Source: UNIV OF MINNESOTA submitted to
MOLECULAR BASES OF PLANT DEFENSES AGAINST FUNGAL DISEASES - SMALL GRAIN CEREALS
Sponsoring Institution
State Agricultural Experiment Station
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0065945
Grant No.
(N/A)
Project No.
MIN-22-070
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Jul 1, 2003
Project End Date
Jun 30, 2008
Grant Year
(N/A)
Project Director
Zeyen, R. J.
Recipient Organization
UNIV OF MINNESOTA
(N/A)
ST PAUL,MN 55108
Performing Department
PLANT PATHOLOGY
Non Technical Summary
This research is aimed at understanding the molecular and biochemical events that plants use to defend themselves against disease, and tailoring cereal plants that are better able to resist fungal diseases. The first phase of testing antifungal transgenes to provide resistance to wheat and barley headblight fungus is complete. Greenhouse and field testing phases are now being done to determine efficacy of these tested antifungal genes in transformed wheat and barley.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2121550104020%
2121560104060%
2121599104020%
Goals / Objectives
Fungal diseases of small grain cereals are an economic and societal problem in Minnesota, adjoining states and Canada. For example, in the 1990's and early 2000's a single fungal disease of wheat and barley, Fusarium head blight alone, cost millions of dollars in economic and societal damage. The objective of this research is to improve our fundamental knowledge of the molecular bases of fungal disease resistance, and use this knowledge for improving and extending existing strategies for fungal disease resistance in cereal plants.
Project Methods
The approaches will be: a) To use known physiological and biochemical inhibitors to gain fundamental information about cereal plant defenses against fungi; and combine inhibition studies with microarrays of ESTs from cereal plants to determine up and down regulated plant genes related to defense. b) To determine, using non-biochemical means, key signal transduction and transcription factors used by cereal plants during defense against fungi; and c) To improve an existing biolistic microassay procedure for up regulating plant defense related in transiently transformed plant cells and plant tissue culture.

Progress 07/01/03 to 06/30/08

Outputs
OUTPUTS: The first objective of this project was to investigate naturally occurring plant defense mechanisms against fungal disease agents in cereals. Initially we investigated the barley powdery mildew disease. The pathogen is the fungus Blumeria graminis. We correlated the temporal sequencing of cytological evidence with temporal transcription of defense response genes. We obtained full-length copies of several antifungal defense response genes and made ballistic cassettes for transforming oat, barley and wheat in order to up regulate antifungal defense response genes used by cereal plants to resist disease. First we test antifungal response genes against powdery mildew disease in oat and got good responses. We also tested disease defense response genes in transformed wheat and barley plants against Fusarium Head Blight disease (FHB). Transformation cassettes were placed into wheat and, with the assistance of several collaborators; these transformed plants were subjected to glasshouse and field evaluations relative to FHB. Bobwhite wheat over expressing defense response genes e-1-purothionin, thaumatin-like protein 1 (tlp-1), and B-1, 3-glucanase were used. One, two, and four lines carrying the a-1-purothionin, tlp-1, and B-1, 3-glucanase transgenes, respectively, had statistically significant reductions in FHB severity in greenhouse evaluations. Seven transgenic lines were tested under field conditions for percent FHB disease severity, deoxynivalenol (DON) mycotoxin accumulation, and percent visually scabby kernels (VSK). Six of the seven differed from the non transgenic parental Bobwhite line for at least one of the traits associated with FHB disease. A B-1, 3-glucanase transgenic line had enhanced resistance, exhibited lower FHB severity and lower DON concentrations, and percent VSK compared to Bobwhite. Taken together, results showed that over expression of defense response genes transcription in wheat can enhance FHB resistance in both greenhouse and field conditions. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Basic knowledge gained from transgenic wheat plants over expressing naturally occurring plant defense response genes proved the principle that these transgenes can and do, enhance resistance in oat to Powdery Mildew Disease and in wheat to Fusarium Head Blight under field conditions. This project pointed the way for an alterative method of genetic control of fungal diseases. It established that over expression defense response genes using transformation technology could contribute to fungal disease resistances.

Publications

  • Mackintosh, C.A., Lewis, J., Radmer, L.E., Shin, S., Heinen, S.J., Smith, L.A., Wyckoff, M.N., Dill-Macky, R., Evans, C.K., Kravchenko, S., Baldridge, G.D., Zeyen, R.J. and G.J. Muehlbauer, G.J. 2007. Overexpression of defense response genes enhances the resistance of wheat to Fusarium Head Blight. Plant Cell Reports 26:479-488.
  • Miller, C.V., Zeyen, R.J., Samac, D.A. 2007. Infection and development of Phoma medicaginis on moderately resistant and susceptible alfalfa genotypes. Canadian Journal of Plant Pathology 29:290-298.
  • Caroline A. Mackintosh, Janet Lewis, Lorien E. Radmer, Sanghyun Shin, Shane J. Heinen, Lisa A. Smith, Meagen N. Wyckoff, Ruth Dill-Macky, Conrad K. Evans, Sasha Kravchenko, Gerald D. Baldridge, Richard J. Zeyen, Gary J. Muehlbauer. 2006. Plant Cell Reporter
  • Elena Prats, Timothy L.W. Carver, Michael F. Lyngkjaer, Pete C. Roberts and Richard Zeyen. 2006. Induced inaccessibility and accessibility in the oat powdery mildew system: insights gained from the use of metabolic inhibitors and silicon nutrition. Molecular Plant Pathology 7(1) 47-59.
  • Kruger WM, Szabo LJ, Zeyen RJ. 2003. Transcription of the defense response genes chitinase IIb, PAL and peroxidase is induced by the barley powdery mildew fungus and is only indirectly modulated by R genes. Physiological and Molecular Plant Pathology 63:167-178.
  • C.A. Mackintosh, S.J. Heinen, L.A. Smith, M.N. Wyckoff, G.D. Baldridge, R.J. Zeyen and G.J. Muehlbauer (2003) Over-expression of Anti-fungal Proteins Increases Resistance of Wheat to Fusarium Head Blight. ASA-CSSA-SSSA Annual Meeting, Denver, CO.
  • C.A. Mackintosh, L.E. Radmer, S.L. Jutila, A.C. Cyrus, G.D. Baldridge, R.J. Zeyen and G.J. Muehlbauer (2003) A Transgenic Approach to Enhancing the Resistance of Wheat to Fusarium Head Blight. 2003 National Fusarium Head Blight Forum Proceedings, Bloomington, MN.
  • C.A. Mackintosh, L.E. Radmer, S.L. Jutila, A.C. Cyrus, L.A. Smith, M.N. Wyckoff, S.J. Heinen, G.D. Baldridge, R.J. Zeyen and G.J. Muehlbauer (2003) Over-expression of Antifungal Proteins Increases the Resistance of Wheat to Fusarium Head Blight. 2003 National Fusarium Head Blight Forum Proceedings, Bloomington, MN.
  • Nadiya A, Al-Saady, Kimberly A. Torber, Lisa Smith Irina Makarevitch, Gerald Baldridge, Richard J. Zeyen, Gary J. Muehlbauer, Neil E. Olszewski and David A. Somers. 2004. Tissue specificity of the sugarcane bacilliform virus promoter in oat, barley and wheat. Molecular Breeding 14:331-338.
  • Zeyen RJ, Carver TLW, Lyngkjaer. 2002. Epidermal Cell Papillae. In: Belanger RR, Bushnell WR, Dik AJ, Carver TLW (eds). The Powdery Mildews: A Comprehensive Treatise. APS Press. Pp. 107-125. St. Paul, Minnesota USA.
  • Kruger WM, Carver TLW, Zeyen RJ. 2002. Effects of inhibiting phenolic biosynthesis on penetration resistance of barley isolines containing seven powdery mildew resistance genes or alleles. Physiological and Molecular Plant Pathology 61(1):41-51.
  • Zeyen RJ. 2002. Silicon in Plant Cell Defenses Against Cereal Powdery Mildew Disease. In: Matoh T, Ma JF, Takashashi E (eds). Silicon in Agriculture: The 2nd International Conference on Silicon in Agriculture, The Japanese Society of Soil Science and Plant Nutrition. Pp 15-21. Tsuruoka, Japan
  • Lyngkjaer MF, Carver TLW, Zeyen, RJ. 2001. Virulent Blumeria graminis infection induces penetration susceptibility and suppresses race-specific hypersensitive resistance against avirulent attack in Mla1-barley. Physiological and Molecular Plant Pathology 59:243-256.
  • Dariush Danesh, Narpat Shekhawat, Gaelle Cardinet, Philippe Thoquet, Joann Mudge, Silvia Penuela, Dongjin Kim, Gyorgy Kiss, Hongkyu Choi, Eric Limpens, Richard Zeyen, Thierry Huguet, Douglas R. Cook, Nevin Dale Young . 2001. Integrated Microsatellite Mapping and Powdery Mildew Resistance in Medicago truncatula. 13th International Genome Sequencing and Analysis Conference, San Diego, CA
  • J.M. Lewis, C.A. Mackintosh, S.H. Shin, L.A. Smith, M.N. Wyckoff, A. Elakkad, K. Wennberg, S.J. Heinen, L. E. Radmer, G.D. Baldridge, R.J. Zeyen, C.K. Evans, S. Kravchenko, R. Dill-Macky, G. J. Muehlbauer. 2005. Overexpression of antifungal proteins increases resistance of wheat to Fusarium Head Blight in the field. National Fusarium Head Blight Forum. December 11-13, Milwaukee, Wisconsin
  • Zeyen RJ, Clark TA, Kruger WM. 2004. In situ spatial distribution of chitinase IIB transcripts in attacked barley leaves differs between Mla1 and mlo5 powdery mildew R gene resistances. Phytopathology S114


