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Introduced Microbes Enhance Root Health and Plant Growth
INTRODUCED MICROBES ENHANCE ROOT HEALTH AND PLANT GROWTH Hancock J.G. ’, Weinhold A.R1, VanGundy S.D.’,
Schroth M.N.I
IDepartment of Plant Pathology, University of California, Berkeley 9 4 7 2 0 , USA %epartment of Nematology, University of California, Riverside 9 2 5 2 1 , USA
ABSTRACT Growth stimulation and root health, dynamics of root colonization by microorganisms, altered root physiology as the result of infection, rootlet turnover and root health, impact of introduced microbes on plant growth, and future research needs in the root health field are the main topics mentioned and discussed in the paper.
INTRODUCTION Root health is a topic of utmost importance to plant agriculture. It is such a poignant topic today because increased practices of monoculture in many parts of the world has led to an observable decline in root health. Our views of root-microbe interactions are often compartmentalized which has impeded progress in studies of root health. The overwhelming complexity of this subject understandably invites narrow research approaches. From the standpoint of scientific experimentation, it seems logical to study single root-microbe interactions or even to study root development and physiology in the abaence of microbes. While these approaches have unquestionably advanced our knowledge of root biology, the problems of root health are complex and must be addressed more comprehensively. A better definition of root microbial ecology should be one of the principal modern goals in plant biology. Without a foundation of information on root biology, our quest for improvement of root health will be largely empirical simply because we have few alternatives. Within plant pathology, the stage for an appreciation of general root health may have first been set when observations were made that soil fumigation with general biocides promoted significant plant growth responses (Wilhelm, 1 9 6 6 ) . This phenomenon has been observed with many -427-
crops in many parts of the world since the nineteenth century. More recently, the so-called plant growth promotion associated with the introduction of selected soil microbes on seed or roots of propagation materials or directly to soil has galvanized this interest and stimulated further research on the bases of these plant responses (Cook and Baker, 1983; Schroth and Weinhold, 1986). Perhaps most importantly, this latter work has given credence to the idea that root health is managable by biological and cultural means. GROWTH STIMULATION AND ROOT HEALTH Cook (R.J. Cook, personnel communication) distinguished between direct growth stimulation and growth enhancement via protection from disease. This is an important distinction and should be kept in mind when considering mechanisms of growth promotion. In addition to the rhizobial and mycorrhizal relationships, there are several reports of microbe-root interactions where plant growth is directly stimulated (Windham et al., 1986). However, in practice it is exceedingly difficult at the stage of development of this field to differentiate between growth stimulation and elimination of some of the restraints on the genotypic growth potential of plants as a result of beneficial microbial activities in the rhizosphere. We simply do not have the techniques to differentiate between these possibilities, especially under field conditions. In our research, we are screening microbes that are antagonistic in culture against soilborne plant pathogens (fungi and nematodes). Thus, our approach is geared toward biological control rather than growth stimulation per se. We are interested in whether and how antagonists interfere with root infection. Protection from root infection by plant pathogens by microbial antagonists is one possible basis for improved root health. Work with soil fumigants and certain selective pesticides indicates that mild but chronic types of root diseases retard plant growth and that relief from these diseases by treatment results in improved plant growth. It is tempting to draw conclusions on cause-effect relations in biological control investigations on the basis of studies with pesticides. Because there are so many effects of pesticides on the soil microbial communities (and even directly on root physiology), including mineral nutrient releases, we should be cautious about linking these different phenomenona.
DYNAMICS OF ROOT COLONIZATION BY MICROORGANISMS We are striving to gain a better understanding of the dynamics of the interactions between microbes and roots. Our initial investigations are directed at obtaining a better picture of the processes of root infection and/or colonization by soilborne plant pathogenic fungi and nematodes and by those microbes that interfere with infection and how these activities are modified by the physical environment of the soil. Most of our research is with minor pathogens. We are not overly strict or enthusiastic about the distinction between the so-called minor and major pathogens, but, we think it is practical to make this distinction in root-microbe ecology. Salt (1979) described minor pathogens as being restricted to juvenile tissues and widely distributed in cultivated soils, having a wide host range, depending on favorable environmental conditions and the disposition of the host, not causing distinctive symptoms and occurring in mixed infections. We put less emphasis on tissue age and we feel minor pathogens include a wide variety of microorganisms, some of which may be innocuous or even beneficial at certain stages in plant development. In studies of patterns of feeder root infection of alfalfa by minor pathogens such as species of Fusarium, Pythium, and Rhizoctonia, it was found that there were strong seasonal influences from both the qualitative and quantitative view (Hancock, 1985). Infection by these three genera were not correlated. Each apparently operated independently of the other. Pythium infection was tied most closely to the periods of the year when feeder root formation or growth was greatest. Plant pathogenic members of this genus usually respond rapidly to exogenous sources of nurients and are pioneer colonizers of fresh organic materials or juvenile plnat tissues such as seeds and young roots. The infection patterns by the other two genera were more difficult to explain. Fusarium was found to colonize roots early in their development but apparently not as rapidly as Pythium (Gerik et al., 1987; Hancock, 1985). On the other hand, the cortical colonization abilities of Fusarium, especially z. oxysporum, and of Rhizoctonia solani, may be stronger than those Of Pythium. The development of these organisms in roots is of great interest to us because they closely fit the description of minor pathogens. It appears from our studies and those of others that initial infection by several of the root infecting fungi is immediately behind the root tip (Gerik et al., 1987; Smith and Walker, 1981). This region is known to be a major site of metabolite exudation and of mucigel extrusion (Schroth and Hildebrand, 1964; Russell, 1977). Exudates stimulate germination of dormant propagules and attract hyphal growth and -429-
