Prioritized Listing of Microbial Biological Control Agents to be Included  on the

APS Microbial Genome Sequencing List

 updated 08/19/03 - draft revision

The members of the APS Biological Control Committee, in consultation with a broad contingent of scientists studying the biological control of plant pathogens and weeds, are putting forward the following list of microbes for inclusion on the APS microbial genome sequencing priority list. Representative strains from several prokaryotic and one eukaryotic genera are included.   Those strains considered to be the highest priority for genomic sequencing are listed in italics, and additional arguments for their inclusion are presented below.  This document begins with a general justification for the inclusion of biological control agents that target plant pathogens.  In summary, the members of the committee assert that significant investment in genomic analyses of biological control agents will lead to increased efficacy and application of biological controls of plant pathogens.

Economic relevance of microbial biological controls agents to agriculture.

Meeting the goal of improving plant disease control for efficient and sustainable production systems will require an increased reliance on biologically-based pest controls.  Every year, plant pathogens are responsible for the loss of 10 to 20 percent of agricultural production world wide despite the several billion dollars spent controlling them with synthetic chemicals.  While synthetic toxins have their place in disease control, there is a growing awareness that integrated pest management (IPM) (or biologically-based pest management, BBPM) strategies provide more environmentally sound and economically viable alternatives for agriculture. Thus, biological controls of plant pathogens have been identified as integral components of IPM strategies.  Primary targets for control are fungi, nematodes, and oomycete pathogens of field, nursery, and glasshouse crops.  Approximately two dozen products are currently on the market, but sales of such products represent only a small fraction of the total pesticides sold for controlling plant pathogens in the United States.  Despite years of research and development, significant questions regarding the physiological and ecological constraints that limit biological controls remain unanswered.  New molecular and genomic tools offer new possibilities for improving the selection, characterization, and management of biological controls. 

Unique biological or environmental features.

            Genomic studies of biological control agents will provide insights into the basic biology of both the microbes and their plant hosts.  The biocontrol strains identified below come from microbial genera that are both well studied and widely distributed in nature.  BCAs are typically applied as epiphytic colonists, but in many cases they also colonize internal tissues.  Systematic investigations of the molecular mechanisms by which BCAs colonize and protect plants from pathogens can now be done with genomic tools. In addition, genomic studies of biological control agents will provide fundamental insights into the microbial ecology of the phytosphere (i.e. the environment immediately surrounding and including the plant), which encompasses the primary loci of biological control. Whether acting by competitive exclusion, biochemical antagonism, or induction of host defenses, biological control agents must be well adapted for survival and functional activity in the phytosphere.

Broad interest to a significantly sized community of scientists and agriculturists.

Biological control is an important area of focus in the discipline of Plant Pathology. Every major university with a department of Plant Pathology has one or more faculty members conducting basic and/or applied research on biological control organisms.  This emphasis has led to the publication of over 4500 articles in peer-reviewed journals during the past 20 years.  There is also continued interest among conventional and organic producers in developing effective biorational controls.  Because of the development of new diseases and the shifting patterns of management and regulation, much research still focuses on the isolation and testing of new biocontrol agents.  However, a large number of researchers are also engaged in fundamental studies of the molecular genetics, physiology, and ecology of biocontrol microbes.  The goals of such research are to improve the efficacy and applicability of biological controls in agricultural production systems of all types.

Genetic tractability of selected microorganisms

The molecular tools required for genetic analyses of the selected organisms already exist and are described in peer-reviewed journals. In some cases, extensive genetic analyses and manipulations have been conducted by biocontrol researchers.  This is true for all strains identified as top priorities for sequencing.  However, in some cases, the tools and expertise exist but not among researchers focused primarily on biological control of plant pathogens. This committee has decided that in order to elevate a strain to priority status, the contact individual(s) must demonstrate that such expertise exists in their laboratory or with a named collaborator. In this way, no substantive technical barriers will hinder further investigations based on the genomic sequence information obtained.

McSpadden Gardener, B., Fravel, D. 2002. Biological control of plant pathogens: Research, commercialization, and application in the USA. Online. Plant Health Progress doi:10.1094/PHP-2002-0510-01-RV.

