Progress 09/01/23 to 08/31/24
Outputs
Target Audience:The target audience for this work is the apple and pear grower community, organic growers, extension agents, representatives for chemical companies and disease scouting consultants, in states where fire blight is a serious problem, which includes both humid environment regions east of the Mississippi river and the more arid growing regions of the Pacific Northwest and California. Additional target audience is the research community of plant pathologists that work on bacterial diseases of tree fruit and bacterial pathogens. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Training opportunities include mentoring of 12 Ph.D. students, 1 M.S. graduate student, and four postdoctorals. How have the results been disseminated to communities of interest? Results have been disseminated to grower audiences through extension presentations held in California, Michigan, New York, North Carolina, Oregon, and Washington. In addition, we have held three webinars and two meetings during the reporting period with information delivered to a total of 461 participants. What do you plan to do during the next reporting period to accomplish the goals? Field experiments will be conducted in the new replicated plantings in Michigan, New York, North Carolina, and Washington, and in existing plantings in California and Oregon for a fourth year in 2025. Analyses of the effect of host resistance inducers on marker gene expression in apple will be conducted to determine the effect of weather parameters and apple cultivar on host resistance induction and fire blight efficacy. Experimental work on shoot blight will be conducted assessing effects of host resistance inducers on pathogen systemic movement through apple trees.
Impacts
What was accomplished under these goals?
Objective 1. In 2024, a replicated field experiment evaluating several candidate non-antibiotic control materials for blossom blight was conducted in California, Michigan, New York, North Carolina, Oregon, and Washington. These materials include Agriphage, alum, Blossom Protect, Cinnerate, Serenade Opti, and Thyme Guard. E. amylovora populations were enumerated from flowers the day after inoculation (1-4 hrs after 2nd spray), 4 days after inoculation (1-4 hrs after 3rd spray) and 3 to 4 days later (7-8 days after inoculation) from a bulk sample of 10 flowers per tree for each treatment and location. Statistical analysis of infection and relative control data was performed using general linear mixed models (GLIMMIX) analysis of variance ANOVA with rep and treatment as fixed effects, and multiple means comparison Tukey's HSD (honestly significant difference) test SAS v 9.4. Due to treatment by location interaction, analysis was performed by location (State). Statistical analysis of E. amylovora counts was performed using general linear mixed models (GLIMMIX) analysis of variance ANOVA with treatment as fixed effect and rep as random effect, and multiple means comparison Tukey's HSD (honestly significant difference) test SAS v 9.4. Due to treatment by date and location interactions analysis was performed by date and by location (State). We are currently finishing up analyses of results from 2024. We completed a project aimed at utilizing lower rates of a growth inhibitor prohexadione calcium (ProCa) and a plant resistance inducer acibenzolar-S-methyl (ASM) to achieve effective management of the shoot blight phase of fire blight. This work included an RNA transcriptome experiments on apple trees treated with this lower rate combination of ProCa and ASM. Various low rate combinations of ProCa and ASM have been screened on multiple apple cultivars at field sites in Michigan, New York, North Carolina, and Washington in a larger experiment assessing the effect of weather conditions, location, and apple cultivar on the performance of host resistance inducers and on fire blight efficacy in years 2, 3, and 4 of the project. Four applications of spray materials were done, using weekly intervals and starting at petal fall. Apple leaves were sampled immediately prior to treatment, and then every third day after each spray application, and also 10 days after the fourth and final spray (6 total samples). These samples were maintained on ice in the field and then immediately placed at -80 C. Gene expression analysis, using the marker genes, will be done in the spring of 2025. Objective 2. The main goal of this objective was to uncover novel large chromosomal inversion (LCI) types in the genome of E. amylovora by combining genome sequencing and comparative genomic analysis as well as by PCR-based molecular markers. Strains with different LCI types found will then be examined to determine if LCI type correlates with differences in virulence. We identified 10 large chromosomal