Agricultural Development and Environmental Research: American and Czechoslovak Perspectives: Proceedings of a Bilateral Workshop (1987)

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Biotechnology and the Development of Microbial Products for Agriculture SUSAN s. BROWN Pioneer Hi-Bred International, Inc. BIOTECHNOLOGY: AN ANCIENT PRACTICE AND A NEW SET OF TOOLS Agriculture is the original biotechnology, the technical applica- tion of biological systems in the selection of plants and animals and in controlled fermentations. The "new" biotechnology rests on recent major developments in biochemistry especially related to recombi- nant DNA and monoclonal antibodies. Recognition at the molecular level is a fundamental characteristic of biological systems which is highly evolved in the genetic material to promote accurate replication through generations, and in the animal immune system to recognize foreign and potentially dangerous antigens. Understanding the bio- chemical aspects of these recognition systems has now also provided more precise tools to study cells and molecules. Public statements create an image that this technology is be- ing directed toward the creation of "new" organisms. Over 1,200 commercial firms are presently using recombinant and monoclonal techniques in product research and development, and the U.S De- partment of Agriculture has 180 projects underway to study appli- cations in agriculture. The genetic engineering of plants by recom- binant DNA and tissue culture and advances in biomedical products from the fermentation industry can be viewed as natural outgrowths of the old biotechnology that apply the new molecular techniques. This paper addresses another equally important role for this new technology—to improve understanding of the ecology of naturally occurring microorganisms. 184

185 TABLE 1 Applications of microbial products for agriculture Application Silage fermentation Nitrogen fixation Animal intestinal inoculants (probiotics) Soil improvement, plant growth Stimulators Biological control - fungicides Insecticides Nematicides Herbicides Preservation of seed Stored grain Hay Types of Organisms Lactic acid bacteria Rhizobium Azospirillum, Azotobacter, Bacilli, Pseudomonas Lactic acid bacteria, yeasts, Bacilli Cyanobacteria, microalgae Mycorrhizal fungi, various bacteria Various fungi as parasites or predators, bacteria Bacilli, viruses, fungal insect pathogens B. sphaericus Viruses, fungi Non-toxic antagonists of fungi Table 1 contains examples of microbial inoculants presently being sold or tested for agriculture. Most of them involve a fundamentally new practice of biotechnology—the domestication of microbes for use in the open environment. Beneficial uses of microbial inoculants were suggested at the turn of the century in Metchnikoff's work with lactobacilli as animal probiotics. Attempts to use rhizobium and other biofertilizers began in the early 1950s and use of insecticidal bacilli followed about ten years later. Thousands of years were pre- viously required to domesticate plants and animals, but scientists are currently attempting to do the same with certain microbes in a single generation of directed research (8). Microbial inoculants for agriculture have attracted the attention of researchers and companies throughout the world because they hold the promise of providing lower cost, ecologically sound alternatives to many chemical applications. The costs of chemical inputs to meet the needs described in Table 1 are rising steadily, but the value

186 of agricultural commodities is not. Lowering the cost of inputs is recognized as an important strategy for raising the profitability of farming. If they could perform consistently and be cheaply produced, microbial inoculants would also present lower risks to animals and the environment. The confidence of both basic and applied researchers in the future of inoculants is indicated in a recent survey of 405 biotechnol- ogy firms by the U.S. publication Genetic Engineering News. Over 30 percent of the firms identified agriculture as a potential market, even though only four percent had products other than animal di- agnostics presently for sale or testing. These other products include cyanobacterial fertilizers, frost protectants, Bacillus thuringiensis- and virus-based insecticides, improved rhizobium inoculants, plant growth regulators, waste processing treatments, and fungicides. INCONSISTENT PERFORMANCE OF INOCULANTS: THE NEED FOR AUTECOLOGICAL DATA There are an estimated 10,000 to 20,000 strains of any given bacterial species. This represents a huge, almost untapped poten- tial for inoculants to affect processes such as those listed in Table 1. Empirical research has shown that control of insects and plant pathogens, enhancement of nitrogen fixation, and protection of an- imals from enteric disease can be accomplished with selected fungi or bacteria. However, few products have given consistent results in the field. Forty years of research on microbial products for agricul- ture have demonstrated that not enough is known about microbial ecology of the agricultural environment to exploit it in an optimal manner. The use of inoculants for agriculture involves their release into the environment where they compete with large and diverse indige- nous populations. As is already known for Bacillus thuringiensis and rhizobia, they usually lose this competition. The survival and func- tion of individual species of organisms in the natural environment are subject to many influences beyond our present understanding or control. When maintained in culture, even organisms isolated from the environment often lose qualities for which they were selected. Thus, natural conditions cannot be easily duplicated. The study of the individual microbial species within an ecosys- tem is termed autecology. Autecological research on pathogenic

