Effect of Plant Systemic Resistance Elicited by Biological and Chemical Inducers on the Colonization of the Lettuce and Basil Leaf Apoplast by Salmonella enterica.

Mitigation strategies to prevent microbial contamination of crops are lacking. We tested the hypothesis that induction of plant systemic resistance by biological (induced systemic resistance [ISR]) and chemical (systemic acquired resistance [SAR]) elicitors reduces endophytic colonization of leaves by Salmonella enterica serovars Senftenberg and Typhimurium. S. Senftenberg had greater endophytic fitness than S. Typhimurium in basil and lettuce. The apoplastic population sizes of serovars Senftenberg and Typhimurium in basil and lettuce, respectively, were significantly reduced approximately 10- to 100-fold by root treatment with microbial inducers of systemic resistance compared to H2O treatment. Rhodotorula glutinis effected the lowest population increases of S. Typhimurium in lettuce and S. Senftenberg in basil leaves, respectively 120- and 60-fold lower than those seen with the H2O treatment over 10 days postinoculation. Trichoderma harzianum and Pichia guilliermondii did not have any significant effect on S. Senftenberg in the basil apoplast. The chemical elicitors acidobenzolar-S-methyl and dl-ß-amino-butyric acid inhibited S. Typhimurium multiplication in the lettuce apoplast 10- and 2-fold, respectively, compared to H2O-treated plants. All ISR and SAR inducers applied to lettuce roots in this study increased leaf expression of the defense gene PR1, as did Salmonella apoplastic colonization in H2O-treated lettuce plants. Remarkably, both acidobenzolar-S-methyl upregulation and R. glutinis upregulation of PR1 were repressed by the presence of Salmonella in the leaves. However, enhanced PR1 expression was sustained longer and at greater levels upon elicitor treatment than by Salmonella induction alone. These results serve as a proof of concept that priming of plant immunity may provide an intrinsic hurdle against the endophytic establishment of enteric pathogens in leafy vegetables. IMPORTANCE Fruit and vegetables consumed raw have become an important vehicle of foodborne illness despite a continuous effort to improve their microbial safety. Salmonella enterica has caused numerous recalls and outbreaks of infection associated with contaminated leafy vegetables. Evidence is increasing that enteric pathogens can reach the leaf apoplast, where they confront plant innate immunity. Plants may be triggered for induction of their defense signaling pathways by exposure to chemical or microbial elicitors. This priming for recognition of microbes by plant defense pathways has been used to inhibit plant pathogens and limit disease. Given that current mitigation strategies are insufficient in preventing microbial contamination of produce and associated outbreaks, we investigated the effect of plant-induced resistance on S. enterica colonization of the lettuce and basil leaf apoplast in order to gain a proof of concept for the use of such an intrinsic approach to inhibit human pathogens in leafy vegetables.

Occupational respiratory allergy to lettuce in lettuce farmers.

BACKGROUND: Lettuce-associated respiratory allergy has never been reported before. The aim of this study was to clarify the clinical condition of lettuce-associated respiratory allergy and to identify the lettuce antigen which induces allergic symptoms. METHODS: We distributed questionnaires to 1168 lettuce farmers and performed medical examinations in those who exhibited respiratory symptoms related to occupational exposure to lettuce. We analysed specific IgE-binding proteins in the sera of patients through immunoblotting analysis and determined molecular characterization of the IgE-binding bands using liquid chromatography-mass spectrometry. RESULTS: A total of 932 farmers (80%) responded to the questionnaire. Of those, 7% exhibited lettuce-associated respiratory symptoms, during harvesting and packaging. Thirteen patients were diagnosed with allergy to lettuce and agreed to undergo further examinations. The percentage of activated basophils in these patients was significantly higher compared with that reported in negative controls (P < .05). Lettuce-specific IgE (ImmunoCAP® ) and skin prick testing was positive in 46% and 62% of patients, respectively. Notably, occupational lettuce-allergic asthma was detected in one patient through specific bronchial provocation testing. The IgE-binding bands recognized in the sera of >50% of patients were identified as epidermis-specific secreted glycoprotein EP1-like (51 kDa). CONCLUSION: The present analysis identified a novel lettuce allergen. This allergen may have clinically useful applications, such as specific IgE testing and allergen-specific immunotherapy.

