Ginette Séguin-Swartz

Hybridization between transgenic Brassica napus L. and its wild relatives: Brassica rapa L., Raphanus raphanistrum L., Sinapis arvensis L., and Erucastrum gallicum (Willd.) O.E. Schulz

Abstract. The frequency of gene flow from Brassica napus L. (canola) to four wild relatives, Brassica rapa L., Raphanus raphanistrum L., Sinapis arvensis L. and Erucastrum gallicum (Willd.) O.E. Schulz, was assessed in greenhouse and/or field experiments, and actual rates measured in commercial fields in Canada. Various marker systems were used to detect hybrid individuals: herbicide resistance traits (HR), green fluorescent protein marker (GFP), species-specific amplified fragment length polymorphisms (AFLPs) and ploidy level. Hybridization between B. rapa and B. napus occurred in two field experiments (frequency approximately 7%) and in wild populations in commercial fields (approximately 13.6%). The higher frequency in commercial fields was most likely due to greater distance between B. rapa plants. All F1 hybrids were morphologically similar to B. rapa, had B. napus- and B. rapa-specific AFLP markers and were triploid (AAC, 2n = 29 chromosomes). They had reduced pollen viability (about 55%) and segregated for both self-incompatible and self-compatible individuals (the latter being a B. napus trait). In contrast, gene flow between R. raphanistrum and B. napus was very rare. A single R. raphanistrum × B. napus F1 hybrid was detected in 32,821 seedlings from the HR B. napus field experiment. The hybrid was morphologically similar to R. raphanistrum except for the presence of valves, a B. napus trait, in the distorted seed pods. It had a genomic structure consistent with the fusion of an unreduced gamete of R. raphanistrum and a reduced gamete of B. napus (RrRrAC, 2n = 37), both B. napus- and R. raphanistrum-specific AFLP markers, and had <1% pollen viability. No hybrids were detected in the greenhouse experiments (1,534 seedlings), the GFP field experiment (4,059 seedlings) or in commercial fields in Québec and Alberta (22,114 seedlings). No S. arvensis or E. gallicum × B. napus hybrids were detected (42,828 and 21,841 seedlings, respectively) from commercial fields in Saskatchewan. These findings suggest that the probability of gene flow from transgenic B. napus to R. raphanistrum, S. arvensis or E. gallicum is very low (<2–5 × 10–5). However, transgenes can disperse in the environment via wild B. rapa in eastern Canada and possibly via commercial B. rapa volunteers in western Canada.

GENE FLOW IN COMMERCIAL FIELDS OF HERBICIDE‐RESISTANT CANOLA (BRASSICA NAPUS)

Multiple herbicide resistance to glyphosate, glufosinate, bromoxynil, or imidazolinone in volunteer plants of canola (Brassica napus) has been attributed to pollen flow among cultivars with different resistance traits. A study was conducted in Saskatchewan, Canada, in 1999 and 2000 to assess gene flow in space and time in adjacent commercial fields of glyphosate- and glufosinate-resistant canola, including (1) estimation of gene flow with distance; (2) frequency and distribution of volunteers, and effect on gene flow; (3) effect of adventitious double herbicide-resistant seed presence in seedlots planted; and (4) a comparison of various marker systems to track gene flow events. At 11 sites in 1999, gene flow was determined by sampling seeds from plants located at 0, 50, 100, 200, 400, 600, or 800 m along a transect perpendicular to the common border in the paired fields, spraying seedlings with glyphosate and glufosinate, and confirming the presence of the transgenes using commercial test strips and PCR analysis. In the spring of 2000, putative double herbicide-resistant volunteers that survived sequential herbicide applications were mapped at three of the sites using GPS and resistance in sampled plants was characterized. In 1999, gene flow between the paired fields was detected to a maximum distance of 400 m. Values ranged from 1.4% outcrossing at the border common to the paired fields to 0.04% at 400 m. In 2000, gene flow as a result of pollen flow in 1999 was detected to the limits of the study areas (800 m). Large variation in gene flow levels and patterns among the three sites was evident. Adventitious presence of double herbicide-resistant seed in glyphosate-resistant seedlots planted at two of the sites in 1999 contributed to the occurrence of double herbicide-resistant volunteers in 2000. The results of this study suggest that gene stacking in B. napus canola volunteers in western Canada may be common, and reflects pollen flow between different herbicide-resistant canola, presence of double herbicide-resistant off-types in seedlots, and/or agronomic practices typically employed by Canadian growers.

Comparison of nuclear ribosomal DNA sequences from Alternaria species pathogenic to crucifers

The sequences coding for the nuclear 18 s rRNA, 5·8 s rRNA, and the internal transcribed spacers (ITS1 and ITS2) were amplified by the polymerase chain reaction and sequenced for one isolate each of Alternaria brassicae, A. brassicicola, A. raphani, A. alternata and Pleospora herbarum . The 5·8 s rDNA sequences from the four Alternaria species were identical and differed at only one base pair from that of P. herbarum . The internal transcribed spacer sequences, especially ITS1, were very variable in both base composition and length. The 18 s rDNA sequences were highly conserved, but enough variability was present to distinguish genera clearly. Phylogenetic analysis of the sequence data sets by both parsimony and maximum likelihood methods clearly separated genera and species. All of the Alternaria species were closely related. Pleospora also appeared to be more closely related to Alternaria than to Leptosphaeria .

