Progress 07/01/16 to 06/30/17
Outputs
Target Audience:During the past year we have developed collaborations with entomologists as well as a chemical engineer in order to develop strategies for engineering flavonoid metabolites for both fungal and insect resistance. Previously we started collaborations with sorghum breeders from Kansas State University for thedevelopment of suitable sweet and grain sorghum germplasm for Pennsylvania and Kentucky. We are also making connections with nutrition experts to measure antioxiadant activities of sorghum flavonoids. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two undergraduate students are receiving training under this project. First, Cullen Dixon performed research on the aspects of flavonoid phytoalexins and presented this research at several meetings. Second student Janelle Thompson, a minority student recently started working on this project ans she is developing Colletotrichum fungal strains tagged with GFP. These strains will be used to follow the fungal infection process in the cell. One graduate student, Dinakaran Elango attended meetings to present results of his research project. How have the results been disseminated to communities of interest?1. Publications of research articles in peer reviewed journals. 2. Presentations at scientific conferences. 3. Presentations at conferences and meetings open to public and local community. What do you plan to do during the next reporting period to accomplish the goals?Obj. 1.We are finally getting 16S rDNA sequencing completed for all the collected fungal strains from different locations/sorghum fields. Results obtained from these suveys will be highly informative to study the prevalence of Colletotrichum fungus in sorghum and maize fields states of PA and KY. Obj. 2.A manuscript describing the biosynthesis of 3-DAs in sorghum and maize is being prepared for submission. Obj. 3. A manuscript describing the disease survey results on various sorghum germplasm lines grown in PA is in preparation. Obj. 4.In 2013 we received a set of 28 anthracnose resistant/susceptible sorghum lines (based on results of screens conducted at ICRISAT, India) along with the susceptible and resistant checks from ICRISAT. These lines were multiplied in the greenhouse as per APHIS regulations/conditions specified in the import permit. Seeds collected from these greenhouse-grown ICRISAT lines were included in our sorghum trials for 2014 and 2015 to evaluate their disease susceptibility under field conditions. Unfortunately, the 2015 trial was damaged. We will need to repeat this trial in 2016 and 2017, in order to have 3 years of data for selected sorghum lines, so we can develop the disease management strategy and select lines for the breeding program. A manuscript describing and cataloguing the novel sources of resistance will be prepared from these results. Obj. 5. 1.A manuscript describing the above-mentioned GWAS work is in preparation. 2. Selected resistant and susceptible lines will be subjected to expression profiling using high throughput deep sequencing. 3. We have also screened the NAM parents and narrowed down to two parents (out of 10) that produce 3-DA and show a resistant phenotype. RILs obtained from these crosses will be evaluated by association mapping. Obj. 6.In progress: We still have some bioinformatics work to do for the paper on the genome comparisons, the manuscript should be ready to submit later this year.We propose to further investigate the role of stress, co-infection, and tissue specificity in recognition of and susceptibility to sorghum anthracnose in maize, and vice versa. Our long-term goal is to identify the plant targets of the pathogen effectors. Modification or blocking of these receptors, or even "swapping" them between maize and sorghum, could make 3-DA-mediated resistance in sorghum more effective and more durable, and also prevent the buildup of inoculum over time in a maize-sorghum rotation. Obj. 7.In progress: We still need to replicate the larger experiment, and we need to confirm the race-specific interactions in larger trials. We are conducting studies of these putative race-specific interactions in leaf sheaths in order to describe the cellular events that characterize compatible versus incompatible interactions. Once these experiments are complete, we will finish the statistical analysis, finish writing the manuscript, and send it for publication. Obj. 8.In progress: We need to replicate the greenhouse experiments and lab assays and finish the statistical analysis for this study. This work will then be sent for publication.
Impacts
What was accomplished under these goals?
Progress on the proposed objectives of this project is summarized below: Obj. 1. Fungi belonging to the genus Colletotrichum cause anthracnose on both sorghum and maize. We have built a strain collection consisting of more than 400 single-spored fungal isolates preserved on silica granules.The collection, which is still growing, currently includes 91 isolates from johnsongrass; 256 isolates from cultivated sorghum (sweet, forage, or grain varieties); and 80 isolates of C. graminicola obtained from maize. We developed and used several repetitive fingerprinting probes to characterize this collection. We surveyed eight fields in eastern Pennsylvania for the presence of C. sublineola on sorghum debris. Colletotrichum isolates from the State College location were recovered, but Colletotrichum strains from debris from other locations were not observed, although several other fungi were documented. Fungal strains collected from PA are being fingerprinted and genotyped (PCR based) to confirm their identity. Based on the sequencing results we will be able to assess the prevalence of Colletotrichum fungal strains after 5 years of sorghum cultivation. We have prepared and submitted a manuscript describing the results from part 1, including formal naming of the new species,C. halepenseae. Obj. 2. Evaluation of ASR resistance with respect to sorghum lines that differ in their antifungal compound profile. We have focused on the 3-deoxyanthocyanidin (3-DA) phytoalexins that are induced in sorghum in response to pathogens including Colletotrichum. To directly test the role of sorghum genes providing anthracnose resistance in maize, we have developed transgenic maize lines carrying the sorghum y1 gene, and showed that this maize is tolerant to the anthracnose fungus. A manuscript describing this work was published in 2015. Further, to elucidate the pathway of biosynthesis of 3-DA's we used the powerful genetics of maize where, unlike sorghum, several mutants are available. We have now completed the evaluation of all introgression lines carrying a selected flavonoid gene mutant plus the Y1 transgene (required for resistance and 3-DA biosynthesis). Based on the results obtained from these evaluations we have identified additional genes that are needed for the biosynthesis of 3-DAs in maize and sorghum. A publication describing this work is being prepared now for submission by the end of 2017. Obj. 3. Evaluate Colletotrichum spp. on 3-DA producers and non-producers for survival, ability to sporulate, and the virulence of these spores.1. We established that sorghum's ability to produce 3-DA confers resistance to ALB by restricting fungal proliferation. 2. We used sorghum near-isogenic lines (NILs) obtained from Jeff Pederson, USDA, Lincoln NE, and performed greenhouse and field studies. These NILs contain 3-DA producers and non-producers and our results are quite striking, showing the lines with the dominant y1 gene are able to produce 3-DAs, while lines carrying loss of function alleles of y1 are defective in their 3-DA synthesis. A manuscript describing this work is being prepared now. Obj. 4. Evaluation of diverse bioenergy sorghum lines for resistance to anthracnose. 1. We have previously set up a sorghum-testing program to develop bioenergy feedstock as well as to study the effect of cropping systems, management practices, and genetics of sorghum lines on disease prevalence. These trials of grain, sweet, forage and bioenergy sorghums are currently underway at Pennsylvania State University. Two fields have been utilized for these trials, and we have obtained reproducible trial data from three years. 