The LsVe1L allele provides a molecular marker for resistance to Verticillium dahliae race 1 in lettuce.

BACKGROUND: Verticillium wilt caused by the fungus Verticillium dahliae race 1 is among the top disease concerns for lettuce in the Salinas and Pajaro Valleys of coastal central California. Resistance of lettuce against V. dahliae race 1 was previously mapped to the single dominant Verticillium resistance 1 (Vr1) locus. Lines of tomato resistant to race 1 are known to contain the closely linked Ve1 and Ve2 genes that encode receptor-like proteins with extracellular leucine-rich repeats; the Ve1 and Ve2 proteins act antagonistically to provide resistance against V. dahliae race 1. The Vr1 locus in lettuce contains a cluster of several genes with sequence similarity to the tomato Ve genes. We used genome sequencing and/or PCR screening along with pathogenicity assays of 152 accessions of lettuce to investigate allelic diversity and its relationship to race 1 resistance in lettuce. RESULTS: This approach identified a total of four Ve genes: LsVe1, LsVe2, LsVe3, and LsVe4. The majority of accessions, however, contained a combination of only three of these LsVe genes clustered on chromosomal linkage group 9 (within ~ 25 kb in the resistant cultivar La Brillante and within ~ 127 kb in the susceptible cultivar Salinas). CONCLUSIONS: A single allele, LsVe1L, was present in all resistant accessions and absent in all susceptible accessions. This allele can be used as a molecular marker for V. dahliae race 1 resistance in lettuce. A PCR assay for rapid detection of race 1 resistance in lettuce was designed based on nucleotide polymorphisms. Application of this assay allows identification of resistant genotypes in early stages of plant development or at seed-level without time- and labor-intensive testing in the field.

Lettuce-derived secretory IgA specifically neutralizes the Shiga toxin 1 activity.

MAIN CONCLUSION: An edible plant was tested as a host for the production of secretory monoclonal IgA against Shiga toxin 1 (Stx1). The lettuce-derived IgA completely protected Vero cells from Stx1. Secretory immunoglobulin A (SIgA) is thought to control mucosal infections and thus it may be applicable to oral passive immunotherapy. Edible plants are candidate hosts for producing oral formulations with SIgA against pathogenic agents. We previously established a recombinant IgA specific for the B subunit of Shiga toxin 1 (Stx1B) consisting of the Fab fragment of Stx1B-specific monoclonal IgG and the Fc region of IgA (hyIgA). Here, we developed transgenic lettuce (Lactuca sativa) that produces hyIgA in a secretory form (S-hyIgA). An Arabidopsis-derived light-harvesting complex II (LHCB) promoter was used for the expression of all four transgenes (hyIgA heavy, light and j chains, and secretory component). Agrobacterium-mediated transformation was carried out to introduce genes into lettuce leaf discs by means of a single vector harboring all four transgenes. Consistent with the tissue specificity of the LHCB promoter, the expression of hyIgA transgenes was observed in leaf and stem tissues, which contain chloroplasts, at the mRNA and protein levels. The leaves produced hyIgA in a more than tenfold higher yield as compared with stems. The lettuce-derived S-hyIgA was found to bind to Stx1B in a dose-dependent manner by means of ELISA. A leaf extract of the transgenic lettuce completely neutralized the cytotoxicity of Stx1 against Vero cells, which are highly susceptible to Stx1. In conclusion, we established a transgenic lettuce producing a secretory form of hyIgA that can bind bacterial toxin. The results indicate that edible practical plants containing S-hyIgA will provide a possible means for immunotherapy for food poisoning.

The genetics of resistance to lettuce drop (Sclerotinia spp.) in lettuce in a recombinant inbred line population from Reine des Glaces × Eruption.

