DEVELOPMENT AND INTERACTION BETWEEN MICROBIAL COMMUNITIES ON THE ROOT SURFACE Lynch M.J. Microbiology Department, AFRC Institute of Horticultural Research, Littlehampton, West Sussex, BN17 6LP, UK
-ABSTRACT Microbial biomass formation on root surfaces can be measured in plants growing in solution with or without an inert solid support. Carbon flow to the biomass can be measured by growing plants in solution or soil on a continuous source of 14C02 and the expected biomass formation predicted. The lack of correlation between measured and predicted biomass can be explained by oligotrophic growth of the micro-organisms. Microbial species within communities on the root surface can interact with each other, and a target for root inoculation is to elev%te the PFOllation of beneficial organisms within the community.
RHIZOSPHERE ANATOMY The rh.izosphere is today regarded as the zone of microbial proliferation in and around roots. A variety of light and electron microscopic techniques have been used to observe bacteria and fungi around roots (the ectorhizosphere), on the root surface (the rhizoplane) and within the root (the endorhizosphere) (Lynch, 1982). Bacteria develop as discrete colonies on the root surface, leaving large areas of the root surface uncolonized (probably greater than 80 % ) . There tends to be a proliferation of colonization at the junctions between the intercellular spaces of the epidermis. Bacterial colonization of the cortex has also been found but it has not been reliably estimated. The major interest in fungi of the rhizosphere has been focussed on mycorrhizas. In some tree species ectomycorrhizas are formed so extensively that they can be separated from the roots and weighed. Endomycorrhizal colonization however is usually assessed by staining with lactophenol and trypan blue and examining the roots microscopically. However root colonization following fungal inoculation of the rhizosphere with nonsymbiotic fungi has not usually been assessed. Rather the success of inoculated Organisms has been determined by measuring
the number of C 0 l O W - f ~ propagules of the inoculant. This does not necessarily relate to the fungal biomass present.
CARBON FLOW TO THE RHIZOSPHERE Growth of micro-organisms in the rhizosphere is dependent on root derived carbon which includes exudates (leaked from living roots), secretions (actively pumped from the roots), lysates (passively released from the roots during autolysis) and mucilage (giving rise to mucigel which is of both plant and microbial origin). The C:N ratio of these materials has not been measured with precision but it has been estimated to be around 40:l (Lynch, 1986). Plants can be grown on a source of uniformly-labelled 14C02 to assess the flow of carbon from roots to the microbial population; this flow can account for up to 40 % of the plant photosynthate produced (Whipps and Lynch, 1985). It is unclear however if carbon or nitrogen are the growth-limiting substrates to specific components of the rhizosphere population. It can be expected that the C:N ratio of bacterial cells will usually be between 5:l and 1O:l (Barber and Lynch, 1977) but it is not known what proportion of the total N available to roots is intercepted by rhizosphere micro-organisms. The contribution of substrates exogenous to the rhizosphere to the nutrition of rhizosphere organisms is also unclear. For example, fungi may colonize plant residues and continue to use them as substrates while hyphae spread to colonize roots. In natural systems there may therefore be a two-way flow of carbon into the rhizosphere. This consideration could be crucial in attempts deliberately to colonize the rhizosphere by beneficial organisms, viz it may be necessary to introduce the organism on a substrate which will give it a competitive advantage over other organisms which are only present as slow-growing or dormant propagules.
RHIZOSPHERE NUTRITION Table 1 defines some terms which have traditionally been used to describe the nutrition of aoil organisms, and compares them with modern ecological terminology.There is a similarity in meaning between autochthonous and oligotrophic, and zymogenous and copiotrophic, but the terms certainly do not equate. Traditionally rhizosphere organiams have been regarded as zymogenous, and this would imply that they will disappear from the rhizosphere when the substrate supply becomes exhausted. In practice the truly successful rhizosphere inoculant would be expected to exhibit copiotrophic growth on the substrate base on which
Nutrition of soil organisms
Autochthonous low but steady level of activity on native soil
Zymogenous rapid metabolism of soil organic matter
MODERN ECOLOGICAL TERMINOLOGY scavenging of Copiotrophic growth on coOligotrophic scarcesupply of nutrients such pious nutrients such as fresh organic manures as trace carbon compourds in the soil atmosphere
it is introduced to soil, it would be zymogenous on the carbon products available from the substrate base and from the root-derived carbon, it would become oligotrophic when the supply of these substrates is reduced and then it would remain in the soil in the autochthonous mode until fresh substrate became available again. For organisms which might pose some risk to the environment this latter mode would not be a desirable trait.