Recommended List of Biological Control Agents for Genomic Sequencing:

Assembled October, 2002 by the APS Biological Control Committee

Revised August, 2003 by the APS Biological Control Committee

Strains noted as "Immediate Priority" and "High Priority" for genome sequencing are listed in italics

Detailed Justifications:

Bacillus cereus strain UW85

            Bacillus cereus strain UW85 is an effective biocontrol agent of damping-off and root-rot diseases of soybeans and alfalfa under diverse field conditions.  This species is common and present in the field soils we have tested, occurring at approximately 10⁴ to 10⁵ cfu/g soil.  The mechanism of disease suppression has been extensively studied, and led to discovery of a novel antibiotic, zwittermicin A, that prevents normal development of several plant pathogens.  Sequencing of the B. cereus UW85 genome could provide insight into the genes involved in the production of this new class of antibiotics.  B. cereus phylogeny is complex, as this species is a member of the “Bacillus cereus group,” which is comprised of B. cereus, B. thuringiensis, and B. anthracis.  These species have recently been shown to resemble a single species based on diverse phylogenetic criteria, but have retained their names for historical reasons and because they engage in very different relationships with eukaryotic hosts.  Genomic studies of B. cereus UW85 would afford genome-wide comparisons between members of the “Bacillus cereus group”.    Such comparisons could be used by scientists with diverse interests in microbiology,  including biocontrol and the prevention of bioterrorism.

Ash, C., Farrow, J. A. E., Wallbanks, S. & Collins, M. D. 1991b.  Phylogenetic heterogeneity of the genus Bacillus revealed by comparative analysis of small-subunit-ribosomal RNA sequences.  Letters in Appl. Microbiol. 13:202-206.

Kazmar, E.R., Goodman, R.M., Grau, C.R., Johnson, D.W., Nordheim, E.V., Undersander, D.J., and Handelsman, J.  2000.  Regression analyses for evaluating the influence of Bacillus cereus on alfalfa yield under variable disease intensity.  Phytopathology  90:657-665.

Osburn, R.M., Milner, J.L., Oplinger, E.S., Smith, R.S., and Handelsman, J.  1995.  Effect of Bacillus cereus UW85 on the yield of soybean at two field sites in Wisconsin.  Plant Dis. 79:551-556.

Stabb, E. V., Jacobson, L. M., and Handelsman, J.  1994. Zwittermicin A producing strains of Bacillus cereus from diverse soils. Appl. Environ. Microbiol.  60, 4404-4412.

Bacillus subtilis GB03

            One of the most ubiquitous bacterial species found in agricultural systems is Bacillus subtilis. commonly found in agricultural systems. These bacteria can be readily cultured from soils, food stuffs, plant and animal tissues and they are known to be genotypically and phenotypically diverse. Numerous strains of this species have been shown to express activities that suppress plant pathogens and promote plant growth.  Several Bacillus-containing products are available to growers who are interested in using biological seed treatments or drench applications as part of their integrated pest management programs. While the utility of these products has been validated in a number of studies, relatively little is known about the genetic basis for their beneficial effects on plant health. Mechanistic descriptions have been offered including direct antagonism of pathogens (i.e. through antibiosis and competition for resources) and indirect stimulation of plant growth (i.e. by promoting nodulation and secreting growth factors).  All of these activities have been observed with applications of B. subtilis GB03. This strain is the active ingredient in one of the most widely distributed biofungicides (Kodiak, Gustafson LLC) which is applied millions of acres of cotton and other field crops every year.  The commercial success of this strain is a testament to the phenotypic characteristics of the strain that make it a successful biological control agent.  The strain is known to effectively colonize plant roots, produce antifungal compounds, and secrete volatile substances that can directly stimulate plant growth. Much of what is known about the physiology and genetics of this species comes from studies of B. subtilis strain 168, a well-studied laboratory strain for which the genomic sequence is already available.  Because strain 168 does not express the properties of effective biological control agents, genomic comparisons with strain GB03 would provide an effective means for determining the molecular basis for the useful properties of Bacillus biocontrol strains.

Brannen, P. M.; Kenney, D. S.  1997.  Kodiak: A successful biological-control   product for suppression of soil-borne plant pathogens of cotton. J. Ind. Microbiol. Biotech. 19:169-171.

Kokalis-Burelle, N., Vavrina, C.S., Reddy, M.S., and Kloepper, J.W. 2003. Amendment of muskmelon transplant media with plant growth-promoting rhizobacteria: Effects on seedling quality, disease, and nematode resistance. Hortechnology 13: 476-482.

Mahaffee, W.F. and Backman, P.A. 1993. Effects of seed factors on spermosphere and rhizosphere colonization of cotton by Bacillus subtilis GB03. Phytopathology 83:1120-1125.

Ryu, C-M, Farag, M.A., Hu C-H, Reddy, M.S., Wei, H-X., Pare, P.W., and Kloepper, J.W. 2003. Bacterial volatiles promote growth in Arabidopsis. Proc. Nat. Acad. Sci (USA) 100:4927-4932.

Wipat, A., and Harwood, C.R. 1999. The Bacillus subtilis genome sequence: the molecular blueprint of a soil bacterium. FEMS Microbiol. Ecol. 28:1-9.