Progress 01/01/07 to 12/31/07

Outputs
OUTPUTS: This is a research project designed to increase plant disease resistance in cereal plants using molecular techniques. This year we tested overexpression of antifungal genes (disease defense response genes from plants) in transformed wheat and barley plants against Fusarium Head Blight disease(FHB). Transformation cassettes (DNA plasmids with a ubiquitin promoter and selected plant defense response genes from barley) were placed into wheat and, with the assistance of several collaborators these transformed plants were subjected to glasshouse and field evaluations relative to FHB. Bobwhite wheat overexpressing defense response genes αlpha-1-purothionin, thaumatin-like protein 1 (tlp-1), and beta-1,3-glucanase were used. Wheat lines carrying the alpha-1-purothionin, tlp-1, and beta-1,3-glucanase transgenes had statistically significant reductions in FHB severity in greenhouse evaluations. We tested seven transgenic lines under field conditions and found six of them differed from the nontransgenic parental Bobwhite line for at least one disease trait. A β-1,3-glucanase transgenic line lowered FHB severity, DON concentration, and percent VSK compared to Bobwhite. Taken together, results demonstrate that overexpression of plant defense response genes in wheat can enhance FHB resistance under both greenhouse and field conditions. PARTICIPANTS: This year's major participants were all Minnesota Agricultural Experiment Station Scientists or USDA ARS collaborators, and their support staffs and graduate students. Dr. Richard J. Zeyen - professor of Plant Pathology, MAES Saint Paul, MN (project leader) Dr. Gary J. Muehlbauer - professor of Agronomy and Plant Breeding, MAES Saint Paul MN Dr. Ruth Dill-Macky - professor of Plant Pathology, MAES Saint Paul, MN Dr. Deborah Samac - adjunct professor of Plant Pathology - USDA, ARS Saint Paul, MN TARGET AUDIENCES: Wheat and barley growers in the upper Great Plains of the U.S. and Canada, agronomists, plant pathologists, plant scientists, agricultural research administrators, agricultural policy makers . PROJECT MODIFICATIONS: This is the final year of this project. The project terminates on June 30, 2008. There will be no modifications to this project in its final 6 months.

Impacts
Transgenic wheat plants overexpressing naturally occurring plant defense response genes (derived from barley) had enhanced disease resistance to Fusarium Head Blight and helped lower the level of fungal toxins in grain. Genetic control of plant disease resistance in crop plants, whether by advanced breeding or transgenic approaches, reduces fungicide and pesticide usage. It protects farm workers, the consumer and is the most environmentally friendly method of plant disease control. This type of transgenic approach toward otherwise difficult to control fungal caused plant diseases has merit and should continue to be pursued.

Publications

  • Mackintosh, C.A., Lewis, J., Radmer, L.E., Shin, S., Heinen, S.J., Smith, L.A., Wyckoff, M.N., Dill-Macky, R., Evans, C.K., Kravchenko, S., Baldridge, G.D., Zeyen, R.J. and G.J. Muehlbauer, G.J. 2007. Overexpression of defense response genes enhances the resistance of wheat to Fusarium Head Blight. Plant Cell Reports 26:479-488. Miller, C.V., Zeyen, R.J., Samac, D.A. 2007. Infection and development of Phoma medicaginis on moderately resistant and susceptible alfalfa genotypes. Canadian Journal of Plant Pathology 29:290-298.


Progress 01/01/06 to 12/31/06

Outputs
Under field conditions, spores of airborne pathogenic fungi land on and attack the leaf and stem surfaces of plants causing disease. However if the initial attack is unsuccessful then the plant's defenses are 'turned on'. These defenses make the stem and leaf surfaces become much more resistant to subsequent attack. Experimentally we used double inoculation experiments with the powdery mildew fungus of barley to show that the outcome of an initial, inducer attack, dictates what plant epidermal cells responses to subsequent challenger attack will be. If the inducer attack failed, the attacked epidermal cell became totally resistant to later challenger fungal attack and penetration. If the initial inducer attack succeeded, the epidermal cell became totally susceptible to any fungal challenger penetration. We devised experiments aimed at discovering what basic biochemical and molecular processes contribute to these phenomena. We discovered that induced inaccessibility to the powdery mildew fungus, Blumeria graminis, of wheat, barley and oat required active epidermal cell processes. Both phenylpropanoid biosynthesis inhibitors and phosphate scavengers strongly suppressed induced inaccessibility. Silicon (Si) nutrition had no effect on this phenomenon. Thus, active reiterative de novo cell processes, requiring cell memory, are involved in induced inaccessibility. We published these results in the international journal Molecular Plant Pathology.

Impacts
Basic knowledge in understanding the cell processes involved in induced inaccessibility (a form of field resistance to fungi) gave us insights into where genetic engineering might be best applied to produce durable and long-lasting fungal disease resistance. It resulted in using specific antifungal genes like the enzyme chitinase IIb in genetic engineering efforts with oat, wheat and barley. Preliminary results of transformations look promising and a manuscript has been accepted in Plant Cell Reports relative to a degree of resistance to Fusarium head blight of wheat.