inversion (LCI) types in the Spiraeoideae-infecting (SI) E. amylovora strains by combining whole genome sequencing and PCR-based molecular markers. We sequenced and obtained genomes of six new LCI type strains, and we designed PCR primers to identify new LCI types without sequencing. We have collected more than 200 E. amylovora isolates throughout Washington state from 2022 to 2024 and conducted genotyping of those strains using the PCR-based molecular markers and primers. The work is still ongoing. Our results further indicated on-going chromosomal recombination events in the E. amylovora population, which may have the potential to forecast disease management. The 3rd year of horticultural research on chemical (PGRs and SAR inducers) and cultural inputs (rootstocks and nitrogen inputs) was completed in NC. To determine cumulative effects, treatments were applied to the same plots in a newly planted orchard for three consecutive years. Data are currently being analyzed for this project. Objective 3. We have continued to make progress in developing advanced fire blight resistant pre-breeding lines. The compatibility of plants involved in pre-breeding line development was determined through fluorescent M13-tailed PCR amplification of S-alleles in order to ensure maximum success of crosses made using first-generation fire blight resistant pre-breeding lines. The S-allele genotype of the transgenic parent and first-generation fire blight resistant pre-breeding lines was determined in order to perform compatible crosses. Over the past year we have performed self-incompatibility allele (S-allele) characterization of the early-flowering transgenic parent (T1190), fire blight resistance donors (Malus × Robusta 5 PI588825, Malus fusca PI589228, Malus 'Enterprise' PI590210, Malus 'Evereste'), and the first-generation fire blight resistant pre-breeding lines in order perform compatible crosses between plants. Additionally in these second generation of crosses, we introduced a fourth fire blight resistance locus, Mfu10 from Malus fusca PI589228 into the early-flowering transgenic background using donor pollen from M. fusca PI589228 and flowers from the first-generation fire blight resistant pre-breeding lines. Crosses between first-generation plants or between Malus 'Evereste' flowers and pollen from first-generation pollen were made as flowers became available in the field and in the greenhouse. The cross between Robusta 5 × T1190_4 and Malus 'Evereste' was the first second-generation cross made in the greenhouse and yielded 31 seed that are currently being germinated. Crosses were made in the field using flowers from Malus 'Evereste' and pollen from first-generation fire blight resistant plants in order to pyramid resistance loci; these crosses have already yielded 31 total seed that are currently being germinated. A total of 21 fire blight resistant early-flowering transgenic lines have been developed so far, with 9 first-generation fire blight resistant lines and 11 second-generation fire blight resistant lines. Objective 4. Webinars conducted included (1) Current resistance status of antibiotics for fire blight management - April 4, 2024 - 75 participants; (2) Fire Blight Moves Fast in the Plant. Prune Fast to Stay Ahead. - April 18, 2024 - 52 participants; Plant Defense Products Actigard and Kudos/Apogee - Nov 6, 2024 - 86 participants. A total of 461 participants have joined webinars from around the country. Of participants in end of training evaluations participants stated that they learned a good or great deal (82%, N=65) and planned to adopt practices that they learned (75%, N=48). Participants in 2023 noted learning for example '12-18 inch from infection optimal and no added benefit with more aggressive pruning, worse with branch breaking, and best to leave about 5 inches from leader'; 'Doing nothing on dwarf trees is not an option for me.' In 2024 multiple participants learned 'the speed that Erwinia spreads.' A fire blight summer sanitation field day was conducted in Washington in June 2024, in which participants learned about the biology of fire blight and best practices for fire blight control. The field day included demonstrations on proper cutting techniques and plant defense applications to effectively control fire blight infections. A total of 20 participants attended in person, and they had an opportunity to work in small groups to look at fire blight infections, and practice cutting real fire blight infections. Respondents to post training evaluation stated that they learned a great deal (86%, N=14) and planned to adopt practices that they learned (93%, N=14). Commonly participants indicated that they learned when and how to cut for fire blight prevention, about Actigard use for prevention of fire blight and new products that are being researched.