Burkholderia ambifaria strain AMMDR1

            The Burkholderia cepacia complex is a group of closely related, gram-negative, non-fluorescent bacteria that includes strains useful in biological control and bioremediation, as well as strains that are plant pathogens or opportunistic pathogens of humans with cystic fibrosis.  Certain members of this group are naturally abundant in the plant rhizosphere and appear to play an important role in managing root health.  The ecological versatility of these bacteria is likely due to their unusually large genomes (typically two to three large replicons) and to their ability to use a large array of compounds as sole carbon sources.  The original species B. cepacia has been split into ten genetic species (genomovars), with many of the human pathogenic strains falling into genomovar III or II, and most of the rhizosphere and biocontrol strains placed in genomovar VII (now called B. ambifaria). However, taxonomic distinctions have not enabled rhizosphere or biological control strains to be clearly distinguished from human pathogenic strains.  The capability of this bacterial complex to cause disease in plants and humans, as well as to control plant diseases, affords a rare opportunity to explore traits that may function in all three environments. Genome sequencing will allow a unique opportunity to make genetic comparisons of human pathogenic and plant-associated strains, and could lead to an understanding of the genes required for pathogenesis, potentially enabling us to distinguish between human pathogenic and non-pathogenic strains.  A human pathogenic strain  (B. cepacia genomovar III, J2315) is currently being sequenced in the U. K.  We recommend that a representative of the rhizospheric biocontrol strains, Burkholderia ambifaria AMMDR1 strain (formerly B. cepacia genomovar VII, now the type strain for B. ambifaria), be selected for sequencing to allow comparison of these genomes.  This strain has three large replicons (3.4, 2.8, and 1.0 Mb) for an overall genome size of 7.2 Mb.  It is the subject of several publications concerning biological control, it is amenable to genetic analysis, and it is the type strain for the new species which comprises most of the rhizospheric Burkholderia cepacia complex bacteria. 

Parke, J. L. and Gurian-Sherman, G. 2001.  Diversity of the Burkholderia cepacia complex and implications for risk assessment of biological control strains.  Ann. Rev. Phyopathol. 39:225-258.

Pantoea agglomerans strain C9-1

            Pantoea agglomerans is a member of the Enterobacteriaceae found commonly in association with plants or as saprophytes from soil, water and insects.  Strains of P. agglomerans capable of causing disease in humans have also been isolated.  The genomic differences between clinical, environmental and insect isolates are unknown even though attempts have been made at their discovery. Since P. agglomerans can compete successfully with the indigenous flora of a variety of microenvironments, it is perhaps not surprising that strains of this organism produce a variety of potent antimicrobial agents.  Some examples are two peptide antibiotics termed herbicolin A and B that inhibit fungi and sterol-requiring mycoplasmas, novel b-lactam antibiotics, and a new class of antibiotics called pantocins that inhibit Erwinia amylovora.  P. agglomerans strain C9-1, secretes at least three antibiotics (herbicolin O, I, and 2C) when grown in a chemically defined medium.  C9-1 was isolated from apple tissue and has been used effectively as a biological control of fire blight.  It is amenable to genetic manipulation by standard techniques applied to E. coli.  Many of the antibiotics produced by P. agglomerans are inactive in the presence of amino acids, such as histidine or arginine.  Herbicolin O, which is not active in the presence of histidine, inhibits growth of several species of enteric bacteria and Bacillus cereus, whereas herbicolin 2C and I inhibit growth of only E. amylovora and Staphylococcus aureus. The genome size of P. agglomerans C9-1 is not known, although it is estimated to be about 4 MB.  Complete genome sequencing of a strain of P. agglomerans C9-1 would provide an opportunity for genome-scale comparison with enterobacterial pathogens, would identify genes required for antibiotic production and regulation, and provide a basis for determining habitat-specific sequences in P. agglomerans.

Ishimaru, C.A., Klos, E.J., Brubaker. R.R.1988. Multiple antibiotic production by Erwinia herbicola. Phytopathology  78: 746-750

Gavini, F., et al. 1989. Transfer of Enterobacter agglomerans (Beijerinck 1888) Ewing and Fife 1972 to Pantoea gen. nov. as Pantoea agglomerans comb. nov. and description of Pantoea dispersa sp. nov.  Int. J. Syst. Bacteriol.  39: 337-345.

Johnson K.B, Stockwell V.O.  1998. Management of fire blight: A case study in microbial ecology. Annu. Rev. Phytopath.  36: 227-248 1998

