were also evaluated in each cross of plants to Xcc race 1 was analyzed using different combination for confirmation of segregation pattern in generations derived from two cross combinations F2 population. In the Pusa Himjyoti (S) 9 BR-207 [Pusa Himjyoti (S) 9 BR-207 (R) and Pusa Sharad (R) cross, the backcross populations (BC1P1;F1 (S) 9 BR-207 (R)]. All the crosses among susceptible backcrossed with susceptible Pusa Himjyoti) segre- and resistant genotypes were successful and there was gated in 43 resistant and 47 susceptible. The BC1P2 (F1 no sterility in the hybrid generations. The results backcrossed with resistant BR-207) population did not obtained in each of the two crosses are presented in segregate (exhibiting only resistant plants), and con- Tables 4 and 5. On individual cross analysis, the tained 87 resistant plants and three susceptible plants. perusal of data (Table 4) showed that in F2 population Of the 220 F3 families derived from the F2 plants, 57 of Pusa Himjyoti (S) 9 BR-207 (R), plants segregated were resistant and 115 segregated for resistance, while in 85 resistant and 35 susceptible with good fit to 3:1 48 F3 plants were susceptible. The segregation ratio in

Table 4 Disease reactions of all the generations to black rot along with estimates of Chi square values and their probability for classical Mendelian ratio in F2,F3 and backcross generations of the Pusa Himjyoti (S) 9 BR-207 (R) combination Cross combination Generation/families Number of plants (lines) Expected ratio v2 P value at 5 % Total R Seg. S

Pusa Himjyoti 9 BR-207 P1 30 0– 30 30

P2 30 30 – 0

F1 30 30 – 0

F2 120 85 – 35 3:1 0.18 0.67

BC1P1 90 43 – 47 1:1 1.11 0.29

BC1P2 90 87 – 3 1:0

F3 220 57 115 48 1:2:1 1.19 0.55

P1 Pusa Himjyoti susceptible parent, P2 BR-207 resistant parent, BC1P1 the F1 backcrossed to susceptible parent, BC1P2 the F1 backcrossed to resistant parent, R Resistant, Seg segregating, S Susceptible

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Table 5 Disease reactions of all the generations to black rot along with estimates of Chi square values and their probability for classical Mendelian ratio in F2, F3 and backcross generations of the Pusa Sharad (S) 9 BR-207 (R) combination Cross combination Generation/families Number of plants (lines) Expected ratio v2 P value at 5 % Total R Seg S

Pusa Sharad 9 BR-207 P1 30 0 – 30

P2 30 30 – 0

F1 30 30 – 0

F2 120 88 – 32 3:1 0.18 0.53

BC1P1 90 42 – 48 1:1 0.4 0.67

BC1P2 90 87 – 3 1:0

F3 220 62 107 51 1:2:1 1.91 0.38

the F2 generation deduced from the progeny test gave a and Lema et al. (2012). Strong and uniform resistance good ﬁt to the 1 R:2 Seg. : 1 S ratio (v2 = 1.19, for race 1 has been found in the botrytis accession BR- P = 0.55), reinforcing the hypothesis of the existence 207 (100 % plant resistant) and AL-15 (80 % plant of a single gene controlling the resistance (Table 4). resistant). The resistance of these accessions has been

The phenotypic values of the F2,F3 and backcross conﬁrmed in re-evaluating them in the next season. generations from Pusa Sharad (S) 9 BR-207 (R) are Various genotypes of B. oleracea with resistance to presented in Table 5. Total 120 F2 plants were black rot disease were identiﬁed previously (Hunter obtained, of them 88 individual plants were resistant et al. 1987; Sharma et al. 1995; Ignatov et al. 1998; (R), and 32 individual plants were susceptible (S), they Taylor et al. 2002; Griesbach et al. 2003; Jensen et al. exhibited a ratio of 3(R):1(S) when inoculated with 2005) but the use of these resistant lines have limited

Xcc race 1. Backcrosses (BC1P1;F1 backcrossed to the success. Here, we found 100 % resistance in B. susceptible Pusa Sharad) produced progeny that oleracea accession BR-207 to Xcc race 1, though showed a 1(R):1(S) ratio (P = 0.67). The backcross many report by several works established that resis-

(BC2P2;F1 backcrossed to the resistant parent BR- tance to race 1 is only present in Brassica crops 207) were all resistant. Total 220 F3 families were carrying the B genome (B. carinata, B. juncea and B. obtained from F2 of the Pusa Himjyoti (S) 9 BR- nigra) (Taylor et al. 2002; Soengas et al. 2007). The 207(R), of them 62 lines were resistant (R), 107 lines ﬁnding of partial resistant plants in accessions BR- were segregating (Seg.), and 51plants were susceptible 202-2 (90 % plants) and AL-27 (10 %) of botrytis,

