PNNL-13501 - Pacific Northwest National Laboratory

Plant Root Exudates and Microbial Gene Expression in the Rhizosphere
Study Control Number: PN99056/1384
Mark T. Kingsley
Nucleic acid arrays are widely used in the pharmaceutical industry to provide information on differentially expressed
genes when screening drugs and drug candidate compounds. Nucleic acid, DNA array analyses provide an automatable,
compact, discrete, reproducible format for analyzing genomic information of microbes and higher organisms. Gene chips
are also being developed for the specific identification of organisms by both government and industry. This project
explored the use of an array based on short oligonucleotide targets, for identifying agricultural pathogens by differential
pattern development and recognition.
Project Description
This research involved 1) developing DNA microarray
analytical methods for 16S rRNA from environmental
samples to rapidly identify members of microbial
communities, and 2) genotyping arrays for environmental
organisms and potential food pathogens, and
“fingerprinting” arrays for discriminating closely related
organisms. We are developing fluorescent polymerase
chain reaction analytical methods for agricultural crop
pathogens (Kingsley 2000; Kingsley and Fritz 2000) that
could have the potential to be employed in agricultural
bioterrorism. DNA microarrays offer a unique ability to
provide inexpensive, rapid, and specific identification of
microbes. Since productivity of renewable energy
biomass sources (crop, forest) can be drastically limited
by bacterial, fungal, or viral pathogen attack, rapid
methods for the specific identification of the causitive
agent(s) is desirable. The high density of diagnostic
probes achievable on microarrays provides for a highly
robust analytical tool. The array technology can be
integrated into online analytical devices currently under
development. A goal of this project was to develop
appropriate DNA diagnostic suites of oligonucleotides for
agricultural and forest pathogens. As part of this initial
proof-of-concept demonstration, we tested microarrays
for analysis of bacterial pathogens in the genus
Xanthomonas and fungal pathogens in the genus
Fusarium. Some members of the genus Fusarium, in
addition to leading to crop productivity losses, produce
mycotoxins which are toxic and/or carcinogenic to
humans or animals.
Introduction
The development and use of nucleic acid arrays in
detection, genotyping, and analysis of gene expression, is
increasing rapidly (Behr et al. 1999; Cockerill 1999;
Tao et al. 1999; Westin et al. 2000). Currently, many
groups are developing DNA arrays for microbial
detection and identification. Genetic arrays, once
prototyped and developed, offer extremely robust, yet
relatively inexpensive means (the array itself is cheap) for
identifying organisms. Arrays offer the ability to create
multiplexed (combined) assays providing for the analysis
of multiple targets simultaneously. Exploiting this
powerful analytical tool for analysis of phytopathogens
has not been reported. This project aims to explore the
use of DNA microarrays for differentiating and
identifying various Xanthomonas (bacterial) and
Fusarium (fungal) phytopathogens.
Since agricultural and forest productivity can be
compromised by rhizosphere inhabiting pathogens such as
the Fusarium species, and foliar pathogens such as
Xanthomonas, rapid and sensitive methods for analysis of
seeds, plant tissues, and soils by DNA microarrays offers
the benefit of a highly accurate method of detection and
pathogen identification. Often these pathogens remain
undetected until the plants are symptomatic, which often
is too late. Preventing crop losses due to early pathogen
detection could become an important issue for DOE’s
Bioenergy initiative where the goal is to triple the use of
renewable crops for fuels and feedstock by the year 2010.
Phytopathogen analysis, especially of orgranisms in the
bacterial genus Xanthomonas and the fungal genus
Fusarium would benefit greatly from the power of nucleic
acid array analysis. Numerous clonally derived species
and subspecies are known within both Fusarium and
Xanthomonas. Historically, these organisms were classed
according to their pathogenicity to particular hosts. Issues
of genetic relatedness to other members of the genus that
attacked different hosts were ignored. Only with the
advent and power of nucleic acid analysis have a number
of issues of taxonomic position and relatedness been
Biosciences and Biotechnology 91