Grantee Research Project Results

Final Report: Chemical Induction of Disease Resistance in Trees

EPA Grant Number: R829479C025
Subproject: this is subproject number 025 , established and managed by the Center Director under grant R829479
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: Water Innovation Network for Sustainable Small Systems
Center Director: Reckhow, David A.
Title: Chemical Induction of Disease Resistance in Trees
Investigators: Davis, John M.G.
Institution: University of Florida
EPA Project Officer: Aja, Hayley
Project Period: October 1, 2004 through September 30, 2007 (Extended to December 31, 2007)
RFA: The Consortium for Plant Biotechnology Research, Inc., Environmental Research and Technology Transfer Program (2001) RFA Text |  Recipients Lists
Research Category: Hazardous Waste/Remediation , Targeted Research

Objective:

The overall objective of this project was to determine if pine genotypes differ in their competence to respond to putative chemical signaling molecules that may be involved in disease resistance.

The specific aims of this project were to:

1.1 Screen > 1,000 clonally propagated genotypes of loblolly pine for resistance to the commercially important diseases fusiform rust and pitch canker. The work was to be done in conjunction with ongoing studies by the Forest Biology Research Cooperative and the Resistance Screening Center (RSC) in Asheville, North Carolina (Completed).

1.2 Select > 25 genotypes that exhibit the most extreme response types (highly resistant and highly susceptible) and repeat the screen by hand to confirm the disease resistance and susceptibility of phenotypes (Completed).

1.3 Evaluate the competence of highly susceptible genotypes to acquire resistance to disease after pre treatment with the putative chemical signaling molecule salicylic acid and correlate this response to anatomical/physiological parameters (Completed).

1.4 Compare and contrast the gene expression response of highly resistant and highly susceptible genotypes to salicylic acid treatment (Modified and Completed).

Summary/Accomplishments (Outputs/Outcomes):

Specific Aim 1.1

Disease screening for both fusiform rust and pitch canker was completed. There were four separate screening tests that were accomplished in a common blocked and replicated design (Table 1):

(1) “Ten-gall” inoculum of Cronartium quercuum f.sp. fusiforme, collected from ten galls at several locations in the lower coastal plain;

(2) “One-gall” inoculum of C. quercuum f.sp. fusiforme, collected from a single gall in the lower coastal plain;

(3) Single-isolate (designated S45) inoculum of Fusarium circinatum, with screening performed at the RSC ; and

(4) Single-isolate (S45) inoculum of F. circinatum, with screening performed at the University of Florida (UF) in Gainesville, Florida .

Screens (1) and (2) were carried out using the concentrated basidiospore suspension system that is standard practice at the RSC. Screens (3) and (4) differed in that the former screen was performed at the RSC using standard clipping and spray inoculation of plants after removal of severed shoots, whereas in the latter screen at UF we used a tip inoculation method as well as a micro-inoculation technique on the sides of succulent shoots.

Table 1. The Design for Fusiform Rust and Pitch Canker Experiments.

Design	Number of Families	Number of Clones	Range of Clone/Family	Number of Blocks
Ten-gall Mix fusiform rust	63	1360	17-31	5
One-gall fusiform rust	63	698	2-30	5
RSC pitch canker	63	1065	7-31	5
UF pitch canker	60	362	1-24	5

Prior to analysis, datasets were standardized by dividing each data point by the standard deviation of the dataset. The following linear model was used to calculate the variance components and genetic parameters for each trait:

Y = μ + rep + tray(rep) + GCA + SCA + clones(family) + family*rep + error

Variance components and genetic parameters were estimated by GAREML, which employs restricted maximum likelihood (REML) estimation and best linear unbiased prediction (BLUP). The clonal means were calculated by summing GCA + SCA + clone(family).

Pitch Canker. Data were collected on the UF pitch canker experiment in August 2002 and on the RSC pitch canker experiment in early December 2002. Data collection is not trivial in a study of this size. For example, disease lesion length and total shoot length were measured on a randomly chosen shoot on each ramet in the RSC trial. Since there were 3-5 ramets per clone, and approximately 1,203 different clones, we obtained lesion data on roughly 4,500 shoots in this single experiment.

Genetic parameters, including clonal and family means along with their rankings, heritabilities, and Type B genetic correlations were calculated for the RSC and UF trials. The broad sense heritabilities of pitch canker resistance were 0.44 (narrow sense = 0.27) in the RSC screen, and 0.35 (narrow sense = 0.27) in the UF screen. The heritabilities estimate the proportion of the trait variation that is attributable to genes, with the remainder being explained by environmental noise.

Fusiform Rust. Data were collected on the broad rust screen in March 2003. Phenotypic measurements included the presence or absence of galls, the number of galls, the number of receptive shoots inferred to be present at the time of inoculation, the number of receptive shoots with galls, and gall dimensional characteristics (gall length, gall width, and width of the healthy stem for two randomly chosen galls per ramet) for a total of nine measurements per ramet. Since the total number of ramets measured was approximately 5,500, this experiment has yielded 49,500 data points.