motile states of root colonizing microbes. Microscopic examinations are made after retrieving roots and attempting to reconstruct the sequence of events, Rhizoplane microbes have often been studied in this way, usually with no taxonomic distinction. Immunological methods show promise in this regard. Using a specific type enzyme-linked immunosorbent assay (ELIZA) stain, Huisman and his students.found that initial colonization of the root cortex by Verticillium dahliae and E . oxysporum occurred near the root tip (Gerik et al.,1987). The details of succession of fungi and other microbes in the root apex region remain to be described but are critical to our understanding of root infection by both "minor" and "major" plant pathogens and, consequently, to root health. As noted by Salt (19791, disease symptoms caused by minor pathogens are difficult to discern. However, Pythium and other minor pathogens can effect direct damage to the root tip which can slow root growth and increase root branching. Root length densities in fumigated soils can be several fold higher than untreated soils, suggesting that when roots are protected from certain microorganisms, their development and/or persistence is greater (unpublished data). Studies with mycorrhizal and nonmycorrhizal fungi indicate that infection patterns established at root tips persist and that further growth of colonies of these organisms in the endorhizosphere or root cortex frequently occurs (Gerik et al., 1987; Smith and Walker, 1981). Fusarium and other nonmycorrhizal fungi can be abundant in feeder roots (Gerik et al., 1987; Hancock, 1985; Yuen and Schroth, 1986). ALTERED ROOT PHYSIOLOGY An aspect of concern in root health is the relationship between root infection by minor pathogens and the function of these organs in the absorption of mineral nutrients and water and the influence of these associations on other plant physiological processes. Absorption of water and calcium occur most readily at processes. Absorption of water and calcium occur most readily at or near root apices (Russell, 1977). Other mineral nutrients are absorbed along a greater length of the feeder root system. Mycorrhizae contributer to absorption of phosphorus but how do other microbial associations influence water and mineral nutrient uptake by roots? Could interference in these processes be factors in root health? Ayres (1984) reviewed some of the problems major plant parasites cause in regard to root growth and function and noted that, indeed, infection may reduce the efficiency of root absorption processes and transport of water and nutrients to shoots. Minor pathogens may disturb water and nutrient relations of roots but the
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study by Fitt and Hornby (1978) concluded that stelar penetration is an important prerequisite for impeding nutrient transport. However, they also identified a group of pathogens which affected wheat growth before they penetrated the stele and suggested that toxin production could account for these results. The host can be drastically altered as the result of infection, with host metabolism and respiratory processes altered both qualitatively and quantitatively (Goodman et al., 1986). Microbes in the ectoand endorhizosphere could affect uptake processes and general root physiology. For example, Nissen (1971) found that certain bacteria induced uptake of choline sulfate by roots which may have wider implications for root microbe associations. Stimulation of host cell membrane transport processes by plant parasites, including necrotrophs, is well known (Hancock and Huisman, 1981). Toxins might certainly alter normal root uptake functions; it is also possible that changes in root physiology could be induced by infection by minor root pathogens to the detriment of the host in the absence of toxins. Cross-protection or acquired resistance occurs if infection by a weakly virulent pathogen causes a reduction in the severity of disease development after subsequent inoculation with a highly virulent pathogen. In the area of root health, most of the studies of cross-protection have involved vascular wilt pthogens (Hillocks, 1986). The role of cross-protection should be pursued with a broader array of root-microbe associations including minor pathogens. ROOTLET TURNOVER AND ROOT HEALTH In their studies on the meaning of rootlet health of strawberries, Wilhelm and Nelson (1970) concluded that the "health and productiveness of the plant depend upon the ability of the root system to maintain a favorable equilibrium between rates of death and rates of replacement of rootlets". Rootlet turnover is a feature of root biology that must be included in any consideration of root health. There are obvious advantages to the plant in renewing it's feeder root system, especially for perennial plants. A continually growing root system is required for water uptake and soil exploration. However, the feeder root system cannot expand unchecked. An excessively large root system could be a serious burden to the plant in terms of fixed carbon required for maintenance respiration. In some of our studies in the greenhouse, we have observed little relationship between root length densities and shoot growth. However, this topic deserves a more critical review. Where crop plants are stressed, such as with drought or harvest pressures, the size of the feeder root system may bolster plant productivity.