187 organisms both in culture and the environment has been pursued be- cause of the importance of these organisms to man and the relative ease with which they can be identified. However, the vast majority of naturally-occurring microbes are not readily identified and have been studied only as components of processes such as the nitrogen cycle. The autecology of these organisms must now be studied in order to harness their potential as inoculants (9). What are the actual num- bers, functions, and fates of indigenous microbes in agroecosystems? The current public concern over the release of genetically engi- neered organisms stems from a lack of the same type of information. A high priority has been placed on research in microbial autecology at this time in the United States because of the desire to monitor proposed field tests of such organisms. The U.S. Environmental Pro- tection Agency (EPA) is currently funding 17 extramural research programs on related problems of environmental effects and on de- velopment of a methodology for estimating the fate of introduced organisms. Until now, methods for identification of specific strains of bacteria among large populations of similar strains of the same species have lacked adequate precision or have been prohibitively difficult. The large numbers and variety of natural populations also led to the concept that a physiological niche in an ecosystem was more important than the actual species which occupied the niche. Enhanced molecular recognition by the new techniques now allows researchers to reach into natural environments and locate partic- ular organisms with a sensitivity that was not possible before. It should be possible to study gene expression specifically related to both persistence and beneficial characteristics of microbes in situ. A good example of the promise and problems of microbial inocu- lants is competitiveness for root nodulation by rhizobium. Enhanced symbiotic nitrogen fixation by these organisms would greatly benefit agriculture (2). Understanding of the molecular biology and bio- chemistry of the nitrogen-fixing enzyme and the physiology of the symbiosis is far advanced after about 20 years of intensive research. However, most of this potential still has not been harnessed for crops because of the unsolved problem of determining what makes individual strains competitive for nodulation in the rhizosphere (4). Strains with superior nitrogen-fixing and nodulating capability lose out in the competition for saprophytic growth in soil or infection sites on the roots of the host. A similar problem presumably exists in the attempt to exploit associative nitrogen fixation by Azospirilla or Azotobacter for cereals.

188 TRACKING NATURALLY-OCCURRING STRAINS OF BACTERIA Identification of specific strains of rhizobia for studying their ecological interactions has been a priority for years because of the desire to supplant indigenous field strains with strains selected in the laboratory. While effective at the species and serogroup level, fluorescent antibody labelling and determination of antibiotic resis- tance patterns lack precision for tracking strains, especially at low population densities. Some direct and much circumstantial evidence suggests that genetic exchange among rhizobia is common in soil and can change the determinants which are the basis of the assay methods. Rhizobium is also usually baited out of soil onto the host plant prior to identification. Since it apparently survives well as a soil saprophyte, a further source of inaccuracy exists. One measure of the difficulty of tracking rhizobium strains is the very large number of methods that have been published over the years. As suggested above, identification methods that rely on first culturing the organisms have many sources of error. In an adaptation which is not yet understood, sporulated gram positive organisms as well as some gram negative organisms can enter a resting state in soil where they cannot be cultured onto laboratory media. About 100 billion bacteria per gram can be cultured from soil, while from 10 to 100 times more can be seen in samples prepared for direct microscopy. There are similar cryptic populations in other natural environments. Additionally, the colonies which do grow on agar media may not represent the cells that were physiologically most active or significant in the ecosystem sample. An example of a new biotechnology methodology to address these problems is the current study of DNA extraction directly from soil by Drs. Holben, Chelm, and Tiedje of Michigan State University (6). Similar work is being pursued for sediment and other environmental materials. The extraction of DNA, with the development of appropriate probes, will allow a direct measurement of the negative microbes which are actually present. Using Bradyrhizobium japonicum con- taining a recombinant gene, Holben has demonstrated the persistence of two separate strains simultaneously inoculated in natural soil by probing a restriction enzyme digest of total DNA for the recom- binant sequence. Products of genetic recombination involving the gene or nearby sequences would also be easily distinguishable if they occurred in the soil. In addition, the method is simple enough to