2. We have screened the performance of several sorghum lines when grown under conditions of high inoculum pressure both in the greenhouse and in field conditions, and identified novel germplasm resistant to anthracnose. Another set of selection experiments was done at ICRISAT, India. Disease data obtained from the past 5 years of growout and disease testing on the selected germplasm of sorghum is being packaged into a manuscript. Obj. 5. Genetic analysis of anthracnose resistance through deep sequencing. We have screened two diverse sorghum collections, one known as Sorghum American Panel (SAP, 377 lines) and the second one called the ICRISAT minicore panel (242 lines). We have now completed the 3-DA and disease analysis from these two diversity panels, and have a reproducible result using GWAS. We have identified genes that have associations with anthracnose resistance and 3-DAs. This work was done in collaboration with Geoff Morris, Kansas State University, KS, USA. A graduate student is currently working on finalizing the results and developing a manuscript as well as part of his thesis. Obj. 6. Evaluate potential for cross-infection of sweet sorghum by maize anthracnose and vice versa under stress conditions. We have tested 25 different varieties of modern and heritage sweet sorghum in the greenhouse for their susceptibility to C. graminicola. None of them supported any growth or colonization by this pathogen, under any condition. We also did three years of field studies in which susceptible, infected, sporulating sorghum was juxtaposed with susceptible field corn inbreds. In leaf sheaths, C. graminicola induced a rapid response in all sorghum varieties tested, in which the plant accumulated vesicles filled with red material within 48 hours, and the maize fungus never colonized or even entered these sheaths. When C. sublineola was applied to maize sheaths, it generally failed to enter the cells, and induced the rapid production of papillae beneath the attempted penetration sites. Less than 1% of the time, C. sublineola did succeed in penetrating maize sheath epidermal cells, but it never managed to move beyond the initially colonized cell. Obj. 7. Evaluate susceptibility of elite and heirloom sweet sorghum varieties to local populations of sorghum anthracnose. Seed for 25 different heritage and improved varieties of sweet sorghum were obtained from the USDA collection, and seed was increased for our experiments in the first year of the project. Fifteen genetically diverse isolates of C. sublineola were tested on 12 of these heritage and improved sweet sorghum varieties in the greenhouse and in the laboratory. There was a range of reactions among the lines, from highly susceptible to all isolates (e.g. Chinese Amber), to highly resistant to all isolates (e.g. Dale). There were significant line-by-isolate interactions in some cases, indicating the presence of race structure in the population. With two notable exceptions, isolates from S. halapense were not pathogenic on any of the sweet sorghum varieties. Strong evidence was found, however, of rare cases of cross-infection of sweet sorghum by C. halepenseae, and also of johnsongrass by C. sublineola, and this could be expected to complicate efforts to develop and deploy resistant sweet sorghum varieties in areas where johnsongrass is common. Obj. 8. Evaluate susceptibility of male sterile and deheaded sweet sorghum to sorghum anthracnose. The pathogenicity of four Colletotrichum isolates from cultivated sorghum and from Johnson grass was compared on the susceptible sweet sorghum inbred Sugar Drip in the field. An isolate that had originally been recovered from sweet sorghum was the most aggressive, while two isolates recovered from johnsongrass caused only minimal disease symptoms. An isolate of C. graminicola from maize used as a control did not cause any symptoms. There was no correlation between the levels of disease and sorghum biomass or juice yield or sugar content.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Xavier, K., Pfeiffer, T., Parreira, D. F., Chopra, S., & Vaillancourt, L. (2017). Aggressiveness of Colletotrichum sublineola strains from Sorghum bicolor and S. halepense to sweet sorghum variety Sugar Drip, and their impact on yield. Plant Disease, Volume 101, Number 9, Pages 1578-1587. https://doi.org/10.1094/PDIS-09-16-1238-RE
- Type:
Journal Articles
Status:
Under Review
Year Published:
2017
Citation:
Xavier, K.V, Queiroz, M. V., Chopra, S., and Vaillancourt, L. (2017). Genotypic and pathogenic diversity of Colletotrichum sublineola isolates from Sorghum bicolor and S. halepense in the Southeastern United States. Phytopathology (under review)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Elango, D; Gaffoor, I; Chopra; S. 2016. Improving anthracnose disease resistance through phytoalexins in sorghum (Sorghum bicolor (L.) Moench) by Genome Wide Association Studies. Poster presented at the Graduate and Undergraduate Gamma Sigma Delta Research Expo. March 24, 2016, Penn State University.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Elango, D., Xue, W., Gaffoor, I., Roth,, G., and Chopra, S. 2016. Genome wide mapping of anti-fungal phytoalexins in sorghum (Sorghum bicolor (L.) Moench) . Presented at 2016 Sorghum Improvement Conference of North America (SICNA) The New Faces of Sorghum September 19-21, 2016 Manhattan Conference Center & Hilton Garden Inn Manhattan, Kansas.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Liu, B; Ibraheem, F; Gaffoor, I; Chopra, S. 2016. Regulation of Flavonoid Biosynthesis Pathway for Antifungal compound in Maize. Poster presented at the Graduate and Undergraduate Gamma Sigma Delta Research Expo. March 24, 2016, Penn State University.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2016
Citation:
INDUCTION AND REGULATION OF FUNGAL DEFENSE RELATED COMPOUNDS IN SORGHUM BICOLOR. A Thesis in Plant Biology By Bin Liu. Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science May 2016. Retrieved from http://etda.libraries.psu.edu
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Dixon, Cullen W., Gaffoor, Iffa., Chopra, Surinder (2017) Testing the role of Sorghum 3-deoxyanthocyanidin phytoalexins as potential biopesticides in combating foliar diseases in Zea mays. P153. Presented at the 59th Annual Maize Genetics Conference, March 9-12, 2017. The Union Station Hotel, St. Louis, Missouri.
- Type:
Books
Status:
Published
Year Published:
2016
Citation:
Rao PS, Vinutha KS, Kumar GS, Chiranjeevi T, Uma A, Lal P, Prakasham RS, Singh HP, Rao RS, Chopra S, Jose S. Sorghum: A multipurpose bioenergy crop. Sorghum: State of the art and future perspectives. 2016 Jun 1. Sorghum: State of the Art and Future Perspectives, Ignacio Ciampitti and Vara Prasad, editors. Agronomy Monograph 58. doi:10.2134/agronmonogr58.2014.0074
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Dinakaran Elango, Iffa Gaffoor, Weiya Xue, Gregory W. Roth and Surinder Chopra. 2016. Genome wide mapping of sorghum anti-fungal compounds. In: The 6th Annual DuPont Plant Sciences Symposium at University of Wisconsin-Madison, USA, 4, November 2016.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Dinakaran Elango, Iffa Gaffoor, Weiya Xue, Gregory W. Roth and Surinder Chopra. 2016. Improving anthracnose disease resistance through phytolexins in sorghum by genome wide association studies. In: The 19th Annual Environmental Chemistry and Microbiology Student Symposium at Penn State University, USA, 8-9, April 2016.
|
Progress 07/01/11 to 06/30/17
Outputs
Target Audience:Collaborated with sorghum breeders from ICRISAT, India and Kansas State University for thedevelopment of suitable sweet and grain sorghum germplasm for Pennsylvania and Kentucky. Worked with nutrition researchers to measure antioxiadant activities of sorghum flavonoids and bioactive compounds. In recent years we have collaborated with entomologists as well as a chemical engineer in order to develop strategies for engineering flavonoid metabolites for insect resistance. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?1. Ms. Janelle Thompson, a minority undergraduate student presented her research work at a Biomedical conference for minority students in December 2017. 2. other graduate and undergraduate students presented their research poster at the annual maize genetics conference in St. Louis , MO in March 2017. How have the results been disseminated to communities of interest?Journal Publications and Scientific Conferences What do you plan to do during the next reporting period to accomplish the goals?We are extending our findings from this project into a new project and testing the role of flavonoids against insects and herbivores. we have so far received positive results towards development of biopesticides. A new project proposal has been submitted thus to NIFA competitive grant program this year.
Impacts
What was accomplished under these goals?