Publications

  • Prats E, Carver TLW, Lyngkjaer MF, Roberts PC, Zeyen RJ. 2006. Induced inaccessibility and accessibility in the oat powdery mildew system: insights gained from use of metabolic inhibitors and silicon nutrition. Molecular Plant Pathology (1): 47-59 JAN 2006


Progress 01/01/05 to 12/31/05

Outputs
In the field, spores of airborne pathogenic fungi like the powdery mildew fungus Blumeria graminis are continuously deposited on plant epidermal cells from the atmospheric pool, thus leaves face repeated attack. We used double inoculation experiments to show that the outcome of an initial, inducer attack, dictates what plant epidermal cells responses to subsequent challenger attack will be. If the inducer attack failed, the attacked epidermal cell became totally resistant to later challenger fungal attack and penetration. If the initial inducer attack succeeded, the epidermal cell became totally susceptible to any fungal challenger penetration. Thus, normally avirulent fungi can attack and live long enough to reproduce spores, and if mutations ocurre new races with increased virulence can arise. We studied these two phenomena, induced penetration resistance and induced penetration susceptibility. We devised experiments aimed at discovering what basic biochemical and molecular processes contribute to these phenomena. We discovered that induced inaccessibility to the powdery mildew fungus, Blumeria graminis, of wheat, barley and oat required active epidermal cell processes. Both phenylpropanoid biosynthesis inhibitors and phosphate scavengers strongly suppressed induced inaccessibility. Silicon (Si) nutrition had no effect on this phenomenon. Thus, active and apparently reiterative de novo cell processes, requiring cell memory, are involved in induced inaccessibility. Induced accessibility however appears to be solely modulated by fungal haustoria in infected plant cells. Once the initial fungal inoculum attacks and establishes an infection structure (fungal haustoria) the fungus produces powerful suppressors of epidermal cell defence responses and the attacked cell is then reprogrammed to be highly susceptible to attack by other fungi. Neither inhibitor treatments mentioned above nor Si nutrition affected induced accessibility. Our findings (my collaborators were from Spain, Wales and Denmark) are now accepted for publication the January 2006 issue of the international journal. Molecular Plant Pathology.

Impacts
Basic knowledge gained from this research helps explain how certain "minor" races and variants of fungal pathogens remain in fungal populations. The history of major plant disease epidemics is full of examples where "minor races" have mutated to become major, dominant races causing great damage. On another level, understanding the cell processes involved in induced inaccessiblity gives us insights into where genetic engineering might be best applied to produce durable and long-lasting plant disease resistance to certain fungi.

Publications

  • Zeyen, R.J. and Kruger, W,M. 2005. Fast, intermediate and slow acting barley powdery mildew R genes influence transcription patterns of basal defense response genes. Phytopathology S pp. 122.
  • J.M. Lewis, C.A. Mackintosh, S.H. Shin, L.A. Smith, M.N. Wyckoff, A. Elakkad, K. Wennberg, S.J. Heinen, L. E. Radmer, G.D. Baldridge, R.J. Zeyen, C.K. Evans, S. Kravchenko, R. Dill-Macky, G. J. Muehlbauer. 2005. Overexpression of Antifungal Proteins Increases Resistance of Wheat to Fusarium Head Blight in the Field. National Fusarium Head Blight Forum. Milwaukee, Wisconsin.


Progress 01/01/04 to 12/31/04

Outputs
In 2004 we concentrated on transcriptional regulation of three fungal defense response related genes in barley plants with differing Mendelian ratio powdery mildew resistance genes. These defense response genes were a specific chitinase IIb that attacks fungal cell walls, a fungal induced phenyalanine ammonia lyase gene that is the gateway to many phenylpropanoid based antifungal compounds, and a specific inducible peroxidase involved with cellular oxidative bursts found in most active defenses. In addition we investigated the cellular localization chitinase IIb transcripts during defense responses, using autoradiographic techniques. Finally, we published information on the tissue specificity of a virus promoter, discovered and refined at the University of Minnesota, that we used to drive transcription of our antifungal gene constructs in transgenic plants.

Impacts
Transgenic plants of barley and wheat were produced using information learned though this project. These transgenic plants are in the process of being screened for fungal disease resistance.

Publications

  • Al-Saady NA, Torbert KA, Smith L, Makarevitch I, Baldridge G, Zeyen RJ, Muehlbauer GJ, Olszewski NE, Somers DA. 2004. Tissue specificity of the Sugarcane Bacilliform Virus promoter in oat, barley and wheat. Molecular Breeding 14:1-8.
  • Kruger WM, Szabo LJ, Zeyen RJ. 2003. Transcription of the defense response genes chitinase IIb, PAL, and peroxidase is induced by the barley powdery mildew fungus and is only indirectly modulated by R genes. Physiological and Molecular Plant Pathology 63:167-178.
  • Zeyen RJ, Clark TA, Kruger WM. 2004. In situ spatial distribution of chitinase IIB transcripts in attacked barley leaves differs between Mla1 and mlo5 powdery mildew R gene resistances. Phytopathology 94(6): S114


Progress 01/01/03 to 12/31/03

Outputs
The objective of the project is to understand the physiology and molecular biology of intrinsic fungal disease resistance of cereal crops. In 2003 we concentrated on the molecular bases of penetration and hypersensitive cell death resistances of barley, wheat and oat to powdery mildew fungal pathogens. We used the metabolic inhibitors D-mannose and 2-deoxy-D-glucose and found that they had similar effects on lowering penetration resistance but had strikingly dissimilar effects on hypersensitive cell death responses. Our results suggest that regulation of endonuclease enzymes in intrinsic hypersensitive cell death requires inducible nitric oxide synthase for its expression.

Impacts
We will try to exploit this knowledge to improve our approaches to genetic engineering of fungal disease resistance.

Publications

  • Zeyen RJ, Kruger WM, Lyngkjaer MF, Carver TLW. 2002. Differential effects of D-mannose and 2-deoxy-D-glucose on attempted powdery mildew fungal infection of inappropriate and appropriate Gramineae. Physiological and Molecular Plant Pathology 61(1):315-323.


Progress 01/01/02 to 12/31/02

Outputs
Data analysis if our rapid, plant suspension cell-based protocols for testing antifungal proteins (AFPs) revealed that all AFP's were being expressed maize cells. F. graminearum contact was repelled by the AFP's - Arabidopsis PR5, Fusarium Tri 101 and wheat WIR 2. This repulsion was not evidenced in all transformed cells, and was only moderately so in most transformed cells. Thus, both genetically engineered AFP containing maize suspension cells and adult barley and wheat plants had a degree of Fusarium Head Blight Disease (FHB). Whether the methodology used produces a FHB resistance that is strong enough to do well in field-testing remains unknown. To gain more basic information on general aspects of gene based fungal disease resistance we studied strong naturally occurring resistances in barley and wheat to powdery mildew disease to gain better insight into defense processes used by barley and wheat.

Impacts
Genetic engineering of AFP transgenes enhances FHB resistance. At present genetically engineered FHB resistance, expressed by using constitutive plant viral promoters, may not be strongly enough expressed under field conditions for use in cultivar production. To date genetically engineered FHB resistance using AFPs gives results in glasshouse tests but is "weak" during field tests.