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Information on root biology such as that provided by Deacon and his associates on root cortex death (RCD) must be considered a major new topic in root health (Henry and Deacon, 1981). The implications of these findings in root turnover is of interest even though their meaning for root health are still very unclear. Feeder root death and renewal (root turnover) may somehow depend on microbial-root interactions and RCD may be an initial aspect of this phenomenon. IMPACT OF INTRODUCED MICROBES ON PLANT GROWTH A better knowledge of the biology and dynamics of root growth, infection by pathogenic organisms (minor and major), the development of these organisms in the rhizosphere and their impact on the physiology, growth and development of the host should allow more incisive studies of interactions between the wider rhizosphere microflora and microfauna nad deleterious organisms and contribute enormously to be science of root biology. Empirical studies in soil microbiology and plant pathology indicate that there is much to be learned about microbial processes in the rhizosphere. Why is the growth of crop plants improved when their root systems are exposed to certain microbes but not others? What are the roles of antibiosis, competition for nutrient and space, and hyperparasitism in rhizosphere ecology? Are rhizosphere inhabiting microbes altering root physiology? The literature on the effects of microbes introduced on plant materials or in soil or plant growth media on plant growth is growing rapidly (Brown, 1974; Cook and Baker,1983; Schroth and Weinhold, 1986). In spite of the difficulty in explaining results of some of this work, there is now considerable effort on the part of a number of laboratories in this field. We believe that the difficulties encountered with reproducibility of results are explainable and should not deter work on ways to alter the rhizosphere to improve root health. Precedents of erratic results with chemical control of soilborne pathogens are also abundant. Tailoring the biocontrol agent or soil pesticide to fit peculiar site needs may be necessary. Brown (1974) reviewed the early literature and several others have reviewed more recent investigations on modifying the rhizosphere to improve plant growth (Baker, 1986; Burr and Caesar, 1984; Cook and Baker, 1983; Schroth and Weinhold, 1986). There is general agreement that the rhizosphere, once thought immutable to change, is vulnerable to human manipulation (Cook and Baker, 1983). Alterations in the natural rhizosphere populations can be effected by changing the physical or chemical properties of the soil through various normal agricultural practices (Linderman, 1986). Definition is needed but use of broad management
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practices to favor the development of beneficial organisms in the rhizosphere has a legitimate place in the field of plant health (Cook and Baker, 1983). Current interest in modifying the rhizosphere with selective nutrient amendments is an extension of the use of cultural methods for rhizosphere modification (Van Gundy and Schroth, personnel communication). Application of ndividual strains of rhizosphere inhabiting organisms to plants is a promising area of research in biological control (Schroth and Hancock 1981). However, the most clearly effective and consistent biocontro to date has been with seed treatments and control of damping-off where efficacy in many cases is equal to the very best fungicides (Harman et al., 1981; Howell and Stipanivic, 1980; Martin and Hancock, 1987; Veselg and Hejddnek, 1984). There are still many important questions such as better defining the environmental limitations of these control activities, mechanisms of activity, practical production of inoculum and it's longevity, genetic variation of isolates, strain genetic stability, practical delivery methods, m., before we can expect commercial development. Spatially and temporally focused biological control systems, such as with damping-off or the already commercially available crown-gall biological control system, have the best chance of immediate development and application (Schroth and Hancock, 1981). The process of colonization of the rhizosphere by beneficial organisms is poorly understood. Direct microscopic observations indicate that rhizoplane colonization by bacteria is surprisingly sparce, with no more than 2 to 8 % of the root surface colonized (Rovira et al., 1983). Weller (1983) found that wheat roots were colonized when seeds were coated with a fluorescent pseudomonad and that the greatest colonization occurred near the seed, declining toward the root tip. When seed pieces of potato were treated with a rhizosphere colonizing strain of Pseudomonas fluorescens with populations of about lo8 cfu per seed piece, colonization of roots of field grown plants was nonuniform and Schippers (1980) found that when wheat roots were grown in soil, the root tip was usually nearly uncolonized. Bahme (1987) found root colonization was improved if root colonizing strains were added to soil prior fo planting in a granular preparation or suspended in water and added to soil with a drip irrigation system developed by Van Gundy and Garabedian (1984). Pythium oligandrum protects sugar beet from damping-off by p. ultimum when seeds are coated but does not grow beyond the base of the radicle (Martin and Hancock, 1987). Wild-type strains of Trichoderma harzianum are effective biocontrol agents against damping off but are poor root colonizers. However, Ahmad and Baker (1987)
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found that benomyl resistant mutants of this species are capable of colonizing roots of seedlings of several species of plants. Knowledge of the characters that allow bacteria and fungi to colonize roots are crucial to biocontrol applications. Clearly, this is a capacity that deleterious microbes possess. It must also be a characteristic of potential biological control agents if they are to compete effectively with root infecting pathogens. Baker and Scher (1987) wrote that "root pathogen controlling agents must colonize the rhizosphere". The results of Ahmad and Baker (1987) indicate that improvement of the rhizosphere colonizing ability of biocontrol agents is attainable by genetic alteration. Riocontrol agents must colonize root apices if they are to interfere with infection by minor pathogens. Soil amendments are also a possible means of improving rhizosphere colonization (Baker, 1986). Improved delivery methods, as found by Bahme (1987) in his investigations with potato, are also practical means of improving rhizosphere colonization. Drip irrigations is increasing as a means of supplying water in semiarid regions and offers an opportunity to apply biocontrol agents and other additives (Van Gundy and Garabedian, 1984). Mechanisms of biocontrol of root infecting organisms by beneficial rhizosphere inhabiting microbes seem to take several forms, with no clearcut patterns. This topic has been intensively reviewed. The principal conclusion is that biocontrol agents very often produce inhibitory substances, either antibiotics, toxic metabolites or siderophores (Ah1 et al., 1986; Cook and Baker, 1983; Schroth and Weinhold, 1986). There are considerable numbers of reports that siderophores are a major means by which soil inhabiting fluorescent pseudomonads compete in the rhizoplane (Loper and Schroth, 1986). However, in nature, the "mechanism of action", is likely to be plural. Loper and Schroth (1986) observed that "the role of siderophores in microbial interactions cannot be considered in isolation of other factors that influence the colonization and other activities of bacterial antagonists". This observation can be applied to other mechanisms. Although less well documented, competition for nutrients and hyperparasitism are also considered as ways in which rhizosphere inhabiting beneficial organisms protect seeds or roots from pathogenic microbes (Cook and Baker, 1983; Martin and Hancock, 1987). A firmer understanding of the mechanisms responsible for biocontrol should assist in selection of better wild strains of biocontrol agents as well as form a foundation for genetic engineering.
