Anne K. Vidaver is professor and head of the Department of Plant Pathology at the University of Nebraska, Lincoln.

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Historical Events in Plant Microbiology
Challenges and Prospects in Plant Microbiology

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Plant Microbiology: Century of Discovery, with Golden Years Ahead

Increased knowledge of plant microbiology is essential for addressing important problems we will face in the next century

Anne K. Vidaver

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Green plants, which are so critical to most life, depend on the microorganisms associated with them. These microorganisms are fascinating, broadly affecting planthealth and, thereby, the entire biosphere. Not only does life on earth depend on green plants; any effort to establish colonies on other planets will certainly hinge on the ability to cultivate plants there. Thus, it behooves microbiologists to study the full range of microbial interactions that maintain plant health-from roots to treetops.

Recent estimates of microorganism numbers and diversity associated with plants are certainly too low. This is but one indication of how little we know either about benign plant-associated microbes or plant pathogens, particularly compared with our understanding of the infectious disease agents of humans. Plant pathogens are diverse in character and type, and the class includes viroids (thus far only associated with plant disease), viruses, bacteria, fungi, protozoa, and nematodes or eel worms. The benign microorganisms associated with plants, including those that are symbiotic or endophytic, are also highly diverse.

The relative meagerness of our knowledge about plant-associated microorganisms reflects in part the considerable diversity of these microorganisms. For instance, little is known about the beneficial bacteria and fungi that act as symbionts and endophytes. Moreover, the microbiology that accompanies aquatic plants is particularly neglected, even though unique viruses are known to infect algae, while yet other uncharacterized viruses are found in abundance in both fresh- and seawater.

However, that meagerness also reflects the relatively poor support that traditionally has been provided for studying plant microbiology. Resources available to investigate both beneficial and detrimental plant-associated microorganisms are barely perceptible amid overall funding for biological research, especially medical microbiology.

Nevertheless, researchers studying and, in some cases, trying to manage plant-microbe interactions are making progress. For one thing, the recent lure of interest in transgenic plants has proved a powerful new driving force. Transgenic techniques enable researchers to incorporate microbial genes, including those specifying pathogen resistance, insecticidal properties, and other useful traits, into plants. Because this ability to develop plant lines that produce new metabolites is of powerful fundamental and commercial interest, it is stimulating a broader study of interactions between microorganisms and plants.

Past Achievements in Plant Microbiology Highlighted

Beneficial and pathogenic microorganisms associated with plants were first described in the mid- to late 1800s. Not surprisingly, morphology or simple metabolic features were used to distinguish these microorganisms from one another, making taxonomy appear simple! Throughout the 1900s, the complexity of the microbial world has become increasingly apparent as new methods of analysis, primarily molecular and computational, have enabled investigators to build and interpret ever larger data sets. Thus, newly discovered and recently reclassified microorganisms have been compiled into an extensive system describing microorganisms associated with plants from Acidovorax to Ralstonia.

New classes of microorganisms associated with plants or parasites of those microorganisms continue to be discovered. These range from Bdellovibrio and the uncultivable insect-vectored phytoplasmas to the double-stranded RNA bacteriophage omega 6 and the largest viruses known, which infect Chlorella. Many microorganisms associated with plants are yet to be discovered and classified, especially those found underground and internally as normal flora.

Special terms are used to describe the types of relationships that develop between microorganisms and specialized plant surfaces. For instance, epiphytes are those microorganisms that live on above-ground parts of plants, whereas rhizobacteria associate with below-ground parts of plants. Microorganisms that live on leaves-on the phylloplane-differ in type and population from those that live in flowers, stems, or along the root system, which is called the rhizoplane. The mechanisms of associations, such as beneficial underground endo- or ectomycorrhizal fungi, and the gene-specified signal compounds that regulate the virulence of pathogens, are increasingly better understood.

Historically, plant-associated microorganisms have been put to multiple uses themselves, such as for controlling deleterious microorganisms. Plant-associated microorganisms also are used directly for producing valuable and useful compounds, such as xanthan gum. This extracellular polysaccharide, which is produced by avirulent Xanthomonas campestris, is widely used in products ranging from cake frostings to shampoos.