KEY MESSAGE: Two QTLs for resistance to lettuce drop, qLDR1.1 and qLDR5.1, were identified. Associated SNPs will be useful in breeding for lettuce drop and provide the foundation for future molecular analysis. Lettuce drop, caused by Sclerotinia minor and S. sclerotiorum, is an economically important disease of lettuce. The association of resistance to lettuce drop with the commercially undesirable trait of fast bolting has hindered the integration of host resistance in control of this disease. Eruption is a slow-bolting cultivar that exhibits a high level of resistance to lettuce drop. Eruption also is completely resistant to Verticillium wilt caused by race 1 of Verticillium dahliae. A recombinant inbred line population from the cross Reine des Glaces × Eruption was genotyped by sequencing and evaluated for lettuce drop and bolting in separate fields infested with either S. minor or V. dahliae. Two quantitative trait loci (QTLs) for lettuce drop resistance were consistently detected in at least two experiments, and two other QTLs were identified in another experiment; the alleles for resistance at all four QTLs originated from Eruption. A QTL for lettuce drop resistance on linkage group (LG) 5, qLDR5.1, was consistently detected in all experiments and explained 11 to 25% of phenotypic variation. On LG1, qLDR1.1 was detected in two experiments explaining 9 to 12% of the phenotypic variation. Three out of four resistance QTLs are distinct from QTLs for bolting; qLDR5.1 is pleiotropic or closely linked with a QTL for early bolting; however, the rate of bolting shows only a small effect on the variance in resistance observed at this locus. The SNP markers linked with these QTLs will be useful in breeding for resistance through marker-assisted selection.

Sweet Immunity: Inulin Boosts Resistance of Lettuce (Lactuca sativa) against Grey Mold (Botrytis cinerea) in an Ethylene-Dependent Manner.

The concept of "Sweet Immunity" postulates that sugar metabolism and signaling influence plant immune networks. In this study, we tested the potential of commercially available inulin-type fructans to limit disease symptoms caused by Botrytis cinerea in lettuce. Spraying mature lettuce leaves, with inulin-type fructans derived from burdock or chicory was as effective in reducing grey mold disease symptoms caused by Botrytis cinerea as spraying with oligogalacturonides (OGs). OGs are well-known defense elicitors in several plant species. Spraying with inulin and OGs induced accumulation of hydrogen peroxide and levels further increased upon pathogen infection. Inulin and OGs were no longer able to limit Botrytis infection when plants were treated with the ethylene signaling inhibitor 1-methylcyclopropene (1-MCP), indicating that a functional ethylene signaling pathway is needed for the enhanced defense response. Soluble sugars accumulated in leaves primed with OGs, while 1-MCP treatment had an overall negative effect on the sucrose pool. Accumulation of γ-aminobutyric acid (GABA), a stress-associated non-proteinogenic amino acid and possible signaling compound, was observed in inulin-treated samples after infection and negatively affected by the 1-MCP treatment. We have demonstrated for the first time that commercially available inulin-type fructans and OGs can improve the defensive capacity of lettuce, an economically important species. We discuss our results in the context of a possible recognition of fructans as Damage or Microbe Associated Molecular Patterns.

A Preliminary Study of a Lettuce-Based Edible Vaccine Expressing the Cysteine Proteinase of Fasciola hepatica for Fasciolosis Control in Livestock.

Oral vaccination with edible vaccines is one of the most promising approaches in modern vaccinology. Edible vaccines are an alternative to conventional vaccines, which are typically delivered by injection. Here, freeze-dried transgenic lettuce expressing the cysteine proteinase of the trematode Fasciola hepatica (CPFhW) was used to orally vaccinate cattle and sheep against fasciolosis, which is the most important trematode disease due to the parasite's global distribution, wide spectrum of host species and significant economic losses of farmers. In the study, goals such as reducing the intensity of infection, liver damage and F. hepatica fecundity were achieved. Moreover, we demonstrated that the host sex influenced the outcome of infection following vaccination, with female calves and male lambs showing better protection than their counterparts. Since differences occurred following vaccination and infection, different immunization strategies should be considered for different sexes and host species when developing new control methods. The results of the present study highlight the potential of oral vaccination with plant-made and plant-delivered vaccines for F. hepatica infection control.

