Downy Mildew Caused by Bremia lactucae on Strawflower (Helichrysum bracteatum) in California.

In 2006, field-planted strawflower (Helichrysum bracteatum (Venten.) Andr.) grown for cutflowers in coastal San Mateo County was severely infected with a downy mildew disease. Initial symptoms consisted of irregularly shaped chlorotic lesions. Lesions were typically vein delimited, and in particular, did not cross the central longitudinal vein of the lanceolate leaves. Lesions were large and could exceed 6 cm long. Profuse white sporulation developed on the abaxial sides of the lesions. As disease progressed, lesions became gray brown and necrotic. Cutflower stems having symptomatic foliage were unmarketable. Hyaline conidiophores emerged from stomata, branched dichotomously, and had branch tips ending in swollen vesicles bearing sterigmata. Conidia were hyaline and ovoid to globose in shape. The pathogen was identified as Bremia lactucae Regel. To establish pathogenicity, strawflower plants were spray inoculated with a conidial suspension of 1 × 104 conidia/ml, incubated for 24 h in a dew chamber (18 to 20°C), and then maintained in a greenhouse (22 to 24°C). After 14 days, symptoms and signs of downy mildew developed on inoculated plants and the pathogen was confirmed to be B. lactucae. Untreated control plants did not develop downy mildew. Because lettuce (Lactuca sativa L.) is grown extensively in this coastal region, we investigated the pathogenicity of B. lactucae from strawflower on lettuce by inoculating a lettuce downy mildew differential series that includes 20 lettuce lines, four wild L. serriola lines, and strawflower. A conidial suspension of 1 × 106 conidia/ml was applied to all plants; plants were then incubated in a growth chamber with irradiation at 15°C. After 14 days, only the strawflower plants showed downy mildew lesions and sporulation. Collected from diseased field-grown strawflower, 100 downy mildew conidia were measured and had dimensions of 24.4 × 17.4 µm. These conidia were significantly longer than the B. lactuca conidia collected from lettuce (21.8 × 18.1 µm) grown in a nearby area (LSD [P = 0.05] = 2.2), though dimensions were within the reported length range for B. lactucae (12 to 31 µm) (2). To our knowledge, this is the first report of downy mildew caused by B. lactucae on strawflower in California. Inoculation studies indicate that these strawflower and lettuce pathogens from California likely belong to distinct formae speciales (2), though a B. lactucae isolate in Italy was able to infect both hosts (1). The pathogen has been reported on strawflower in Florida, Egypt, Italy, and the United Kingdom (1). References: (1) A. Garibaldi et al. Plant Dis. 87:315, 2003. (2) W. M. Morgan. CMI Descriptions of Pathogenic Fungi and Bacteria. No. 682. CAB International, Wallingford, UK, 1981.

Recombination and spontaneous mutation at the major cluster of resistance genes in lettuce (Lactuca sativa).

Two sets of overlapping experiments were conducted to examine recombination and spontaneous mutation events within clusters of resistance genes in lettuce. Multiple generations were screened for recombinants using PCR-based markers flanking Dm3. The Dm3 region is not highly recombinagenic, exhibiting a recombination frequency 18-fold lower than the genome average. Recombinants were identified only rarely within the cluster of Dm3 homologs and no crossovers within genes were detected. Three populations were screened for spontaneous mutations in downy mildew resistance. Sixteen Dm mutants were identified corresponding to spontaneous mutation rates of 10(-3) to 10(-4) per generation for Dm1, Dm3, and Dm7. All mutants carried single locus, recessive mutations at the corresponding Dm locus. Eleven of the 12 Dm3 mutations were associated with large chromosome deletions. When recombination could be analyzed, deletion events were associated with exchange of flanking markers, consistent with unequal crossing over; however, although the number of Dm3 paralogs was changed, no novel chimeric genes were detected. One mutant was the result of a gene conversion event between Dm3 and a closely related homolog, generating a novel chimeric gene. In two families, spontaneous deletions were correlated with elevated levels of recombination. Therefore, the short-term evolution of the major cluster of resistance genes in lettuce involves several genetic mechanisms including unequal crossing over and gene conversion.

A transgenic mutant of Lactuca sativa (lettuce) with a T-DNA tightly linked to loss of downy mildew resistance.

One hundred and ninety-two independent primary transformants of lettuce cv. Diana were obtained by co-cultivation with Agrobacterium tumefaciens carrying constructs containing maize Ac transposase and Ds. R2 families were screened for mutations at four genes (Dm) for resistance to downy mildew. One family, designated dm3t524, had lost resistance to an isolate of Bremia lactucae expressing the avirulence gene Avr3. Loss of resistance segregated as a single recessive allele of Dm3. The mutation was not due to a large deletion as all molecular markers flanking Dm3 were present. Loss of Dm3 activity co-segregated with a T-DNA from which Ds had excised. Genomic DNA flanking the right border of this T-DNA was isolated by inverse polymerase chain reaction. This genomic sequence was present in four to five copies in wild-type cv. Diana. One copy was missing in all eight deletion mutants of Dm3 and altered in dm3t524, indicating tight physical linkage to Dm3. Three open reading frames (ORFs) occurred in a 6.6-kb region flanking the insertion site; however, expression of these ORFs was not detected. No similarities were detected between these ORFs and resistance genes cloned from other species. Transgenic complementation with 11-to 27-kb genomic fragments of Diana spanning the insertion site failed to restore Dm3 function to two ethyl methanesulfonate (EMS)-induced mutants of Dm3 or to cv. Cobham Green, which naturally lacks Dm3 activity. Therefore, either the T-DNA inserted extremely close to, but not within, Dm3 and the mutation may have been caused by secondary movement of Ds, or Dm3 activity is encoded by a gene extending beyond the fragments used for complementation.

Mutants of downy mildew resistance in Lactuca sativa (lettuce).

As part of our investigation of disease resistance in lettuce, we generated mutants that have lost resistance to Bremia lactucae, the casual fungus of downy mildew. Using a rapid and reliable screen, we identified 16 distinct mutants of Latuca sativa that have lost activity of one of four different downy mildew resistance genes (Dm). In all mutants, only a single Dm specificity was affected. Genetic analysis indicated that the lesions segregated as single, recessive mutations at the Dm loci. Dm3 was inactivated in nine of the mutants. One of five Dm 1 mutants was selected from a population of untreated seeds and therefore carried a spontaneous mutation. All other Dm1, Dm3, Dm5/8 and Dm7 mutants were derived from gamma- or fast neutron-irradiated seed. In two separate Dm 1 mutants and in each of the eight Dm3 mutants analyzed, at least one closely linked molecular marker was absent. Also, high molecular weight genomic DNA fragments that hybridized to a tightly linked molecular marker in wild type were either missing entirely or were truncated in two of the Dm3 mutants, providing additional evidence that deletions had occurred in these mutants. Absence of mutations at loci epistatic to the Dm genes suggested that such loci were either members of multigene families, were critical for plant survival, or encoded components of duplicated pathways for resistance; alternatively, the genes determining downy mildew resistance might be limited to the Dm loci.