Publications
|
Progress 09/01/22 to 08/31/23
Outputs
Target Audience:The target audience for this work is the apple and pear grower community, organic growers, extension agents, representatives for chemical companies and disease scouting consultants, in states where fire blight is a serious problem, which includes both humid environment regions east of the Mississippi river and the more arid growing regions of the Pacific Northwest and California. Additional target audience is the research community of plant pathologists that work on bacterial diseases of tree fruit and bacterial pathogens. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Training opportunities include mentoring of 12 Ph.D. students, 1 M.S. graduate student, and four postdoctorals. How have the results been disseminated to communities of interest? Results have been disseminated to grower audiences through extension presentations held in California, Michigan, New York, North Carolina, Oregon, and Washington. In addition, we have held three webinars and two meetings during the reporting period with information delivered to a total of 789 participants. Research results have also been delivered to the research community through the publication of 6 refereed publications and also through presentations given at Plant Health, the annual meeting of the American Phytopathological Society and at the 3rd International Fire Blight meeting in Dresden, Germany. What do you plan to do during the next reporting period to accomplish the goals? Field experiments will be conducted in the new replicated plantings in Michigan, New York, North Carolina, and Washington, and in existing plantings in California and Oregon for a third year in 2024. Analyses of the effect of host resistance inducers on marker gene expression in apple will be conducted to determine the effect of weather parameters and apple cultivar on host resistance induction and fire blight efficacy. Experimental work on shoot blight will be conducted assessing effects of host resistance inducers on pathogen systemic movement through apple trees. Work will continue on identifying genomic signatures of virulence in the fire blight pathogen Erwinia amylovora. This will involve genome sequencing and analysis of more E. amylovora strains, and correlation with virulence in inoculated trees. F2 crosses will be made in breeding experiments and progeny will be evaluated for fire blight resistance. Webinars and in-person extension meetings focused on fire blight management were initiated in 2022. Two full-day grower meetings are planned (Michigan and Washington) to be conducted in early 2024 and additional webinars are planned for 2024.
Impacts
What was accomplished under these goals?
Objective 1. In 2023, a replicated field experiment evaluating several candidate non-antibiotic control materials for blossom blight was conducted in California, Michigan, New York, North Carolina, Oregon, and Washington. These materials include Agriphage, alum, Blossom Protect, Cinnerate, Serenade Opti, and Thyme Guard. E. amylovora populations were enumerated from flowers the day after inoculation (1-4 hrs after 2nd spray), 4 days after inoculation (1-4 hrs after 3rd spray) and 3 to 4 days later (7-8 days after inoculation) from a bulk sample of 10 flowers per tree for each treatment and location. Statistical analysis of infection and relative control data was performed using general linear mixed models (GLIMMIX) analysis of variance ANOVA with rep and treatment as fixed effects, and multiple means comparison Tukey's HSD (honestly significant difference) test SAS v 9.4. Due to treatment by location interaction, analysis was performed by location (State). Statistical analysis of E. amylovora counts was performed using general linear mixed models (GLIMMIX) analysis of variance ANOVA with treatment as fixed effect and rep as random effect, and multiple means comparison Tukey's HSD (honestly significant difference) test SAS v 9.4. Due to treatment by date and location interactions analysis was performed by date and by location (State). We are currently finishing up analyses of results from 2023. We followed up on RNA transcriptome experiments conducted in Yrs 1 and 2 with further RNA transcriptome experiments on apple trees treated with a combination of acibenzolar-S-methyl and prohexadione calcium. These growth chamber experiments enabled the selection of a group of 8-10 genes that will be used as markers to screen a panel of apple cultivars from plots in Michigan, New York, North Carolina, and Washington in a larger experiment assessing the effect of weather conditions, location, and apple cultivar on the performance of host resistance inducers and on fire blight efficacy. In 2023, field experiments were conducted for the second year in which apple trees from multiple cultivars were treated with acibenzolar-S-methyl, prohexadione calcium, a combination of acibenzolar-S-methyl and prohexadione calcium, or water. Field samples were maintained on ice in the field and then immediately placed at -80 C. Gene expression analysis, using