Survival and interaction of Escherichia coli O104:H4 on Arabidopsis thaliana and lettuce (Lactuca sativa) in comparison to E. coli O157:H7: Influence of plant defense response and bacterial capsular polysaccharide.

Shiga toxin-producing Escherichia coli (STEC) has been associated with illnesses and outbreaks linked to fresh vegetables, prompting a growing public health concern. Most studies regarding interactions of STEC on fresh produce focused on E. coli O157:H7. Limited information is available about survival or fitness of E. coli O104:H4, non-O157 pathogen that was linked to one of the largest outbreaks of hemolytic uremic syndrome in 2011. In this study, survival of E. coli O104:H4 was evaluated on Arabidopsis thaliana plant and lettuce for 5 days compared with E. coli O157:H7, and expression of pathogenesis-realted gene (PR1; induction of plant defense response) was examined by reverse transcription quantitative PCR, and potential influence of capsular polysaccharide (CPS) on the bacterial fitness on plant was investigated. Populations of E. coli O104:H4 strains (RG1, C3493, and LpfA) on Arabidopsis and lettuce were significantly (P < 0.05) greater than those of E. coli O157:H7 strains (7386 and sakai) at day 5 post-inoculation, indicating E. coli O104:H4 may have better survival ability on the plants. In addition, the E. coli O104:H4 strains produced significantly (P < 0.05) higher amounts of CPS compared with the E. coli O157:H7 strains. RG1 strain (1.5-fold) initiated significantly (P < 0.05) lower expression of PR1 gene indicating induction of plant defense response compared with E. coli O157:H7 strains 7386 (2.9-fold) and sakai (2.7-fold). Collectively, the results in this study suggests that different level of CPS production and plant defense response initiated by each STEC strain might influence the bacterial survival or persistence on plants. The present study provides better understanding of survival behavior of STEC, particularly E. coli O104:H4, using a model plant and vegetable under pre-harvest conditions with plant defense response.

Influence of surface polysaccharides of Escherichia coli O157:H7 on plant defense response and survival of the human enteric pathogen on Arabidopsis thaliana and lettuce (Lactuca sativa).

This study aimed to determine the influence of bacterial surface polysaccharides (cellulose, colanic acid, and lipopolysaccharide; LPS) on the colonization or survival of Escherichia coli O157:H7 on plants and the plant defense response. Survival of E. coli O157:H7 were evaluated on Arabidopsis thaliana and romaine lettuce as a model plant and an edible crop (leafy vegetable), respectively. The population of the wild-type strain of E. coli O157:H7 on Arabidopsis plants and lettuce was significantly (P < 0.05) greater compared with the colanic acid-deficient and LPS-truncated mutants on day 1 and day 5 post-inoculation. This result indicates that colanic acid and LPS structures may contribute to the ability of bacterial survival or persistence on plants. The wild-type strain of E. coli O157:H7 produced approximately twice the amount (P < 0.05) of capsular polysaccharide (CPS) than the colanic acid and LPS-truncated mutants. The significantly lower production of CPS was associated with significantly greater (2-fold) expression of pathogenesis-related gene (PR1) compared with the wild-type and cellulose-deficient mutant (P < 0.05). Collectively, the results of this study may suggest that specific surface polysaccharides of E. coli O157:H7 differentially induce the plant defense response, consequently affecting the survival of the human pathogen on plants. The survival and persistence of E. coli O157:H7 was similar on Arabidopsis and lettuce regardless of day post-inoculation.

Rapid Transient Production of a Monoclonal Antibody Neutralizing the Porcine Epidemic Diarrhea Virus (PEDV) in Nicotiana benthamiana and Lactuca sativa.

Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea, vomiting, dehydration, weight loss, and high mortality rate in neonatal piglets. Porcine epidemic diarrhea (PED) has been reported in Europe, America, and Asia including Thailand. The disease causes substantial losses to the swine industry in many countries. Presently, there is no effective PEDV vaccine available. In this study, we developed a plant-produced monoclonal antibody (mAb) 2C10 as a prophylactic candidate to prevent the PEDV infection. Recently, plant expression systems have gained interest as an alternative for the production of antibodies because of many advantages, such as low production cost, lack of human and animal pathogen, large scalability, etc. The 2C10 mAb was transiently expressed in Nicotiana benthamiana and lettuce using geminiviral vector. After purification by protein A affinity chromatography, the antibody was tested for the binding and neutralizing activity against PEDV. Our result showed that the plant produced 2C10 mAb can bind to the virus and also inhibit PEDV infection in vitro. These results show excellent potential for a plant-expressed 2C10 as a PEDV prophylaxis and a diagnostic for PEDV infection.

Human Norovirus and Its Surrogates Induce Plant Immune Response in Arabidopsis thaliana and Lactuca sativa.

Human norovirus is the leading cause of foodborne illness worldwide with the majority of outbreaks linked to fresh produce and leafy greens. It is essential that we thoroughly understand the type of relationship and interactions that take place between plants and human norovirus to better utilize control strategies to reduce transmission of norovirus in the field onto plants harvested for human consumption. In this study the expression of gene markers for the salicylic acid (SA) and jasmonic acid (JA) plant defense pathways was measured and compared in romaine lettuce (Lactuca sativa) and Arabidopsis thaliana Col-0 plants that were inoculated with Murine Norovirus-1, Tulane Virus, human norovirus GII.4, or Hank's Balanced Salt Solution (control). Genes involving both the SA and JA pathways were expressed in both romaine lettuce and A. thaliana for all three viruses, as well as controls. Studies, including gene expression of SA- and JA-deficient A. thaliana mutant lines, suggest that the JA pathway is more likely involved in the plant immune response to human norovirus. This research provides the first pieces of information regarding how foodborne viruses interact with plants in the preharvest environment.

Lettuce Allergy Is a Lipid Transfer Syndrome-Related Food Allergy With a High Risk of Severe Reactions.

BACKGROUND AND OBJECTIVE: Lipid transfer protein (LTP) sensitization is the most common cause of food allergy in the Mediterranean area, with peach allergy acting as the primary sensitizer in most cases. Lettuce has been described as a common offending food in patients with LTP syndrome. The aim of the study was to investigate the frequency and clinical expression of LTP syndrome in a sample of lettuceallergic patients. METHODS: We determined specific IgE to Pru p 3 and lettuce in a sample of 30 patients with a diagnosis of lettuce allergy. Symptoms elicited by other LTP-containing plant-derived foods and the presence of cofactors were assessed. RESULTS: The clinical symptoms of lettuce allergy were frequently severe, with 18 of the 30 patients experiencing anaphylaxis. All the patients had allergic reactions to other plant foods. Cofactors were involved in the clinical reactions of 13 of the 30 patients. Sensitization to pollens was found in 90% of patients. CONCLUSIONS: Lettuce allergy is found not as an isolated condition but in the context of LTP syndrome and it is characterized by severe reactions and frequent cofactor association.

Lettuce allergy is a lipid transfer syndrome-related food allergy with a high risk of severe reactions

Background and Objectives: Lipid transfer protein (LTP) sensitization is the most common cause of food allergy in the Mediterranean area, with peach allergy acting as the primary sensitizer in most cases. Lettuce has been described as a common offending food in patients with LTP syndrome. The aim of the study was to investigate the frequency and clinical expression of LTP syndrome in a sample of lettuceallergic patients. Methods: We determined specific IgE to Pru p 3 and lettuce in a sample of 30 patients with a diagnosis of lettuce allergy. Symptoms elicited by other LTP-containing plant-derived foods and the presence of cofactors were assessed. Results: The clinical symptoms of lettuce allergy were frequently severe, with 18 of the 30 patients experiencing anaphylaxis. All the patients had allergic reactions to other plant foods. Cofactors were involved in the clinical reactions of 13 of the 30 patients. Sensitization to pollens was found in 90% of patients. Conclusions: Lettuce allergy is found not as an isolated condition but in the context of LTP syndrome and it is characterized by severe reactions and frequent cofactor association (AU)

