Metabolization of Rhizodeposits

Rhizosphere Soil

D. Metabolization of Rhizodeposits

Swinnen (35) added labeled “model” rhizodeposits to planted soils to measure their respiration and residue and compared a number of soil factors and different substrates. He discussed their suitability to represent uctual rhizodeposits. A higher fraction of the soluble substrates (38% of the glucose, or 37% of root extracts) were respired than those of root residues (26%) during the 21 days of incubation in a climate room. Incubation in field soils reduced the respiration of substrates compared to the incubation in climate rooms (23-25% for solubles and 12% for root residues). Naumova and Kuikman (unpublished results) studied the influence of three levels of glucose addition on microbial utilization of ground-root residues (solubles extracted with water) and native soil organic mat- ter and their interactions during an incubation of 24 days (Table 2). The mineral- ization rate of C derived from roots was stimulated during days 0-3 by the low level of glucose addition and during days 3-24 by the high level of glucose addition. The cumulative CO, evolution, the amounts of extractable C (2 pg C/g soil) and the microbial carbon (29 p g C / g soil) produced during the decomposi- tion from added root material were not affected by addition of glucose. However, the addition of glucose increased the mineralization rate of native SOM by l 1 and 22% under low and high levels of glucose addition, respectively (Table 2).

Virtually all residual “C in soil at the low addition rate of glucose was glucose- derived microbial biomass C, whereas at the highest rate, glucose-derived micro- bial biomass represented about 50% of the residual “C in soil (Table 3). This indicates that a high level of soluble substrate stimulates the turnover of newly formed microbial biomass. On the contrary, a low level of substrate may be less well utilized by the microbial population awaiting further mineralization (17) or result in a lower turnover, i.e., induced by bacterial predators (41). It is concluded that the addition of soluble substrates such as glucose accelerates the turnover

Table 2 Cumulative C O I Evolution from Root Residues (RM), Soil Organic Matter

(SOM). and Glucose During 24 Days of Incubation

C source for COz

Total

Treatment“ (% of added

(pg C per g soil) RM SOM Glucose RM

+

glucose)

~ ~~

No glucose 128 253

Glucose (45 pg C) 133 280 14

Glucose (541 pg C) 126 308 383

30 31 53

~ ~~

In all treatments, root material was added at a constdnt rate.

Source: Naumova and Kuikman (unpublished results).

Table 3 Soil C and Microbial C (pg C per gram soil) Derived from Glucose

Ratio

cwd

C n u r r u h l d Cm~'roh~rl-to-C\u~l

Glucose (45 pg C) 12.1 13.1 I .08 Glucose (541 pg C) 156.8 72.5 0.46

Sorrrcw Naumova and Kuikman (unpublished results)

See Table 2 for more details.

of microbial biomass (or soil organic matter) only at relatively high addition

rates. Soluble substrates are respired to a larger extent than particulate substrates and we hypothesize that solubles do not interact or interfere with plant particulate substrates during their decomposition in soil. The latter can be explained by as- suming that fractions with a different physical behavior in soil are decomposed by spatially separated microbial populations. This may lead to different turnover rates of the (microbial) products that are being formed. Clearly differences be- tween soluble and particulate substrates exist in the short term but may not last at longer incubations.

E. Production of Refractory Soil Organic Matter

From Rhizodeposits

So far, we have no answer to the issue whether the nature of the substrate influ- ences its transformation into refractory products. The hypothesis is that decom- posability of rhizodeposits is not simply a characteristic of biochemical quality of the substrate but also of their physical characteristics and the rate at which they are supplied to the soil. Most simulation models that are used in soil organic matter studies will partition residues into two to three pools from easily decom- posable to recalcitrant organic compounds (42,43). Each of these pools will have a distinct turnover rate and a distinct probability to produce refractory soil organic matter. The easily decomposable substrate may very well end up in refractory pools, whereas substrate that initially is decomposed slowly will, in the end, be decomposed to a larger extent and result in less refractory soil organic matter.

Organic matter formed from glucose is metabolized at a fast rate, but the residue is only further mineralized at a low rate (< 150 pg/g soil) and less at a low addition rate of glucose than at higher addition rates of glucose (17).