Publications

  • Dariush Danesh Narpat Shekhawat, Gaelle Cardinet, Philippe Thoquet, Joann Mudge, Silvia Penuela, Dongjin Kim, Gyorgy Kiss, Hongkyu Choi, Eric Limpens, Richard Zeyen, Thierry Huguet, Douglas R. Cook, Nevin Dale Young. 2001. Integrated Microsatellite Mapping and Powdery Mildew Resistance in Medicago truncatula. 13th International Genome Sequencing and Analysis Conference, San Diego, CA.
  • Kruger WM, Carver TLW, Zeyen RJ. 2002. Effects of inhibiting phenolic biosynthesis on penetration resistance of barley isolines containing seven powdery mildew resistance genes or alleles. Physiological and Molecular Plant Pathology 61(1):41-51.
  • Lyngkjaer MF, Carver TLW, Zeyen, RJ. 2001. Virulent Blumeria graminis infection induces penetration susceptibility and suppresses race-specific hypersensitive resistance against avirulent attack in Mla1-barley. Physiological and Molecular Plant Pathology 59:243-256.
  • Zeyen RJ, Carver TLW, Lyngkjaer. 2002. Epidermal Cell Papillae. In: Belanger RR, Bushnell WR, Dik AJ, Carver TLW (eds). The Powdery Mildews: A Comprehensive Treatise. APS Press. pp. 107-125. St. Paul, Minnesota USA.
  • Zeyen RJ. 2002. Silicon in Plant Cell Defenses Against Cereal Powdery Mildew Disease. In: Matoh T, Ma JF, Takashashi E (eds). Silicon in Agriculture: The 2nd International Conference on Silicon in Agriculture, The Japanese Society of Soil Science and Plant Nutrition. pp 15-21. Tsuruoka, Japan


Progress 01/01/01 to 12/31/01

Outputs
We developed two rapid, plant suspension cell based protocols for testing antifungal proteins (AFPs) and tested the growth development of the fungal pathogen of wheat and barley against seven different AFPs. We use the Sugarcane Badna Virus promoter - (ScBV - promoter) to drive the following AFP- biolistic plasmid constructs: 1) pBScBV Rchit- rice chitinase; pBScBV TLP1- oat thaumatin like protein; pBScBV Barchit- barley chitinase; pBScBV Barglu- barley glucanase; pBScBv ArabPR5- Arabidopsis PR5; pAHC WIR 2- wheat thaumatin-like protein; and pUBK Tri101-Trichothecene 3-0-acetyltransferase. In our rapid, plant cell assay (microassay protocol) we determined that F. graminearum was repelled to a significant degree only by the following three AFP's: Arabidopsis PR5, Fusarium Tri 101 and wheat WIR 2. None of the other AFP's, either singly or in combination, affected the FHB fungus.

Impacts
The first phase of testing antifungal transgenes to provide resistance to wheat and barley headblight fungus is complete. Greenhouse and field testing phases are now being done to determine efficacy of these tested antifungal genes in transformed wheat and barley.

Publications

  • Hilburn, KLB. 2001. A Plant Suspension Cell Assay for Anti-Fungal Transgene Efficacy. MS Thesis. University of Minnesota 210 pp (RJ Zeyen advisor).


Progress 01/01/00 to 12/31/00

Outputs
Two avenues of basic research into fungal disease resistance were pursued: 1) We developed a plant cell system for rapidly testing the efficacy of antifungal protein genes (AFP's) against Fusarium head blight of wheat and barley (FHB) caused by Fusarium graminearum (Scab disease). We put seven AFPs in biolistic plasmid constructs and tested them in a plant suspension cell system against the growth of the FHB fungus. Only three of seven demonstrated any activity against the growth of F. graminearum. 2) Six of our AFPs were engineered into the whole plant transformation systems in Gary Muehlbauer's laboratory at Minnesota and transformed lines of wheat and barley were made. Once the molecular screening and transgene stabilization is completed these whole plant lines will be tested for FHB resistance and results correlated with our rapid AFP assay system.

Impacts
There is little or no data on the efficacy of various antifungal proteins (AFP) on the wheat and barley headblight fungus. Our objective was to develop a plant suspension culture rapid assay system to speed the selection of AFP's for whole plant transformation. The ultimate goal is to use AFP's to genetically engineer strong FHB resistance in wheat and barley.

Publications

  • 1. R. J. Zeyen, G.D. Baldridge, W.R. Bushnell, K.L.B. Hilburn. 2000. A Microassay Approach to Rapid Antifungal Protein Gene Pretesting. National Fusarium Headblight Forum -2000. Cincinnati Ohio, Dec. 10-12. 4 pp.
  • 2. Hilburn KLB, Bushnell WR, Baldridge GD, & Zeyen RJ. 2000. Toward a plant suspension cell assay for eukaryotic antifungal protein constructs used in cereal transformation. Phytopathology 90(6): S35.
  • 3. Hilburn KLB, Baldridge GD, Bushnell WR, Zeyen RJ. 2000. A Visible Fungal Growth Approach to Rapid Antifungal Protein Gene Pretesting. National Fusarium Headblight Forum 2000. Cincinnati Ohio, Dec. 10-12. 4 pp.
  • 4. Smith, L, M. Wyckoff, G. Baldridge, R. Zeyen and G.J. Muehlbauer. 2000 Antifungal protein gene expression in transgenic wheat (Triticum aestivum). Agronomy Society of America Abstracts


Progress 01/01/99 to 12/31/99

Outputs
Basic research into fungal disease resistance was continued, however emphasis was put on molecular and disease screening of transgenic oat. Oat was transformed singly with barley or rice chitinase antifungal genes, using a plasmid constructs (made in our laboratory). Constructs contained the CaMV 35S promoter and the maize alcohol dehydrogenase I intron to drive transgene transcription. Transgenic lines were selfed after selection by positive Southern, reverse transcriptase polymerize chain reaction (RT-PCR) and Northern blot analysis for transcription of the chitinase transgenes, and by ELISA testing for NPTII selectable marker protein. Transgenic plants (360) and parental controls were screened for powdery mildew resistance with a Minnesota glasshouse isolate of Blumeria graminis f.sp. avenae. The parent oat line, GAF/Park was totally susceptible (4 on a 0-4 scale) while selected transgenic rice or barley chitinase containing lines, in T3 to T5 generations, were rated at 2 to 3 for apparent partial powdery mildew resistance. Disease screening, Northern blotting and RT PCR revealed the existence of barley and rice chitinase transcripts in plants with partial powdery mildew resistance.

Impacts
The objective is to gain better understanding the physiology and molecular biology of genetic-based disease resistance in plants. We use this knowledge to genetically engineer new disease resistance. Engineered genetic resistance is an economical and sustainable method of disease control. It reduces pesticide use, human health risks to farmers and environmental pollution by pesticides now used for plant disease control.

Publications

  • Bushnell WR, Somers DA, Giroux RW, Szabo LJ, & Zeyen R.J. 1998. Genetic engineering of disease resistance in cereals. Canadian Journal of Plant Pathology 20(2):137-220.
  • Van de Mortel, M., Bladridge, G., Bushnell, W., Somer, D., Tobert, K., and Zeyen R. 1999. Apparent partial powdery mildew resistance in transgenic oat genetically engineered to express rice or barley chitinases. Phytopathology 89 (Supplement) 6:S80.
  • Zeyen, R.J. "Chemical elements in epidermal cell defense against Blumeria graminis". Symposium - Plant Cell Biology Related to Defense Response Pathways. American Phytopathological Society and Canadian Phytopathological Society. Montreal, Canada. August 9, 1999. Program Book p. 78.
  • Zeyen, R.J. Papillae and localized inorganic chemical elements in defense. P. 66. First International Powdery Mildew Conference. Avignon, France - August 29-September 2, 1999.
  • Zeyen, R., Kruger, W., Carver T., and Lyngkjaer, M. 1999. Breaking nonhost powdery mildew penetration resistance in small grain cereals. Phytopathology 89 (Supplement) 6:S89.


Progress 01/01/98 to 12/31/98

Outputs
The objective is understanding the physiology and molecular biology of ephemeral(short-lived) and durable (long-lasting) genetic-based fungal disease resistance of cereal crops. We use this knowledge to improve genetic engineering of fungal disease resistance. In 1998 we conducted fundamental research on the mechanism(s) of powdery mildew resistance in barley and oat, including the need for elemental silicon, calcium and manganese as well as contributions of the phenylpropanoid biosynthesis pathway. We used oat and wheat tissue culture to create transgenic lines expressing antifungal genes cloned from alfalfa, rice and barley. These antifungal genes code for hydrolytic enzymes, that attack fungal cell walls. We used the sugarcane bacilliform virus promoter in our plasmid constructs. Fertile adult oat and wheat transformants are expected in 1999.