the marker genes, will be done in the spring of 2024. Objective 2. We identified 10 large chromosomal inversion (LCI) types in the Spiraeoideae-infecting (SI) E. amylovora strains by combining whole genome sequencing and PCR-based molecular markers. We sequenced and obtained genomes of six new LCI type strains and the sequences were deposited in NCBI (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA970729/). We designed PCR primers to identify new LCI types without sequencing. Subsequently, we evaluated 117 SI strains and found that 48%, 21.4% and 15.4% of the strains belong to the LCI types of Ea1189, CFBP1430, and Ea273, respectively. These three LCI types were the most common with world-wide distribution, whereas the remaining seven LCI types were restricted to North America. Objective 3. We have made significant progress in developing fire blight resistant pre-breeding lines. Last year, we successfully crossed early flowering transgenic apples with fire blight-resistant accessions from wild and domesticated apples. We have created F1 lines that combine at least four fire blight resistance loci/genes i.e., MR5 (Malus robusta 5), FBF7 (Enterprise, Cox's Orange Pippen, Mar12 (Malus floribunda, Malus evereste), and Mfu10 (Malus fusca). This year, we extracted DNA and genotyped all F1 pre-breeding lines using DNA markers for both resistance loci and the early flowering transgene. Now, we have multiple F1 pre-breeding lines with confirmed resistance loci and the early flowering transgene. We planted these F1 lines in the greenhouse from seeds, and they started flowering within one year. Additionally, we created 5-10 clonal replicates of each line by budding on G.935 apple rootstocks to bulk up plant material to make pollinations to develop F2 and to perform fire blight inoculation assays to confirm the presence of resistance loci. Early in 2023, these grafted F1 plants began flowering, and we collected pollen to make crosses for developing the F2 generation of pre-breeding lines with multiple resistance genes in one background. We are now preparing to make F2 crosses and conduct fire blight inoculation assays. Objective 4. Traveling Teams. On 23 February, and 1 March, 2023 Fire Blight Fruit Schools were hosted in Wenatchee Washington and Traverse City Michigan. Participants include 40 in person and 140 online growers on 23 February and 36 in person and 237 online on 1 March. Presentations were given by Ken Johnson, OSU, Sara Villani, NCSU, Frank Zhao and Ricardo Delgado Santander, WSU Plant Pathology, Tianna DuPont and Aina Baro, WSU Extension; George Sundin, Katherine Olive, Xiaochen Yuan, Michigan State University; Kerik Cox, Cornell University. The biology of the pathogen, antibiotic resistance, biopesticides efficacy and their use, tree response to plant defense inducers and their use, the management of fire blight by pruning and sanitation, and the pathogen survival in cankers during winter were the main topics discussed during the fruit school. As a result of the workshop attendees learned a good or great deal (83% N=77) and more than half planned to apply the information they learned in their orchards (68%, N=47). Webinar Series. A webinar series was conducted with three webinars during 2022-2023 including Models for prediction: Cougar Blight, Maryblyt, NEWA on December 12, 2022, Biopesticides for Fire Blight Management on March 15, 2023 and Fire Blight Pruning & Sanitation on June 13, 2023. A total of 334 participants joined from around the country (57 on 12 Dec 2022, 131 on 15 Mar 2023, and 146 on 13 Jun 2023). Respondents to post training evaluation stated that they learned a good or great deal (76%, N=40) and planned to adopt practices that they learned (71%, N=31). Conduct demonstration trials to answer long-standing questions in fire blight management. The first set of demonstration trials was conducted to demonstrate best management practices for cutting blight. Ten on-farm experiments were conducted to evaluate the efficacy of methods to therapeutically remove fire blight cankers from apple trees with different age, vigor, training system and rootstock combinations in Washington, Oregon, Pennsylvania and New York, United States. Removal methods included pruning at varying distances from visible symptoms, utilization of a sanitizing agent and the use of a stub cut to minimize cankers on structural wood. As a result of demonstration trials the team was able to document that fire blight removal resulted in reductions in rootstock blight and tree death. Removal treatments resulted in fewer additional cankers compared to the no-treatment control in most trial sites and years. The standard best management practice (BMP) for removing fire blight was branch removal at 30 cm below the proximal edge of visibly cankered tissue with sanitized loppers. This practice significantly reduced the number of new systemically-caused symptoms compared to the no-treatment control in seven of nine experiments with significant reductions in five experiments. Baseline survey. A baseline survey was conducted to identify what the fire blight management practices are which had 92 participants. These data will be used to compare to practices after the project in order to measure impact. In the survey, participants described their standard control practices for apple and pear in high, medium and low risk blocks. They also described the cost of these management programs as well as the resulting losses due to cutting infected material and tree loss.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