Obj. 1 We surveyed eight fields in eastern Pennsylvania for the presence of C. sublineola on sorghum debris. Colletotrichum isolates from the State College location were recovered, but Colletotrichum strains from debris from other locations were not observed, although several other fungi were documented. Fungal strains collected from PA are being fingerprinted and genotyped (PCR based) to confirm their identity. Based on the sequencing results we will be able to assess the prevalence of Colletotrichum fungal strains after 5 years of sorghum cultivation. We have prepared and submitted a manuscript describing the results from part 1, including formal naming of the new species,C. halepenseae. We are further getting 16S rDNA sequencing completed for all the collected fungal strains from different locations/sorghum fields. Results obtained from these suveys will be highly informative to study the prevalence of Colletotrichum fungus in sorghum and maize fields states of PA and KY. Obj. 2. To directly test the role of sorghum genes providing anthracnose resistance in maize, we have developed transgenic maize lines carrying the sorghum y1 gene, and showed that this maize is tolerant to the anthracnose fungus. A manuscript describing this work was published in 2015. Further, to elucidate the pathway of biosynthesis of 3-DA's we used the powerful genetics of maize where, unlike sorghum, several mutants are available. We have now completed the evaluation of all introgression lines carrying a selected flavonoid gene mutant plus the Y1 transgene (required for resistance and 3-DA biosynthesis). Based on the results obtained from these evaluations we have identified additional genes that are needed for the biosynthesis of 3-DAs in maize and sorghum. A publication describing this work is being prepared now for submission by the end of 2018. Obj. 3. We used sorghum near-isogenic lines (NILs) obtained from Jeff Pederson, USDA, Lincoln NE, and performed greenhouse and field studies. These NILs contain 3-DA producers and non-producers and our results are quite striking, showing the lines with the dominant y1 gene are able to produce 3-DAs, while lines carrying loss of function alleles of y1 are defective in their 3-DA synthesis. A manuscript describing this work is being prepared for submission. Obj. 4. In 2013 we received a set of 28 anthracnose resistant/susceptible sorghum lines (based on results of screens conducted at ICRISAT, India) along with the susceptible and resistant checks from ICRISAT. These lines were multiplied in the greenhouse as per APHIS regulations/conditions specified in the import permit. Seeds collected from these greenhouse-grown ICRISAT lines were included in our sorghum trials for 2014 and 2015 to evaluate their disease susceptibility under field conditions. Unfortunately, the 2015 trial was damaged. We repeated this trial in 2016 and 2017, in order to have 3 years of data for selected sorghum lines, so we can develop the disease management strategy and select lines for the breeding program. A manuscript describing and cataloguing the novel sources of resistance will be prepared from these results. Obj. 5. We have screened two diverse sorghum collections, one known as Sorghum American Panel (SAP, 377 lines) and the second one called the ICRISAT minicore panel (242 lines). We have now completed the 3-DA and disease analysis from these two diversity panels, and have a reproducible result using GWAS. We have identified genes that have associations with anthracnose resistance and 3-DAs. This work was done in collaboration with Geoff Morris, Kansas State University, KS, USA. A graduate student is currently working on finalizing the results and developing a manuscript as well as part of his thesis. Two manuscripts are their final stages: 1. A manuscript describing the above-mentioned GWAS work is in preparation. 2. Selected resistant and susceptible lines will be subjected to expression profiling using high throughput deep sequencing. 3. We have also screened the NAM parents and narrowed down to two parents (out of 10) that produce 3-DA and show a resistant. Obj. 6. We have tested 25 different varieties of modern and heritage sweet sorghum in the greenhouse for their susceptibility to C. graminicola. None of them supported any growth or colonization by this pathogen, under any condition. We also did three years of field studies in which susceptible, infected, sporulating sorghum was juxtaposed with susceptible field corn inbreds. In leaf sheaths, C. graminicola induced a rapid response in all sorghum varieties tested, in which the plant accumulated vesicles filled with red material within 48 hours, and the maize fungus never colonized or even entered these sheaths. When C. sublineola was applied to maize sheaths, it generally failed to enter the cells, and induced the rapid production of papillae beneath the attempted penetration sites. Less than 1% of the time, C. sublineola did succeed in penetrating maize sheath epidermal cells, but it never managed to move beyond the initially colonized cell. We have finished the sequencing work of identification of different fungal strain from PA fields and this work tis being packaged into a manuscript on the genome comparisons. This manuscript will be ready to submit later this year. Obj. 7. Evaluate susceptibility of elite and heirloom sweet sorghum varieties to local populations of sorghum anthracnose. Seed for 25 different heritage and improved varieties of sweet sorghum were obtained from the USDA collection, and seed was increased for our experiments in the first year of the project. Fifteen genetically diverse isolates of C. sublineola were tested on 12 of these heritage and improved sweet sorghum varieties in the greenhouse and in the laboratory. There was a range of reactions among the lines, from highly susceptible to all isolates (e.g. Chinese Amber), to highly resistant to all isolates (e.g. Dale). There were significant line-by-isolate interactions in some cases, indicating the presence of race structure in the population. With two notable exceptions, isolates from S. halapense were not pathogenic on any of the sweet sorghum varieties. Strong evidence was found, however, of rare cases of cross-infection of sweet sorghum by C. halepenseae, and also of johnsongrass by C. sublineola, and this could be expected to complicate efforts to develop and deploy resistant sweet sorghum varieties in areas where johnsongrass is common. A publication describing this work has been accepted in Plant Disease this year (2018). Obj. 8. Evaluate susceptibility of male sterile and deheaded sweet sorghum to sorghum anthracnose. The pathogenicity of four Colletotrichum isolates from cultivated sorghum and from Johnson grass was compared on the susceptible sweet sorghum inbred Sugar Drip in the field. An isolate that had originally been recovered from sweet sorghum was the most aggressive, while two isolates recovered from johnsongrass caused only minimal disease symptoms. An isolate of C. graminicola from maize used as a control did not cause any symptoms. There was no correlation between the levels of disease and sorghum biomass or juice yield or sugar content.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Xavier, K.V, Mizubuti, ESG., Queiroz, M. V., Chopra, S., and Vaillancourt, L. (2018). Genotypic and pathogenic diversity of Colletotrichum sublineola isolates from sorghum (Sorghum bicolor) and Johnsongrass (S. halepense) in the Southeastern United States. Plant Disease https://doi.org/10.1094/PDIS-04-18-0562-RE.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Thompson J., Gaffoor, I., Chopra, S. (2017). Developing Fluorescent Tagged Plant Pathogenic Fungi to Study Plant-Fungal Interactions in Maize and Sorghum. Presented at the Annual Biomedical Research Conference for Minority Students, Phoenix Convention Center, Phoenix, Arizona, Nov 1-4 2017.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2018
Citation:
Keriyat, R., Gaffoor, I., Frock, N., Moen, J., Sattar, S., DeMoraes, C.S., Mescher, M.C., Thompson, G. A., and Chopra, S. Resistance against corn leaf aphid is mediated by yellow seed1-regulated 3-deoxyanthocyanidins in sorghum Submitted. J of Chemical Ecology.
|
Progress 07/01/15 to 06/30/16
Outputs
Target Audience:Developed collaborations with sorghum breeders from Kansas State University for thedevelopment of suitable sweet and grain sorghum germplasm for Pennsylvania and Kentucky. Developed collaborativeefforts with sorghum food science and nutrition experts to measure antioxiadant activities of sorghum flavonoids. Changes/Problems:1. One of the primary goals of this research project was assessing the survival of the fungus during overwintering. Our goal was to perform five trials in PA, one in each year, starting with 2011. The summer of 2015 would have been the last year of the field trials. However, we need to repeat the field trials of 2012 and 2015 that were severely damaged by high winds during the late crop stages of the trial. This substantial crop damage did not allow us to collect the disease data from these two years of trials. It is thus essential for the goals of this project to repeat the trials in the summers of 2016 and 2017. 2. As mentioned above, our essential fifth year of the disease trials will be complete in Fall 2017. Thus, we will definitely need some time in Fall 2017 to gather all the results and compile the final and cumulative trials data for report and publication. 3. This research project being a collaborative one, involving over 22 researchers and students, has led to some complexity for compiling all the results data. There are thus a number of manuscripts, reports, and presentations remaining to be prepared. Several data analyses are ongoing, and thus more time is required to prepare and submit manuscripts. It can take a year from submission to page proofs, so sufficient time needs to be allowed to ensure designated the funds will be available for publication fees. 4. We had an administrative delay in hiring a graduate student from ICRISAT, India because of immigration and visa issues. This graduate student thus joined a year later to the Penn State project. He is contributing to an important genome-wide sorghum-fungal interaction aspect of this project. The student will finish his research thesis and write the research manuscript in the Fall 2017. What opportunities for training and professional development has the project provided?Following professionals received training in the field of plant science and they attended professional meetings. one undergraduate one graduate student one post-doctoral fellow. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?Obj1. One of our goals was to assess the survival of the fungus during overwintering. In PA, our plan was to perform five trials, one in each year starting with summer 2011. The summer of 2015 would have been the last year of the field trials. However, we need to repeat the field trials of 2012 and 2015 because the crop was severely damaged by high winds during the late crop stages of the trial in these years. This substantial crop damage did not allow us to collect disease and feedstock yield data. It is thus essential for the goals of this project to repeat the trials in the summers of 2016 and 2017. In Kentucky, neitherC. graminicolanorC. sublineolacould be recovered from stalk or leaf debris of either maize or sorghum that had been inoculated and buried in the field for the winter season. This experiment was repeated with the same results in the winters of 2013-14 and 2014-15. 2. Fungal strains from PA remain to be fingerprinted and genotyped (PCR based) to confirm their identity. 3. We are currently preparing a manuscript describing the results from part 1, including formal naming of the new species,C. halepenseae. The only work that remains is to prepare and submit a sample to a recognized herbarium to serve as the holotype for the species. Obj2.To directly test the role of sorghum genes providing anthracnose resistance in maize, we have developed transgenic maize lines carrying the sorghum y1 gene, and showed that this maize is tolerant to the anthracnose fungus. A manuscript describing this work was published in 2015. Further, to elucidate the pathway of biosynthesis of 3-DA's we used the powerful genetics of maize where, unlike sorghum, several mutants are available. We are currently evaluating lines carrying the sorghum y1 gene (required for resistance and 3-DA biosynthesis), introgressed with different mutant genes of the putative pathway that give rise to 3-DA's in sorghum and maize. Obj3. Currently we are performing HPLC and LC-MS to identify the novel compounds produced by the sorghum NILs. A manuscript describing these results is in preparation. Obj4.In 2013 we received a set of 28 anthracnose resistant/susceptible sorghum lines (based on results of screens conducted at ICRISAT, India) along with the susceptible and resistant checks from ICRISAT. These lines were multiplied in the greenhouse as per APHIS regulations/conditions specified in the import permit. Seeds collected from these greenhouse-grown ICRISAT lines were included in our sorghum trials for 2014 and 2015 to evaluate their disease susceptibility under field conditions. Unfortunately, the 2015 trial was damaged. We will need to repeat this trial in 2016 and 2017, in order to have 3 years of data for selected sorghum lines, so we can develop the disease management strategy and select lines for the breeding program. A manuscript describing and cataloguing the novel sources of resistance will be prepared from these results. Obj5. 1.A manuscript describing the above-mentioned GWAS work is in preparation. 2. Selected resistant and susceptible lines will be subjected to expression profiling using high throughput deep sequencing. 3. We have also screened the NAM parents and narrowed down to two parents (out of 10) that produce 3-DA and show a resistant phenotype. RILs obtained from these crosses will be evaluated by association mapping. Obj6.We still have some bioinformatics work to do for the paper on the genome comparisons, the manuscript should be ready to submit later this year.We propose to further investigate the role of stress, co-infection, and tissue specificity in recognition of and susceptibility to sorghum anthracnose in maize, and vice versa. Our long-term goal is to identify the plant targets of the pathogen effectors. Modification or blocking of these receptors, or even "swapping" them between maize and sorghum, could make 3-DA-mediated resistance in sorghum more effective and more durable, and also prevent the buildup of inoculum over time in a maize-sorghum rotation. Obj7.We still need to replicate the larger experiment, and we need to confirm the race-specific interactions in larger trials. We are conducting studies of these putative race-specific interactions in leaf sheaths in order to describe the cellular events that characterize compatible versus incompatible interactions. Once these experiments are complete, we will finish the statistical analysis, finish writing the manuscript, and send it for publication. Obj8.We need to replicate the greenhouse experiments and lab assays and finish the statistical analysis for this study. This work will then be sent for publication.