Transgenic Plants and Deletion Mutants: Dramatic Applications of Microbes

The most dramatic practical application of microorganisms is in plant genetic engineering, which relies heavily on inserting genes of interest into the transfer region of plasmids of Agrobacterium tumefaciens to deliver and integrate these same desirable genes into recipient plant cells. This tumor-inducing bacterial pathogen transfers genes naturally to a wide range of host plants and also to fungi.

In studying A. tumefaciens, researchers developed the concept of "genetical colonization" to explain the selective advantage of this and other bacteria that are naturally capable of genetically engineering their hosts. For instance, when agrobacteria infect plants, they reprogram their hosts to produce opines. Over the past two decades, this natural capacity has been carefully studied-and usefully harnessed. Thus, most transgenic plants owe their origin to this well-studied bacterium, which is used extensively for transferring a wide variety of genes into plants.

Removal of the transfer (tra+) gene from a plasmid of Agrobacterium radiobacter to maintain its longevity as a biocontrol agent was a first in environmental microbiology. It is the first genetically engineered microbe sold commercially-in Australia, where it is especially useful in protecting transplants of fruit trees and roses from the crown gall disease.

Another, somewhat earlier effort to harness a genetically altered, plant-associated microorganism also proved interesting-and controversial. Indeed, these efforts led to what was perhaps the most highly publicized experiments in the history of the biological sciences. That series of experiments entailed producing deletion mutants of the ice-nucleation (ina+) gene from an epiphytic pseudomonad. The goal was to control frost damage on plants such as strawberries and potatoes.

The researchers wanted to test whether such mutants could competitively exclude ice-nucleating, pathogenic pseudomonads from growing on those plants under field conditions. However, a lawsuit and court injunction temporarily halted the field tests in 1987 while activists argued that genetically modified microorganisms should not be released into the environment. Although testing was eventually permitted, high costs, commercial application problems, and concerns over public acceptance of this technology halted further commercial development of such mutants.

Nonetheless, it represented the first deliberate release of genetically engineered deletion mutants in the environment. Subsequently, wild-type, ice-nucleating bacteria that cause frost damage to plants came to be used to improve snow-making procedures at ski resorts and also to improve the texture of ice cream.

History of Plant Pathology- Rarely Ornamented

The microorganisms associated with plants have only rarely inspired artists, novelists, or those in comparable disciplines to dramatize their manifold impact on history, literature, and art. To be sure, most readers of history know something about the Irish potato famine, but probably very little about the pathogenic fungus that gave rise to it.

A citrus canker outbreak early in this century helped to unleash a scorched-earth policy in orchards in Florida for several decades. More recently, a television documentary dramatized the effects of such a canker outbreak on a family of citrus fruit growers. But such dramatizations are rare. Similarly, some 18th- and 19th-century painters included recognizable plant diseases in their depictions of plants. For instance, the exquisite glass flowers and fruits at Harvard Museum of Natural History depict plant diseases in some of the objects within this collection. However, those representations are mainly inadvertent.

Periodically, plant disease outbreaks become newsworthy events, such as the Southern corn leaf blight fungus in the 1970s, the reemergence of late blight of potato, the wide dissemination of a fungus of wheat (scab) associated with newer agricultural (tillage) practices in the Midwest, and an outbreak of insect-transmitted virus of tomato (tomato yellow leaf curl) in the South.

In general, however, although plant diseases exact a considerable toll in terms of crop losses and impacts on farm families, their tales are rarely told. Perhaps some of the microbiologists who have focused exclusively on human health will reconsider also studying plants and plant-associated microorganisms as they come to appreciate the remarkable commonalities among infectious agents of plants and animals.