Sarensen et al. (44) hypothesized that finer plant residues (i.e., upon grind- ing) may have better contact between substrate and decomposer organisms within the soil matrix and would decompose less extensively than coarser (i.e., intact,

unground) plant residues. They tested this hypothesis by measuring decomposi- tion and biomass formation in soils with ground and unground plant residues and with glucose. The results led them to conclude that glucose-derived C was re- tained more in soil than residue-retained C; this confirms data reported by Ladd et al. (45). Sorensen et al. (44) also suggested that grinding plant residues, upon addition to soil, favors, on their addition to soil, a more intimate contact between the plant residues and the soil matrix, thereby enhancing opportunities for the colonization by decomposer organisms that are more protected against predation and further decomposition and result in higher retention.

F. Advantages and Limitations of Applying C Isotopes

The methodology to study the C economy and C fluxes of plant-soil interfaces is fraught with problems (46). Studies employing isotopes of C, particularly “C.

seem to provide the only feasible way to obtain accurate figures for root-derived C. Pulse-labeling is relatively simple and cheap and there is 110 need for compli- cated apparatus in the field (35). This approach provides an estimate of gross C fluxes under ecologically realistic conditions but has the disadvantage that C fluxes are derived by fitting data to a mathematical model rather than by direct measurement (35,47). The decomposition of roots was measured by leaving pulse-labeled plants in the field. The dynamics of the remaining carbon was fol- lowed during 1.5 years following the addition of shoots and labeling of roots, respectively. This approach has the advantage that root decomposition was stud- ied in situ (35,47).

The major assumption in pulse labeling is that the distribution of the carbon assimilated during labeling with “C-C02 is representative for the carbon assimi- lated during that period. Jensen (26) addressed some of these assumptions by measuring the distribution of photoassimilated C in spring barley plants at differ- ent times after the onset of light and at different light intensities during assimila- tion. Higher proportions (15-20% more) of assimilates tended to be transported below ground at lower light intensities and after labeling early in the morning (40-60% more). These results were confirmed by Swinnen et a l . (27,28).

G. Scientific Relevance

Thus, there is evidence that the methodology of pulse-labeling plants under field conditions can very well be applied to study carbon dynamics i n both the short term (root and microbial respiration and root exudation) and longer term (turn- over and decomposition of roots). The release of organic compounds from roots of growing plants may have substantial effects on vital soil ecosystem processes, such as organic matter dynamics, structure formation, and nutrient cycling. Yet, proper quantification of the release of organic and inorganic C compounds from

roots or seasonal dynamics in various ecosystems is still lacking, and the funda- mental mechanisms involved in their metabolism in soil are not fully understood.

It is important to realize that any changes in the proportional allocation of carbon that form coarse and fine roots or in the rate of exudation may affect overall C sequestration in terrestrial ecosystems. Changes in the carbon distribution pattern over exudation, fine roots, and coarse roots may strongly affect subsequent de- composition patterns; some materials may be protected within the soil matrix while others remain unprotected (45). Even though exudates consist of simple sugars that are generally qualified as easily decomposable, Martin and Merckx (47) showed that some of this carbon is recovered from very recalcitrant organic materials in soil shortly after their release from roots. Swinnen (48) showed that exudation can amount to 25% or more of the total carbon translocated below ground in annuals such as barley and wheat. Thus, the contribution of rhizodepo- sition to the recalcitrant organic pool can be substantial.

W. ENZYME ACTIVITIES IN THE RHIZOSPHERE

A. General Concepts

All soil metabolic processes are driven by enzymes. The main sources of enzymes in soil are roots, animals, and microorganisms; the last are considered to be the most important (49). Once enzymes are produced and excreted from microbial cells or from root cells, they face harsh conditions; most may be rapidly decom- posed by organisms (SO), part may be adsorbed onto soil organomineral colloids and possibly protected against microbial degradation (SI), and a minor portion may stand active in soil solution (52). The fraction of extracellular enzyme activ- ity of soil, which is not denaturated and/or inactivated through interactions with soil fabric ( 5 l) , is called ncrt~rrcllly sttrbilized or immobilized. Moreover, it has been hypothesized that immobilized enzymes have a peculiar behavior, for they might not require cofactors for their catalysis.