(S). The heterogeneity test of this F3 population DJ8012 of capitata (20 %), 005426 (10 %) of italica showed a good ﬁt to the 1(R):2(Seg):1(S) ratio. is very relevant because resistance to race 1 is very unusual as reported by Lema et al. (2012). In our case, a preliminary mean disease score was helpful to select Discussion the most interesting genotypes for breeding purposes. We re-evaluated on the accessions which are resistant Inoculated plants started showing V shaped symptom to Xcc race 1, and none of the accessions was found to as light yellowish spot near the cut portion of leaves at be resistant to Xcc race 4 and hence were not used for 10 days after inoculation (DAI). As the disease further study against race 4. The resistant accessions progressed, the yellowing became more pronounced showed very sporadic and fewer symptoms than the and exhibited black veins symptom. The genotypes susceptible ones. The appearance of disease symptoms employed in this study showed variable response to in the resistant accessions was delayed relative to the the races of Xcc. In our study, race 4 was more virulent susceptible ones. Based on this study, we selected BR- than race 1 in the accessions evaluated and it 207 as resistant pollen parent. Some of the commercial contradict the previous report of Taylor et al. (2002) varieties (Pusa Sharad and Pusa Himjyoti) which are 123 46 Euphytica (2016) 207:35–48 susceptible but otherwise having desirable horticul- when inoculated with Xcc race 1, and phenotypic tural traits were used as female parent. values were obtained, respectively. It clearly demon-

The resistance reaction showed that the F1s of both strated that the results from F2 and F3 populations were the cross combinations (Pusa Himjyoti 9 BR-207, consistent basically, and BR-207 showed the inherited Pusa Sharad 9 BR-207) were resistant. The pheno- mode of the single dominant gene for resistance to Xcc type of F1 progenies confirms the dominance nature of race 1. The segregation ratio in the F2 generation black rot resistance. Dominance nature of inheritance deduced from the progeny test gave a good fit to the was reported by Sharma et al. (1972), Thakur et al. 1(R):2(Seg):1(S) ratio reinforcing the hypothesis of (2003) in temperate late snowball cauliflower (botrytis the existence of a single gene controlling the group). Besides, the stable performance of parents and resistance.

F1 generation for disease resistance during crossing for The present investigation was undertaken with two developing different generations showed consistency cross combination involving one resistant and two of black rot resistance in parents and F1s. Individual susceptible accessions based on their reaction to Xcc plants in all F2 and F3 generations were scored for race 1 and desirable horticultural characters. The black rot resistance to Xcc race 1 and classified as results revealed that the resistance in B. oleracea black rot resistant (R) and black rot susceptible accession BR-207 (botrytis group) is governed by a (S) plants. single dominant gene which can be used to develop For any monogenic trait, the segregation of plants black rot resistant varieties/hybrids. We report here in F2 generation should follow 3:1 ratio (resistant: other resistant accessions (AL-15, BR-1, BR-202-2) in susceptible). The goodness of fit was used to calculate B. oleracea var. botrytis which can be utilized in v2 for 3:1 ratio with 5 % probability value. The ratio of intraspecific hybridization for the improvement of susceptible and resistant plants in F2 generation of cauliflower without using other costly approaches like susceptible 9 resistant cross combination were tested protoplast fusion (Hansen and Earle 1995), embryo for goodness of fit to the expected segregation and all rescue (Tonguc and Griffiths 2004; Dey et al. 2015). the crosses were in agreement with the expected 3:1 ratio with high degree of confidence (P = 0.29–0.67). This confirms the presence of one gene in the resistant parent BR-207. However, reports of Sharma et al. References (1972), Tewari et al. (1987), Thakur et al. (2003), Tonu et al. (2013)inbotrytis group were contrary to Bain D (1952) Reaction of Brassica seedlings to black rot. Phytopathology 42:316–319 the present findings as they reported polygenic control Camargo LEA, Williams PH, Osborn TC (1995) Mapping of of resistance. Ignatov et al. (1998) attributed race quantitative trait loci controlling resistance of Brassica specific resistance (possibly race 3) to a single oleracea to Xanthomonas campestris pv. campestris in the dominant locus. Vicente et al. (2002) reported resis- field and greenhouse. Phytopathology 85:1296–1300 Chen LY, Price TV, Park-Ng Z (2003) Conidial dispersal by tance to race 3 in DH line derived from cv. Bohmer- Alternaria brassicicola on Chinese cabbage (Brassica waldkohl was controlled by single dominant gene. pekinensis) in the field and under simulated conditions. Mau et al. (2011) reported polygenic control of Plant Path 52:536–545 resistance in resistant DH line ‘Reiho P01’ (B. Dey SS, Sharma K, Dey RB, Kumar GMS, Singh D, Kumar R, Parkash C (2015) Inter specific hybridization (Brassica oleracea subsp. capitata). The deviation of the present carinata 9 Brassica oleracea) for introgression of black findings may be due to influence of different genetic rot resistance genes into Indian cauliflower (B oleracea var background of the accession BR-207. In course of botrytis L.). Euphytica. doi:10.1007/s10681-015-1352-0 time, the genetic constitution of cross pollinated crop Dhar S, Singh D (2014) Performance of cauliflower genotypes for yield and resistance against black rot (Xanthomonas may get changed which may also affects its reaction campestris pv. campestris). Indian J Hort 71:197–201 against pathogen. Furthermore, resistant plants of the Fargier E, Manceau C (2007) Pathogenicity assays restrict the two crosses, F3 plant progenies resulted in true species Xanthomonas campestris into three pathovars and resistant segregating and susceptible progenies which reveal nine races within X. campestris pv. campestris. Plant Pathol 56:805–818 had good fit to the expected 1(R):2(Seg):1(S). The Gill HS (1993) Improvement of cole crops. In: Chadha KL, seeds of the individual plants in F2 populations from Kalloo G (eds) Advances in Horticulture-Vegetable Crops, the two crosses were planted and advanced to F3 lines vol 5. Malhotra Publishing House, New Delhi, pp 287–303 123 Euphytica (2016) 207:35–48 47