1. Yang, H-W, Thapa, R., Johnson, K., Dupont, T., Khan, A., and Zhao, Y. F. 2023. Examination of large chromosome inversions in the genome of Erwinia amylovora strains reveals worldwide distribution and north America-specific types. Phytopathology (in press) https://doi.org/10.1094/PHYTO-01-23-0004-SA
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Gdanetz, K., Dobbins, M.R., Villani, S.M., Outwater, C.A., Slack, S.M., Nesbitt, D., Svircev, A.M., Lauwers, E.M., Zeng, Q., Cox, K.D., and Sundin, G.W. 2023. Multisite field evaluation of bacteriophages for fire blight management: incorporation of UVR protectants, and impact on the apple flower microbiome. Phytopathology (in press) https://doi-org.proxy1.cl.msu.edu/10.1094/PHYTO-04-23-0145-KC
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Yuan, X., Gdanetz, K., Outwater, C.A., Slack, S.M., and Sundin, G.W. 2023. Evaluation of plant defense inducers and plant growth regulators for fire blight management using transcriptome studies and field assessments. Phytopathology (in press) https://doi-org.proxy1.cl.msu.edu/10.1094/PHYTO-04-23-0147-KC
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Dupont, S.T., Munir, M., Cox, K., Johnson, K., Peter, K., and Baro, A. 2023. Evaluation of pruning therapies in apple trees with fire blight. J. Plant Pathol. (in press) https://doi.org/10.1007/s42161-023-01447-5
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Dupont, S.T., Cox, K., Johnson, K., Peter, K., Smith, T., Misbakhul, M., and Baro, A. 2023. Evaluation of biopesticides for the control of Erwinia amylovora in apple and pear. J Plant Pathol. (in press) https://doi.org/10.1007/s42161-023-01372-7
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Sundin, G.W., Peng, J., Brown, L.E., Zeng, Q., Forster, H., and Adaskaveg, J.E. 2023. A novel IncX plasmid mediates high-level oxytetracycline and streptomycin resistance in Erwinia amylovora from commercial pear orchards in California. Phytopathology (in press) https://doi-org.proxy1.cl.msu.edu/10.1094/PHYTO-06-23-0190-SA
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Progress 09/01/21 to 08/31/22
Outputs
Target Audience:The target audience for this work is the apple and pear grower community, organic growers, extension agents, representatives for chemical companies and disease scouting consultants, in states where fire blight is a serious problem, which includes both humid environment regions east of the Mississippi river and the more arid growing regions of the Pacific Northwest and California. Additional target audience is the research community of plant pathologists that work on bacterial diseases of tree fruit and bacterial pathogens. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Training opportunities include mentoring of seven Ph.D. students, one M.S. graduate student, and four postdoctorals. How have the results been disseminated to communities of interest?Results have been disseminated to grower audiences through extension presentations held in California, Michigan, New York, North Carolina, and Washington. What do you plan to do during the next reporting period to accomplish the goals?Field experiments will be conducted in the new replicated plantings in Michigan, New York, North Carolina, and Washington, and in existing plantings in California and Oregon. Assessments of the effect of host resistance inducers on marker gene expression in apple will be done at multiple locations as the second year of experiments designed to determine the effect of weather parameters and apple cultivar on host resistance induction and fire blight efficacy. Experimental work on shoot blight will be conducted assessing effects of host resistance inducers on pathogen systemic movement through apple trees. Work will continue on identifying genomic signatures of virulence in the fire blight pathogen Erwinia amylovora. This will involve genome sequencing and analysis of more E. amylovora strains, and correlation with virulence in inoculated trees. Currently, seeds from crosses conducted in 2022 are stored in cold to stratify and grow in early next year for marker-assisted selection of seedlings. We have obtained apple trees that are sources of fire blight resistance QTLs/genes from commercial nurseries and established a new orchard to make more crosses with transgenic early flowering lines next year. Webinars and in-person extension meetings focused on fire blight management were initiated in 2022. Two full-day grower meetings are planned (Michigan and Washington) to be conducted in early 2023.
Impacts
What was accomplished under these goals?