Multiple herbicide-resistant canola can be controlled by alternative herbicides

Abstract Unintentional herbicide resistance gene stacking in canola may alter the sensitivity of volunteers to herbicides of alternative modes of action commonly used for their control. Greenhouse experiments were conducted to investigate the response of three single-herbicide–resistant (HR) cultivars (glyphosate, glufosinate, imidazolinone), one non-HR cultivar, and seven multiple (double or triple)–HR experimental lines to 2,4-D (amine and ester), MCPA ester, and metribuzin applied at the two- to three-leaf stage and of one non-HR and four HR cultivars (glyphosate, glufosinate, imidazolinone, bromoxynil) to 2,4-D amine applied at two growth stages (two- to three-leaf stage and five- to six-leaf stage). All canola cultivars or lines treated at the two- to three-leaf stage responded similarly to increasing doses of each of the three herbicides. At the five- to six-leaf stage, however, the bromoxynil HR cultivar was less sensitive to 2,4-D than the other cultivars. The results of this study suggest that canola with multiple-herbicide–resistance traits does not differ from cultivars that are non-HR or single HR in its sensitivity to herbicides commonly used to control volunteers. All volunteers, whether non-HR, single HR, or multiple HR, should be treated when plants are most sensitive to herbicides (two- to four-leaf stage) to reduce their interference against crops and their perpetuation of gene flow. Nomenclature: bromoxynil; 2,4-D; glufosinate; glyphosate; MCPA; metribuzin; canola, Brassica napus L.

Sexual hybridisation in crosses of cultivated Brassica species with the crucifers Erucastrum gallicum and Raphanus raphanistrum : Potential for gene introgression

Studies were conducted to investigate the crossability of the cultivated Brassica species, Brassica napus (oilseed rape), B. rapa (turnip rape), and B. juncea (brown and oriental mustard), with two related cruciferous weeds that are abundant in certain regions of Canada, Erucastrum gallicum (dog mustard) and Raphanus raphanistrum ssp. raphanistrum (wild radish). Seed was produced without recourse to embryo rescue from all reciprocal crosses except R. raphanistrum × B. juncea. Four hybrid plants were recovered, namely B. napus × E. gallicum, B. napus × R. raphanistrum (two plants), and B. rapa × E. gallicum. The hybrids were characterized by their morphology, RAPD analysis, and cytological examination. The B. rapa × E. gallicum hybrid was extremely vigourous and fertile, and would likely grow in natural habitats. This hybrid produced self-seed and backcrossed readily with the B. rapa parent and, to a lesser extent, with the E. gallicum parent. The B. napus × E. gallicum hybrid was a weak plant, but produced fertile backcross progeny with the E. gallicum parent. The B. napus × R. raphanistrum hybrids were vigourous but mostly sterile. Because of their low vigour and/or sterility, hybrids produced from crosses of B. napus with the cruciferous weeds would not likely be an environmental concern. However, the potential of the B. napus × E. gallicum and B. rapa × E. gallicum hybrids to backcross with E. gallicum may be of concern.

Monolignol biosynthesis is associated with resistance to Sclerotinia sclerotiorum in Camelina sativa

The ascomycete Sclerotinia sclerotiorum is a necrotrophic plant pathogen with an extremely broad host range. It causes stem rot in Camelina sativa, a crucifer with great potential as an alternative oilseed crop. Lignification is a common phenomenon in the expression of resistance against necrotrophs, but the molecular mechanisms underlying this defence response are poorly understood. We present histochemical, gene expression and biochemical data investigating the role of monolignols in the resistance of C. sativa to S. sclerotiorum. Comparative studies with resistant and susceptible lines of C. sativa revealed substantial differences in constitutive transcript levels and gene regulation patterns for members of the gene family encoding cinnamoyl-CoA reductase (CCR), the first enzyme specifically committed to the synthesis of lignin monomers. These differences were associated with anatomical and metabolic factors. While the induction of CsCCR2 expression after inoculation with S. sclerotiorum was associated with the deposition of lignin mainly derived from guaiacyl monomers, high constitutive levels of CsCCR4 paralleled a high syringyl lignin content in healthy stems of resistant plants. The results provide evidence that plant cell wall strengthening plays a role in the resistance of C. sativa to S. sclerotiorum, and that both constitutive and inducible defence mechanisms contribute to reduced symptom development in resistant germplasm. This study provides the first characterization of quantitative resistance in C. sativa to S. sclerotiorum.