Current State of Plant Microbiology

The molecular interactions between plants and microorganisms are under intensive study, with many researchers taking advantage of systems in which the genetics of a plant host and its associated microbes are reasonably well understood. Researchers continue to identify some of the plant and microbial genes that code for attachment of one to the other, that regulate important metabolic pathways, or that specify critical virulence factors, toxins, and other novel plant and bacterial natural products. Meanwhile, heightened interest in genomic sequencing has led investigators to analyze the genomes of several plant viruses, while others are sequencing plant pathogens, including those of specific bacteria, fungi, and nematodes.

One striking realization is that many functional molecular components are similar, if not identical, from one microbe-plant pairing to another. For example, products of several plant genes that confer on those plants resistance to specific pathogens apparently share functional domains, regardless of the type of pathogen involved. Thus, certain leucine-rich repeat (LRR) units inside plasma membranes, others outside such membranes, and certain protein kinases interact with a variety of fungal and bacterial gene products to protect the host plants. Broader comparisons of the pathobiology of infectious disease agents from different kingdoms are also being made. Progress is somewhat hampered because there are no programs to fund cross-kingdom research. Moreover, traditional discipline-determined boundaries tend to keep investigators from recognizing commonalities in infectious diseases caused by plant and animal pathogens.

Indeed, although this field is in its infancy, some surprising similarities among plant, animal, and human pathogens have been found-notably the type III secretion mechanisms. Other similarities, involving replication mechanisms used by the separate viruses and also the fungi that infect both plants and animals, are now recognized.

Also on the biochemical level, reactive oxygen and nitric oxide are formed in infected cells of both plants and animals. This highly conserved intracellular signaling system is considered part of an innate system that p rovides the first active line of defense against microbial pathogens. Nitric oxide acts together with or through additional signaling agents (e.g., salicylic acid, hydrogen peroxide) to elicit the hypersensitive or programmed cell death response and activation of defense genes in plants; similar responses occur in animals.

Plants, Associated Microbes Produce Rich Array of Chemicals

The microorganisms associated with plants can produce a wide array of novel chemical compounds, including plant hormones such as gibberellin, auxins, and ethylene, that are produced normally by plants. Genes coding for these compounds may be on plasmids or on a chromosome.

Some of these microbes produce bacteriocins, which are specialized, narrow-host-range antibiotics. At least one such bacteriocin, produced by Agrobacterium radiobacter, consists of a "fraudulent" adenosine nucleotide that inhibits most strains of A. tumefaciens, the crown gall bacterium, by acting as a DNA chain inhibitor. Another bacteriocin, called nisin, is being considered for use as a preservative of fresh foods. It is produced by Lactococcus lactis, which associates with grasses.

Another burgeoning field-involving biocontrol of pathogens-is leading researchers who specialize in plants and their associated microorganisms to examine how some of these microbes may help in controlling other specific deleterious microorganisms or pathogens. Biocontrol agents act in various ways, such as through competitive exclusion of pathogens, by producing metabolites that are lethal only to certain organisms, or by inducing resistance in plants to specific pathogens. One notable example of a biocontrol agent with potential commercial value is a group of host-range bacteriophage mutants that are being tested as a means of counteracting bacteria that cause spots in tomato fruit.

Signaling compounds from plants, such as a nematode egg-hatching factor, are being analyzed and synthesized for potential commercial applications in disease management. Although many additional signaling compounds produced by microorganisms are now being widely studied, none has yet moved into commercial use.

Mycotoxins produced by plant pathogens in infected plants are among the most potent toxins and carcinogens known. Based on genes isolated from saprophytic microorganisms and other undisclosed sources, researchers are genetically engineering plants to inhibit toxin production. If such efforts prove successful, the suffering of animals that were fed contaminated plant materials, economic losses, and the costly regulatory requirements associated with establishing tolerances for human consumption of potentially contaminated products, such as peanut butter and corn chips, may someday be overcome.

Some Challenges and Predictions

Most investigators focus on microorganisms associated with terrestrially based plants of economic significance. Moreover, the main plant-associated microorganisms being investigated are pathogens, including fungi, bacteria and viruses, or symbionts such as fungi and bacteria. The restricted scope of these studies leaves plenty of room for high expectations and bold predictions for the future (see table). For example, aquatic plant microbiology will soon thrive.