Objective 1. Multi-cultivar replicate apple plantings were established in Michigan, New York, North Carolina, and Washington in 2020, and maintained for field research that was initiated in 2022. A replicated field experiment evaluating several candidate non-antibiotic control materials for blossom blight was conducted in California, Michigan, New York, North Carolina, Oregon, and Washington. These materials include Agriphage, alum, Blossom Protect, Cinnerate, Serenade Opti, and Thyme Guard. Of these, alum and Blossom Protect consistently showed significantly better efficacy for blossom blight in all experiments. These non-antibiotic control materials will be evaluated again in 2023 at the six locations. In 2021, an RNA transcriptome experiment was conducted on apple trees maintained under growth chamber conditions that were treated with known and potential host resistance inducers including acibenzolar-S-methyl, prohexadione calcium, and the biological control materials LifeGard and Regalia. We followed up on this experiment in 2022 with further RNA transcriptome experiments on apple trees treated with a combination of acibenzolar-S-methyl and prohexadione calcium. These growth chamber experiments enabled the selection of a group of 8-10 genes that will be used as markers to screen a panel of apple cultivars from plots in Michigan, New York, North Carolina, and Washington in a larger experiment assessing the effect of weather conditions, location, and apple cultivar on the performance of host resistance inducers and on fire blight efficacy. In 2022, field experiments were conducted in which apple trees from multiple cultivars were treated with acibenzolar-S-methyl, prohexadione calcium, a combination of acibenzolar-S-methyl and prohexadione calcium, or water. Four applications of spray materials were done, using weekly intervals and starting at petal fall. Apple leaves were sampled immediately prior to treatment, and then every third day after each spray application, and also 10 days after the fourth and final spray (6 total samples). These samples were maintained on ice in the field and then immediately placed at -80 C. Gene expression analysis, using the marker genes, will be done in the spring of 2023. Objective 2. The main goal of this objective was to uncover novel large chromosomal inversion (LCI) types in the genome of E. amylovora by combining genome sequencing and comparative genomic analysis as well as by PCR-based molecular markers. Strains with different LCI types found will then be examined to determine if LCI type correlates with differences in virulence. MAUVE alignment of known E. amylovora genome sequences has uncovered four LCIs. In addition, four more novel LCI types were identified by complete genome sequences of 14 strains and verified by PCR. In the subsequent determination of LCI types in 117 strains by PCR, we further discovered two novel LCI types and confirmed by whole genome sequencing. In total, we have now identified 10 LCI types in the Spiraeoideae-infecting (SI) E. amylovora strains by combining whole genome sequencing of 16 strains and PCR-based molecular markers. It was found that LCIs were mainly caused by homologous recombination events among seven rRNA operons (rrns) in SI E. amylovora strains. Among the 117 SI strains evaluated, the LCI types of Ea1189, CFBP1430, and Ea273 were the most common ones. These three LCI types had world-wide distribution, whereas the remaining seven LCI types were restricted to North America (or certain regions of the US). Objective 3. We are currently incorporating four different fire blight resistance loci from four donors into pre-breeding lines. In 2022, we conducted controlled cross pollinations between early flowering transgenic 'T1190' and 'Florina', 'Enterprise', and 'G.41' as resistance donors. From the Florina × T1190 cross, we harvested 44 fruit and collected 202 seeds. The G.41 × T1190 crosses resulted in a total of three fruit and four seeds. We also repeated the Enterprise × T1190 cross and obtained 11 fruit and 40 seeds. We developed and optimized a protocol for using fluorescently labeled simple sequence repeat (SSR) markers flanking fire blight resistance loci and markers specific to the early flowering transgene for marker-assisted selection of seedlings. Afterwards, we genotyped F1 plants from the 2021 crosses to confirm the presence of the early flowering transgene and targeted fire blight resistance allele. This resulted in three genotypes from the Enterprise × T1190 cross, two genotypes from the Evereste × T1190 cross, and two genotypes from the Robusta 5 × T1190 cross that had three different fire blight resistance loci. In total, we have developed seven pre-breeding lines that potentially carry fire blight resistance loci on chromosomes 3, 7, and 12, as well as the early flowering transgene. Some of these seed grown pre-breeding lines with fire blight resistance and the early flowering transgene have started to flower in just 4-5 months, rather than the typical 5-7 years. Currently, we are bulking up these pre-breeding lines through vegetative propagation (chip-bud grafting) onto G.935 rootstocks to make cross pollinations next year to combine multiple resistance loci in one genetic background and perform controlled fire blight inoculation tests on the pre-breeding lines.