Impacts
What was accomplished under these goals?
Our project represents a major collaboration among scientists from ICRISAT, India; University of Kentucky, KY, USA; and The Pennsylvania State University, PA, USA. Anthracnose is one of the most important diseases on both maize and sorghum worldwide. It has been reported in the literature that C. sublineola from sorghum can colonize, cause symptoms, and sporulate on maize, and that similarly C. graminicola from maize can colonize sorghum, especially under conditions of plant stress. As sorghum acreage increases, and as climatic patterns change, we anticipate that diseases will become a significant limiting factor for biomass production in the U.S. and elsewhere. This project was designed to study the effect of maize-sorghum cropping systems on anthracnose disease pressure and prevalence. Our goals were to study survival of the fungus, as well as to increase our understanding of the genetics and molecular biology of plant-pathogen interactions. We have made substantial progress on the proposed objectives of this project, as summarized below: Obj. 1. Fungi belonging to the genus Colletotrichum cause anthracnose on both sorghum and maize. We have built a strain collection consisting of more than 400 single-spored fungal isolates preserved on silica granules. The isolates from sorghum and johnsongrass are mostly from Kentucky and the Southeastern U.S. (Alabama, Texas, Florida, and Georgia). The collection, which is still growing, currently includes 91 isolates from johnsongrass; 256 isolates from cultivated sorghum (sweet, forage, or grain varieties); and 80 isolates of C. graminicola obtained from maize. We developed and used several repetitive fingerprinting probes to characterize this collection. 3. We surveyed eight fields in eastern Pennsylvania for the presence of C. sublineola on sorghum debris. Colletotrichum isolates from the State College location were recovered, but Colletotrichum strains from debris from other locations were not observed, although several other fungi were documented. Obj. 2. Evaluation of ASR resistance with respect to sorghum lines that differ in their antifungal compound profile. We have focused on the 3-deoxyanthocyanidin (3-DA) phytoalexins that are induced in sorghum in response to pathogens including Colletotrichum. We carried out disease trials in 2012-2015 using sorghum accessions that produced 3-Das at varying concentrations. Plants were infected with local isolates of Colletotrichum. We selected 22 resistant and 9 susceptible lines for further testing in a replicated trial in 2014 and 2015. Obj. 3. Evaluate Colletotrichum spp. on 3-DA producers and non-producers for survival, ability to sporulate, and the virulence of these spores.1. We established that sorghum's ability to produce 3-DA confers resistance to ALB by restricting fungal proliferation. 2. We used sorghum near-isogenic lines (NILs) obtained from Jeff Pederson, USDA, Lincoln NE, and performed greenhouse and field studies. These NILs contain 3-DA producers and non-producers and our results are quite striking, showing the lines with the dominant y1 gene are able to produce 3-DAs, while lines carrying loss of function alleles of y1 are defective in their 3-DA synthesis. Obj. 4. Evaluation of diverse bioenergy sorghum lines for resistance to anthracnose. Sorghum is a relatively new crop in PA, most of it is grown for fodder and the rest for research purposes. Therefore not much is known about how it will perform under increased anthracnose disease pressures, once it becomes more widely cultivated. 1. We have set up a sorghum-testing program to develop bioenergy feedstock as well as to study the effect of cropping systems, management practices, and genetics of sorghum lines on disease prevalence. These trials of grain, sweet, forage and bioenergy sorghums are currently underway at The Pennsylvania State University. Two fields have been utilized for these trials, and we have obtained reproducible trial data from three years. 2. We have screened the performance of several sorghum lines when grown under conditions of high inoculum pressure both in the greenhouse and in field conditions, and identified novel germplasm resistant to anthracnose. Another set of selection experiments was done at ICRISAT, India. Obj. 5. Genetic analysis of anthracnose resistance through deep sequencing. We have screened two diverse sorghum collections, one known as Sorghum American Panel (SAP, 377 lines) and the second one called the ICRISAT minicore panel (242 lines). We have now completed the 3-DA and disease analysis from these two diversity panels, and have a reproducible result using GWAS. We have identified genes that have associations with anthracnose resistance and 3-DAs. This work was done in collaboration with Geoff Morris, Kansas State University, KS, USA. Obj. 6. Evaluate potential for cross-infection of sweet sorghum by maize anthracnose and vice versa under stress conditions. We have tested 25 different varieties of modern and heritage sweet sorghum in the greenhouse for their susceptibility to C. graminicola. None of them supported any growth or colonization by this pathogen, under any condition. We also did three years of field studies in which susceptible, infected, sporulating sorghum was juxtaposed with susceptible field corn inbreds. In leaf sheaths, C. graminicola induced a rapid response in all sorghum varieties tested, in which the plant accumulated vesicles filled with red material within 48 hours, and the maize fungus never colonized or even entered these sheaths. When C. sublineola was applied to maize sheaths, it generally failed to enter the cells, and induced the rapid production of papillae beneath the attempted penetration sites. Less than 1% of the time, C. sublineola did succeed in penetrating maize sheath epidermal cells, but it never managed to move beyond the initially colonized cell. Obj. 7. Evaluate susceptibility of elite and heirloom sweet sorghum varieties to local populations of sorghum anthracnose. Seed for 25 different heritage and improved varieties of sweet sorghum were obtained from the USDA collection, and seed was increased for our experiments in the first year of the project. Fifteen genetically diverse isolates of C. sublineola were tested on 12 of these heritage and improved sweet sorghum varieties in the greenhouse and in the laboratory. There was a range of reactions among the lines, from highly susceptible to all isolates (e.g. Chinese Amber), to highly resistant to all isolates (e.g. Dale). There were significant line-by-isolate interactions in some cases, indicating the presence of race structure in the population. With two notable exceptions, isolates from S. halapense were not pathogenic on any of the sweet sorghum varieties. Strong evidence was found, however, of rare cases of cross-infection of sweet sorghum by C. halepenseae, and also of johnsongrass by C. sublineola, and this could be expected to complicate efforts to develop and deploy resistant sweet sorghum varieties in areas where johnsongrass is common. Obj. 8. Evaluate susceptibility of male sterile and deheaded sweet sorghum to sorghum anthracnose. The pathogenicity of four Colletotrichum isolates from cultivated sorghum and from johnsongrass was compared on the susceptible sweet sorghum inbred Sugar Drip in the field. An isolate that had originally been recovered from sweet sorghum was the most aggressive, while two isolates recovered from johnsongrass caused only minimal disease symptoms. An isolate of C. graminicola from maize used as a control did not cause any symptoms. There was no correlation between the levels of disease and sorghum biomass or juice yield or sugar content.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
S. Chopra. Biosynthesis, Regulation and Genetic Associations for Phytoalexins Induced during Sorghum-Colletotrichum interactions. Jan 9-13, 2016. In Plant and Animal Genome XXIV Conference. San Diego, CA. (Chair: Y. Huang)
- Type:
Other
Status:
Published
Year Published:
2015
Citation:
S. Chopra. Genetics & Genomics of Flavonoid Compounds: Their Defensive Role Against Fungal Pathogenes & Insect Pests in Sorghum & Maize. March 11, 2015. Punjab Agric. University, Ludhiana, India. (Org: Drs. S. Banga and D. Brar)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Echols, Kayla A; Gaffoor, Iffa; Chopra, Surinder. 2015. Genetic control of 3-Deoxyanthocyanidins in maize. P94. Presented at the 57th Annual Maize Genetics Conference, March 12 March 15, Pheasant Run, St. Charles, Illinois.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Gaffoor, I., and Chopra, S. 2015. Transfer of an antifungal metabolic pathway from sorghum to breed maize resistant to foliar fungal pathogens. 69th Northeastern Corn Improvement Conference, University Park, PA. 2015.