189 allow processing of sufficient samples to complete a well designed ecological study. This technique will permit researchers to follow not only individual strains of microorganisms but also specific genes and the fate of co-inoculated mixtures of bacteria or fungi in the natu- ral environment. The fate of specific plasmids within the microbial population can also be determined. THE IMPORTANCE OP PLASMIDS Tracking plasmids in the environment is arguably more impor- tant than tracking individual strains of bacteria themselves. Plas- mids are extra-chromosomal, self-replicating genetic elements by which bacterial populations maintain biochemical flexibility in adapt- ing to their environments. Plasmid-borne functions such as antibi- otic resistance or catabolism of uncommon carbon sources may be magnified as needed in a population by increasing plasmid copy number or expression. Plasmids may be lost from cells when there is no selective pressure to maintain them, and they may transfer to other cells, occasionally mobilizing parts of the chromosome or other, non-transmissable plasmids along with them. Thus, the ecology of bacteria depends fundamentally on the ecology of their plasmids (7). Some of the traits selected as useful for agriculture in bacteria have been found to occur only—or naturally—on plasmids. These include the nitrogen-fixation enzymes, symbiotic determinants, and probably competitiveness in rhizobia; the Ti and Hi plasmids of Agrobacterium which cause disease by incorporating into plant chro- mosomes and have become important vectors for plant genetic engi- neering; and the insecticidal crystal proteins of Bacillus thuringiene- sis. Many of the bacteriocin and antibiotic factors selected in typical screening for biological control agents are probably also plasmid- associated. A recent report by Carney et al. from the University of California on metabolism of xenobiotics by pseudomonads suggests that microbes may even recombine parts of their plasmids to develop new pathways for degradation of novel waste compounds (3). The phenotypic expression and the reason for retention of most plasmids in nature are still totally unknown. Increased understanding of plasmids in natural populations is necessary for several reasons: • to determine when select characters are associated with them as a basis for increasing their stability in commercial inoculants;

190 to control their transfer to other organisms in the environment, especially in the case of recombinant genes; to maintain laboratory cultures under the appropriate conditions in order to retain desired plasmid-associated characters; to cure or replace them in the case of strains in which this is desirable but difficult. PLASMID PROFILING FOR ECOLOGICAL RESEARCH At Pioneer, plasmid characterization has been used in two areas of microbial ecological research pertinent to silage inoculant and seed inoculant products. Silage is presently the world's largest fermenta- tion industry, and it is growing in importance in every agricultural economy. Ensiling preserves more nutrients in more crops than other methods of storage, and its use is increasing in beef and dairy cattle feed. Silage is also the feed component which varies most in quality. Addition of lactic acid bacteria to improve silage fermentation has become a well-recognized practice over the last 30 years (1). There are 91 producer/distributors of over 100 different live-culture addi- tives in the United States alone. Strains are selected on the basis of their ability to rapidly lower the pH in the ensiled crop. Ecologi- cal research on the populations of fermenting bacteria would form a rational basis for further improving these selection criteria. Drs. Hanna and John Hill have found that the Lactobacillus plan- tarum strains important to silage fermentation often have multiple plasmids, and that these are stably maintained both in the laboratory and the natural environment (5). Therefore, plasmid profiles—or ex- tracted plasmids separated electrophoretically by molecular weight— show characteristic patterns which identify individual strains. Strains which do not have plasmids have been identified by their biochemical profiles. Figure 1 presents an example of plasmid profiles of L. plantarum strains isolated from 32-day-old uninoculated high moisture corn silage. The pH had been stable at about 4.0 for at least 3 weeks, and overall lactic acid bacteria numbers were continuing to slowly decrease from an initial peak reached in the first week. The variable banding pattern in the gel represents plasmids of different molecular weight. There are eight distinct types, indicating that at least eight strains of L. plantarum are present. It is also apparent that two are dominant. In Figure 2 the same analysis has been made of inoculated silage. In this case the only L. plantarum strains recovered were those