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FUTURE RESEARCH NEEDS IN THE ROOT HEALTH FIELD Research on root health in the future must have an interdisciplinary perspective and be comprehensive in outlook. We believe the best approach to improving root health is by integrated crop and pest management. But, we cannot effectively manage a system that we do not fully understand. Erratic and unexplained results from chemical and biological control practices are intolerable and show our ignorance of root and soil biology. The goal of future research on root health must be directed toward attaining a basic understanding of root-microbe interactions as they occur in the field. Progress toward this goal will depend on major improvements in techniques for studying there interactions and improved analyses of the systems. This goal is attainable. Much progress has been made in the last decade in showing that introduced microbes can improve crop growth. Studies of an applied nature are important also and should be incorporated into future research needs. With adequate governmental financial support and involvement from biotechnology interests, we are convinced that progress in root health knowledge and applications will be rapid.
REFERENCES AHL, P., VOISARD, C.,DEFAGO, G.: Iron bound-siderophores, cyanic acid, and antibiotics involved in suppression of Thielaviopsis basicola by a Pseudomonas fluorescens strain. J. Phytopathol. 166: 121 -134, 1986. AHMAD, J.S., BAKER, R.: Rhizosphere competence of Trichoderma harzianum. Phytopathology 77: 182-189, 1987. AYRES, P.G.: Effects of infection on root growth and function; consequences for plant nutrient and water relations. In: WOOD, R.K.S., JELLIS, G.J. (Eds.): Plant Diseases: Infection, Damage, and Loss. Pp. 105-117. Blackwel Scientific Publications, Oxford 1984. BAHME, J.B.: Colonization of plants by rhizobacteria and the effect on associated microflora: Ph.D. Thesis, Univ. Calif., Berkeley 1987. BAKER, R.: Biological control: an overview. Can. J. Plant Pathol. 8: 218-221, 1986. BAKER, R., SCHER, F . M . : Enhancing tho activity of biological control agents. In: CHET, I. (Ed.): Innovative Approaches to Plant Disease Control. Pp. 1-16. Wiley, New York 1987. BROWN, M.: Seed and root bacterization. Ann. Rev. Phytopathol. 12: 181-197, 1974.
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BURR, T.J., CAESAR, A . : Beneficial plant bacteria. Critical Rev. Plant Sci. 2: 1-20, 1983. COOK, R.J., BAKER, K.F.: The Nature and Practice of Biological Control of Plant Pathogens. Amer. Phytopathol. SOC., St. Paul, MN 1983. FITT, B.D.L., HORNBY, D.: Effects of root-infecting fungi on wheat transport processes and growth. Physiol. Plant Pathol. 13: 335-346, 1978. GERIK, J . S . , LOMMEL, HUISMAN, O.C.: A specific serological staining procedure O f Verticillium dahliae in cotton root tissue. Phytopathology 77: 261-265, 1987. HANCOCK, J.G.: Fungal infection of feeder rootlets of alfalfa. Phytopathology 75: 1112-1120, 1985. HANCOCK, J.G., HUISMAN, O.C.: Nutrient movement in host-pathogen systems. Ann. Rev. Phytopathol. 19: 309-331, 1981. HENRY, C.M., DEACON, J.W.: Natural (non-pathogenic) death of the cortex of wheat and barley seminal roots, as evidenced by nuclear staining with acridine orange. Plant and Soil 60: 255-274, 1981. HILUXKS, R.J.: Cross protection between strains of Fusarium oxysporum f. sp. vasinfectum and its effect on vascular resistance mechanisms. J. Phytopathol. 117: 216-225, 1986. HOWELL, C.R., STIPANOVIC, R.D.: Suppression of Pythium ultimum induced damping-off of cotton seedlings by Pseudomonas fluorescens and its antibiotic, pyoluteorin. Phytopathology 70: 712-715, 1980. LINDERMAN, R.G.: Managing rhizosphere microorganisms in the production of horticultural crops. Hort Sci. 21: 1299-1302, 1986. LOPER, J.E., SCHROTH, M.N.: Importance of siderophores in microbial interactions in the rhizosphere. In: SWINBURNE, T.R. (Ed.): Iron, Siderophores, and Plant Diseases. Pp. 85-98. Plenum Publ. Corp., London 1986. MARTIN, F.N., NAHCOCK, J.G.: The use of Pythium oligandrum for biological control o f preemergence damping-off caused by g. ultimum. Phytopathology 77: (in press) , 1987. NISSEN, P.: Uptake mechanisms: inorganic and organic. Ann. Rev. Plant Physiol. 25: 53-79, 1974. ROVIRA, A.D., NEWMAN, E.I., BOWEN, H.J., CAMPBELL, R.: Quantitative assessment of the rhizosphere microflora by direct microscopy. Soil Biol. Biochem. 6: 211-216, 1974. RUSSELL, R.S.: Plant Root Systems: Their Function and Interaction with the Soil. McGraw-Hill Book Co., Ltd., London 1977. SALT, G.A.: The increasing interest in "minor pathogens". In: SCHIpPERS, B., GAMS, W. (Eds.): Soil-Borne Plant Pathogens. Pp. 289312. Academic Press, London 1979.