|
Progress 07/01/14 to 06/30/15
Outputs
Target Audience:Professional training to graduate and undergraduate students post-doctoral fellows in the fields of plant pathology, genetics and plant molecular biology. Developed collaborations with sorghum breeders from Kansas State University for the development of suitable sweet and grain sorghum germplasm for Pennsylvania and Kentucky. Developed collaborative efforts with sorghum food science and nutrition experts to measure antioxiadant activities of sorghum flavonoids. International research efforts with ICRISAT, India to develop anthracnose resistant germplasm. Changes/Problems:We will be repeating the sorghum-anthracnose disease nursery evaluation trial in 2016 and thus we will be requesting for NCE. What opportunities for training and professional development has the project provided?Four undergraduate students received training in the area of plant genetics and biochemistry. Threegraduate students are currently working on their dissertation reserarch. One post-doctoral fellow is performing multi year disease evaluationsto study mitigation stragies of anthracnose disease and gene expression studies on selected germplasm accessions. One Ph.D. visiting scholar is getting training in GWAS. How have the results been disseminated to communities of interest?Published in peer reviewed journal, book chapters, edited book and presented research posters at scientific meetings. What do you plan to do during the next reporting period to accomplish the goals?We will be repeating the field trails with a subset of the accessions from the previous year to confirm the results. In addition we will include several null lines for y1 to better understand the role of 3-deoxyanthocyanidins in disease resistance under field conditions. In addition to evaluating these lines for resistance to anthracnose leaf blight, we will also quantify the phenolic, flavonoid and 3-deoxyanthocyanidins in the leaf tissue to better understand the role of these compounds in disease development. Evaluation of anthracnose stalk rot in five sorghum lines - Evaluation of anthracnose stalk rot in five sorghum lines - PAR3, PAW1, PAW4, BTx623 and H112. The rating scale used to evaluate the spread of disease along the stalk will be modified to reflect the movement across several internodes. Infected debris from the 2014 field season will be evaluated for survival of the inoculum and also tested on leaves of H112 (anthracnose susceptible) for their ability to cause disease. This debris will also be analyzed for flavonoid compounds. We are in the process of identifying sorghum germplasm that produces copious amounts of 3-deoxyanthocyanidins. We will analyze these extracts to determine the 3-deoxyanthocyanidins derivatives and their relative abundance and also determine the effect of these extracts on spores of foliar pathogens including Colletotrichum sublineolum, C. graminicola, and C. heterostrophus. Use primers to amplify sequences from a variety of strains, and sequence a selection of the polymorphic and non-polymorphic bands to analyze sequence variation, and determine if non-polymorphic bands are more conserved than polymorphic ones.Screen strains that are genetically similar to those to test the hypothesis that they are also more similar in their effector profiles.Amplify and sequence more conserved effectors to test the hypothesis that the ones that are conserved among the three species are not diverging as rapidly (evaluate gain-loss polymorphisms).
Impacts
What was accomplished under these goals?
In KY, our strain collection consists of 402 isolates including: 238 from sweet sorghum (from Kentucky, Georgia, Alabama, and Florida); 49 from forage sorghum (all from Alabama); 18 from grain sorghum (from Kentucky, Indiana, Texas, Brazil, and Africa); and 97 from Johnson grass (from Kentucky, Florida, Alabama, Georgia and Indiana). RAPD analyses to identify haplotypes: We analyzed 375 isolates using RAPD to separate haplotypes. RAPD fingerprinting patterns divided the isolates into two main groups, one from S. halapense (johnsongrass) and the other from S. bicolor (grain, forage, and sweet sorghum). The isolates from S. halapense (JG isolates) seem to vary more within the group than isolates from S. bicolor. There did not appear to be a correlation with host (grain, forage, or sweet sorghum) among the isolates from S. bicolor. From 375 isolates analyzed, 330 haplotypes were identified. There were 202 haplotypes out of 221 isolates from SS that were analyzed. The JG isolates also were very variable, with 86 haplotypes among the 97 isolates analyzed. All 18 of the GS isolates that I analyzed represented different haplotypes. The FS isolates had 24 haplotypes, among the 42 isolates analyzed. In PA, fifteen strains of Colletotrichum were collected from two fields where sorghum had been grown over a three year period. Several fields were surveyed over the past three years to recover Colletotrichum isolates from sorghum debris. These isolates were used for field trials of sorghum accessions. In PA, we have evaluated several sorghum accessions over the past four years for resistance to anthracnose leaf blight. During the 2014 field season, we evaluated a total of 165 accessions encompassing grain, forage, biomass and sweet sorghums in a replicated trial. This included germplasm tested for anthracnose resistance at ICRISAT and both resistant and susceptible controls. We also monitored the plant height and days to flowering when grown in central PA. Our results indicate that the relative marginal effects of disease were greatest in the taller varieties (>10 ft) and the two susceptible maize lines (B73 and MO940) were affected by the disease similar to the susceptible control. These varieties were mainly sweet sorghums and Lines identified as anthracnose resistant by ICRISAT maintained their phenotype under PA conditions too. A subset of these lines will be evaluated in the 2015 field season to confirm the above results. This will enable us to identify varieties suitable to be grown in PA either for use as feedstock or as a multiuse crop. Our standard sorghum lines were evaluated in PA for anthracnose stalk rot. The disease was evaluated using a scale developed for anthracnose stalk rot in maize. Therefore the experiment will be repeated during the 2015 field season and the scale will be modified to better evaluate the spread of disease along the stalk. Twenty sister lines that differ in their seed color and plant body color were evaluated for their ability to produce 3-deoxyanthocyanidins. Eight select lines from this family were further evaluated for performance against disease resistance and it's correlation to the de novo biosynthesis of 3-deoxyanthocyanidins and other phenolic and flavonoid compounds. Evaluation of the responses of 12 varieties of sweet sorghum to C. sublineola. In grain sorghum resistance to Colletotrichum is determined mostly by single genes and there is assumed to be a gene-for-gene relationship with the pathogen. In this chapter I investigated the cytological response of 12 varieties of sweet sorghum to 14 representative strains of C. sublineola and one of C. graminicola to determine if they varied in their ability to resist colonization at a cellular level, and if there is evidence for existence of vertical resistance (that is high levels of resistance to some isolates but not others), and pathogenic races. The hypothesis is that heritage varieties are less resistant than the newer varieties and also that there would be evidence for race specificity, i.e., quantitative interactions.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Reese L, Gaffoor I, and Chopra S. Partial purification of effectors from Colletotrichum sublineolum. Poster presented at Annual Gamma Sigma Delta graduate and undergraduate research expo, Pennsylvania State University, University Park, PA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Wu Y, Gaffoor I, and Chopra S. Role of sorghum 3-deoxyanthocyanidins in resisting gamma irradiation caused damages. Poster presented at Annual Gamma Sigma Delta graduate and undergraduate research expo, Pennsylvania State University, University Park, PA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Clauer P, Gaffoor I, and Chopra S. 3-Deoxyanthocyanidins in Sorghum bicolor Roots upon Fungal Infection. Poster presented at: Annual Gamma Sigma Delta graduate and undergraduate research expo, Pennsylvania State University, University Park, PA.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Ibraheem, F, Gaffoor, I., Tan, Q., Shyu, CR., Chopra, S. 2015. A sorghum MYB transcription factor induces 3-deoxyanthocyanidins and enhances resistance against leaf blights in maize." Molecules 20.2: 2388-2404.