Impacts
(N/A)

Publications

  • Carver TLW, Thomas BJ, Robbins MP, Zeyen RJ. 1998. Phenylalanine ammonia-lyase inhibition, autofluorescence, and localized accumulation of silicon, calcium and manganese in oat epidermis attacked by the powdery mildew fungus Blumeria graminis (DC) Speer. Physiological and Molecular Plant Pathology 52:223-243.
  • Carver TLW, Robbins MP, Thomas BJ, Troth K, Raistrick N, Zeyen RJ. 1998. Silicon deprivation enhances localized autofluorescent responses and phenylalanine ammonia-lyase activity in oat attacked by Blumeria graminis. Physiological and Molecular Plant Pathology 52:245-257.
  • Zeyen RJ, Kruger WM, Carver TLW. 1998. Barley powdery mildew resistance genes control two different cell death pathways. Phytopathology 88(9) S103.
  • Kruger WM, Zeyen RJ, Saab LJ, Smith AG. 1998. Barley powdery mildew resistance genes exert differential temporal transcription control over two chitinase genes. Phytopathology 88(9) S50.


Progress 01/01/97 to 12/31/97

Outputs
The long-term goal is to produce cereal plants with genetically controlled durable and sustainable fungal disease resistance. In 1997, we continued fundamental physiology and molecular biology research into resistance of biotrophic fungal disease agents. We also extended our genetic engineering efforts in oat for fungal disease resistance. A study of the transcription of genes coding for specific enzymes of the phenylpropanoid biosynthetic pathway, relative to powdery mildew resistance in oat was published. This information is useful for future genetic engineering of disease resistance involving portions of the phenylpropanoid biosynthetic pathway. Progress was made in the molecular screening of transgenic oat plants containing copies of barley and rice chitinase transgenes genes and an alfalfa glucanase transgene. It appears that stable, fertile oat transformants containing the barley chitinase transgenes for putative fungal disease reistance will be produced in 1998 using a popular, midwest adapted, public oat variety as parental material. Collaborative research efforts in the UK continue with NATO funding, and a new collaboration with investigators from Spain was proposed.

Impacts
(N/A)

Publications

  • Lyngjaer, M.F., Carver, T.L.W., Zeyen, R.J. 1997. Suppression of resistance to Erysiphe graminis f. sp. hordei by the mlo5 barley powdery mildew resistance gene. Physiological and Molecular Plant Pathology 50:17-36.
  • Zhang, L., Robbins, M.P., Carver, T.L.W., Zeyen, R.J. 1997. Induction of phenypropanoid gene transcripts in oat attacked by Erysiphe graminis at 20C and 10C. Physiological and Molecular Plant Pathology 51:17-33.
  • Bushnell, W.R., Somers, D.A., Zeyen, R.J. 1997. Genetic engineering of disease resistance in cereals. CPS Winnipeg Conference, June 1997. 1 page.


Progress 01/01/96 to 12/30/96

Outputs
Goals are to produce plants with genetically controlled disease resistance. We couple understanding of the molecular biology & physiology of genetically controlled fungal dis. resistance with genetic engineering research. We continued research & extended our genetic engineering of oat plants. We discovered the resistance of barley/oat to the fungus causing powdery mildew dis. has 2 distinct physiological components. 1 is based on cell death & can be inhibited by stopping specific places in the biochemical pathway of phenylpropanoid synthesis. 2nd is based on failure of the fungus to breach the cell wall of barley/oat. This component isn't affected by blocking biosynthesis in the phenypropanoid pathway. It's broken by using sugar analogues as inhibitors. 2nd component of resistance needs energy because the sugar analogues used sequester phosphate & lower the energy levels of cells trying to mount a penetration defense. Exp. are underway to determine how wide spread this 2nd component of resistance is among the grasses. Incidental to the res. was the finding that one sugar analogue, dexoy-D-glucose has fungicidal properties & may be useful in that role. Exp. will be conducted to determine if dexoy-D-glucose can be used for dis. control in glasshouses. Genetic eng. res. moves forward we are beginning to screen lines of transformed oat for powdery mildew/rust resistance. The prog. is underfunded & relies on grant funding. Res. was enhanced by collab. with res. progs. in the UK through NATO funding.

Impacts
(N/A)

Publications

  • Carver, T.L.W., Zhang, L., Zeyen, R.J., and Robbins, M.P. 1996. Phenolic biosynthesis inhibitors suppress adult plant resistance to Erysiphe graminis in oat at 20C & 10C. Physiological & Molecular Plant Pathology 49:121-142.
  • Bastian, E., Zeyen, R., Kruger, W., and Carver, T. 1996. Breaking in appropriate host powdery mildew resistance using mannose and 2-deoxy-d-glucose. 8th Inter. Cong. - Molecular Plant Microbe Interactions, p. B-8, Knoxville, TX, 14-19 July.
  • Kruger, W., Zeyen, R., Clark, T., Bastian, E., Smith, A., Conicella, C. 1996. Mendelian resistance alleles to barley powdery mildew exert differential spatial control over response gene trans. 8th Int. Cong. Mole. Plt-Microbe Int. p. B-58,
  • Lyngkjaer, M.F., Carver, T.L.W., Zeyen, R.J. 1996. Suppression of mlo5 powdery mildew res.in barley. Proc. 9th Euro. & Med. Cer. Rusts & Powdery Mildew Conf. pp. 2-6. Sept. 1996, Luthern, The Netherlands.


Progress 01/01/95 to 12/30/95

Outputs
The long-term goal is to produce cereal plants with genetically controlled durable & sustainable fungal disease resistance. We couple the understanding of the molecular biology & physiology of genetically controlled fungal disease resistance with genetic engineering research. In 1995, we continued fundamental physiology & molecular biology research & extended our genetic engineering of oat plants. We used microprojectile bombardment of regenerable oat tissue culturs to introduce & "up regulate" genes for rice & barley chitinases & alfalfa B-1,3-glucanase. These are hydrolytic enzymes known to digest walls of fungi and may contribute to fungal disease resistance. Transformations were successful as measured by molecular genetic techniques. If extramural funding levels increase we will begin to further screen oat progeny with molecular probes & test transformed plants & their progeny for resistance to powedery mildew & rust. To "drive" over expression of genes for hydrolytic enzymes, we used a plant virus promoter from the cauliflower mosaic virus (the 35S CaMV promoter). Transformation using 35S leads to gene expression in most plant tissues & may not be particularly effective at the site of fungal attack. An ideal promoter would "turn on & drive" gene expression early during fungal attack, & only in attacked cells, much like these controlling natural response genes.

Impacts
(N/A)

Publications


    Progress 01/01/94 to 12/30/94

    Outputs
    The ultimate goal of this project is to produce cereal plants with genetically controlled durable & sustainable fungal disease resistance, especially those caused by powdery mildew & rust fungi. We have done fundamental studies into the physiology & molecular biology of resistances to powdery mildew in barley & oat. In genetic engineering research, we used microprojectile bombardment of regenerable oat tissue cultures to introduce & "up regulate" genes for rice and barley chitinases and alfalfa B-1,3-glucanase. These are hydrolytic enzymes known to digest walls of fungi & may contribute to fungal disease resistance. Transformations were successful, & fertile oat transformants are now undergoing selection & testing for expression of these introduced genes. In 1995, we will begin to test transformed plants & their progeny for resistance to powdery mildew and rust. To "drive" over expression of genes for hydrolytic enzymes, we used a plant virus promoter (the 35S CaMV promoter). Transformation using 35S leads to gene expression in most plant tissues & may not be particularly effective at the site of fungal attack. An ideal promoter would "turn on & drive" gene expression early during fungal attack, & only in attacked cells. Therefore, we researched response genes whose transcription occurred early and specifically at fungal attack sites in plant tissues. Using northern blot analysis & in situ hybridization, we tested several plant "defense response genes" from barley.