Introducción y Objetivo: La sensibilización a la LTP es la causa más frecuente de alergia alimentaria en el área mediterránea, siendo la alergia al melocotón el sensibilizador primario en la mayoría de los casos. La alergia a la lechuga ha sido descrita como un manifestación frecuente en los pacientes que sufren síndrome de LTP. El objetivo del estudio fue investigar la frecuencia del síndrome de LTP en una muestra de pacientes alérgicos a lechuga y evaluar su patrón clínico. Métodos: Se determinó la IgE específica a Pru p 3 y a lechuga en una muestra de 30 pacientes con un diagnóstico de alergia a la lechuga. Se evaluaron los síntomas con otras LTPs de alimentos de origen vegetal y la presencia de cofactores. Resultados: Los síntomas clínicos de la alergia a lechuga fueron frecuentemente graves, ya que 18/30 pacientes experimentaron anafilaxia. Todos los pacientes experimentaron reacciones alérgicas a otros alimentos vegetales. En 13/30 pacientes, los cofactores estaban implicados en las manifestaciones clínicas. Se observó que el 90% de los pacientes estaban sensibilizados a pólenes. Conclusiones: La alergia a la lechuga más que de forma aislada, ocurre en el contexto del síndrome LTP y se caracteriza por su frecuente asociación a cofactores y la gravedad de sus reacciones (AU)

Molecular Mapping of High Resistance to Bacterial Leaf Spot in Lettuce PI 358001-1.

Lettuce (Lactuca sativa L.) is a diploid (2n = 18) with a genome size of 2,600 Mbp, and belongs to the family Compositae. Bacterial leaf spot (BLS), caused by Xanthomonas campestris pv. vitians, is a major disease of lettuce worldwide. Leaf lettuce PI 358001-1 has been characterized as an accession highly resistant to BLS and has white seed. In order to understand inheritance of the high resistance in this germplasm line, an F3 population consisting of 163 families was developed from the cross PI 358001-1 × 'Tall Guzmaine' (a susceptible Romaine lettuce variety with black seed). The segregation ratio of reaction to disease by seedling inoculation with X. campestris pv. vitians L7 strain in the F3 families was shown to be 32:82:48 homozygous resistant/heterozygous/homozygous susceptible, fitting to 1:2:1 (n = 162, χ2 = 3.19, P = 0.20). The segregation ratio of seed color by checking F2 plants was 122:41 black/white, fitting to 3:1 (n = 163, χ2 = 0.002, P = 0.96). The results indicated that both BLS resistance and seed color were inherited as a dominant gene mode. A genetic linkage map based on 124 randomly selected F2 plants was developed to enable molecular mapping of the BLS resistance and the seed color trait. In total, 199 markers, comprising 176 amplified fragment length polymorphisms, 16 simple-sequence repeats, 5 resistant gene candidate markers, and 2 cleaved amplified polymorphic sequences (CAPS) markers were assigned to six linkage groups. The dominant resistance gene to BLS (Xcvr) was mapped on linkage group 2 and the gene locus y for seed color was identified on linkage group 5. Due to the nature of a single gene inheritance, the high-resistance gene should be readily transferred to adapted lettuce cultivars to battle against the devastating disease of lettuce.

Cyclic Lipopeptides of Bacillus amyloliquefaciens subsp. plantarum Colonizing the Lettuce Rhizosphere Enhance Plant Defense Responses Toward the Bottom Rot Pathogen Rhizoctonia solani.

