Mark W. Farnham

Genetic combining ability of glucoraphanin level and other horticultural traits of broccoli

Broccoli (Brassica oleracea L., Italica Group) is a source of glucosinolates and their respective isothiocyanate metabolites that are believed to have chemoprotective properties in humans. Glucoraphanin (4-methylsulfinyl-butyl glucosinolate) is a predominant glucosinolate of broccoli. Its cognate isothiocyanate, sulforaphane, has proven a potent inducer of phase II detoxification enzymes that protect cells against carcinogens and toxic electrophiles. Little is known about the genetic combining ability for glucosinolate levels or the types of genetic variation (i.e., additive vs. dominance) that influence those levels in broccoli. In this study, a diallel mating design was employed in two field experiments to estimate combining abilities for glucoraphanin content. The diallel population was developed by crossing nine doubled-haploid (inbred) parents in all possible combinations (36), excluding the reciprocals. Horticultural traits of all entries were assessed on a plot basis. In fall 2001, glucoraphanin concentration of broccoli heads ranged from 0.83 to 6.00 μmol/gdw, and in spring 2002, ranged from 0.26 to 7.82 μmol/gdw. In both years, significant general combining ability was observed for glucoraphanin concentration and total head content, days from transplant to harvest, head weight, and stem diameter. Conversely, no significant specific combining ability was observed for any trait in either year. Results indicate that a given inbred will combine with others to make hybrids with relatively predictable levels of head glucoraphanin as well as, other important horticultural traits. This should allow identification of inbreds that typically contribute high glucoraphanin levels when hybridized with others.

Impact of thermal processing on sulforaphane yield from Broccoli (Brassica oleracea L. ssp. italica)

In broccoli, sulforaphane forms when the glucosinolate glucoraphanin is hydrolyzed by the endogenous plant thiohydrolase myrosinase. A myrosinase cofactor directs hydrolysis away from the formation of bioactive sulforaphane and toward an inactive product, sulforaphane nitrile. The cofactor is more heat sensitive than myrosinase, presenting an opportunity to preferentially direct hydrolysis toward sulforaphane formation through regulation of thermal processing. Four broccoli cultivars were microwave heated, boiled, or steamed for various lengths of time. Production of nitrile during hydrolysis of unheated broccoli varied among cultivars from 91 to 52% of hydrolysis products (Pinnacle > Marathon > Patriot > Brigadier). Boiling and microwave heating caused an initial loss of nitrile, with a concomitant increase in sulforaphane, followed by loss of sulforaphane, all within 1 min. In contrast, steaming enhanced sulforaphane yield between 1.0 and 3.0 min in all but Brigadier. These data are proof of concept that steaming for 1.0-3.0 min provides less nitrile and more sulforaphane yield from a broccoli meal.

Internal Transcribed Spacer Regions 1 and 2 and Random Amplified Polymorphic DNA Analysis of Didymella bryoniae and Related Phoma Species Isolated from Cucurbits.

ABSTRACT Didymella bryoniae (anamorph Phoma cucurbitacearum) is the causal agent of gummy stem blight, although other Phoma species are often isolated from cucurbit plants exhibiting symptoms of the disease. The molecular and phylogenetic relationships between D. bryoniae and these Phoma species are unknown. Isolates of D. bryoniae and Phoma obtained from cucurbits grown at various geographical locations in the United States were subjected to random amplified polymorphic DNA (RAPD) analysis and internal transcribed spacer (ITS) sequence analysis (ITS-1 and ITS-2) to determine the molecular and phylogenetic relationships within and between these fungi. Using RAPD fingerprinting, 59 isolates were placed into four phylogenetic groups, designated RAPD group (RG) I, RG II, RG III, and RG IV. D. bryoniae isolates clustered in either RG I (33 isolates), RG II (12 isolates), or RG IV (one isolate), whereas all 13 Phoma isolates clustered to RG III. There was greater than 99% sequence identity in the ITS-1 and ITS-2 regions between isolates in RG I and RG II, whereas isolates in RG III, P. medicaginis ATCC 64481, and P. exigua ATCC 14728 clustered separately. On muskmelon seedlings, a subset of RG I isolates were highly virulent (mean disease severity was 71%), RG II and RG IV isolates were slightly virulent (mean disease severity was 4%), and RG III isolates were nonpathogenic (disease severity was 0% for all isolates). The ITS sequences indicate that RG I and RG II are both D. bryoniae, but RAPD fingerprints and pathogenicity indicate that they represent two different molecular and virulence subgroups.