191 ^3.4 -1.4 FIGURE 1 Plasmid profiles of Lactobacillus plantarum isolates from uninoculated silage. Reference plasmids from E. coli V517 lanes 14 and 28; MGD inoculant product (MGD286) lane 27; Chr, contaminating chromosomal DNA (Hill and Hill 1986).

192 which were introduced. This proves that the natural population of fermenting organisms can be altered by inoculation of competitive strains. Physiological studies coupled with plasmid analysis on all the lactic acid bacteria isolated in this experiment also showed that populations of homofermentative and heterofermentative organisms shifted during the fermentation. Heterofermentative organisms pro- duce gas as well as acid, and thus more of the nutrients in the original crop are lost due to their activity. Not only did the desirable homofermentative organisms, L. plantarum, predominate sooner in the inoculated silage, but after two months, the numbers of L. plan- tarum declined in both the control and the inoculated silos. A large amount of field data is available to show that these particular or- ganisms produce a high quality, stable silage. The Hills' work also shows, therefore, that isolation of organisms from older silage would not necessarily produce the strains which were originally responsible for the successful fermentation. This is an excellent example of the way in which plasmid profiling can be used to study microbial ecol- ogy and also how understanding that ecology can lead to strategies for improving research approaches to inoculants. A final example is an experiment on the persistence of Bacillus sp. strain (MGD311) on soybean roots. The organism tested in this experiment had shown positive effects on plant vigor in greenhouse trials and improved yield in some field trials. The gram positive bacilli are attractive for product development of microbial seed in- oculants because they frequently elaborate and excrete products in- hibitory to bacteria and other plant pests and could thus be useful for biological control. Also, their ability to sporulate and survive heat and drying would simplify product formulation. However, among the common soil microorganisms, the gram negative pseudomonads are widely regarded as active plant root colonizers and candidates for protective inoculants, while bacilli are thought to be saprophytes that do not selectively live in association with roots. The exper- iment was conducted to determine whether a particular organism with known beneficial effects might in fact persist in the rhizosphere and, if so, for how much of the growing season. Polyclonal antibodies to MGD311 were raised by hyperimmuniz- ing rabbits. Cross reactivity to this antiserum was the preliminary selection criterion. Roots were dug in the field and cultured on var- ious media. Colonies which resembled the inoculant were tested,

193 10 13 Chr*- 3.4* 3.4* 1.4* 26 FIGURE 2 Plasmid profiles of Lactobactiliu plantamm isolates from silage in- oculated with MGD286 (reference lane 15) and MGD287. Reference plasmids from E. coli V517, lanes 1 and 14. MGD287 does not contain plasmids. Chr, contaminating chromosomal DNA (Hill and Hill 1986).