MEASURED AND CALCULATED MICROBIAL BIOMASS By measuring the flow of carbon to the rhizosphere using the 14C method and assuming a growth yield of 0.35 g of bianass per g of carbon (glucose)substrate consumed (ignoring any carbon used in maintenance of the population), the rhizosphere biomass can be calculated. Further by counting the number of cells associated with the root using a washing technique and by determining the mean cell weight of the members of the population by growing them in luxuriant media, the biomass of organisms actually present on the root can be determined. Table 2 indicates that the biomass observed is usually greater than would be expected than that calculated from the carbon flow measuyements. This must mean that cellp which colonize the roots shrink under the (natural)conditions of substrate limitation compared with their growth on luxuriant nutrients or that the organisms colonizing roots grow as oligotrophs and acquire a proportion of their carbon for growth by utilizing trace carbon compounds. This concept was tested by growing wheat roots in soil, isolating a dominant Gram-negative rod from the roots (Enterobacter cloacae M0/1) and comparing its growth on gnotobiotic wheat roots with E . cloacae C2/4 which had been isolated as a colonist of straw (Chapman and Lynch,
Calculated and measured substrate inputs to the rhizoaphere
Barber and Whipps and Lynch Lynch (1977) (1984) Barley Microbial biomass Mean cell weight, 10-l’ g Bacterial biomass, 9.mg-l dry root Substrate input Calculated, gC.mg-’ dry root Measured, 9C.rng-l dry root
1985). The two bacterial strains were of similar size but in a population of each strain there was a very large variation in cell length (1-4 ,um) and this was independent of the substrate availability (nutrient broth or root-derived carbon). To account for the inadequacy of the budgets with a mixed bacterial population on roots described in Table 2,the cell size during rhizosphere growth would have to decrease by 3 and 10 times on average compared with growth on nutrient broth. Therefore if all rhizosphere bacteria behaved like E . cloacae, it seems most likely that there is substantial oligotrophic growth in the rhizosphere. The trace amounts of carbon for this growth could be introduced from forced aeration of roots in experimental systems. The results also indicate the difficulty in analysing soil population biology generally even though not all bacteria may vary in cell size to the extent of E. cloacae. It is increasingly common to use antibiotic marking to trace the fate of soil organisms and it is frequently assumed that biomass can be calculated from counts of viable cells. This could only be valid if the mean cell size or weight under natural soil conditions is known.
COMMUNITY INTERACTIONS When two organisms come together in vitro or on the root surface they can potentially interact in several ways depending on their physiological characteristics. However, those characteristics are often dependent on the substrate base on which the organism grows. For example a potential antagonist may produce an antibiotic on a nutrient-rich agar contained in a Petri dish but the root itself may not pro-
vide the necessary substrates. Then again, even if one plant species provides the substrate, another may not. Thus caution must be exercised in extrapolating in vitro laboratory screens of potential biocontrol agents to the microbial interaction occurring in the field. Chemical
(a.g. pH) and physical (e.g. temperature) factors will likely govern any interaction and such factors can be investigated in the laboratory. Whereas screening procedures for indentifying microbial antagonism in the laboratory must be as quick and simple as possible, they should consider wherever possible field factors which could influence the interaction. Enterobacter cloacae is a bacterium which is a natural colonist O f the endorhizosphere (Kleeberger et al., 1983) and has proved to be effective in controlling damping-off diseases of pea and cucumber (Hadar et al., 1983). In vitro studies showed that the sugar composition of the growth medium determined the inhibitory effect of E. cloacae on Pythium ultimum and that growth inhibition was linked to binding of the bacteria to the hyphae, thus indicating that a lectin-type interaction is probably involved (Nelson et al., 1986). This interaction may not however be the exclusive mode of action in the biological control. In addition to lectin interactions, the following have been proposed as modes of action which could be involved in biological control: competition for available substrates, production of antibiotics, production of cell walldegrading enzymes, physical restriction of pathogens to reduce site occupancy, ionophore production by antagonists to impede ion uptake by the pathogen and cross protection or induced resistance in the host. Many of the investigations of practical biocontrol systems have paid little attention to