Extracellular enzymes are essential for the initial degradation of high-mo- lecular-weight substrates like lignin, cellulose, pectins, chitin, or humic mole- cules, which cannot enter microbial cell envelopes to be processed (53). The presence of immobilized extracellular enzymes in soil has been hypothesized to be necessary for the liberation of low-molecular-weight monomers that subse- quently may diffuse into microbes for signaling the (temporary) presence of par- ticulate substrates in the rhizosphere environment. This process induces an aimed enzyme biosynthesis for a consequent excretion and save the microbial biomass the high metabolic expense of secreting continuously and blindly a wide array of different hydrolytic enzymes (53). The ecological meaning of this crucial mechanism could get over the simple nutritional requirements of roots if we consider recent hypotheses on the positive influence on plasma membrane H + -

ATPase and specific modification of root cell membrane permeability directly mediated by low-molecular-weight (<5000 Da) fulvic acid-like compounds deriving from native soil organic matter (54-56) (see also Chap. 5).

Assays of soil enzyme activities are usually carried out in soil slurries, since efficiencies of enzyme extraction from soil and purification are still low (49). Such assays, under these conditions, will only give a measure of potential rather than actual activities; moreover, they constitute integrated measures of activity as enzymes come from a variety of sources and are in several states in the soil (50). Enzyme activities may vary substantially with the season according to the synthesis, release into soil, and persistence of plant, animal, and microbial enzymes (57).

B. Specific Enzyme Activities

It is expected that the enzyme activity is related to microbial activity and produc- tion and will be higher in rhizosphere than in bulk soils. This may be due to ( l ) the possible release of root-derived enzymes and (2) enhanced microbial biomass and/or activity due to the greater substrate availability. Thus far these relation- ships have not been quantified well (58). Similarly, other organic matter inputs

such as plant litter (59) or amendments with green manures or composts (60) have been shown to increase soil enzyme activities. For example, protease activity has been demonstrated to be significantly higher in the wheat rhizosphere than in the bulk soil (61,62). As a direct result of the higher rhizosphere enzyme activities, it is possible to markedly reduce the size of the soil rhizosphere samples needed for assays to only tenths of grams (61-63). This then makes it possible to investi- gate on the specific enzyme dynamics in the very proximity of roots.

In general, a close spatial relationship between microorganisms and plant roots may result in activities that are greater than the sum of the two components:

phosphatase activity was significantly greater in the rhizospheres of rape, wheat, and onion in the presence of the michorrhizal fungus Glomus sp. than in noninoc- ulated plants (64). The increases were shown to be due to a stimulation of root rather than fungal phosphatase activity.

Intrinsic difficulties in sampling truly representative rhizospheric soil (Ta- ble 4) have hampered workers searching rhizosphere soil for a number of different enzyme activities as high as in the bulk soil.

Many more papers deal with rhizosphere phosphatase activity (63-83) in the presence of a number of different plant species; this will partly be due to the simplicity of the enzyme activity assay (85,86) and the generally reported, well- correlated variation trends among organic and inorganic phosphorus content and phosphatase activity. More precisely, closer to the roots, the inorganic P depletion zone in comparison with bulk soil is more pronounced; in addition, organic and inorganic P contents are inversely correlated, and the mineralization rate of or-

173

ganic P seems in such a way directly proportional to the rhizospheric phosphatase activity. In details, Tarafdar and Jungk (63) studied the distribution of both acid and alkaline phosphatase activity and of phosphate fractions in the proximity of roots of four plants (Brassica Olemcea, Allium cepa, Triticum uestivum, Trifolium dexandrinunl). A great increase in both acid (up to 9 I 1 % at 17 days of rape growth) and alkaline (up to 262%) phosphatase activity was measured i n all the four soil-root interfaces. Closer to the roots the phosphatase activity is higher;

acid phosphatase activity was found to be increased over bulk soil up to a distance of 2.0-3.1 mm irrespective of plant species and soil type. Both phosphatase activ- ities increased in the rhizosphere with plant age. In clover and wheat root surfaces the content of organic P was strongly depleted and the depletion zone extended to 0.8 mm in clover and 1.5 mm in wheat. On the contrary, the inorganic P levels adjacent to the root surface were higher than in the bulk soil measured. In the rhizospheres of clover and wheat, a significant correlation ( p

<

0.01) was ob- served between the depletion of organic P and the sum of both phosphatase activi- ties (63). Because concurrently an increase in both fungal and bacterial biomasses was observed in the rhizosphere compared to bulk soil, Tarafdar and Jungk (63) could not definitely conclude whether these diverse phosphatases were mostly of plant or microbial origin. Experiments carried out with axenic plants, however, have established that plant-borne phosphatases are excreted into the rhizosphere, probably as an adaptative response to P deficiency (83). A major role in the hydrolysis of organic P and in the subsequent acquisition of soil P is also played by phytase (84).