AFLP analysis of a worldwide collection of Didymella bryoniae

Didymella bryoniae (anamorph Phoma cucurbitacearum) is an ascomycete that causes gummy stem blight, a foliar disease that occurs on cucurbits in greenhouses and fields throughout the world. In a previous study using RAPD analysis, little genetic diversity was found among isolates of D. bryoniae from New York and South Carolina, USA. Here we report the use of amplified fragment length polymorphism (AFLP) analysis to assess the genetic variation within a worldwide collection of D. bryoniae, 102 field and greenhouse isolates from ten states in the USA (California, Delaware, Florida, Georgia, Indiana, Maryland, Michigan, Oklahoma, South Carolina, and Texas) and seven other countries (Australia, Canada, China, Greece, Israel, Sweden, and The Netherlands) were examined. Seven different AFLP primer-pair combinations generated 450 bands, of which 134 were polymorphic (30%). Using cluster analysis, two groups and a total of seven subgroups were delineated. Representative isolates varied in their virulence on muskmelon and watermelon seedlings, but the degree of virulence was not strongly associated with AFLP groupings. However, isolates from the northern USA grouped separately from isolates originating from the southern USA.

A single dominant gene for downy mildew resistance in broccoli

Downy mildew, incited by Peronospora parasitica (Pers.: Fr.) Fr., is a destructive disease of broccoli (Brassica oleraceaL., Italica Group). Resistant cultivars represent a desirable control method to provide a practical, environmentally benign, and long-term means of limiting damage from this disease. Doubled-haploid (DH) lines developed by us exhibit a high level of downy mildew resistance at the cotyledon stage. To determine the mode of inheritance for this resistance, a resistant DH line was crossed to a susceptible DH line to make an F1, from which F2 and backcross (BC) populations were developed. All populations were evaluated for response to artificial inoculation with P. parasitica at the cotyledon stage. All F1 plants (including reciprocals) were as resistant as the resistant parent, indicating no maternal effect for this trait. F2 populations segregated approximately 3resistant to 1 susceptible, BC populations using the resistant parent as the recurrent parent contained all resistant plants, and the BC to the susceptible parent segregated 1 resistant to 1 susceptible. These results indicate that resistance is controlled by a single dominant gene. This gene should be easily incorporated into F1 hybrids and used commercially to prevent downy mildew at the cotyledon stage.

Differential Cultivars and Criteria for Evaluating Resistance to Rhizoctonia solani in Seedling Brassica oleracea

Growth-room and field experiments were conducted to develop methods of studying resistance in Brassica oleracea crops to Rhizoctonia solani anastomosis groups (AG) 2-1 and 4, causal agents of wirestem. Seedlings of 12 cultivars (3 each of broccoli, cauliflower, cabbage, and collard) at the four- to five-leaf stage were transplanted to trays in a growth room and covered with steamed soil infested with cornmeal-sand cultures or sclerotia of R. solani or to fumigated field plots infested with sclerotia. The percent healthy, diseased, and dead plants was assessed every 3 to 5 days for 2 weeks in the growth room and for 3 weeks in field trials. At harvest, plants were dug out with roots intact and rated for wirestem severity. In most experiments, wirestem incidence (percent diseased and dead plants) stabilized within 10 to 14 days after inoculation. Inoculation with cornmeal-sand cultures of both AGs and sclerotia of AG-4 resulted in severe wirestem in all experiments, whereas sclerotia of AG-2-1 were less effective in the growth room and not effective in the field. Percent healthy and surviving (healthy plus diseased) plants, area under the disease progress curve (AUDPC), and wirestem severity all separated the most susceptible from the partially resistant cultivars more consistently than fresh weight of inoculated plants expressed as a percentage of noninoculated plant weight. Wirestem severity and AUDPC were always negatively and significantly (P ≤ 0.01) correlated with percent healthy plants. Although genotype by environment interactions were observed, the cauliflower cvs. Snowcone and Snow Crown were severely diseased in all experiments, whereas collard cv. Blue Max was consistently and significantly (P ≤ 0.05) less diseased.