- Type:
Books
Status:
Published
Year Published:
2014
Citation:
Plant Biotechnology: Experience and Future Prospects. Eds: Ricroch, A., Chopra, S., & Fleischer, S. J. (2014). Springer International Publishing. Wageningen, Netherlands. ISBN: 978-3-319-06891-6 (Print) 978-3-319-06892-3 (Online)
- Type:
Book Chapters
Status:
Published
Year Published:
2014
Citation:
Chopra, Surinder. Techniques and Tools of Modern Plant Breeding: Field Crops. Plant Biotechnology. Eds: Ricroch, A., Chopra, S., & Fleischer, S. J. Springer International Publishing, 2014. 25-33. ISBN: 978-3-319-06891-6 (Print) 978-3-319-06892-3 (Online)
- Type:
Book Chapters
Status:
Published
Year Published:
2014
Citation:
Gaffoor, Iffa, and Surinder Chopra. Role of Biotechnology to Produce Plants Resistant to Fungal Pathogens. Plant Biotechnology. Eds: Ricroch, A., Chopra, S., & Fleischer, S. J. Springer International Publishing, 2014. 169-177. ISBN: 978-3-319-06891-6 (Print) 978-3-319-06892-3 (Online)
- Type:
Book Chapters
Status:
Published
Year Published:
2015
Citation:
P . Srinivasa Rao, Reddy Shetty Prakasham, P . Parthasarathy Rao, Surinder Chopra, and Shibu Jose. Sorghum as a Sustainable Feedstock for Biofuels. Biomass and Biofuels. Advanced Biorefineries for Sustainable Production and Distribution. Eds. Shibu Jose and Thallada Bhaskar
CRC Press 2015. Pages 2748. Print ISBN: 978-1-4665-9531-6. eBook ISBN: 978-1-4665-9532-3.
|
Progress 07/01/13 to 06/30/14
Outputs
Target Audience: International research efforts with ICRISAT, India to develop anthracnose resistant germplasm. Professional training to graduate students post-doctoral fellows in fields of plant pathology, genetics and plant molecular biology. Developed collaborations with sorghum breeders for the development of suitable sweet and grain sorghum germplasm for Pennsylvania and Kentucky. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? 1. At Penn State University undergraduate students Shereen Elmaghrabi, Laura Reese and Yenting Wu. Shereen have been carrying out their independent research projects to characterize the infection process of Colletotrichum sublineolum on sorghum and identification of putative effector molecules in C. sublineolum. Students have presented this work at university wide exhibitions. 2. At Penn State University Graduate student Dinakaran Elango is involved in the projet. He has began screening of sorghum germplasm for the synthesis of antifungal compounds in seedling leaves. He will now perform a field based Association mapping study using diversity sorghum lines. 3. At University of KY this project involves a Ph.D. student, Katia Xavier. Katia is beginning her third year of her graduate program and has been making excellent progress. She passed her qualifying examinations last week, and will now concentrate 100% of her time on research for this project. Katia has a Masters degree in Plant Pathology from the Federal University at Lavras in Brazil, where she was studying induced resistance to pathogens in plants (coffee). This project is giving her an opportunity to learn about the pathogen side of disease interactions. She has been learning many new techniques, including phylogenetic analysis, genomic analysis, and population genetics, all new areas for her. 4. Shidad Gaffoor, postdoctoral fellow at Penn State visited ICRISAT, India to attend a Sweet Sorghum workshop. She also received there training to perform controlled crosses in sorghum. How have the results been disseminated to communities of interest? A. Presentation of our research at the Sweetfuel workshop held at ICRISAT, India in a talk entitled “Anthracnose tolerance in sorghum: new insights”. Presented information on the sorghum cultivation, specially as a suitable biofuel feedstock to students following classes in international agriculture Presentation of results at the Genetics of maize-microbe interactions workshop held at the Donald Danforth Plant Science Center and the annual post-doc exhibition held at Penn State. B. At ICRISAT center in India, the anthracnose resistant sweet sorghum hybrid parental lines and cultivars has been exposed to the participants of field day on 17th February 2014 (about 40 participants) and delegates of International Sweet sorghum Workshop in March 2014. The selected lines by the participants will be multiplied and sharednext year. C. We are currently working on few a manuscripts that are listed below: 1. Manuscript describing the comparative genomics of C. graminicola and C. sublineola. We hope to submit that manuscript for publication within the next month. 2. Manuscript describing testing of sorghum germplasm for Anthracnose severity. 3. Manuscript describing generation of maize plant lines that are resistant against Colletotrichum graminicola. What do you plan to do during the next reporting period to accomplish the goals? 1. Generation and evaluation of sorghum and maize germplasm to evaluate near-isogenic sorghum lines that differ in their 3-DA profile for anthracnose stalk rot resistance. Evaluated Colletotrichum sp. –plant interactions for 3-deoxyanthocyanidins synthesis and observed the effect of 3-DA on for survival, ability to sporulate, and the virulence of these fungal spores. 2. We will continue to build our collection of strains, concentrating now on obtaining isolates from Johnsongrass from Alabama and Florida, and increasing our representation of sweet sorghum strains from Kentucky. 3. We will continue to evaluate diversity among the isolates by fingerprinting, sequencing, and pathogenicity tests. 4. We will evaluate diversity of pathogenicity determinants (SM genes and secreted protein effectors) by hybridizing the entire collection with probes designed against identified genes. 5. This summer we will repeat our field experiment to test the effect of deheading on sugar content and on susceptibility to anthracnose, and to test susceptibility in the field to isolates from johnsongrass and maize. We will add one additional isolate from sweet sorghum.
Impacts
What was accomplished under these goals?