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SCHROTH, M.N., HILDEBRAND, D.C.: Influence of plant exudates on root-infecting fungi. Ann. Rev. Phytopathol. 2: 389-393, 1964. SCHROTH, M.N., HANCOCK, J.G.: Selected topics in biological control. Ann. Rev. Microbiol. 35: 453-476, 1981. SCHROTH, M.N., WEINHOLD, A.R.: Root-colonizing bacteria and plant health. Hort Sci. 21: 1295-1298, 1986. SMITH, S.E., WALKER, N.A.: A quantitative study of mycorrhizal infection in Trifolium: separate determination of rates of infection and of mycelial growth. New Phytol. 89: 225-240, 1981. VAN GUNDY, S.D., GARABEDIAN, S.: Application of nematocides through drip irrigation systems. Med. Fac. Landboww. Rijksuniv. Gent, 49/2b: 629-634, 1984. VAN W U R D E , J.W.L., SCHIPPERS, B.: Bacterial colonization of seminal wheat roots. Soil Biol. Biochem. 12: 559-565, 1980. VESEL?, D., HEJDhEK, S.: Microbial relations of Pythium oligandrum and problems in the use of this organism for the biological control of damping-off of sugar beet. Zentralbl. Mikrobiol. 139: 254-265, 1984. WELLER, D.M.: Colonization of wheat roots by a fluorescent pseudomonad suppressive to take-all. Phytopathology 73: 1548-1553, 1983. WILHELM. S.: Chemical treatments and inoculum potential of soil. Ann. Rev. Phytopathol. 4: 53-78, 1966. WILHELM, S., NELSON, P.E.: A concept of rootlet health of strawberries in pathogen-free field soil achieved by fumigation. In: TOUSSOUN, T.A., BEGA, R.V., NELSON, P.E. (Eds.): Root Diseases and Soil-Borne Pathogens. Pp. 208-214. Univ. Calif. Press, B e r keley 1970. WINDHAM, M.T., ELAD, Y., BAKER, R.: A mechanism for increased plant growth induced by Trichoderma spp. Phytopathology 76: 518-521, 1986. YUEN, G.Y., SCHROTH, M.N.: Ihteractions of Pseudomonas fluorescens strain E 6 with ornamental plants and its effect on the composition of root-colonizing microflora. Phytopathology 76: 176-180, 1986.
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Schroth M.N.I
IDepartment of Plant Pathology, University of California, Berkeley 9 4 7 2 0 , USA %epartment of Nematology, University of California, Riverside 9 2 5 2 1 , USA
ABSTRACT Growth stimulation and root health, dynamics of root colonization by microorganisms, altered root physiology as the result of infection, rootlet turnover and root health, impact of introduced microbes on plant growth, and future research needs in the root health field are the main topics mentioned and discussed in the paper.