Genetic parameters were measured for rust traits. The most heritable trait was gall score, indicating gall presence or absence. Broad sense heritabilities for gall score were 0.43 (narrow sense = 0.19) for the ten-gall inoculum, and 0.50 (narrow sense = 0.24) for the one-gall inoculum. It is notable that the epistatic component of the broad sense heritability was quite high for the ten-gall screen (0.18) and the one-gall screen (0.21), which means that the additive and epistatic components were about equal in magnitude in both screens. This large epistatic component may be due to the presence of multiple avirulence genes in the inocula, which are revealing corresponding R genes in the host population. We have confirmed through simulation modeling that the observed epistatic components can be generated if at least three avirulence genes are present in the pathogen population. These data are consistent with the known existence of multiple R genes in loblolly pine that confer specific resistance to C. quercuum f.sp. fusiforme.

The second most heritable trait was gall length. For the ten-gall screen, the broad sense heritability for gall length was 0.23 (narrow sense = 0.13), whereas for the one-gall screen the broad sense heritability was 0.26 (narrow sense = 0.22). Gall length may reflect partial resistance in the host, and if this model is correct (as has been observed in analogous pathosystems), then gall length may be inherited as a quantitative trait that is race non specific. Therefore, when we conducted our acquired resistance experiments in 2004, we measured both gall score and gall length to determine if acquired resistance had occurred. Based on the known lack of race (and pathogen) specificity of systemic acquired resistance, we anticipate that gall length will be a more reliable indicator of its onset in pines.

Type B genetic correlations of clonal ranks showed that resistance to fusiform rust and pitch canker diseases were completely uncorrelated. Thus, breeding of pine trees for resistance to one disease does not affect resistance to the other disease.

Specific Aim 1.2

Based on the high genetic correlation (0.88) between the RSC and UF pitch canker studies, data were merged to identify the 25 most susceptible and 25 most resistant clones. The genotypes that comprise these “tails” were transported to UF, up-potted, and used in a confirmatory inoculation study that was conducted in October of 2003. The tissue samples from this confirmatory study were flash frozen in liquid nitrogen and placed in a -80°C freezer, then used later to create complementary DNA (cDNA) libraries (see Specific Aim 1.4).

Specific Aim 1.3

Spray and root drench treatments of salicylic acid (SA) were toxic to pine seedlings and made it difficult to determine appropriate doses that would mimic physiologically relevant endogenous levels. Toxicity was manifested by browning of leaf tips resulting from apparent tissue desiccation. Acquired resistance studies did not reveal differences between genotypes with respect to response to exogenous SA, which led us to alter Specific Aim 4. We reasoned that since the overarching goal of this project was to understand the molecular basis of pine genotype differences in response to pathogen challenge, we would generate cDNA libraries from highly resistant and highly susceptible genotypes after pathogen challenge instead of after treatment with SA.

Specific Aim 1.4

To meet this objective, we carried out a gene expression array study that focused on the susceptible host response to pathogen challenge (Morse, et al., 2004) and generated cDNA libraries to further quantify the molecular characteristics that distinguish resistant and susceptible pine genotypes (see below).

cDNA libraries create a “snapshot” of gene expression at the time of tissue harvest. In this case, we captured tissues after infection by the pathogen but prior to the manifestation of full-blown disease symptoms in the susceptible host. A comparison of the genes expressed in the resistant and susceptible hosts would then reveal differences in the molecular machinery that ultimately conditions these distinct responses to pathogen challenge.

The libraries were prepared from polyA+ RNA isolated from juvenile stem material of loblolly pine (Pinus taeda) hedges challenged with the necrotrophic fungus F. circinatum. Hedge plants were fertilized monthly and inoculated in November 2003. Shoot tips of succulent stems were inoculated with 1 μL of a 500 spores/μL solution after excision. The terminal 1 cm of each challenged stem was harvested for mRNA isolation 10 days after challenge. The 10 -day time point was selected because the beginning of tissue necrosis was observed in the region within 1 mm of the inoculation site in the susceptible host. Thus it was clear that disease progression had been initiated in the susceptible host, and by extension pathogen infection would have occurred (or been initiated) in the resistant host. Samples for the susceptible library, STRS1 (for STem Response Susceptible), were collected from genotypes 40149, 40036, and 40177. Samples for generating the resistant library, STRR1 (STem Response Resistant), were harvested from genotypes 40414, 40092, 46102, and 40071. Tissue samples were bulked prior to RNA extraction and library synthesis. The library was made at the University of Georgia by Walt Lorenz as a collaboration between our laboratory and the laboratories of Jeff Dean, Lee Pratt, and Marie-Michele Cordonnier-Pratt.

Sequence information is collected from these types of projects in the form of expressed sequence tags (ESTs). ESTs are strings of DNA sequence that represent genes actively expressed, or turned “ on” in the tissue at the time of harvest. ESTs can be obtained from either the upstream portion of an expressed gene (a 5’ EST) or the downstream portion of an expressed gene (a 3’ EST). In total, 5,067 3’ ESTs and 5,466 5’ ESTs were obtained from the resistant library, whereas 5,011 3’ ESTs and 5,631 5’ ESTs were obtained from the susceptible library, for a total of 21,175 ESTs. These ESTs collapsed into a total of 8,961 unique transcripts, after alignment of the EST sequences was performed to adjust for redundant sampling of the same expressed genes.