The question of what contributes to the stability of a taxon is unknown-virtually all attention is being given to change. Yet stability of microbial taxa is perhaps key to understanding how a species survives and disseminates, as well as how it evolved. Molecular systematics, coupled with informatics, promises to provide some answers, which can best be tested with whole organisms in managed or natural ecosystems.

Failures to adequately manage or eradicate some diseases, such as fire blight of apples and pears, which limits where these fruits can be grown, remain a challenge. The causative agent, Erwinia amylo-vora, is closely related to gram-negative bacteria of medical interest.

One of the greatest challenges to those who study plants and their associated microorganisms is developing accurate and complete descriptions and understanding of such complex systems. Biofilm formation, microbial communities of seaweed, and root-colonizing microorganisms, both beneficial and detrimental, are examples of complex systems that are now amenable to comprehensive study.

Meanwhile, the rare pathogens that cross kingdom barriers are intriguing. What contributes to their selection, survival, dissemination, and unique properties? Although investigators acknowledge the presence of vast numbers of uncultivable microorganisms in soil systems, the role of those microbes in mediating sustainable systems is poorly understood.

Needs and Projections

We can anticipate that new microbes and microbial relationships with naturally occurring and cultivated plants will continue to be discovered. These discoveries will accelerate as researchers work together in multidisciplinary teams and in cooperative programs that move across current boundaries (consilience).

Understanding of host-parasite and symbiotic interactions will be well advanced and used for maintaining a sustainable environment in both managed and natural ecosystems. Trees for urban areas, for stream bank stabilization, and as habitats for conserving biodiversity will require microbiological analysis and management. Amenity plants for home gardens, public grounds, parks, and golf courses will be an expanding area, along with concomitant microbial challenges.

A skilled work force will be needed for integrating information into the task of growing and using plants for direct and indirect consumption, and producing valued-added plants or plant-derived products. Bioinformatics will show the need for increased attention to physiological, biochemical, and comparative studies of plant-microbe interactions. Molecular genomics and biosphere sustainability must be brought together for maximum benefits.

Microorganisms associated with plants affect plant, animal, and human health; the food supply and its safety; national security; nutrient cycling on land and in water; and global warming. Increased knowledge of plant microbiology is absolutely essential if we are to solve important problems looming in the next century. The golden age of plant microbiology is in its ascendancy.

SUGGESTED READINGS

Baron, C., and P. C. Zambryski. 1995. The plant response in pathogenesis, symbiosis, and wounding: variations on a common theme? Annu. Rev. Genetics 29:107-129.

Hueck, C. J. 1998. Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol. Mol. Biol. Rev. 62:379-433.

Kelman, A. 1995. Contributions of plant pathology to the biological sciences and industry. Annu. Rev. Phytopathol. 33:1-21.

Kommedahl, T. and P. H. Williams (ed.). 1983. Challenging problems in plant health. The American Phytopathological Society, St. Paul, Minn.

R. E. Lee, Jr., G. J. Warren, and L. V. Gusta (ed.). 1995. Biological ice nucleation and its applications. APS Press, St. Paul, Minn.

Smith, S. E., and D. J. Read. 1997. Mycorrhizal symbiosis, 2nd ed. Academic Press, San Diego, Calif.

Sutherland, I. W. 1993. Xanthan, p. 363-388. In J. G. Swings and E. L. Civerolo (ed.), Xanthomonas. Chapman & Hall, London.

Vidaver, A. K. 1996. Emerging and reemerging infectious diseases. ASM News 62:583-585.

Wilson, E. O. 1998. Consilience: the unity of knowledge. Alfred A. Knopf, New York.

Ye, X. S., N. Strobel, and J. Kuc'. 1995. Induced systemic resistance (ISR): activation of natural defense mechanisms for plant disease control as part of integrated pest management (IPM), p. 95-113. In R. Reuveni (ed.), Novel approaches to integrated pest management. CRC Press, Boca Raton, Fla.