The commercially available inoculant Bacillus amyloliquefaciens FZB42 is able to considerably reduce lettuce bottom rot caused by Rhizoctonia solani. To understand the interaction between FZB42 and R. solani in the rhizosphere of lettuce, we used an axenic system with lettuce bacterized with FZB42 and inoculated with R. solani. Confocal laser scanning microscopy showed that FZB42 could delay the initial establishment of R. solani on the plants. To show which secondary metabolites of FZB42 are produced under these in-situ conditions, we developed an ultra-high performance liquid chromatography coupled to time of flight mass spectrometry-based method and identified surfactin, fengycin, and bacillomycin D in the lettuce rhizosphere. We hypothesized that lipopeptides and polyketides play a role in enhancing the plant defense responses in addition to the direct antagonistic effect toward R. solani and used a quantitative real-time polymerase chain reaction-based assay for marker genes involved in defense signaling pathways in lettuce. A significant higher expression of PDF 1.2 observed in the bacterized plants in response to subsequent pathogen challenge showed that FZB42 could enhance the lettuce defense response toward the fungal pathogen. To identify if surfactin or other nonribosomally synthesized secondary metabolites could elicit the observed enhanced defense gene expression, we examined two mutants of FZB42 deficient in production of surfactin and the lipopetides and polyketides, by expression analysis and pot experiments. In the absence of surfactin and other nonribosomally synthesized secondary metabolites, there was no enhanced PDF 1.2-mediated response to the pathogen challenge. Pot experiment results showed that the mutants failed to reduce disease incidence in lettuce as compared with the FZB42 wild type, indicating, that surfactin as well as other nonribosomally synthesized secondary metabolites play a role in the actual disease suppression and on lettuce health. In conclusion, our study showed that nonribosomally synthesized secondary metabolites of FZB42 are actually produced in the lettuce rhizosphere and contribute to the disease suppression by mediating plant defense gene expression toward the pathogen R. solani.

Resistance to Downy Mildew in Lettuce 'La Brillante' is Conferred by Dm50 Gene and Multiple QTL.

Many cultivars of lettuce (Lactuca sativa L.) are susceptible to downy mildew, a nearly globally ubiquitous disease caused by Bremia lactucae. We previously determined that Batavia type cultivar 'La Brillante' has a high level of field resistance to the disease in California. Testing of a mapping population developed from a cross between 'Salinas 88' and La Brillante in multiple field and laboratory experiments revealed that at least five loci conferred resistance in La Brillante. The presence of a new dominant resistance gene (designated Dm50) that confers complete resistance to specific isolates was detected in laboratory tests of seedlings inoculated with multiple diverse isolates. Dm50 is located in the major resistance cluster on linkage group 2 that contains at least eight major, dominant Dm genes conferring resistance to downy mildew. However, this Dm gene is ineffective against the isolates of B. lactucae prevalent in the field in California and the Netherlands. A quantitative trait locus (QTL) located at the Dm50 chromosomal region (qDM2.2) was detected, though, when the amount of disease was evaluated a month before plants reached harvest maturity. Four additional QTL for resistance to B. lactucae were identified on linkage groups 4 (qDM4.1 and qDM4.2), 7 (qDM7.1), and 9 (qDM9.2). The largest effect was associated with qDM7.1 (up to 32.9% of the total phenotypic variance) that determined resistance in multiple field experiments. Markers identified in the present study will facilitate introduction of these resistance loci into commercial cultivars of lettuce.

Genome-Enabled Phylogeographic Investigation of the Quarantine Pathogen Ralstonia solanacearum Race 3 Biovar 2 and Screening for Sources of Resistance Against Its Core Effectors.

Phylogeographic studies inform about routes of pathogen dissemination and are instrumental for improving import/export controls. Genomes of 17 isolates of the bacterial wilt and potato brown rot pathogen Ralstonia solanacearum race 3 biovar 2 (R3bv2), a Select Agent in the United States, were thus analyzed to get insight into the phylogeography of this pathogen. Thirteen of fourteen isolates from Europe, Africa, and Asia were found to belong to a single clonal lineage while isolates from South America were genetically diverse and tended to carry ancestral alleles at the analyzed genomic loci consistent with a South American origin of R3bv2. The R3bv2 isolates share a core repertoire of 31 type III-secreted effector genes representing excellent candidates to be targeted with resistance genes in breeding programs to develop durable disease resistance. Toward this goal, 27 R3bv2 effectors were tested in eggplant, tomato, pepper, tobacco, and lettuce for induction of a hypersensitive-like response indicative of recognition by cognate resistance receptors. Fifteen effectors, eight of them core effectors, triggered a response in one or more plant species. These genotypes may harbor resistance genes that could be identified and mapped, cloned, and expressed in tomato or potato, for which sources of genetic resistance to R3bv2 are extremely limited.