1. Our goal is to evaluate diversity among isolates of C. sublineola from sweet and grain sorghum, and from wild sorghum relatives (johnsongrass and shattercane) in Kentucky and the Southeast, in order to assess the risk to sweet sorghum if it were to be widely planted, in rotations with maize, for production of biofuel. Our strain collection now consists of 327 single-spored fungal isolates preserved on silica granules. This includes: 48 isolates from johnsongrass (from Kentucky and Indiana); 160 isolates from sweet or forage sorghum (from Kentucky, Alabama, and Florida); 17 isolates from grain sorghum (from North and South America and Africa); 3 isolates from shattercane (from Kentucky and Indiana); and 99 isolates of C. graminicola from maize (from North and South America). 2. We surveyed five fields in eastern Pennsylvania for the presence of Colletotrichum sublineolum on sorghum debris. Colletotrichum isolates from State College location were recovered for further testing of their aggressiveness. Colletotrichum strains from debris from other locations were not observed although several other fungi were abundant. Further, we carried out disease trials on 135 accessions of sorghum. These lines were obtained from Dr. Lisa Vaillancourt, University of Kentucky; Dr. John J. Toy USDA-ARS, Lincoln, NE; Dr. Chad Hays, USDA-ARS, Lubbock, TX and Dr. Greg Roth, Pennsylvania State University. Plants were infected with local isolates of Colletotrichum. While a majority of the lines showed minor symptoms corresponding to an anthracnose resistant classification, there were nine lines including a commercial line that were found to be susceptible. We also received 28 anthracnose resistant (as screened at ICRISAT) sorghum seeds and the susceptible and resistant checks from ICRISAT. These lines were multiplied in the green house as per APHIS regulations/conditions specified in the import permit. Seeds collected from these greenhouse grown ICRISAT lines will be included in the Sorghum trial for 2014 to evaluate the disease susceptibility under field conditions. 3. We have developed methods for analysis of genetic diversity of these isolates (RAPD and RFLP fingerprinting, ITS and MAT locus sequencing). Our results continue to show that the isolates from sorghum are highly diverse, in comparison to the isolates from maize, and that the johnsongrass isolates, in particular, are extremely variable, even among isolates from the same plant. 4. We conducted a field experiment using a highly susceptible variety of sweet sorghum (“Sugar Drip”) inoculated with four different fungal isolates including: two isolates of C. sublineola from Johnsongrass; one isolate of C. sublineola from sweet sorghum; and one C. graminicola isolate from maize. Results showed that the sweet sorghum isolate was statistically more aggressive to the Sugar Drip plants than either of the two johnsongrass isolates. This isolate even spread beyond the inoculated plants into the border rows. This result agreed with our greenhouse whole plant and sheath inoculation results. 5. The two JG isolates in the field experiment were more aggressive than C. graminicola, which did not differ from the water controls. Inoculation with any of the strains had no significant effect on the yields of seeds, plant biomass, sap, or the BRIX of the sap. Deheading the sorghum had no effect on either disease or yields. 6. We were unable to recover C. graminicola from inoculated sorghum tissues either at the end of the growing season, or after the tissues had been overwintered buried one inch deep in the soil. The three C. sublineola isolates could be recovered from the fresh inoculated tissues at the end of the season, but not from the stalks that had been buried. We did have an extraordinarily harsh winter. 7. We plan to evaluate molecular diversity of relevant pathogenicity genes among the collection of isolates. We have sequenced the genome of a grain sorghum isolate of C. sublineola, and we are in the process of analyzing the genome. Preliminary results are as follows. i. Comparison with the C. graminicola genome, including analysis of synteny and identification of unique and rapidly evolving gene families. Results: the two species share most genes, and are highly syntenous (about 80%). ii. There are 1164 genes in C. graminicola that are not shared with C. sublineola, and 1018 in C. sublineola that are not shared with C. graminicola. Many of these genes are transporters, and many others are secreted proteins, some of which are enzymes, and others are of unknown function. iii. Identification of secondary metabolite genes and gene clusters, and characterization of clusters unique to C. sublineola. iv. C. sublineola has 33 PKS genes, 10 NRPS genes, 5 PKS-NRPS hybrids, 8 DMAT genes, and 14 Terpene synthase genes. Phylogenetic analysis suggests that C. sublineola has 2 DMATs, 6 TS, and 16 PKS genes that are not shared with C. graminicola. v. Identification of secreted protein genes, and characterization of genes encoding putative secreted effectors, especially those unique to C. sublineola. vi. C. sublineola has 824 secreted effector genes, and C. graminicola has 687. C. sublineola has 301 effectors that are not shared with C. graminicola, and C. graminicola has 143 effectors that are not shared with C. sublineola.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Gaffoor, I and Chopra S. 2014. Anthracnose tolerance in sorghum: New insights. Conference organized by ICRISAT, India on Sweet fuel: Sweet Sorghum: an alternative energy crop. ICRISAT, Hyderabad, India, March 3-6, 2014.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Elmaghrabi S, Gaffoor I, and Chopra S. 214. Characterization of disease responses of near isogenic lines of sorghum. Poster presented at: Annual graduate and undergraduate research expo, Pennsylvania State University, University Park, PA. March 19-20, 2014
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Reese L, Gaffoor I, and Chopra S. Partial purification of effectors from Colletotrichum sublineolum. Poster presented at the 2014 undergraduate exhibition, Pennsylvania State University, University Park, PA. April 8-9, 2014.
- Type:
Other
Status:
Other
Year Published:
2013
Citation:
Chopra, S. 2014. Plant-defense against pathogens and pests through phytoalexins.
Presented at ICRISAT, India, on Jan 7, 2014. An invited lecture.
- Type:
Book Chapters
Status:
Published
Year Published:
2013
Citation:
Xin, Z., Wang, M., Chopra, S., and Wang, P. 2013. Gene mutagenesis systems and
resources for the Saccharinae. Plant Genetics and Genomics. Crops and Models vol 11,
pp 169-285. Ed. Andrew Paterson. Springer press.
|
Progress 07/01/12 to 06/30/13
Outputs
Target Audience: 1. Professional training to graduate students, post-doctoral fellows in the fields of plant pathology, genetics and plant molecular biology. 2. Collaborations with sorghum breeders for the development of suitable sweet and grain sorghum germplasm for Pennsylvania and Kentucky. 3. Extension activities with small farmers in Pennsylvania who are interested in growing and testing sorghum crop for forages abd feedsock. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? At Penn State University, Graduate student Nicholas McGinty began evaluation of sorghum germplasm by PCR methods to identify genes that are dominant or recessive in the pathway of flavonoid biosynthesis. Specifically, he will focus on the identification of alleles of the y1 gene. Four undergraduate students have been carrying out their independent research projects in Chopra lab to characterize the foliar flavonoid compounds induced in response to C. sublineolum infection. Post-doctoral fellow Iffa Gaffoor worked with Bioenergy extension specialist Dr. Greg Roth and present information on the cultivation of forage and sweet sorghum cultivars in regions of Pennsylvania. Surinder Chopra and Iffa Gaffoor presented sorghum bioenergy program research topics to the International Ag Development students. Specific focus was on the suitable development of biofuel feedstock. At University of KY, Graduate student, Katia Xavier is working on identification of strains of the fungus from different field sites. She is also analyzing the genome of Colletotrichum graminicola and C. sublineolum for the identification of genes that are required for the synthesis of fungal secondary metabolites How have the results been disseminated to communities of interest? Several scientific talks and poster have been presented at International, National and on campus meetings. Speaking to local farmers about the advantages of growing sorghum. What do you plan to do during the next reporting period to accomplish the goals? We have obtained additional sorghum germplasm and have included these lines along with the sorghum diversity panel and existing lines for our field trial. We have also included MO940 (anthracnose susceptible check) and B73 maize lines in the trial. The plants are being grown on a field where maize was grown the previous year. This stand of maize was heavily infected with several diseases including anthracnose. Plants will be infected with locally isolated C. sublineolum since we have not detected the pathogen in the vicinity of the field. Our standard lines have also been included in this trial for a preliminary anthracnose stalk rot trial. We have also obtained 30 forage sorghum lines (both resistant and susceptible to anthracnose leaf blight). These lines are being grown in the sorghum nursery and preliminary tests will be carried out to determine their flavonoid profile, specifically in response to infection with C. sublineolum. Seeds will be either multiplied in the field this season or in the greenhouse if the flowering is delayed and will be included in the 2014 trial. We will continue to build our collection of strains, and have made arrangements to receive additional strains from johnsongrass, grain, and sweet sorghum from Pennsylvania, Kentucky, Florida, Georgia, and Alabama. We will continue to evaluate diversity among the isolates by fingerprinting, sequencing, and pathogenicity tests. We will continue our comparative analysis of the C. sublineola and C. graminicola genomes. We will then evaluate diversity of pathogenicity determinants (SM genes and secreted protein effectors) by hybridizing the entire collection with probes designed against identified genes. This summer we have set up our first field experiment to test the effect of deheading on sugar content and on susceptibility to anthracnose, and to test susceptibility in the field to isolates from johnsongrass and maize.
Impacts
What was accomplished under these goals?