    Impacts
    (N/A)

    Publications


      Progress 01/01/93 to 12/30/93

      Outputs
      The goal of this project is to help breeders and genetic engineers to produce plants with genetically controlled durable and sustainable disease resistance. Two genetically conditioned powdery mildew disease resistances in barley were investigated for sensitivity to blockage of portions of phenolic synthesis pathways. The ephermeral (short-lived) resistance conditioned by the Ml-a locus (to which "races of mildew form to overcome resistance) was "broken" by blockage of the enzyme cinnamyl alcohol dehydrogenase. The durable (long-lived) resistance conditioned by the ml-o locus was not affected by phenolic synthesis inhibition. Thus, there are fundamental differences between genetically conditioned disease resistances, and sensitivity to phenolic synthesis inhibition may have predictive value for breeding better, more durable resistances. Fundamental research into plant defense response gene localization at the site of powdery mildew attack showed that some plant response genes are localized at fungal attack sites in plants. These response genes may have promoter regions that could be utilized in more coherent genetic engineering efforts to engineer new and novel resistances to disease in cereals. Fundamental research was enhanced by collaboration with programs in Wales and Australia whose goals are to produce more durable disease resistances in oat and barley.

      Impacts
      (N/A)

      Publications


        Progress 01/01/92 to 12/30/92

        Outputs
        Powdery mildrew disease resistance phenomena in barley and oats were investigated for their sensitivity to phenolic compound inhibition. The enzymes phenylalanine ammonia lyase -PAL (necessary for general phenolic synthesis) and cinnamyl alcohol dehydrogenase - CAD (necessary for synthesis of lignin precursors) were blocked by specific inhibitors. Inhibition of both enzymes caused certain types of disease resistance to "break", but not all resistances. This indicates that different disease resistances have differing requirements for phenolic compound synthesis and some may have no requirement for phenolic compound synthesis. Specific response gene mRNAS were located in tissues by in situ hybridization procedures. Preliminary indications are that some response genes are tissue-specific while others are not. Basic research was enhanced by collaboration with programs in Denmark, Germany, Australia and in the U.K. The U.K. collaboration was strengthened by a renewal of a NATO collaborative research grant in conjunction with the Welsch Plant Breeding Station. Research findings were shared and collaborative work done on wild rice pathology and sweet corn pathology.

        Impacts
        (N/A)

        Publications


          Progress 01/01/91 to 12/30/91

          Outputs
          Papilla and cell death disease resistance responses in Gramineae (barley and oats) were reinvestigated. cDNA probes of resistance response genes (1 known function and 5 unknown) were used to screen cRNA from discrete time series extractins of total RNA. Times used were 0, 2, 4, 6, 8, 10, 12, 15, 18, 21, 24, 30, 36 & 48 h post inoculation with powdery mildew (PM) pathogens on near isogenic barley lines differing in resistance genes to PM (papilla resistance or cell death resistance). Expression of response genes was much earlier than previously suspected (2 to 4 h after inoculation), and was closely correlated with host cytoplasmic responses to fungal spore development on leaves. Some response genes were consecutively expressed while others were not. The role of phenolics in papilla based and cell death resistancs was tested using enzyme inhibitors of phenolic synthesis. Cell death and "generalized" resistances were "turned off" by phenolic inhibitors. Thus, some resistance response genes may be involved with phenolic synthesis. Work on mineral composition of cells undergoing resistance responses, using x-ray microanalysis and scanning electron miroscopy continues. Collaboration with research programs in Australia and the United Kingdom were strengthened, and a NATO grant made travel between the U.K. and Minnesota possible.

          Impacts
          (N/A)

          Publications


            Progress 01/01/90 to 12/30/90

            Outputs
            Regulation of the papilla and hypersensitive cell death resistance responses in Gramineae (barley and oats) are being investigated. Resistance response genes associated with both cell responses to infection by powdery mildew fungi are being screened using Northern blots. This screening is being done in a very carefully controlled time-course experimental design where RNA is extracted from inoculated plants at 2, 4, 6, 8, 10, 12, 18, 24 and 36 hours after inoculation. The inoculated plant materials are histologically monitored to ascertain what visible cell responses are correlated with timed RNA extractions. Resistance response gene probes include messages for key enzymes in phenylpropanoid synthesis, and some sequenced but as yet unidentified response genes believed responsible for either the papilla or hypersensitive cell death response. Research is being done in collaboration with programs in Australia and the United Kingdom. A key biological question is why some resistance responses in Gramineae confer "durable" (long lasting resistance under field conditions), while other resistances are ephemeral (fail in a gene-for-gene manner, under field conditions). Durable and ephemeral powdery mildew resistances in barley and oats are being investigated initially, since the Mendelian inheritance is already understood and characterized. A recently funded NATO grant provided for exchange of scientists with the Welsh Plant Breeding Station in the U.K., the U.K.

            Impacts
            (N/A)

            Publications


              Progress 01/01/89 to 12/30/89

              Outputs
              The program is in transition, going from cytochemical investigations centering on X-ray microanalysis and moving towards preparing radioactive and non-radioactive probes for specific mRNAs associated with powdery mildew resistance in barley. cDNA clones of the resistance related mRNAs were obtained and probes were prepared for Northern Blot analysis and for in situ hybridization. In situ hybridization procedures are being worked out using several mRNA probes that may or may not be related to disease resistance (RUBISCO & PAL probes) but are good for practicing hybridizations, and are good controls. mRNA probes specific for M1p12 resistance response genes in barley are now available.

              Impacts
              (N/A)

              Publications


                Progress 01/01/88 to 12/30/88

                Outputs
                The role of elemental silicon (Si) in resistance to plant disease was further investigated during 1988. Whole plant experiments, Si-deficient and Si-supplied, were conducted using a barley-powdery mildew system and a wild rice-fungal brown spot system. In both instances resistance to these diseases were greatly increased when plants had adequate Si for their structural needs. Large-scale field experimentation using Si fertilization of these plant species showed that Si ferilization is site-specific, with the greatest responses being found on peat soils with low ash contents (i.e., low mineral contents). Because Si is found in high levels in plant cells that have actively resisted fungal invasion we followed an hypothesis concerning the reactivity of Si with phenolics synthesized by specific gene expression during resistance. We have synthesized several Si-phenolic complexes in vitro and will be investigating their physical properties relative to what is found in resistant plant cells. During 1988, we also worked on perfecting better techniques of tissue preparation for X-ray microanalysis of individual cells. This research should enable us to better identify the role of water soluble chemical elements involved in resistant responses. In 1988, we began background work on in situ localization of "resistance response genes" in plant tissues and shall continue this work relative to the molecular biology and regulation of the papilla and hypersensitive cell death responses in barley and wheat.