Gomez KA, Gomez AA (1984) Statistical procedures for agri- geminivirus (TYLCV) by the whitefly Bemisia tabaci cultural research, 3rd edn. Wiley, London, p 13 (Gennadius). Crop Prot 23:473–479 Go´mez-Campo C (1999) Biology of Brassica Coenospecies. Mau D, Mohsin GM, Ishikawa K, Hori H, Okazaki K (2011) Developments in plant genetics and breeding. Elsevier Construction of a Linkage Map and QTL analysis for Black Science B. V, Amsterdam, p 490 Rot Resistance in Brassica oleracea L. Int J Nat Sci 1:1–6 Griesbach E, Loptien H, Miersch U (2003) Resistance to Xan- Pandey SC, Naik G, Kishun R, Sridhar TS (1995) Breeding thomonas campestris pv. campestris (Pammel) Dowson in resistant varieties in cauliflower and cabbage. In: cabbage Brassica oleracea L. J Plant Dis Prot 110:461–475 Mukhopadhyay SK, Das Gupta MK, Gupta PK, Majumdar Griffiths PD, Marek LF, Robertson LD (2009) Identification of DK, Mandal NC (eds) Agro-ecosystems management. crucifer accessions from the NC-7 and NE-9 plant intro- Pali-Siksha Bhawan, Sriniketan, pp 144–149 duction collections that are resistant to black rot (Xan- Panse VG, Sukhatme PV (1967) Statistical methods for agri- thomonas campestris pv. campestris) races 1 and 4. cultural workers. ICAR, New Delhi, pp 152–157 HortScience 44:284–288 Pico B, Diez MJ, Nuez F (1996) Viral diseases causing the Hansen LN, Earle ED (1995) Transfer of resistance to Xan- greatest economic losses to the tomato crop. II. The thomonas campestris pv. campestris into Brassica oler- Tomato yellow leaf curl virus- a review. Sci Hort acea L. by protoplast fusion. Theor Appl Genet 67:151–196 91:1293–1300 Ribeiro B, Dias JS (1997) Screening of a Brassica napus col- Hunter JE, Dickson MH, Ludwig JW (1987) Source of resis- lection with two Portuguese isolates of Xanthomonas tance to black rot of cabbage expressed in seedlings and campestris pv. campestris. In: Thomas GG, Monteiro AA adult plants. Plant Dis 71:263–266 (eds), second ISHS symposium on Brassicas, 10th Crucifer Ignatov A, Kuginuki Y, Hida K (1998) Race-specific reaction of Genetics Workshop, 210, Rennes resistance to black rot in Brassica oleracea. Eur J Plant Sharma BR, Swarup V, Chatterjee SS (1972) Inheritance of Pathol 104:821–827 resistance to black rot in cauliflower. Can J Gen Cytol Ignatov A, Kuginuki Y, Hida K (1999) Vascular stem resistance 14:363–370 to black rot in Brassica oleracea. Canadian J Bot Sharma SR, Kapoor KS, Gill HS (1995) Screening against 77:442–446 sclerotinia rot (Sclerotinia sclerotiorum), downy mildew Ignatov A, Kuginuki Y, Hidam K (2000) Distribution and (Peronospora parasitica) and black rot (Xanthomonas inheritance of race-specific resistance to Xanthomonas campestris) in cauliflower (Brassica oleracea var. botrytis campestris pv. campestris in Brassica rapa and B. napus. subvar cauliflora. Indian J Agri Sci 65:916–918 J Russ Phytopathol Soc 1:89–94 Singh D, Dhar S, Yadava DK (2011) Genetic and pathogenic Jamwal RS, Sharma PP (1986) Inheritance of resistance to black variability of indian strains of Xanthomonas campestris pv. rot (Xanthomonas campestris pv. campestris) in cauli- campestris causing black rot disease in crucifers. Curr flower (Brassica oleracea var. botrytis). Euphytica Microbiol 63:551–560 35:941–943 Soengas P, Hand P, Vicente JG, Pole JM, Pink DAC (2007) Jensen BD, Massomo SMS, Ignas Swai S, Hockenhull J, Identification of quantitative trait loci for resistance to Andersen SB (2005) Field evaluation for resistance to the Xanthomonas campestris pv. campestris in Brassica rapa. black rot pathogen Xanthomonas campestris pv. cam- Theor Appl Genet 114:637–645 pestris in cabbage (Brassica oleracea). Eur J Plant Pathol Steel RGD, Torrie JH, Dickey DA (1997) Principles and pro- 113:297–308 cedures of statistics. A biometrical approach, 1st edn. Kamoun S, Kadmar HV, Tola E, Kado CI (1992) Incompatible McGraw-Hill, New York interaction between crucifers and Xanthomonas campestris Taylor JD, Conway J, Roberts SJ, Astley D, Vicente JG (2002) involve a vascular hypersensitive response: role of the Sources and origin of resistance to Xanthomonas cam- hrpX locus. Mol Plant-microbe Int 5:22–33 pestris pv. campestris in Brassica genomes. Phytopathol- Kashyap PL, Dhiman SJ (2010) Ecofriendly strategies to sup- ogy 92:105–111 press the development of Alternaria blight and black rot of Tewari RN, Chatterjee SS, Swarup V (1987) Inheritance of cauliflower. World Appl Sci J 9:345–350 resistance to black rot (Xanthomonas campestris (Pam) Kaur R, Shivani Saxena B, Kanwar HS, Dohroo NP, Majeed S, Dowson) in cabbage. Veg Sci 6:27–36 Sharma DR (2009) Detecting RAPD markers associated Thakur BS, Korla BN, Khosla K (2003) Inheritance of black rot with black rot resistance in cabbage (Brassica oleracea var. resistance in late cauliflower. Ann Agric Res 24:244–248 capitata). Fruit Veg Cereal Sci Biotech 13:12–15 Tonguc M, Griffiths PD (2004) Evaluation of Brassica carinata Lapidot M, Friedmann M, Lachman O, Yehezkel A, Nahon S, accessions for resistance to black rot (Xanthomonas cam- Cohen S, Pilowsky M (1997) Comparison of resistance pestris pv. campestris). HortScience 39:952–954 level to tomato yellow leaf curl virus among commercial Tonu NN, Doullah MA, Shimizu M, Karim MM, Kawanabe T, cultivars and breeding lines. Plant Dis 81:1425– Fujimoto R, Okazaki K (2013) Comparison of Positions of 1428 QTLs Conferring Resistance to Xanthomonas campestris Lema M, Velasco P, Soengas P, Francisco M, Cartea ME (2012) pv. campestris in Brassica oleracea. American J Plant Sci Screening for resistance to black rot in Brassica oleracea 4:11–20 crops. Plant Breed 131:607–613 Vicente JG, Conway J, Roberts SJ, Taylor JD (2001) Identifi- Mason G, Rancati M, Bosco D (2000) The effect of thi- cation and origin of Xanthomonas campestris pv. cam- amethoxam, a second generation neonicotinoid insecticide, pestris races and related pathovars. Phytopathology in preventing transmission of tomato yellow leaf curl 91:492–499 123 48 Euphytica (2016) 207:35–48

Vicente JG, Conway J, King GJ, Taylor JD (2000) Resistance Williams PH (1980) Black rot: a continuing threat to world to Xanthomonas campestris pv. campestris in Brassica crucifers. Plant Dis 64:736–742 spp. Acta Hort 539:1–67 Williams PH, Staub T, Sutton JC (1972) Inheritance of resis- Vicente JG, Taylor JD, Sharpe AG, Parkin IAP, Lydiate DJ, tance in cabbage to black rot. Phytopathology 62:247–252 King GJ (2002) Inheritance of race-speciﬁc resistance to Xanthomonas campestris pv. campestris in Brassica genomes. Phytoathology 92:1134–1141

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