INTRODUCTION Root health is a topic of utmost importance to plant agriculture. It is such a poignant topic today because increased practices of monoculture in many parts of the world has led to an observable decline in root health. Our views of root-microbe interactions are often compartmentalized which has impeded progress in studies of root health. The overwhelming complexity of this subject understandably invites narrow research approaches. From the standpoint of scientific experimentation, it seems logical to study single root-microbe interactions or even to study root development and physiology in the abaence of microbes. While these approaches have unquestionably advanced our knowledge of root biology, the problems of root health are complex and must be addressed more comprehensively. A better definition of root microbial ecology should be one of the principal modern goals in plant biology. Without a foundation of information on root biology, our quest for improvement of root health will be largely empirical simply because we have few alternatives. Within plant pathology, the stage for an appreciation of general root health may have first been set when observations were made that soil fumigation with general biocides promoted significant plant growth responses (Wilhelm, 1 9 6 6 ) . This phenomenon has been observed with many -427-
crops in many parts of the world since the nineteenth century. More recently, the so-called plant growth promotion associated with the introduction of selected soil microbes on seed or roots of propagation materials or directly to soil has galvanized this interest and stimulated further research on the bases of these plant responses (Cook and Baker, 1983; Schroth and Weinhold, 1986). Perhaps most importantly, this latter work has given credence to the idea that root health is managable by biological and cultural means. GROWTH STIMULATION AND ROOT HEALTH Cook (R.J. Cook, personnel communication) distinguished between direct growth stimulation and growth enhancement via protection from disease. This is an important distinction and should be kept in mind when considering mechanisms of growth promotion. In addition to the rhizobial and mycorrhizal relationships, there are several reports of microbe-root interactions where plant growth is directly stimulated (Windham et al., 1986). However, in practice it is exceedingly difficult at the stage of development of this field to differentiate between growth stimulation and elimination of some of the restraints on the genotypic growth potential of plants as a result of beneficial microbial activities in the rhizosphere. We simply do not have the techniques to differentiate between these possibilities, especially under field conditions. In our research, we are screening microbes that are antagonistic in culture against soilborne plant pathogens (fungi and nematodes). Thus, our approach is geared toward biological control rather than growth stimulation per se. We are interested in whether and how antagonists interfere with root infection. Protection from root infection by plant pathogens by microbial antagonists is one possible basis for improved root health. Work with soil fumigants and certain selective pesticides indicates that mild but chronic types of root diseases retard plant growth and that relief from these diseases by treatment results in improved plant growth. It is tempting to draw conclusions on cause-effect relations in biological control investigations on the basis of studies with pesticides. Because there are so many effects of pesticides on the soil microbial communities (and even directly on root physiology), including mineral nutrient releases, we should be cautious about linking these different phenomenona.
DYNAMICS OF ROOT COLONIZATION BY MICROORGANISMS We are striving to gain a better understanding of the dynamics of the interactions between microbes and roots. Our initial investigations are directed at obtaining a better picture of the processes of root infection and/or colonization by soilborne plant pathogenic fungi and nematodes and by those microbes that interfere with infection and how these activities are modified by the physical environment of the soil. Most of our research is with minor pathogens. We are not overly strict or enthusiastic about the distinction between the so-called minor and major pathogens, but, we think it is practical to make this distinction in root-microbe ecology. Salt (1979) described minor pathogens as being restricted to juvenile tissues and widely distributed in cultivated soils, having a wide host range, depending on favorable environmental conditions and the disposition of the host, not causing distinctive symptoms and occurring in mixed infections. We put less emphasis on tissue age and we feel minor pathogens include a wide variety of microorganisms, some of which may be innocuous or even beneficial at certain stages in plant development. In studies of patterns of feeder root infection of alfalfa by minor pathogens such as species of Fusarium, Pythium, and Rhizoctonia, it was found that there were strong seasonal influences from both the qualitative and quantitative view (Hancock, 1985). Infection by these three genera were not correlated. Each apparently operated independently of the other. Pythium infection was tied most closely to the periods of the year when feeder root formation or growth was greatest. Plant pathogenic members of this genus usually respond rapidly to exogenous sources of nurients and are pioneer colonizers of fresh organic materials or juvenile plnat tissues such as seeds and young roots. The infection patterns by the other two genera were more difficult to explain. Fusarium was found to colonize roots early in their development but apparently not as rapidly as Pythium (Gerik et al., 1987; Hancock, 1985). On the other hand, the cortical colonization abilities of Fusarium, especially z. oxysporum, and of Rhizoctonia solani, may be stronger than those Of Pythium. The development of these organisms in roots is of great interest to us because they closely fit the description of minor pathogens. It appears from our studies and those of others that initial infection by several of the root infecting fungi is immediately behind the root tip (Gerik et al., 1987; Smith and Walker, 1981). This region is known to be a major site of metabolite exudation and of mucigel extrusion (Schroth and Hildebrand, 1964; Russell, 1977). Exudates stimulate germination of dormant propagules and attract hyphal growth and -429-
