Crucifer Cultivars Research Articles

Pathogenic and Genetic Characterization of Xanthomonas campestris Pv. campestris Races Based on Rep-PCR and Multilocus Sequence Analysis

Xanthomonas campestris pv. campestris (Pammel) Dowson (Xcc) is the causal agent of black rot disease of crucifers worldwide. Seventy five isolates of Xcc were collected from 12 agro-climatic regions of India to determine the distribution pattern of races and diversity of the population. Based on pathogenic reaction on seven standard differential crucifers, race 1, 4 & 6 were found to be prevalent. For assessing the pathogenic diversity, forty one cultivars of crucifers comprising seven Brassica and coeno species were inoculated artificially under field conditions. Brassica juncea cultivars (Pusa Bold, Varuna, Pusa Vijay, Pusa Mustard 21 and Pusa Mustard 25) showed resistance against all the strains of Xcc, whereas the Brassica olerecea cultivar Pusa Ageti was found to be resistant to races 1 and 4. Genetic characterization of these 75 strains of Xcc was carried out using rep-PCR (ERIC, REP and BOXPCRs) followed by phylogenetic analysis. The strains of Xcc clustered into 6 groups at 50% similarity coefficient and among these groups, 28 strains of Xcc belonging to races 1, 4 & 6 were clustered together under Group 5. Sequences of the 16S rRNA, hrpF and efP genes of five strains representing the races 1, 4 and 6 were used for multilocus sequence analysis. Based on sequence analysis of 16S rRNA and hrpF genes, the Indian strains were found to be very closely related to the strain Xcc ATCC 33913 (race 3, UK), whereas based on efP sequences, they were found to be closely related to strains race 1 Xcc B100 (Italy) and race 9 Xcc 8004 (UK).

Seedling Resistance to Sclerotinia sclerotiorum as Expressed Across Diverse Cruciferous Species.

Sclerotinia stem rot, caused by Sclerotinia sclerotiorum, is a serious disease of many cruciferous crops and frequently poses a threat to the sustainable and profitable production of these crops worldwide. Differences in seedling resistance to S. sclerotiorum across 46 diverse cruciferous genotypes from 12 different species were assessed by comparing the extent of pathogenesis on inoculated cotyledons under controlled conditions. Selections of Brassica carinata, B. incana, B. juncea, B. napus, and B. napus introgressed with B. carinata, B. nigra, B. oleracea, B. rapa var. rosularis, B. rapa var. chinensis, B. tournefortii, Raphanus raphanistrum, R. sativus, and Sinapis arvensis were tested. The average size of lesions on cotyledons 48 h post inoculation varied from 0.8 to 7.3 mm. The three most resistant genotypes with the smallest lesions were all from B. oleracea (viz., B. oleracea var. italica 'Prophet' and B. oleracea var. capitata 'Burton' and 'Beverly Hills'). Representatives of R. raphanistrum, S. arvensis, B. juncea, and B. carinata were the most susceptible to S. sclerotiorum, with the largest lesions. To our knowledge, this is the first report of high levels of resistance to S. sclerotiorum in B. oleracea at the cotyledon stage and also the first report of the host cotyledon reactions against S. sclerotiorum for all tested species except B. napus and B. juncea. The mean lesion size for B. napus introgressed with B. carinata was 5.6 mm, which is midway between the lesion size for the two parent species B. napus (5.1 mm) and B. carinata (5.8 mm). Separate genetic control for cotyledon versus mature plant resistance was demonstrated by the lack of correlation between lesion size from S. sclerotiorum on the cotyledon with the severity of disease initiated by stem inoculation or natural processes in a previous field test. On the most resistant genotypes, B. oleracea var. italica Prophet and var. capitata Burton, growth of S. sclerotiorum on the cotyledon surface prior to penetration was severely impeded, production of appressoria inhibited, and both cytoplasm shrinkage and protoplast extrusion in S. sclerotiorum hyphae prevalent. This is the first report of such resistant mechanisms in B. oleracea. Genotypes with cotyledon resistance identified in this study will be of great value not only in furthering our understanding of resistance mechanisms across different cruciferous species but also could be exploited for developing commercial crucifer cultivars with high-level resistance against S. sclerotiorum.