                Impacts
                (N/A)

                Publications


                  Progress 01/01/87 to 12/30/87

                  Outputs
                  Resistance to powdery mildew attack in barley containing the Mla locus can be expressed as hypersensitive cell death of individual barley leaf epidermal cells. We researched the temporal relationships between epidermal cell death, onset of blue-light stimulated autofluorescence (indicative of phenolic compound accumulation), and accumulation of insoluble silicon (Si). Physiological cell viability or death was determined by concomitant uptake of the vital dye neutral red, and by cell plasmolysis using alpha methyl-D-glucose. We concluded that autofluorescence accompanies cell death due to release of phenolic compounds from cell vacuoles after host cell membranes lose their semipermeable properties. Insoluble Si accumulation (by energy dispersive X-ray microanalysis) does not correspond directly to the initial appearance of fluorescence in dead cells, but accumulated gradually after death and in apparent relation to increasing autofluorescent intensity. Thus, phenolics are most likely released by decompartmentalization following cell death, accumulate in the wall areas of dead cells and Si complexes with these phenolics as Si moves apoplastically in epidermal cell wall areas due to passive, transpiration stream transport. Si accumulation is, therefore, a secondary feature of hypersensitive cell death. The role of Si nutrition in whole plants presents a different view than that found in hyper-sensitive cell death.

                  Impacts
                  (N/A)

                  Publications


                    Progress 01/01/86 to 12/30/86

                    Outputs
                    Previous research on this project showed barley epidermal cells under attack by powdery mildew fungi rapidly accumulate silicon (Si) at the point of attempted penetration, in conjunction with the "papilla response". Insoluble Si accumulated in papillae appears to be related to the effectiveness of papilla in preventing fungal penetration, but whether the relationship is direct or not is still under investigation. Si accumulation may be linked with the accumulation of phenolic compounds which may be the "effective" ingredient in papillae, and Si is known to form insoluble complexes with certain types of phenolic compounds. Two approaches are being taken to attempt to understand the relationship: 1) growing barley plants with known papilla esponses in the presence and absence of Si nutrition and examining the powdery mildew infection process in these plants to determine if absence of Si results in enhanced infection; and 2) phenolic compounds in papillae autofluoresce when exposed to certain wavelengths of blue light, and we are attempting to correlate the amount of fluoresence with the presence of Si in epidermal cells of barley grown with and without available Si, to determine if Si is an important component of effective papillae. We are using a combination of incident light fluoresence microscopy, scanning electron microscopy, and X-ray microanalysis to accomplish these objectives.

                    Impacts
                    (N/A)

                    Publications


                      Progress 01/01/85 to 12/30/85

                      Outputs
                      The use of X-ray microanalysis coupled with SEM or "bulk" specimens of barley leaves under attack by powdery mildew fungi revealed that the papilla response which prohibits fungal infection contains insoluble silicon. The insoluble Si concentrations of effective papillae are significantly higher than concentrations in ineffective papillae, indicating that Si may be playing a major role in the formation of effective papillae. In frozen-hydrated barley epidermal cells Si is accompanied by several soluble ions that appear in different ratios depending upon the effectiveness of papillae in stopping the fungal ingress. Barley lines containing the recessive mlo locus on chromosome 5 apparently allow for more rapid and apparently "unregulated" deposition of the papilla response. mlo - containing barley lines have apparently "durable" resistance to all known races of barley powdery mildew, thus implicating the papilla response as the "mechanism" of "durable" resistance in these lines. Work is underway to look at the role of insoluble Si in effective papillae from mlo containing barleys.

                      Impacts
                      (N/A)

                      Publications


                        Progress 01/01/84 to 12/30/84

                        Outputs
                        The "papilla" response of barley epidermal cells to powderly mildews continues to be of greatest interest. Chemical ions associated with the response appear to be correlated with success or failure of the papilla in prohibiting fungal ingress. Soluble ion concentrations have been studied using frozen hydrated specimens in an SEM instrument fitted with an energy dispersive x-ray microanalysis unit and a cold stage. Good correlations are emerging relative to soluble ion concentrations and fungal failure in situations where the "resistance" in barley is "race specific", single gene resistance. The origins of insoluble ions like silicon which play an apparent role in the papilla response are being investigated using several novel procedures. At present the movement of Si that contributes to the papilla response in cells is under investigation using TEM and ultrathin sections combined with several analytical procedures.

                        Impacts
                        (N/A)

                        Publications


                          Progress 01/01/83 to 12/30/83

                          Outputs
                          The role of soluble and insoluble ion accummulation in the "papilla response" ofbarley was investigated using x-ray microanalysis on frozen hydrated epidermal cells under attack by powdery mildew fungi. Preliminary data indicate that certain ions are present in higher concentrations when the papilla response is successful at preventing the fungal parasite from penetrating the epidermal cell. Whether these ions are correlated with "general resistance" of "race-specific resistance" is unknown at this time. Further progress has been made in development of computer, assisted cataloging of epidermal cell responses correlated with resistance to powdery mildew fungi in nonhost plants. Background data from this procedure has proven valuable in determining cell defense reactions in plant taxa.

                          Impacts
                          (N/A)

                          Publications


                            Progress 01/01/82 to 12/30/82

                            Outputs
                            Plant cell responses that end in failure of powdery mildew fungi to penetrate epidermal cells were studied in detail. Emphasis was placed on detection of insoluble ions, using SEM and X-ray microanalysis, associated with cell wall appositions that prevent ingress of fungal penetration structures. Apparent correlations between fungal failure and insoluble silicon deposition in plant cells were found in both nonhost plants and in so-called "adult plant resistance". In race specific resistance there was no correlation between the concentration of insoluble silicon, suggesting that race specific or "single gene" resistance to epidermal penetrating fungi proceeds differently in a biochemical sequencing of events from those events in "nonhost" or "adult plant" resistance. Thus, success or failure of fungal parasites in race specific resistance relies on incompatibility events occurring very early in attempted fungal penetration. The reason race specific resistance can be readily overcome by race changes in the fungal parasite may be more related to the fungus being the "not to trigger" certain host cell events early in the parasitic process.

                            Impacts
                            (N/A)

                            Publications


                              Progress 01/01/81 to 12/30/81

                              Outputs
                              In 1981 efforts centered upon constructing an interactive computer-based system for transferring large numbers of microscopic observations on stages of fungal development and host cell responses directly to a "logic tree" program that allows for sorting and statistical analysis of thousands of observations. This system when coupled with recent advances in histological technique enables us to determine the critical stages of fungal parasite development where failure resulting from plant immunity acts. In addition, certain host epidermal cell responses, highly correlated with failure of powdery mildew fungi to infect specific grass hosts are being studied in detail using newly developed light microscopic staining procedures, SEM, and energy dispensive X-ray microanalysis on the same cells to determine if certain naturally occurring chemical elements play a definite role in cell defense mechanisms. The goal of this work is to lay the background for the manipulation of plant nutrition as it is related to disease resistance, and to develop analysis procedures for determining the action of exgenously applied chemical agents to plants.

                              Impacts
                              (N/A)

                              Publications


                                Progress 01/01/80 to 12/30/80

                                Outputs
                                In 1980, large amounts of data concerning the defense patterns of monocotyledenous plant species to powdery mildew fungi (adapted to both monocotyledenous and dicotyledenous hosts) were processed and analyzed. The results from 15 monocotyledonous plant species from 4 tribes of Gramineae, the Iridaceae and Lilieceae showed that the defense patterns conferring disease immunity differed according to the adaptiveness of the fungi (Monocotyledenous or dicotyledenous adapted) and patterns also emerged related to the taxonomic relatedness of the plant species tested. Powdery mildews not adapted monocotyledenous plant species have developmental problems related to their ability to cope with monocotyledenous cuticular waxes, cuticle, cell walls and the papilla response of epidermal cells. Powdery mildew adapted tommonocotyledenous hosts (in this case adapted to barley), exhibit developmental problems similar to those found in the dicotyledenous adapted mildew when the Gramineae tested are distantly related to barley; however, as taxonomic distance is narrowed, the defense pattern of Gramineae rests mainly upon the papilla response of the epidermal cells. Applied work on corn rust and certain corn virus problems are also included in this progress report.