Publications
|
Progress 09/01/20 to 08/31/21
Outputs
Target Audience:The target audience for this work is the apple and pear grower community, organic growers, extension agents, representatives for chemical companies and disease scouting consultants, in states where fire blight is a serious problem, especially those east of the Mississippi river. Additional target audience is the research community of plant pathologists that work on bacterial diseases of tree fruit and bacterial pathogens. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Training opportunities include mentoring of six Ph.D. students, one M.S. graduate student, and three postdoctorals. How have the results been disseminated to communities of interest?Results have been disseminated to grower audiences through extension presentations held in Michigan, New York, North Carolina, and Washington. What do you plan to do during the next reporting period to accomplish the goals? Field experiments will be conducted in the new replicated plantings in Michigan, New York, North Carolina, and Washington, and in existing plantings in California and Oregon. Assessments of the effect of host resistance inducers on marker gene expression in apple will be done at multiple locations as the first year of experiments designed to determine the effect of weather parameters and apple cultivar on host resistance induction and fire blight efficacy. Experimental work on shoot blight will be conducted assessing effects of host resistance inducers on pathogen systemic movement through apple trees. Work will continue on identifying genomic signatures of virulence in the fire blight pathogen Erwinia amylovora. This will involve genome sequencing and analysis of more E. amylovora strains, and correlation with virulence in inoculated trees. Currently, seeds from crosses conducted in 2021 are stored in cold to stratify and grow in early next year for marker-assisted selection of seedlings. We have obtained apple trees that are sources of fire blight resistance QTLs/genes from commercial nurseries and established a new orchard to make more crosses with transgenic early flowering lines next year.
Impacts
What was accomplished under these goals?
Objective 1. Multi-cultivar replicate apple plantings were established in Michigan, New York, North Carolina, and Washington for field research that will be initiated in 2022. Preliminary field experiments from 2021 yielded several candidate non-antibiotic control materials for blossom blight. These materials include Agriphage, alum, Blossom Protect, Cinnerate, Serenade Opti, and Thyme Guard, and will be tested in replicated field trials in 2022 in California, Michigan, New York, North Carolina, Oregon, and Washington. Additives aimed at providing sunscreen protection will also be used with Serenade Opti to determine if efficacy is increased. An RNA transcriptome experiment was conducted on apple trees treated with known and potential host resistance inducers including acibenzolar-S-methyl, prohexadione calcium, and the biological control materials LifeGard and Regalia. Apple leaves were sampled immediately prior to treatment, and at 3 and 7 days after treatment (7 and 14 days after treatment for prohexadione calcium). Results from this experiment, once validated, will enable us to select specific genes that will be used to screen a panel of apple cultivars from plots in Michigan, New York, North Carolina, and Washington in a larger experiment assessing the effect of weather conditions, location, and apple cultivar on the performance of host resistance inducers and on fire blight efficacy. Objective 2. We have tested the virulence of 16 Erwinia amylovora strains from Cornell using an immature Bartlett pear assay, and identified differences in virulence of the strains. We have done whole genome sequencing of more than 18 E. amylovora strains using Illumina short-read sequencing and Oxford Nanopore long-read sequencing. This included nanopore sequencing of 16 strains from the Cornell strain collection and 2 strains from Washington. We have assembled de novo genomes of these E. amylovora strains to identify structural variations across the whole genome of E. amylovora for future work. Objective 3. We have prepared a design protocol, and developed an application and obtained a BRS permit to work with the pollens of early flowering transgenic line in the field/orchard. We have collected pollens from early flowering transgenic lines in the greenhouse at Cornell AgriTech for pollinations. During the growing season, we have also collected pollens from orchard from fire blight resistant gene donor accessions to make crosses in the greenhouse as well as in the field. We made crosses in the orchard as well as greenhouse at Cornell AgriTech with transgenic pollens and fire blight donor lines. We have ensured to follow permit requirements to contain transgenic pollens, and fruits. In October-November 2021, we have harvested fruit from these crosses and extracted seeds in the laboratory. Currently, seeds from these crosses are stored in cold to stratify and grow in early next year for marker-assisted selection of seedlings. We have obtained apple trees that are sources of fire blight resistance QTLs/genes from commercial nurseries and established a new orchard to make more crosses with transgenic early flowering lines next year.
Publications
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