Pseudomonas fluorescens strain A506

            Pseudomonas fluorescens strain A506 is one of the most studied members of the Pseudomonas fluorescens group.  This species is a very common colonist of the phytosphere.  Strain A506 is exceptionally active in preemptive competitive exclusion of plant pathogens such as Erwinia amylovora and of ice nucleation active bacteria such as Pseudomonas syringae and Erwinia herbicola.  Extensive field and laboratory studies have shown this strain to provide excellent biological control of fire blight of pear and apple, as well as frost damage to a variety of plant species.  Thus P. fluorescens strain A506 has been registered by the EPA as a biological pesticide and is commercially available as a freeze-dried preparation. Blightban A506® is among the most successful and widely-used biological control agents, being used on over 25% of pear and apple acreage in the US.  Given that extensive research has already been done to indicate that biological control is conferred by both competition for limiting resources and antibiosis, the availability of the genomic sequence for this strain would increase the rate of discovery in studies of the physiology and ecology of this species.  The genomic sequence would also guide the rational development of new formulations of viable cells needed for biological control. Insights into factors controlling long-term survival in this model organism should also greatly enhance the ease of production, delivery, and storage of other bacterial strains useful for other applications.  P. fluorescens strain A506 has received considerable attention as a rhizosphere colonist and has been the focus of extensive physiological studies of cell culturability and stress survival.  Furthermore, its environmental competence has been exploited to enable it to be developed as a “biological sensor” whereby it harbors environmentally-responsive genes linked to reporter genes to report on soil environmental conditions.   

Lindow S.E.,  McGourty G. Elkins R.1996. Interactions of antibiotics with Pseudomonas fluorescens strain A506 in the control of fire blight and frost injury to pear.  Phytopathology. 86:841-848.

Wilson M., Lindow S.E. 1993.  Interactions between the biological control agent Pseudomonas fluorescens A506 and Erwinia amylovora in pear blossoms. Phytopathology. 83:117-123.

Stiner L., Halverson L.J.  2002. Development and characterization of a green fluorescent protein-based bacterial biosensor for bioavailable toluene and related compounds. Appl. Environ. Microbiol.  68:1962-1971.

Lowder M., Unge A., Maraha N.,  Jansson J K., Swiggett J., Oliver J.D. 2000. Effect of starvation and the viable-but-nonculturable state on green fluorescent protein (GFP) fluorescence in GFP-tagged Pseudomonas fluorescens A506. Appl. Environ. Microbiol. 66:3160-3165.

Trichoderma harzianum strain T22

            Trichoderma spp. are highly diverse and ecologically successful fungi. Members of the genera have long been known to act as biocontrol agents of plant pathogens. Recently, these fungi have been used in quite significant amounts in commercial agriculture (e.g., about 30% of the total soil fungicide used in the greenhouse industry in the USA are products based on T. harzianum strain T22).  Until recently, Trichoderma spp. were believed to achieve biocontrol by direct effects on fungal pathogens, particularly via mycoparasitism, antibiosis and competition. While these mechanisms are important, direct effects on plants are no doubt equally important.  For example, T22 effectively colonizes the roots of most crops and, in most soils, increases the density and depth of rooting of many plants, enhances nutrient uptake (including an ability to reduce, in many cases the nitrogen requirement of maize by at least 25%), and solubilizes some mineral plant nutrients. Some Trichoderma strains have been reported to induce systemic resistance in plants, and T22 also possesses this ability. However, since most of these properties have been discovered only recently, their molecular and physiological bases are unknown. It is essential to study the genes of these Trichoderma biocontrol strains and to put their sequences into public databases. Because it demonstrates multiple useful properties, T22 is a most appropriate target for genomic sequencing.

Harman, G.E. 2000. The dogmas and myths of biocontrol. Changes in perceptions based on research with Trichoderma harzianum T-22. Plant Dis. 84:377-393.

Yedidia, I., Benhamou, N. and Chet, I. 1999. Induction of defense responses in cucumber plants (Cucumis sativus L.) by the biocontrol agent Trichoderma harzianum. Appl. Environ. Microbiol. 65:1061-1070.

Yedidia, I., Benhamou, N., Kapulnik, Y. Chet, I. 2000. Induction and accumulation of PR proteins activity during early stages of root colonization by the mycoparasite Trichoderma harzianum strain T-203. Plant Physiol. Biochem. 38:863-873.

Contributors:

B. McSpadden Gardener, Ohio State U. - OARDC

Chair APS Biological Control Committee

bbmg+@osu.edu

G. Harmann, Cornell U.

geh3@nysaes.cornell.edu

H. Hoitink, Ohio State U. - OARDC

hoitink.1@osu.edu

J. Handelsman, U. Wisconsin

joh@plantpath.wisc.edu

D. Kenney, Gufstafson LLC

dkenney@gustafson.com

L. Kinkel, U. Minnesota

kinkel@umn.edu

J. Kloepper, Auburn U.

jkloeppe@acesag.auburn.edu

C. Lawrence, Colorado State U.

clawrenc@lamar.colostate.edu

C. Ishimaru, Colorado State U.

carol.ishimaru@colostate.edu

S. Lindow, UC Berkeley

icelab@socrates.Berkeley.edu

J. Loper, USDA-ARS

loperj@mail.science.oregonstate.edu

J. Parke, Oregon State U.

Jennifer.Parke@orst.edu