motile states of root colonizing microbes. Microscopic examinations are made after retrieving roots and attempting to reconstruct the sequence of events, Rhizoplane microbes have often been studied in this way, usually with no taxonomic distinction. Immunological methods show promise in this regard. Using a specific type enzyme-linked immunosorbent assay (ELIZA) stain, Huisman and his students.found that initial colonization of the root cortex by Verticillium dahliae and E . oxysporum occurred near the root tip (Gerik et al.,1987). The details of succession of fungi and other microbes in the root apex region remain to be described but are critical to our understanding of root infection by both "minor" and "major" plant pathogens and, consequently, to root health. As noted by Salt (19791, disease symptoms caused by minor pathogens are difficult to discern. However, Pythium and other minor pathogens can effect direct damage to the root tip which can slow root growth and increase root branching. Root length densities in fumigated soils can be several fold higher than untreated soils, suggesting that when roots are protected from certain microorganisms, their development and/or persistence is greater (unpublished data). Studies with mycorrhizal and nonmycorrhizal fungi indicate that infection patterns established at root tips persist and that further growth of colonies of these organisms in the endorhizosphere or root cortex frequently occurs (Gerik et al., 1987; Smith and Walker, 1981). Fusarium and other nonmycorrhizal fungi can be abundant in feeder roots (Gerik et al., 1987; Hancock, 1985; Yuen and Schroth, 1986). ALTERED ROOT PHYSIOLOGY An aspect of concern in root health is the relationship between root infection by minor pathogens and the function of these organs in the absorption of mineral nutrients and water and the influence of these associations on other plant physiological processes. Absorption of water and calcium occur most readily at processes. Absorption of water and calcium occur most readily at or near root apices (Russell, 1977). Other mineral nutrients are absorbed along a greater length of the feeder root system. Mycorrhizae contributer to absorption of phosphorus but how do other microbial associations influence water and mineral nutrient uptake by roots? Could interference in these processes be factors in root health? Ayres (1984) reviewed some of the problems major plant parasites cause in regard to root growth and function and noted that, indeed, infection may reduce the efficiency of root absorption processes and transport of water and nutrients to shoots. Minor pathogens may disturb water and nutrient relations of roots but the
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study by Fitt and Hornby (1978) concluded that stelar penetration is an important prerequisite for impeding nutrient transport. However, they also identified a group of pathogens which affected wheat growth before they penetrated the stele and suggested that toxin production could account for these results. The host can be drastically altered as the result of infection, with host metabolism and respiratory processes altered both qualitatively and quantitatively (Goodman et al., 1986). Microbes in the ectoand endorhizosphere could affect uptake processes and general root physiology. For example, Nissen (1971) found that certain bacteria induced uptake of choline sulfate by roots which may have wider implications for root microbe associations. Stimulation of host cell membrane transport processes by plant parasites, including necrotrophs, is well known (Hancock and Huisman, 1981). Toxins might certainly alter normal root uptake functions; it is also possible that changes in root physiology could be induced by infection by minor root pathogens to the detriment of the host in the absence of toxins. Cross-protection or acquired resistance occurs if infection by a weakly virulent pathogen causes a reduction in the severity of disease development after subsequent inoculation with a highly virulent pathogen. In the area of root health, most of the studies of cross-protection have involved vascular wilt pthogens (Hillocks, 1986). The role of cross-protection should be pursued with a broader array of root-microbe associations including minor pathogens. ROOTLET TURNOVER AND ROOT HEALTH In their studies on the meaning of rootlet health of strawberries, Wilhelm and Nelson (1970) concluded that the "health and productiveness of the plant depend upon the ability of the root system to maintain a favorable equilibrium between rates of death and rates of replacement of rootlets". Rootlet turnover is a feature of root biology that must be included in any consideration of root health. There are obvious advantages to the plant in renewing it's feeder root system, especially for perennial plants. A continually growing root system is required for water uptake and soil exploration. However, the feeder root system cannot expand unchecked. An excessively large root system could be a serious burden to the plant in terms of fixed carbon required for maintenance respiration. In some of our studies in the greenhouse, we have observed little relationship between root length densities and shoot growth. However, this topic deserves a more critical review. Where crop plants are stressed, such as with drought or harvest pressures, the size of the feeder root system may bolster plant productivity.
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Information on root biology such as that provided by Deacon and his associates on root cortex death (RCD) must be considered a major new topic in root health (Henry and Deacon, 1981). The implications of these findings in root turnover is of interest even though their meaning for root health are still very unclear. Feeder root death and renewal (root turnover) may somehow depend on microbial-root interactions and RCD may be an initial aspect of this phenomenon. IMPACT OF INTRODUCED MICROBES ON PLANT GROWTH A better knowledge of the biology and dynamics of root growth, infection by pathogenic organisms (minor and major), the development of these organisms in the rhizosphere and their impact on the physiology, growth and development of the host should allow more incisive studies of interactions between the wider rhizosphere microflora and microfauna nad deleterious organisms and contribute enormously to be science of root biology. Empirical studies in soil microbiology and plant pathology indicate that there is much to be learned about microbial processes in the rhizosphere. Why is the growth of crop plants improved when their root systems are exposed to certain microbes but not others? What are the roles of antibiosis, competition for nutrient and space, and hyperparasitism in rhizosphere ecology? Are rhizosphere inhabiting microbes altering root physiology? The literature on the effects of microbes introduced on plant materials or in soil or plant growth media on plant growth is growing rapidly (Brown, 1974; Cook and Baker,1983; Schroth and Weinhold, 1986). In spite of the difficulty in explaining results of some of this work, there is now considerable effort on the part of a number of laboratories in this field. We believe that the difficulties encountered with reproducibility of results are explainable and should not deter work on ways to alter the rhizosphere to improve root health. Precedents of erratic results with chemical control of soilborne pathogens are also abundant. Tailoring the biocontrol agent or soil pesticide to fit peculiar site needs may be necessary. Brown (1974) reviewed the early literature and several others have reviewed more recent investigations on modifying the rhizosphere to improve plant growth (Baker, 1986; Burr and Caesar, 1984; Cook and Baker, 1983; Schroth and Weinhold, 1986). There is general agreement that the rhizosphere, once thought immutable to change, is vulnerable to human manipulation (Cook and Baker, 1983). Alterations in the natural rhizosphere populations can be effected by changing the physical or chemical properties of the soil through various normal agricultural practices (Linderman, 1986). Definition is needed but use of broad management