194 and those that bound the antibody were isolated for further analy- sis. Figure 3 shows antibody reactivity for organisms isolated from inoculated and control soybean roots through the growing season. Roots from treated seed initially had a higher level of cross-reacting organisms, which suggested survival of the inoculant. However, the control plot had a high background of immunopositive organisms, and both plots showed increased numbers at the end of the summer. Therefore, further identification of the isolates was made by plasmid analysis. Bacilli found in soil frequently contain single large plasmids which are too large to be easily isolated or compared on the basis of molecular weight. In this case it was useful to digest the plasmid preparation with restriction enzymes and compare the profiles of the plasmid fragments. Different large plasmids have char- acteristic patterns of fragments as the lactobacilli have characteristic patterns of smaller plasmids. Figure 4 shows plasmid digests of organisms from the treated plot. Early in the growing season, anti- genically similar isolates appeared identical to the inoculant. Late in the season, although a very large number of cross-reaction strains were isolated, none were identical to the inoculant. This was also true for late season isolates from the control plot. This research has shown that soybean roots can be success- fully colonized by a bacillus inoculant during early weeks of seedling growth. Late in the season, organisms in the group related to this bacillus become a major part of the rhizosphere flora as soy roots become senescent. This competition may influence the persistence of the introduced organism. CONCLUSION The transfer of knowledge of microbial physiology into practice as exploitation of inoculants is presently hindered by a lack of fun- damental information. Research on microbial ecology at the level of specific strains should be encouraged in all agroecosystems— host/pathogen interactions, saprophytic life stages of pathogens, legume/rhizobium interactions, colonization of plant roots by non- pathogenic bacteria and fungi, fermentation of silage, decomposition of wastes, and colonization of the rumen and gut. In some cases, plasmid profiles are suitable for tracking individ- ual strains in these environments. In other cases, probes may be developed by inserting unique, nonfunctional DNA fragments into

195 80 -I CONTROL PLOT MAY1 SO1L MAY 25 ROOT JUNE 26 ROOT AUG 1 ROOT SEPT 9 ROOT FIGURE 3 Recovery of organisms cross-reacting with polyclonal antibody to seed inoculant MGD311. Bacterial isolates from roots or soil with similar colony morphology to MGD311 were rated for strong positive reaction to anti-MGD311 by immunoblot assay (Brown and Hendricks, unpublished). the organism. This should be clearly differentiated from the engi- neering of genes into organisms and should be supported as a critical step in expanding our knowledge. Acquisition of this basic knowledge will change what is now thought of as the potential for use of natu- rally occurring microbes as inoculants. This is an exciting promise for biotechnology in the future of agriculture.

196 S85 MAY SEPT «*•«»>*• FIGURE 4 Hindlll restriction fragments of plasmid DNA from MGD311 and cross-reacting field isolates from the treated plot as shown in Figure 3. The month in which the cross-reacting isolates were recovered is indicated above the appropriate lanes (Brown and Hendricks, unpublished). REFERENCES 1. Bolsen, K., and J.I. Heidker. 1985. Silage additives USA. Marlow, Great Britain: Chalcombe Publications. 2. Brill, W.J. 1986. Agricultural opportunities in nitrogen fixation research. In P.O. Augustine, H.D. Danforth, and M.R. Bakst, Eds. Beltsville symposia in agricultural research, 10. Biotechnologyfor solving agricultural problems. Boston: Martinus Nijhoff. pp. 183-193. 3. Carney, B., L. Krockel, D.D. Focht, and J.V. Leary. 1987. Alterations in plasmid DNA after prolonged growth on selective hydrocarbon substrates. In Abstr. 87th Annual Meeting of American Society for Microbiology, p. 232. 4. Dowling, D.N., and W.J. Broughton. 1986. Competition for nodulation of legumes. Ann. Rev. Microbiol. 40:131-157. 5. Hill, H.H., and J.H. Hill. 1986. The value of plasmid profiling in monitoring Lactobaeilliu plantarum in silage fermentations. Current Microbiology 13:91- 94.

197 6. Holben, W.E., J.R. Jansson, B.K. Chelm, and J.M. Tiedje. 1988. DNA probe method for the detection of specific microorganisms in the soil bacterial community. Applied and Environmental Microbiology (in press). 7. Shaw, P.D. 1986. Plasmid ecology. In T. Kosgue and E.W. Nester, Eds. Plant-microbe interactions—molecular and genetic perspectives. Vol.2. New York: Macmillan Publishing Co. pp.3-39. 8. Stellwag, E.J., and J.E. Brenchley. 1986. Genetic engineering of microor- ganisms for biotechnology. Microbial Ecology 12:3-13. 9. Tate, R.L., Ed. 1986. Microbial autecology. A method for environmental studies. New York: John Wiley and Sons.