Characterization of the Sorghum nested association mapping (NAM) diversity parental lines with respect to flavonoid biosynthesis in floral and vegetative tissues and in response to infection with the anthracnose pathogen Colletotrichum sublineolum and a non-pathogenic fungus Cochliobolus heterostrophus. All genotypes tested did not produce elevated levels of flavan-4-ols in the foliar tissue, and no significant induction of this compound was detected when the leaves were infected with either C. sublineolum or C. heterostrophus. We are now in the process of quantifying the levels on 3-deoxyanthocyanidins produced in this tissue. The NAM genotype SC1103 produced appreciable quantities of flavan-4-ols in the pericarp and glume tissue, indicating an active Y1 gene in this floral tissue. SC1345, Segalone, SC971 and SC283 were able to produce high levels of 3-deoxyanthocyanidins when the mesocotyls were infected with C. sublineolum. We have surveyed three fields in PA for the incidence of C. sublineolum. This was the first time that sorghum was grown in these fields although it had been grown in adjacent fields in previous years. We are in the process of identifying new fields to continue the survey. Our goal is to evaluate diversity among isolates of C. sublineola from sweet and grain sorghum, and from wild sorghum relatives (johnsongrass and shattercane) in Kentucky and the Southeast, in order to assess the risk to sweet sorghum if it were to be widely planted, in rotations with maize, for production of biofuel. Our strain collection currently consists of: 23 isolates from johnsongrass (from Kentucky and Indiana), 63 isolates from sweet sorghum (from Kentucky and Florida), 17 isolates from grain sorghum (from North and South America and Africa), 3 isolates from shattercane (from Kentucky and Indiana), and 99 isolates of C. graminicola from maize (from North and South America). We have developed methods for analysis of genetic diversity of these isolates (RAPD and RFLP fingerprinting, ITS and MAT locus sequencing). Our results show that the isolates from sorghum are highly diverse, in comparison to the isolates from maize, and that the johnsongrass isolates, in particular, are extremely variable, even among isolates from the same plant. We have developed methods for analysis of pathogenic diversity using detached sorghum sheath assays and whole plant spray inoculation assays in the greenhouse. We obtained a collection of 25 different varieties of sweet sorghum, including heritage varieties, and planted them in the field for seed increase last season. Some of these heritage varieties are very susceptible, although most of the commercially improved varieties are relatively resistant to the pathogen. Our results so far indicate that johnsongrass isolates are less aggressive to sweet sorghum than isolates from either grain or sweet sorghum. However, these same isolates are quite aggressive on johnsongrass. This suggests a degree of host specialization, and may even suggest that the johnsongrass isolates are a separate species. We will test this hypothesis by conducting a multigene phylogenetic analysis later in the project with a few selected representative isolates. We plan to evaluate molecular diversity of relevant pathogenicity genes among the collection of isolates. We have sequenced the genome of a grain sorghum isolate of C. sublineola, and we are now in the process of analyzing the genome as follows. Comparison with the C. graminicola genome, including analysis of synteny and identification of unique and rapidly evolving gene families. Identification of secondary metabolite genes and gene clusters, and characterization of clusters unique to C. sublineola. Results so far: C. sublineola has 33 PKS genes, 10 NRPS genes, 5 PKS-NRPS hybrids, 8 DMAT genes, and 14 Terpene synthase genes. Phylogenetic analysis suggests that C. sublineola has 2 DMATs, 6 TS, and 16 PKS genes that are not shared with C. graminicola. Identification of secreted protein genes, and characterization of genes encoding putative secreted effectors, especially those unique to C. sublineola.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Chopra, S., and Gaffoor, I. Metabolomics of Phytoalexin-Dependent Anthracnose Resistance in Sorghum. Talk presented at the Sorghum and Millet workshop. Plant and Animal Genome Conference XXI. Jan 12- 16, 2013. San Digo, CA, USA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Gaffoor, I., McGinty, N., Chopra, S. Using sorghum NAM parents to identify resistance to anthracnose. Poster presented at: Genetics of Maize-Microbe Interactions Workshop. 2013 Feb 24-27; Donald Danforth Plant Science Center, Saint Louis, MO.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2013
Citation:
Robbins, M., Roy, A., Wang, P., Gaffoor, I., Sekhon, R., Buanafina, M., Rohila,J., and Chopra, S. Comparative Proteomics Analysis by DIGE and iTRAQ Provides Insight into the Regulation of Phenylpropanoids in Maize. Submitted to J of Proteomics.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Shereen E., Gaffoor, I., and Chopra, S. Characterization of disease responses of near isogenic lines of sorghum. Poster presented at the Annual Undergraduate Exhibition. April 9-10, 2013.Penn State University, University Park, PA 16802.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Choksi, A., Gaffoor, I., and Chopra, S. The Effect of Ufo1 mutation on phenylpropanoids. Poster presented at the Annual Undergraduate Exhibition. April 9-10, 2013.Penn State
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2012
Citation:
Comparison of putative secondary metabolite genes and gene clusters of XAVIER, KV, Torres, MF., E. A. Buiate, EA., Gaffoor, I., Chopra, S., and Vaillancourt, JJ. Colletotrichum graminicola and C. sublineolum. Poster presented at the Annual APS meeting, Aug 4-8, 2012. Providence, RI.
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Progress 07/01/11 to 06/30/12
Outputs
OUTPUTS: Output 1. One of the goals of this project is to study interaction of anthracnose fungus with sweet sorghum. In Pennsylvania, two fields where sorghum had been cultivated for several years were surveyed. Several strains of Colletotrichum sublineolum were isolated from the debris collected from these fields. One of these fields has been sown with maize this year. Once the plants were sufficiently mature they were surveyed for anthracnose symptoms. Lesions were collected and fungal strains were isolated and characterized. Debris was also collected from these fields to isolate strains of C. sublineolum that have survived over the winter. Two more fields where sorghum was grown in the previous season will be surveyed again in a similar manner. We are in the process of identifying more fields where sorghum is being grown during the 2012 season. Output 2. Field trials are in progress for 18 commercial and 24 experimental lines of sorghum. Plants are being monitored for anthracnose symptoms. Plants will be inoculated with field isolates of C. sublineolum. Output 3. Seeds for the association mapping population were obtained from GRIN and multiplied. We are in the process of screening this germplasm for their response to Cochliobolus heterostrophus to identify putative resistant lines. The selected lines will be retested with C. sublineolum prior to deep sequencing of their transcriptome in response to infection. Output 4. To date, six inbred lines of sweet sorghum (Dale, Della, N100, Simon, Sugar Drip and Umbrella) have been screened in a detached sheath assay using 11 isolates of C. sublineolum (CgSl1, JG1001, JG2001, JG3001, JG4001, JG5001, JG6001, JG7001, S26.001, Cstx430, and one isolate from Africa, S3001). Output 5. We have sequenced the genome of the CgSl1 strain of C. sublineolum. Our goals for the project are to compare the genome with the available genome sequence of the closely related C. graminicola strain M1.001, paying particular attention to differences in secreted effectors and secondary metabolites that might be important in determining host specificity. A total of 178 putative effector genes have been identified in the C. graminicola and C. higginsianum genomes. We used O-MCL and C-CL to identify putative orthologues of these genes from C. sublineolum. Output 6. At ICRISAT center in Patancheru, Andhra Pradesh, India, a total of five hundred sorghum lines including grain sorghum hybrid parents, sweet sorghum lines and germplasm accessions were chosen on the basis of plant morphological traits such as plant color, glume color, seed color, previous data on diseases scores in particular anthracnose to retain the diversity. These lines were evaluated in the anthracnose screening nursery at Patancheru, India, during 2011 rainy season. A total of 66 B lines that have recorded disease score less than 2.5 in the rainy season screening were multiplied during post-rainy season 2011. These selected lines are ready for distribution to Penn State University for further studies. PARTICIPANTS: Collaborators: Dr. Lisa Vaillancourt, University of Kentucky,USA. Dr. Belum Reddy, ICRISAT, India. Dr. R.P. Thakur, ICRISAT, India. TARGET AUDIENCES: Professional training to graduate students post-doctoral fellows in fields of plant pathology, genetics and plant molecular biology. Develop collaborations with sorghum breeders for the development of suitable sweet and grain sorghum germplasm for Pennsylvania and Kentucky. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Outcome 1. Screening of sorghum germplasm at ICRISAT, India and lines received from USDA GRIN for identification of sources of resistance to anthracnose fungus. Outcome 2. Isolation of new strains of Colletotrichum graminicola and Colletotrichum sublineolum from filed grown maize and sorghum fields. 3. Our goal is to evaluate diversity among the strains and the sorghum lines, and also to identify a reliable compatible interaction for future use in studies of molecular host-pathogen interactions. Results so far suggest that some isolates are more aggressive than others, and that some lines, for example Sugar Drip are more susceptible than others. We are very interested in the molecular basis for this host specificity. We will continue these studies to include all the isolates we have in hand plus any new isolates that we will identify. Results will be confirmed using whole plant inoculations. Outcome 3. Another outcome will be identification of effectors that are unique to C. sublineolum, which may be important for its specificity to sorghum. Outcome 4. Initiation of a sorghum breeding program for cultivars that inhibit or retard development of the anthracnose in the stalk debris. Outcome 5. Startegies that would enable more extensive cultivation of sorghum alongside and in rotation with maize. It would also enable the use of no-till agricultural practices that are more environmentally friendly. Outcome 6. Identification of targets for potential biotechnological manipulation of sorghum to express a non-host resistance phenotype could lead to durable and highly effective control for ASR and ALB.
Publications
- No publications reported this period
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