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practices to favor the development of beneficial organisms in the rhizosphere has a legitimate place in the field of plant health (Cook and Baker, 1983). Current interest in modifying the rhizosphere with selective nutrient amendments is an extension of the use of cultural methods for rhizosphere modification (Van Gundy and Schroth, personnel communication). Application of ndividual strains of rhizosphere inhabiting organisms to plants is a promising area of research in biological control (Schroth and Hancock 1981). However, the most clearly effective and consistent biocontro to date has been with seed treatments and control of damping-off where efficacy in many cases is equal to the very best fungicides (Harman et al., 1981; Howell and Stipanivic, 1980; Martin and Hancock, 1987; Veselg and Hejddnek, 1984). There are still many important questions such as better defining the environmental limitations of these control activities, mechanisms of activity, practical production of inoculum and it's longevity, genetic variation of isolates, strain genetic stability, practical delivery methods, m., before we can expect commercial development. Spatially and temporally focused biological control systems, such as with damping-off or the already commercially available crown-gall biological control system, have the best chance of immediate development and application (Schroth and Hancock, 1981). The process of colonization of the rhizosphere by beneficial organisms is poorly understood. Direct microscopic observations indicate that rhizoplane colonization by bacteria is surprisingly sparce, with no more than 2 to 8 % of the root surface colonized (Rovira et al., 1983). Weller (1983) found that wheat roots were colonized when seeds were coated with a fluorescent pseudomonad and that the greatest colonization occurred near the seed, declining toward the root tip. When seed pieces of potato were treated with a rhizosphere colonizing strain of Pseudomonas fluorescens with populations of about lo8 cfu per seed piece, colonization of roots of field grown plants was nonuniform and Schippers (1980) found that when wheat roots were grown in soil, the root tip was usually nearly uncolonized. Bahme (1987) found root colonization was improved if root colonizing strains were added to soil prior fo planting in a granular preparation or suspended in water and added to soil with a drip irrigation system developed by Van Gundy and Garabedian (1984). Pythium oligandrum protects sugar beet from damping-off by p. ultimum when seeds are coated but does not grow beyond the base of the radicle (Martin and Hancock, 1987). Wild-type strains of Trichoderma harzianum are effective biocontrol agents against damping off but are poor root colonizers. However, Ahmad and Baker (1987)
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found that benomyl resistant mutants of this species are capable of colonizing roots of seedlings of several species of plants. Knowledge of the characters that allow bacteria and fungi to colonize roots are crucial to biocontrol applications. Clearly, this is a capacity that deleterious microbes possess. It must also be a characteristic of potential biological control agents if they are to compete effectively with root infecting pathogens. Baker and Scher (1987) wrote that "root pathogen controlling agents must colonize the rhizosphere". The results of Ahmad and Baker (1987) indicate that improvement of the rhizosphere colonizing ability of biocontrol agents is attainable by genetic alteration. Riocontrol agents must colonize root apices if they are to interfere with infection by minor pathogens. Soil amendments are also a possible means of improving rhizosphere colonization (Baker, 1986). Improved delivery methods, as found by Bahme (1987) in his investigations with potato, are also practical means of improving rhizosphere colonization. Drip irrigations is increasing as a means of supplying water in semiarid regions and offers an opportunity to apply biocontrol agents and other additives (Van Gundy and Garabedian, 1984). Mechanisms of biocontrol of root infecting organisms by beneficial rhizosphere inhabiting microbes seem to take several forms, with no clearcut patterns. This topic has been intensively reviewed. The principal conclusion is that biocontrol agents very often produce inhibitory substances, either antibiotics, toxic metabolites or siderophores (Ah1 et al., 1986; Cook and Baker, 1983; Schroth and Weinhold, 1986). There are considerable numbers of reports that siderophores are a major means by which soil inhabiting fluorescent pseudomonads compete in the rhizoplane (Loper and Schroth, 1986). However, in nature, the "mechanism of action", is likely to be plural. Loper and Schroth (1986) observed that "the role of siderophores in microbial interactions cannot be considered in isolation of other factors that influence the colonization and other activities of bacterial antagonists". This observation can be applied to other mechanisms. Although less well documented, competition for nutrients and hyperparasitism are also considered as ways in which rhizosphere inhabiting beneficial organisms protect seeds or roots from pathogenic microbes (Cook and Baker, 1983; Martin and Hancock, 1987). A firmer understanding of the mechanisms responsible for biocontrol should assist in selection of better wild strains of biocontrol agents as well as form a foundation for genetic engineering.
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FUTURE RESEARCH NEEDS IN THE ROOT HEALTH FIELD Research on root health in the future must have an interdisciplinary perspective and be comprehensive in outlook. We believe the best approach to improving root health is by integrated crop and pest management. But, we cannot effectively manage a system that we do not fully understand. Erratic and unexplained results from chemical and biological control practices are intolerable and show our ignorance of root and soil biology. The goal of future research on root health must be directed toward attaining a basic understanding of root-microbe interactions as they occur in the field. Progress toward this goal will depend on major improvements in techniques for studying there interactions and improved analyses of the systems. This goal is attainable. Much progress has been made in the last decade in showing that introduced microbes can improve crop growth. Studies of an applied nature are important also and should be incorporated into future research needs. With adequate governmental financial support and involvement from biotechnology interests, we are convinced that progress in root health knowledge and applications will be rapid.
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