Characterization of Fusarium Yellows Resistance in Collard

The yellows disease of cole crops, caused by Fusarium oxysporum f. sp. conglutinans, can be very damaging to collard. Growers in the southeastern United States frequently produce collard in hot, summer months when conditions for yellows development are favorable, and thus, incidence of this disease is increasing. A collection of essentially all U.S. commercial cultivars of collard, various landraces of collard, and other representative cole crops was evaluated for response to artificial inoculation with F. oxysporum f. sp. conglutinans under controlled-temperature conditions. In addition, the same collection was evaluated following transplanting for response to naturally infested soil in the field during summer 1997 and 1998. In all trials, genotype had the most significant effect on percentage of diseased plants, and genotype responses ranged from resistant (0 to 20% diseased) to susceptible (61 to 100% diseased). There was a significant temperature effect on percentage of diseased plants in one growth chamber experiment with five genotypes that resulted primarily from an increase in disease incidence for the cultivar Blue Max at 30°C compared with 25°C. Temperature was not significant in a second experiment with 20 genotypes. In the field, although significant differences were observed among genotypes and between years, a significant genotype × year interaction was not detected for percentage of diseased plants, indicating a similar ranking of genotypes for resistance between years. There was a significant correlation between results from controlled-environment studies and the field. A resistant response to F. oxysporum f. sp. conglutinans was expressed in certain cultivars of collard, including Flash, Heavicrop, and Morris Heading, and also in specific landraces. This resistance was stable in relatively high temperature environments used in evaluations. Results of this research indicate that choice of cultivar is a critical factor in producing collard where conditions favor infection by F. oxysporum f. sp. conglutinans. This information will aid in development of new yellows-resistant cultivars.

Effects of Planting Pattern of Collards on Resistance to Whiteflies (Homoptera: Aleyrodidae) and on Parasitoid Abundance

Abstract Fourteen collard entries, Brassica oleraceae L., Acephala group, were evaluated for resistance to natural populations of Bemisia argentifolii Bellows & Perring in replicated field plots in Charleston, SC. Glossy-leaf phenotypes (‘SC Glaze’, ‘SC Landrace’, ‘Green Glaze’) were the most resistant collard entries and had fewer whiteflies than the nonglossy, open-pollinated cultivars. Also, two F1 hybrid cultivars with normal, nonglossy leaves (‘Blue Max’ and ‘Top Bunch’) were resistant. In laboratory experiments, there were no differences in the intrinsic rate of growth (rs) of B. argentifolii populations on either glossy or nonglossy collard phenotypes. Over a 2-yr period, there were no differences in the abundance of whiteflies on the glossy phenotype of Green Glaze when it was planted in solid 20-plant plots or when it was alternated (every other plant) with the nonglossy phenotype of Green Glaze. In a similarly designed experiment, there was no difference in the resistance of Blue Max in either solid or mixed planting scheme compared with the susceptible ‘Morris Heading’. Higher numbers of whiteflies and parasitoids (primarily Eretmocerus spp.) were collected on yellow sticky cards in the solid plantings of the nonglossy phenotype of Green Glaze than were collected in the solid plantings of the glossy Green Glaze phenotype. Counts on sticky cards in the mixed plots were intermediate. These data show that planting pattern of collard entries is relatively unimportant in the deployment of these sources of host plant resistance. The data also suggest that nonpreference is the primary mode of resistance to whiteflies for certain collard entries.

First report of bacterial leaf blight on broccoli and cabbage caused by Pseudomonas syringae pv. alisalensis in South Carolina.