Key mutations in the cylindrical inclusion involved in lettuce mosaic virus adaptation to eIF4E-mediated resistance in lettuce.

We previously showed that allelic genes mol¹ and mo1² used to protect lettuce crops against Lettuce mosaic virus (LMV) correspond to mutant alleles of the gene encoding the eukaryotic translation initiation factor 4E. LMV resistance-breaking determinants map not only to the main potyvirus virulence determinant, a genome-linked viral protein, but also to the C-terminal region of the cylindrical inclusion (CI), with a key role of amino acid at position 621. Here, we show that the propagation of several non-lettuce isolates of LMV in mo1¹ plants is accompanied by a gain of virulence correlated with the presence in the CI C terminus of a serine at position 617 and the accumulation of mutations at positions 602 or 627. Whole-genome sequencing of native and evolved isolates showed that no other mutation could be associated with adaptation to mo1 resistance. Site-directed mutagenesis pinpointed the key role in the virulence of the combination of mutations at positions 602 and 617, in addition to position 621. The impact of these mutations on the fitness of the virus was evaluated, suggesting that the durability of mo1 resistance in the field relies on the fitness cost associated with the resistance-breaking mutations, the nature of the mutations, and their potential antagonistic effects.

Effects of stacked quantitative resistances to downy mildew in lettuce do not simply add up.

KEY MESSAGE: In a stacking study of eight resistance QTLs in lettuce against downy mildew, only three out of ten double combinations showed an increased resistance effect under field conditions. Complete race nonspecific resistance to lettuce downy mildew, as observed for the nonhost wild lettuce species Lactuca saligna, is desired in lettuce cultivation. Genetic dissection of L. saligna's complete resistance has revealed several quantitative loci (QTL) for resistance with field infection reductions of 30-50 %. To test the effect of stacking these QTL, we analyzed interactions between homozygous L. saligna CGN05271 chromosome segments introgressed into the genetic background of L. sativa cv. Olof. Eight different backcross inbred lines (BILs) with single introgressions of 30-70 cM and selected predominately for quantitative resistance in field situations were intercrossed. Ten developed homozygous lines with stacked introgression segments (double combinations) were evaluated for resistance in the field. Seven double combinations showed a similar infection as the individual most resistant parental BIL, revealing epistatic interactions with 'less-than-additive' effects. Three double combinations showed an increased resistance level compared to their parental BILs and their interactions were additive, 'less-than-additive' epistatic and 'more-than-additive' epistatic, respectively. The additive interaction reduced field infection by 73 %. The double combination with a 'more-than-additive' epistatic effect, derived from a combination between a susceptible and a resistant BIL with 0 and 30 % infection reduction, respectively, showed an average field infection reduction of 52 %. For the latter line, an attempt to genetically dissect its underlying epistatic loci by substitution mapping did not result in smaller mapping intervals as none of the 22 substitution lines reached a similar high resistance level. Implications for breeding and the inheritance of L. saligna's complete resistance are discussed.

VARIETAL RESISTANCE TO CONTROL FUSARIUM WILTS OF LEAFY VEGETABLES UNDER GREENHOUSE.

Fusarium wilts of leafy vegetables are difficult to manage under intensive cropping systems. The objective of this study was to evaluate, in three experimental trials, the susceptibility of commercial cultivars of lettuce, wild and cultivated rocket and lamb's lettuce to Fusarium wilts in orderto provide information to breeders, as well as to growers. Some of the cultivars of lettuce tested were completely resistant to the three races of Fusarium wilt. It is interesting to observe that most of the resistant cultivars were 'Batavia red'. Only few rocket cultivars commercially available show a partial resistant reaction to F. oxysporum f.sp. raphani, while, varietal resistance is not applicable at the moment to control Fusarium wilt of lamb's lettuce. The integration of cultural practices, use of resistant cultivars, when available, chemicals and biological control agents, permit to prevent and manage these important diseases on leafy vegetables for fresh-cut production.