                                Impacts
                                (N/A)

                                Publications


                                  Progress 01/01/79 to 12/30/79

                                  Outputs
                                  Determinative Plant Virology - the entire research emphasis of the program was devoted to maize dwarf mosaic virus (MDMV) and its impact on field corn and sweet corn production in Minnesota. Areas specifically investigated were: a) the impact of MDMV on sweet corn yields, b) grass hosts that may harbor the virus from year to year, c) state-wide survey of sweet corn processing fields for MDMV infection, d) improvement of serological tests used to identify infected plants, and e) preliminary breeding for MDMV resistance in sweet corn. A simple and highly accurate serological test for identification of MDMV and its 2 major strains was developed and utilized. Yield loss experiments demonstrated that loss is related to the stage of growth of the plants when infected, the younger the plant infected the more severe the yield loss. Breeding programs for MDMV resistance showed 2 Mendalian patterns of resistance, oligogenic-dominant for MDMV-"A" and oligogenic recessive for MDMB-"B". The combining ability of field corn resistance to sweet corn inbreds was excellent and F(3) populations showed promise for development of good MDMV resistance in sweet corn. Histopathology - The cytological basis for resistance in barley, to parasitic fungi whose primary mode of attack is through epidermal cells, was clarified and linked to a specific cell response, the papilla response, when the M1/a2 gene for resistance is operative.

                                  Impacts
                                  (N/A)

                                  Publications


                                    Progress 01/01/78 to 12/30/78

                                    Outputs
                                    Determinative plant virology - In 1976 maize dwarft mosaic virus was discovered in Minnesota, in 1977 MDMV reached epidemic proportions in sweet corn causing an estimated 2.5 million dollar loss to the processing growers of the state. The majority of research time in 1978 was spent identifying viral isolates, purifying viral strains, preparing antisera for detecting virus infected plants and for determining the effect on yields caused by MDMV infections of commercial cultivars relative to the stage of plant growth at the time of infection. A survey of field border and roadside grasses, located near fields heavily infected with MDMV in 1977, was completed to see if any of the common annual or perennial grasses act as a reservoir for MDMV. In a separate study, Great Plains weather patterns surrounding the 1977 epidemic are being analyzed to determine if the MDMV inoculum for the 1977 epidemic could have come from massive flights of winged aphid vectors. Histopathology - Microscopically observed host-pathogen events surrounding the phenomena known as plant immunity, or inappropriate host resistance, to powdery mildew fungi were analyzed for 38 plant species. The binary pathway system of analysis used in these investigations was submitted and accepted for publication, and a set of manuscripts describing plant immunity to powdery mildew fungi is in preparation. A study of fungal spore ontogeny in Botryodiplodia theobromae was begun on this important facultative plant parasite.

                                    Impacts
                                    (N/A)

                                    Publications


                                      Progress 01/01/77 to 12/30/77

                                      Outputs
                                      Determinative plant virology - In 1976 maize dwarf mosaic virus (MDMV) was firstfound and characterized in Minnesota. In 1977 MDMV reached epidemic proportions in late planted sweet and field corn. A considerable amount of time was spent in isolating and identifying MDMV in Minnesota and in following the progress of the epidemic. A combination of University of Minnesota and processing industry data indicate that the epidemic in sweet corn cost growers over 2.5 million dollars in reduced yields. Characterization of the hydrangea ringspot virus was completed and published. Additional time was spent on the virus transmitting capabilities of insects (leafhoppers) that survive high level dosages of insecticides. It was determined that insecticide stimulation (carbaryl, DDT and Fenthion) of virus transmission at statistically significant levels occurs regardless of the age or sex of the vectors. Histopathology - Generalized resistance to powdery mildew fungi was studied extensively. Computerization and statistical methods for determining resistance types were refined final analysis of resistance events for 8 families of plants was completed and several manuscripts relative to this work are being prepared. The study of spore ontogeny in the fungus Septoria nodorum, the cause of leaf, stem and head blotch of wheat, was completed.

                                      Impacts
                                      (N/A)

                                      Publications


                                        Progress 01/01/76 to 12/30/76

                                        Outputs
                                        Determinative plant virology - Emphasis was placed on the virus transmitting capabilities of insects (leafhoppers) that survived high level (LD 80) dosages of insecticides. Three insecticides were used; DDT, carbaryl, and Fenthion. All three insecticides stimulated virus transmission to plants by leafhoppers for periods of 10 to 13 days following insecticide exposure at LD 80 levels for 2 hr. In the area of diagnosis of virus diseases found in Minnesota several diseases were investigated. The most important new virus disease found in Minnesota in 1976 was maize dwarf mosaic virus; this disease agent was isolated from 2 separate geographical areas and is the first reported incidence of the disease in Minnesota. Characterization of the strain of MDMV continues in cooperation with Iowa State University. Histopathology - Generalized resistanceto powdery mildew fungi were studied extensively. We succeeded in designing computerized sampling and statistical methods that enabled us to define resistance events operating before hypersensitive collapse of epidermal tissues.Final analysis of resistance events for 8 families of plants is currently underway and should be completed early in 1977. The second year of study of spore formation in Septoria nodorum, that causes leaf, stem and head blotch on wheat demonstrated that this fungus has at least 2 mechanisms of spore formationin its fruiting structures. The ability to form spores continually from affected tissues has led to a much better

                                        Impacts
                                        (N/A)

                                        Publications


                                          Progress 01/01/75 to 12/30/75

                                          Outputs
                                          Determinative plant virology - Emphasis was placed on diagnosis of plant virus disease occuring in Minnesota and on the virus transmission capabilities of leafhoppers. In the area of diagnosis 51 samples (mostly derived from the PlantDisease Clinic of the Dept. of Plant Pathology) were investigated. Most of the diagnosis stressed floricultural plants although samples of corn, soybeans, peasand potatoes were examined. Work on the virus transmission by leafhoppers that survive insecticide applications was instigated and showed that insects surviving insecticides at the LD 80 level were better transmitters than untreated populations. Histopathology - Generalized resistance mechanisms of plants to powdery mildew fungi are being studied. We have succeeded in designing sampling and statistical methods useful in defining resistance mechanisms operating before hypersensitive collapse of epidermal tissues. Fruiting structures of 2 Septoria fungi, important for their attack on wheat, are being investigated relative to spore formation and release. Work on the Septoria species is aimed at a better understanding of spore formation and release relative to the epidemiology of disease caused by these fungi.

                                          Impacts
                                          (N/A)

                                          Publications


                                            Progress 01/01/74 to 12/30/74

                                            Outputs
                                            Determinative plant virology - Major effort of first six months of project concentrated on diagnosis of plant virus diseases occurring in Minnesota. Over 50 clinical cases were handled using limited plant host ranges and electron microscopy as diagnostic procedures. Virus diseases were diagnosed in corn, soybean, tomato, pea, beans, potato and various floricultural crops. Most commonly diagnosed viruses were tobacco mosaic virus in tomato and soybean mosaic virus in soybean. Unexpected numbers of floricultural crops such as chrysanthemum were found to be infected with viruses. On the basis of the typesand diagnosis of diseases, an improved set of differential hosts to identify predominate virus diseases in Minnesota is being assembled. Histopathology - Generalized resistance mechanisms of plants to powdery mildew fungi and Septoriafungi attacking cereal crops is being studied. The observations made are being combined with valid statistical designs to yield information useful in defining resistance mechanisms. Improved observation methods using light and electron microscopy have been stressed.

                                            Impacts
                                            (N/A)

                                            Publications