In May of 2009, leaf spot and leaf blight symptoms were observed on broccoli (Brassica oleracea var. italica) and cabbage (B. oleracea var. capitata) on several farms in Lexington County, the major brassica-growing region of South Carolina. Affected areas ranged from scattered disease foci within fields to entire fields. Initial infection symptoms on leaves of both crops included circular and irregular-shaped necrotic lesions that were 3 to 10 mm in diameter, often with yellow halos and water soaking. As the disease progressed, the lesions tended to coalesce into a general blight of the entire leaf. Diseased leaves from both broccoli and cabbage were collected from each of four fields at different locations in the county. Leaves were surface disinfested, macerated in sterile distilled water, then aliquots of the suspension were spread on Kings medium B (KB) agar. All samples produced large numbers of bacterial colonies that fluoresced blue under UV light after 24 h of growth. In total, 23 isolates (13 from broccoli and 10 from cabbage) were collected. These isolates were gram negative, levan production positive, oxidase negative, pectolytic activity negative, arginine dihydrolase negative, and produced a hypersensitive response on tobacco, thus placing them in the Pseudomonas syringae LOPAT group (2). Two broccoli and two cabbage isolates were selected at random and tested for pathogenicity to cabbage cv. Early Jersey Wakefield, broccoli cv. Decicco, turnip cv. Topper, broccoli raab cv. Spring, collard cv. Hi-Crop, and oat cv. Montezuma in greenhouse tests. Bacteria were grown on KB agar for 24 h and a bacterial suspension was prepared and adjusted to an optical density of 0.1 at 600 nm. Three-week-old plants were spray inoculated to runoff and held at 100% relative humidity for 12 h after inoculation, prior to return to the greenhouse bench (4). P. syringae pv. maculicola strain F18 (4) and the pathotype strain of P. syringae pv. alisalensis BS91 were included as controls, along with a water-inoculated negative control. Plants were evaluated at 14 days postinoculation. The four unknown bacterial isolates and BS91 were pathogenic on all brassica plants tested, as well as on oat. In contrast, the P. syringae pv. maculicola strain F18 was not pathogenic on broccoli raab or oat. Symptoms produced by all isolates and strains tested were similar to those observed in the field. No symptoms were observed on water-inoculated plants. Comparative repetitive sequence-based (rep)-PCR DNA analysis using the BOXA1R primer (3) resulted in a DNA banding pattern of each of the isolates from the South Carolina fields (23 isolates), as well as those reisolated from inoculated plants, that was identical to P. syringae pv. alisalensis BS91 and differed from the P. syringae pv. maculicola F18 strain. On the basis of the rep-PCR assays and the differential host range (1), the current disease outbreak on broccoli and cabbage in South Carolina is caused by the bacterium P. syringae pv. alisalensis. Broccoli is a relatively new, albeit rapidly expanding, production vegetable in South Carolina; this disease may represent a limiting factor to future production. References: (1) N. A. Cintas et al. Plant Dis. 86:992, 2002. (2) R. A. Lelliott et al. J. Appl. Bacteriol. 29:470, 1966. (3) J. Versalovic et al. Methods Mol. Cell. Biol. 5:25, 1994. (4) Y. F. Zhao et al. Plant Dis. 84:1015, 2000.

The Genetics of Brassica oleracea

Brassica oleracea is one of the most important species of the Brassicaceae family because the species includes some of the most economically important vegetables in the world. Common heading cabbage and cauliflower are the most widely grown crops of this species, but broccoli is also now emerging rapidly as a world vegetable. The wide center of origin for this species is the Mediterranean Basin, and primitive forebears of our modern B. oleracea crop forms have been cultivated and selected for several millennia. Undoubtedly, the diverse array of wild forms found in this species and other very closely related species played very important roles in stimulating the occurrence of morphological variation within and among the B. oleracea crops as they underwent development. In the years following the rediscovery of Mendel’s work, many scientists studied the underlying genetic factors controlling the divergent morphologies within the species. This was of interest not only from a basic scientific standpoint, but also due to the practical necessity of understanding the complex sets of genes that combine and give rise to a specific crop form like heading cabbage or cauliflower. This knowledge is crucial in moving genes between crops in the process of breeding improved varieties. Secondary plant metabolites have emerged as key components of crops within this species because they appear to contribute added-value to the various crops by conferring intrinsic healthful effects on populations that consume these vegetables. Among the various components believed to confer a chemoprotective effect in B. oleracea, glucosinolates, and isothiocyanates have received the most attention in recent years and are considered in detail herein. The study of B. oleracea genetics has been greatly advanced during the modern era of gene study at the molecular level. Although the species has presented challenges, scientists focused on these crops are now mapping genes to specific chromosomes and the genome is well on its way to being sequenced. As knowledge advances at the molecular level, a fuller understanding of gene sequences and there relations to morphology, disease resistance, phytochemical make-up, and other important traits are being realized in B. oleracea.