Virulence analysis and genetic diversity of Xanthomonas campestris pv. campestris causing black rot of crucifers

Xanthomonas campestris pv. campestris (Xcc) is a devastating bacterium to cause black rot disease in crucifers. To study the genetic diversity and virulence analysis, 24 isolates of Xcc were collected from cole crops including cauliflower, cabbage, broccoli and knol khol from different agro-climatic regions of India ranging from temperate to subtropical climates. For virulence analysis, 24 isolates of Xcc were tested on 27 cultivars of crucifers including seven species of Brassica spp. (B. campestris, B. carinata, B. juncea, B. napus, B. nigra, B. oleracea and B. rapa), Sinapsis alba, Eruca sativa and Raphanus sativus under field conditions at IARI, New Delhi, during November 2010–March 2011. Maximum disease incidence 85.15% was found in the cultivars of crucifers caused by strains Xcc-C124, Xcc-C6, Xcc-C125, Xcc-C111 and Xcc-C131 after 15 days of inoculation and significantly increased after 30 days. Black rot severity in cultivars of crucifers varied from 0 to 6.9 and 0 to 7.9 out of 9 scale after 15 and 30 days of inoculation, respectively. But, no disease incidence was recorded on all the tested cultivars of B. juncea (Pusa Bold, Varuna, Pusa Mustard-21 and Pusa Vijay) against all the strains of Xcc after 15 days. Genetic diversity of 24 strains of Xcc was studied using REP- and BOX-PCR, indicating the existence of wide range of genetic diversity among the strains. The strains were clustered into two groups at 50% similarity coefficient and designated as Group 1 and Group 2. The majority of the strains (23 strains) were clustered under Group 1 except Xcc-C120, which formed separate group (Group 2). In the present study, genetic diversity and virulence pattern in Indian strains of Xcc were established, which will be helpful in the development of resistant genotypes against this bacterial pathogen.

Classification of crucifer cultivars based on the life-history of diamondback moth (Plutella xylostella)

We compared different cultivars and hybrids of crucifers in relation to development and life-history of diamondback moth (Plutella xylostella) to classify the plants according to their resistance to the pest. The plants used were “Manteiga da Geórgia” kale, “Bola de Neve” cauliflower, “Ramoso Piracicaba Precoce” broccoli, “Chato-de-quintal” cabbage, and the hybrid cabbages “Midori”, “TPC668”, “TPC308”, and “TPC681”. We evaluated performance daily until the pupal stage. Pupae were assessed individually to determine the pupal weight, performance, and pupal period. We determined the sex ratio, fecundity, fertility, and longevity of the emerged adults and calculated their reproductive potential. Cabbage hybrids “TPC668”, “TPC308”, and “TPC681” do not support the development and reproduction of the diamondback moth. These hybrids show a level of resistance that is similar to that found the commercially available hybrid Midori and cultivar Chato de Quintal, which are known to be resistant to the diamondback moth. This finding implies that the capitata (cabbage) cultivars are the most suitable for planting because they are more resistant to pest than the cultivar's moth, acephala (kale).

Pathogenicity of Plasmodiophora brassicae populations from small, spheroid, resistant-type clubroot galls on roots of clubroot-resistant cultivars of Chinese cabbage (Brassica rapa L. subsp. pekinensis)

A resistant type of small, spheroid clubroot galls (SSGs) containing resting spores formed on the root surface of clubroot-resistant (CR) cultivars of Chinese cabbage (Brassicarapa L. subsp. pekinensis) inoculated with an avirulent population of Plasmodiophora brassicae. Populations isolated from the SSGs severely affected a common (susceptible) cultivar but did not have the typical pathogenicity on CR cultivars, indicating similarity in pathogenicity between SSG and original spore populaions. Populations pathogenic on CR cultivars were not detected from SSGs. Therefore, the ability of the avirulent population among the SSGs to form resting spores may need to be considered to achieve clubroot control in common cultivars of crucifers.