the mode of action but rather have concentrated on isolating antagonists from the soil by either in vitro or in vivo study and then evaluating their field effectivness (Cook and Baker, 1983). The search for potential antagonists might prove more rewarding if the modes of action are considered. There is increasing evidence for the range of actions possible (Lynch, 1987a) but the truly successful biocontrol agent is unlikely to act in a single mode. It should be possible to isolate antagonists with one or more antagonist actions, and then introduce the others by genetic engineering with recombinant DNA or protoplasting and using somoclonal variation. A problem that could arise from this approach is that the genetic modification could reduce the ecological competitiveness and rhizosphere colonization by the organism. Furthermore it is likely that regulatory authorities will be far more stringent about the release of such modified organisms into the enviroment. Therefore at this stage it seems most reasonable to search for organisms with
as many of desired traits as possible by isolating them from the environment. Soil-borne diseases which appear to be good candidates for biocontrol include those caused by the sclerotial-forming pathogens, such as Rhizoctonia and Sclerotinia. We have particularly considered Trichoderma spp., Gliocladium spp. and Coniothyrium minitans as potential bicontrol agents. Most studies of the mode of action have been on Trichoderma spp., which are not natural rhizosphere colonists and therefore have to be introduced to soil on a substrate base. It is unclear however under these circumstances if the antagonist then becomes a rhizosphere colonist. # Compared with other potential biocontrol fungi, Trichoderma spp. have a rapid growth rate on agar media and straw (Harper and Lynch, 1985; Lynch 198733). This efficiency of competitive substrate utilization can be decreased at low (5 'C) temperature (Lynch, 1987b) and low (-7.0 MPa) water potential (Magan and Lynch, 1986). The suppression of one organism by another on agar is dependent on the relative inoculum size of the antagonist and pathogen. From in vitro experiments with Trichoderma versus Fusarium (Lynch, 1987b) and experiments in soil with Trichoderma versus Rhizoctonia (C.J. Ridout, J.R. Coley-Smith and J.M. Lynch, unpublished) it seems necessary to have the antagonist present at an inoculum level which is an order of magnitude greater that of the pathogen but at present this can be difficult to achieve because it is difficult to determine the biomass of specific fungi in nature. Trichoderma spp. can become predatory on pathogens (mycoparasitic) and whereas this can enhance the effectiveness of the action of antibiotics or cell-wall degrading enzymes produced by the antagonist, the mycoparasitic action per se may not be an essential requisite for biocontrol. We have analysed the extracellular enzymes of a range of isolates of _T. harzianum and 2. viride using gel electrophoresis, isoelectric focussing, chromatofocussing and fast protein liquid chromatography (Ridout et al., 1986 and unpublished). Proteins produced by the various isolates differ, and more are induced, by growing Trichoderma spp. on the cell walls of the pathogen Rhizoctonia solani. In addition to glucan endo-1,3-~-glucosidase and chitinase, proteases are amongst the major enzymes produced. Whereas all these enzymes may contribute to the biocontrol action,the degree of contribution is unclear. Several antibiotic materials have been isolated from Trichoderma spp. and recently a volatile pyrone, dec-2,4-dien-5-olide possesing antifungal properties has been isolated from 2. harzianum (Claydon et al., 1987). Both the organism and the antibiotic are effective against
R. -solani and a range of other pathogens and this metabolic property of the antagonist could be important in its biocontrol action.
CONCLUSION Microbial communities in the rhizosphere consist of some populations which have beneficial effects on plant growth and others such as pathogens which are harmful. The community structure is governed by environmental, and plant and microbial physiological factors. Present knowledge of these factors and the quantitative analysis of the populations and communities is fragmentary. It is likely that genetic exchange will take place between members of the communities, for example plasmids may be exchanged between bacteria, between bacteria and fungi or even with the plant. This could have consequences in attempts to introduce genetically modified organisms into the rhizosphere but until there is a more complete understanding of natural community structures, this will be difficult to assess. Biological control of root diseases appears to be one of the most useful targets to aim for in the manipulation of the rhizosphere and this might be achieved with organisms which are not generally regarded as rhizosphere organisms.
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