Fitness of double vs. single herbicide-resistant canola

Abstract Since 1995, canola cultivars with herbicide resistance (HR) have been readily adopted by Canadian producers. Gene flow between these cultivars with different HR traits has led to the occurrence of double herbicide–resistant (2HR) volunteers. To evaluate the fitness of canola volunteers with double HR, we compared three 2HR combinations to each of their parent single-HR plants (1HR: glufosinate-R, imidazolinone-R, glyphosate-R) commercial canola lines in separate greenhouse experiments. The replacement series design included five ratios of 2HR vs. 1HR plants at a single density of 129 plants m−2 and three stress treatments: herbicide application with either glufosinate, imazethapyr, or glyphosate; competition with a wheat crop; and a control without herbicide or wheat competition. Fitness indicators included aboveground biomass at 5 and 12 to 16 wk, seed production, and reproductive allocation. The 2HR plants showed delayed reproductive growth but were generally as competitive as 1HR commercial lines. Plant biomass of 2HR canola was comparable to or greater than 1HR canola, whereas seed biomass of 2HR canola was less than that of 1HR canola in half of the cases, likely because of delayed reproductive growth and early harvesting. Glufosinate–glyphosate 2HR was the fittest combination. Herbicide application had little effect on 2HR biomass at harvest, except for imazethapyr, which reduced the biomass and seed production of 2HR plants with imidazolinone-glyphosate resistance by 30%. The latter effect could have been from the unsuspected presence of 2HR plants with only one of the two acetolactate synthase mutations conferring resistance to imidazolinones. Wheat competition reduced fitness values of both 2HR and 1HR canola similarly, but seed production was still 64% that of the controls. Overall, there was little indication of reduced fitness in 2HR canola compared with commercial 1HR varieties. Nomenclature: Canola, Brassica napus L.; wheat, Triticum aestivum L. ‘Voyageur’, ‘AC Pollet’.

Reaction of wild crucifers to Leptosphaeria maculans, the causal agent of blackleg of crucifers

Cotyledons, leaves, and stems of the wild crucifers Arabidopsis thaliana, Diplotaxis muralis, Diplotaxis tenuifolia, and Raphanus raphanistrum and cotyledons and leaves of Sisymbrium loeselii were inoculated with pycnidiospores of an aggressive isolate of Leptosphaeria maculans, the cause of blackleg of crucifers. All species except R. raphanistrum expressed a high level of resistance to L. maculans; the resistance was characterized by rapid cell death, tissue browning, and lignin deposition. In R. raphanistrum, the reaction of cotyledons and leaves ranged from a hypersensitive-like response to extensive tissue collapse and necrosis comparable to that observed in susceptible Brassica napus cv. Westar; stem tissue of R. raphanistrum, however, was highly resistant to L. maculans. The fungus was recovered from necrotic cotyledon and leaf tissue of all the wild crucifers 10 and 20 days postinoculation, respectively, and from stem tissue of D. muralis around the point of inoculation 40 days postinoculation.

Detection of aster yellows phytoplasma DNA in seed and seedlings of canola (Brassica napus and B. rapa) and AY strain identification

Abstract A study was conducted to identify the strains of aster yellows (AY) disease present in crops of Brassica napus and B. rapa grown near Medstead, Saskatchewan. AY phytoplasma DNA was detected in midrib, stem and root tissues of several symptomless plants as well as plants exhibiting typical AY disease symptoms. Most symptomatic and symptomless, AY-infected plants produced normal-looking and misshapen seeds. However, for both Brassica species, symptomatic plants produced significantly more seeds containing phytoplasma DNA than symptomless, AY-infected plants. Also, significantly more misshapen seeds contained phytoplasma DNA than normal seeds. Phytoplasma DNA belonging to subgroups 16SrI-A and 16SrI-B was detected in symptomatic and symptomless, AY-infected plants and in seed of these plants. The new AY strain sequences were registered in Genbank. The study reports for the first time the detection of AY strains in seedling tissues of both Brassica species. The research also showed that spiral cleaning has the potential to remove seeds that contain phytoplasma DNA in B. napus.

A rapid method for assessing the viability of fungal spores1

The LIVE/DEAD® BacLight™ Bacterial Viability Kit (L-7007) manufactured by Molecular Probes, Inc. (Eugene, Oregon, U.S.A.), which provides a two-colour fluorescence assay of bacterial viability, was useful for determining the viability of the hyaline spores of Colletotrichum gloeosporioides f. sp. malvae, Leptosphaeria maculans, and Sclerotinia sclerotiorum and the thick-walled spores of Alternaria brassicae. Under blue light, viable spores fluoresced green and dead spores fluoresced red. Cells of multicellular spores fluoresced green or red according to their viability. In tests performed with S. sclerotiorum, there was no significant difference between viability as determined with the kit and in vitro germinability. With C. gloeosporioides, the proportion of spores that germinated in vitro was lower than the proportion of viable spores, indicating that not all viable spores could germinate.Key words: blackleg, black spot, two-colour fluorescence assay.