우리나라 배추 뿌리혹병 연구 현홍과 향후과제

Clubroot disease of curcifer crops caused by Plasmodiophora brassicae had been first reported in 1928 in Korea, and maintained mild occurrence until 1980s. Since 1990s the disease has become severe in alpine areas of Kyonggi and Kangwon, gradually spread to plain fields throughout the country, and remains as the great-est limiting factor for its production. Researches on the disease has begun in late 1990s after experiencing severe epidemics. Survey of occurrence and etiological studies have been carried out, particularly, on the pathogen physiology, race identification, quantification of soil pathogen population, and host spectrum of the pathogen. Ecology of gall formation and its decay, yield loss assessment associated with time of infection, and relationships between crop rotation and the disease incidence was also studied during late 1990s. In studies of its control, more than 200 crucifer cultivars were evaluated for their resistance to the disease. Lime applica-tion to field soil was also attempted to reduce the disease incidence. Resistant radish and welsh onion were recommended as rotation crops with crucifers after 3-year field experiments. However, so for, most studies on clubroot disease in Korea have been focused on chemical control. Two fungicides, fluazinam and flusulfamide, were selected and extensively studied on their application technologies and combination effects with lime application or other soil treatment. To develop environmentally-friendly control methods, solar-disinfection of soil, phosphoric acid as a nontoxic compound, and root-parasiting endophytes as biocontrol agents were examined for their effects on the disease in fields. In the future, more researches are needed to be done on development of resistant varieties effective to several races of the pathogen, establishment of economically-sound crop rotation system, and improvement of soil-disinfection technique applicable to Korean field condi-tion, and development of methodology of pretreatment of fungicides onto seeds and seedbeds.

Report of Bacterial Leaf Spot on Collards and Turnip Leaves in Ohio.

In 2000, circular water-soaked lesions typical of bacterial leaf spot were observed on leaves of collards (Brassica oleracea L. var. viridis) throughout commercial fields in northwest Ohio. Light brown, rectangular, water-soaked lesions were observed on turnip leaves (Brassica rapa L.). Bacterial streaming from lesions on both crops was observed microscopically. Cream colored, fluorescent colonies were isolated from diseased tissues on Pseudomonas F medium, and eight representative colonies (four from collards and four from turnip) were selected and purified. Fatty acid methyl ester analysis was performed on all of the isolates. Two from collards and two from turnip were identified as Pseudomonas syringae pv. maculicola (mean similarity index = 0.82 [MIDI Inc., Newark, DE]). DNA extracts from pure cultures of the P. syringae pv. maculicola strains were used as template in a polymerase chain reaction (PCR) assay with primers derived from the region of the coronatine gene cluster controlling synthesis of the coronafacic acid moiety found in P. syringae pv. tomato and P. syringae pv. maculicola (CorR and CorF2) (D. Cuppels, personal communication). DNA from P. syringae pv. tomato strain DC3000 and P. syringae pv. maculicola strain 88-10 (2) served as positive controls, while water and DNA from Xanthomonas campestris pv. vesicatoria strain Xcv 767 were used as negative controls. The expected 0.65-kb PCR product was amplified from three of four strains (two from turnip and one from collards) and the positive control DNA, but not from the negative controls. Pathogenicity tests were performed twice on 6-week-old turnip ('Forage Star', 'Turnip Topper', 'Turnip Alamo', 'Turnip 7'), collard ('Champion') and mustard (Brassica juncea L. 'Southern Giant Curl') seedlings using the three PCR-positive strains. Premisted seedlings were spray-inoculated separately with each of the three strains (2 × 108 CFU/ml, 5 ml per plant) and a water control. Greenhouse temperatures were maintained at 20 ± 1°C. For both tests, all strains caused characteristic lesions on all of the crucifer cultivars within 5 days after inoculation; the control plants did not develop symptoms. To satisfy Koch's postulates, one of the turnip strains was reisolated from 'Turnip Topper' plants, and the collard strain was reisolated from 'Champion' plants. The three original and two reisolated strains induced a hypersensitive response in Mirabilis jalapa L. and Nicotiana tabacum L. var. xanthia plants 24 h after inoculation with a bacterial suspension (1 × 108 CFU/ml). The original and reisolated strains were compared using rep-PCR with the primer BOXA1R (1). The DNA fingerprints of the reisolated strains were identical to those of the original strains. To our knowledge, this is the first report of bacterial leaf spot on commercially grown collards and turnip greens in Ohio.

First Report of Plasmodiophora brassicae on Cabbage in Eastern North Carolina.