Comparison of ESTs from each library —resistant host and susceptible host —is continuing in order to identify genes that are differentially regulated in the two libraries. In particular, we are interested in genes represented in the resistant library, but not in the susceptible library, since these are genes expressed in association with a resistance response to pathogen challenge.

Conclusions:

• Pitch canker and fusiform rust resistance are heritable traits in loblolly pine, and there was abundant natural genetic variation for both traits in the study population.
• Clonal propagation allowed precise estimation of disease phenotypes.
• Pitch canker and fusiform rust resistance traits are genetically uncorrelated.
• Gene expression profiles in resistant and susceptible hosts differ, as do gene expression events that occur in response to challenge by the pitch canker and fusiform rust pathogens.

Journal Articles on this Report : 2 Displayed | Download in RIS Format

Publications Views

Other subproject views:	All 2 publications	2 publications in selected types	All 2 journal articles
Other center views:	All 208 publications	48 publications in selected types	All 44 journal articles

Publications

Type	Citation	Sub Project	Document Sources
Journal Article	Kayihan GC, Huber DA, Morse AM, White TL, Davis JM. Genetic dissection of fusiform rust and pitch canker disease traits in loblolly pine. Theoretical and Applied Genetics 2005;110(5):948-958.	R829479 (Final) R829479C025 (Final)	Abstract from PubMed Full-text: SpringerLink Full Text Exit
Journal Article	Morse AM, Nelson CD, Covert SF, Holliday AG, Smith KE, Davis JM. Pine genes regulated by the necrotrophic pathogen Fusarium circinatum. Theoretical and Applied Genetics 2004;109(5):922-932.	R829479 (Final) R829479C025 (Final)	Abstract from PubMed Full-text: SpringerLink Full Text Exit

Supplemental Keywords:

pinus, pitch canker, fusiform rust, disease resistance, acquired resistance,

Relevant Websites:

http://dendrome.ucdavis.edu/adept/ Exit
http://davis_lab.ifas.ufl.edu/ Exit
http://www.fungen.org/Projects/Pine/Pine.htm Exit

Progress and Final Reports:

Original Abstract

2005

2006

2007

Main Center Abstract and Reports:

R829479    Water Innovation Network for Sustainable Small Systems

Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R829479C001 Plant Genes and Agrobacterium T-DNA Integration
R829479C002 Designing Promoters for Precision Targeting of Gene Expression
R829479C003 aka R829479C011 Biological Effects of Epoxy Fatty Acids
R829479C004 Negative Sense Viral Vectors for Improved Expression of Foreign Genes in Insects and Plants
R829479C005 Development of Novel Plastics From Agricultural Oils
R829479C006 Conversion of Paper Sludge to Ethanol
R829479C007 Enhanced Production of Biodegradable Plastics in Plants
R829479C008 Engineering Design of Stable Immobilized Enzymes for the Hydrolysis and Transesterification of Triglycerides
R829479C009 Discovery and Evaluation of SNP Variation in Resistance-Gene Analogs and Other Candidate Genes in Cotton
R829479C010 Woody Biomass Crops for Bioremediating Hydrocarbons and Metals
R829479C011 Biological Effects of Epoxy Fatty Acids
R829479C012 High Strength Degradable Plastics From Starch and Poly(lactic acid)
R829479C013 Development of Herbicide-Tolerant Energy and Biomass Crops
R829479C014 Identification of Receptors of Bacillus Thuringiensis Toxins in Midguts of the European Corn Borer
R829479C015 Coordinated Expression of Multiple Anti-Pest Proteins
R829479C016 A Novel Fermentation Process for Butyric Acid and Butanol Production from Plant Biomass
R829479C017 Molecular Improvement of an Environmentally Friendly Turfgrass
R829479C018 Woody Biomass Crops for Bioremediating Hydrocarbons and Metals. II.
R829479C019 Transgenic Plants for Bioremediation of Atrazine and Related Herbicides
R829479C020 Root Exudate Biostimulation for Polyaromatic Hydrocarbon Phytoremediation
R829479C021 Phytoremediation of Heavy Metal Contamination by Metallohistins, a New Class of Plant Metal-Binding Proteins
R829479C022 Development of Herbicide-Tolerant Energy and Biomass Crops
R829479C023 A Novel Fermentation Process for Butyric Acid and Butanol Production from Plant Biomass
R829479C024 Development of Vectors for the Stoichiometric Accumulation of Multiple Proteins in Transgenic Crops
R829479C025 Chemical Induction of Disease Resistance in Trees
R829479C026 Development of Herbicide-Tolerant Hardwoods
R829479C027 Environmentally Superior Soybean Genome Development
R829479C028 Development of Efficient Methods for the Genetic Transformation of Willow and Cottonwood for Increased Remediation of Pollutants
R829479C029 Development of Tightly Regulated Ecdysone Receptor-Based Gene Switches for Use in Agriculture
R829479C030 Engineered Plant Virus Proteins for Biotechnology