Clubroot, caused by Plasmodiophora brassicae Woronin, has occurred for at least 50 years in three counties in northwestern North Carolina, but has not been reported previously from eastern North Carolina, where most commercial cabbage is produced. In the fall of 1995, clubroot was observed in a direct seeded, commercial cabbage field in Plymouth, NC. Diseased cabbage plants were stunted and roots exhibited clublike swellings. Clubs were randomly harvested from roots of five plants to obtain a composite isolate to determine which race(s) of P. brassicae are infecting cabbage in eastern North Carolina. Three experiments were conducted, using the procedure of Williams (2). Four replicates of 10, 1-week-old seedlings of eight different crucifer cultivars were inoculated by dipping in a spore suspension (1 × 108 cysts/ml) of P. brassicae and planted in pasteurized potting mix. Seedlings dipped in sterile water served as controls. Inoculated seedlings were incubated in a greenhouse at 18 to 28°C for 6 to 8 weeks and assessed for clubroot incidence and severity. The isolate of P. brassicae from eastern North Carolina was most virulent on cabbage (Brassica oleracea var. capitata cv. Jersey Queen), collard (B. oleracea var. acephala cv. Vates), and wild mustard (B. nigra); moderately virulent on canola (B. napus cv. Brutor) and rutabaga (B. napus cvs. Laurentian and Wilhelmsburger); and least virulent on cabbage (cv. Badger Shipper). Canola (B. napus cv. Nevin) and control seedlings were not infected and exhibited no symptoms. Similar results were obtained for all experiments. Based on these results, the isolate of P. brassicae from eastern North Carolina was designated as race 6 and pathotype 5 according to Williams (2) and Some (1), respectively. However, further experiments with single-cyst-derived isolates from individual clubs obtained from different geographic locations are needed to accurately characterize field populations of P. brassicae on cabbage in eastern North Carolina.

Tryptic inhibitory activity in wild and cultivated crucifers

Tryptic inhibitory activity was detected in all species and cultivars of crucifers examined. When comparing the three groups of crucifers tested [(i) foliage of cultivated crucifers, (ii) foliage of wild crucifers and (iii) storage organs of cultivated crucifers], the foliage of cultivated crucifers contained significantly higher levels of tryptic inhibitory activity than the other two groups.

Pathogenicity of Geographic Isolates of Fusarium oxysporum from Crucifers on a Differential Set of Crucifer Seedlings

AbstractPathogenicity tests with 103 isolates of Fusarium oxysporum from crucifers worldwide were conducted using a standard set of six differential crucifer cultivars. Based on their reaction to the standard differentials, all isolates could be grouped into the five major pathotypes in the three formae speciales, f. sp. conglutinans. f. sp. raphani and f. sp. matthioli. When two additional crucifers, Brassica nigra and B. campestris were added to the differe, ntial set, and the reactions of the host differentials were analyzed, an increased range of virulence profiles was observed, implying the existence of more virulence factors in the F. oxysporum population. An isolate from the U.S.S.R., was identified with virulence capable of overcoming dominant monogenic “type A” resistance in Brassica oleracea suggestive of a potentially new location for this pathotype.

An evaluation of Fusarium oxysporum from crucifers based on pathogenicity, isozyme polymorphism, vegetative compatibility, and geographic origin

A global collection of 123 putative isolates of Fusarium oxysporum from crucifers was examined for pathogenicity, isozyme polymorphism, and vegetative compatibility. Of these isolates, 103 were found to be pathogenic on one or more of six differential crucifer cultivars. Three patterns of isozyme polymorphism (electrophoretic types) were found and by means of a nitrate reductase complementation test, three major vegetative compatibility groups were identified that could differentiate among the F. oxysporum pathotypes. Complete correspondence was found among pathotype, electrophoretic type, and vegetative compatibility. It seems appropriate to classify isolates from the Cruciferae into the subspecific taxa, F. oxysporum f.sp. conglutinans, F. oxysporum f.sp. raphani, and F. oxysporum f.sp. matthioli, based on their naturally infected host species, Brassica oleracea, Raphanus sativus, and Matthiola incana, and on estimates of genetic identity. Within formae speciales, races can be identified based on intraspecific host specialization. Geographic origin was not found to be associated with the vegetative compatibility, isozyme phenotype, or pathotype. Isozyme polymorphisms also differentiated among four F. oxysporum formae speciales from other host families and among various Fusarium species.

Resistance of Brassica Lines to the Diamondback Moth (Lepidoptera: Yponomeutidae) in the Field, and Inheritance of Resistance

Selected lines and cultivars of crucifers were evaluated in the field for resistance to the diamondback moth, Plutella xylostella (L.). Larvae failed to develop on dark green, glossy foliage of PI cauliflower 234599 grown in the field, and cabbage inbred lines G 8329, G 9660, and G 9639, although the glossy type was preferred for oviposition. Alteration of waxy material on leaf surfaces of two normal, light green commercial cultivars changed the leaf color to dark green and significantly (P < 0.05) increased oviposition greenhouse experiments. Inheritance studies in the field using one population each of cauliflower and cabbage indicated that the type of resistance exhibited by the darkgreen, glossy foliage used in these experiments was heritable with broad sense heritabilities of 0.75 and 0.88 for cauliflower and cabbage, respectively.