Up to now much less efforts have been devoted to rhizosphere enzyme activities related to organic C or N biochemical transformations. By an interdisci- plinary approach, based on a soil-plant microcosm suitable for studying the “rhi- zosphere effect” over time of plant growth and distance gradient from an induced soil-root interface, both two N-cycle enzymes (caseinase and histidinase) and

bacterial and protozoan population dynamics were monitored in the presence or absence of winter wheat seedlings at 21 and 33 days of growth (61). Both micro- bial populations and enzyme activities were significantly higher i n the presence than in the absence of plants up to 4 mm (microbes) and 2 mm (enzymes) away from the soil-root interface; the closer to it, the higher the numbers and the activi- ties. It was postulated that bacteria were the main source of histidinase activity, while bacteria, protozoa, and root hairs all contributed to the rhizosphere protease activity (61).

In general, one would expect that every enzyme will be released close to the roots either by roots or by microbes and eventually will be immobilized i n rhizosphere soil. If a given enzyme activity has not yet been measured in rhizo- sphere soil, this is likely more due to the lack of specifically devoted studies or suitable methods than to a real absence. For example, a recent study has first developed an assay for myrosinase in soil and, second, has hypothesized the

extracellular preservation of plant-derived myrosinase (P-thioglucoside glucohy- drolase; EC 3.2.3.1) in soil cropped with rapeseed in view of a possible allelo- chemical impact on soil-borne organisms within the rhizosphere (87). Myrosinase is produced in living cruciferous plants and enters the soil during senescence or potentially through root exudation. On the other hand, release of glucosinolates into the rhizosphere from root tissue damage of cruciferous plants seems likely.

Thus the existence of myrosinase activity in the rhizosphere would catalyze glu- cosinolate hydrolysis with the formation of a number of potential allelochemicals (isothiocyanates, nitriles, etc.) against fungi, nematodes, insects, and plants (87).

V. NITROGEN MINERALIZATION-IMMOBILIZATION

IN THE RHIZOSPHERE A. Concepts and Models

The release of organic materials from growing roots represents significant inputs of energy and matter that promote the microbial growth in the rhizosphere soil.

The efficient assimilation of the root-derived C would result in a substantial mi- crobial N-immobilization, since the average C/N ratio of the substrate is remark- ably higher than C/N ratios of microbial populations (88). The stimulation of the microorganisms by growing or decomposing roots may therefore result in a temporary reduction of available mineral N to the plant and a negative overall effect of plant roots on the net N mineralization in the soil (89). Robinson et al.

(88) mathematically analyzed the possibility whether the amount of N resulting from root-induced N mineralization could balance the amount of N lost through rhizodepositions. Even if those theoretical assumptions were chosen in order to maximize the impact of a root on N mineralization, only up to 10% of the plant N requirement seemed to be made available through root-induced N mineralization.

However, many theoretical assumptions of such models (88,89) are questionable:

( I ) the role of fungi in N mineralization and the production of ammonium through extracellular degradation (i.e., by enzymes naturally stabilized) of organic N was not considered and (2) the radius of the rhizosphere and total number of living bacteria per g of dry rhizosphere were supposed to be constant ( 1 mm and IO’, respectively). Despite this, experimental comparison of rhizosphere and nonrhi- zosphere soil has demonstrated that the “rhizosphere effect” on the net N miner- alization from native soil organic matter is positive in many cases but not in all.

Indeed, the microbial N immobilization through growth on root-released organic C (90,91) seems to be counterbalanced by a stimulation of the N mineralization promoted by protozoan grazing, with the release of ammonium derived from assimilated bacteria and/or due to a higher microbial turnover of soil native or- ganic matter (41,92,93). This trend has been also confirmed indirectly by ob- served increased soil enzyme activities related to N mineralization. concomitant

to higher bacterial populations and associated grazing protozoa in the rhizosphere soil (61).

In addition, the ecological advantages in root-soil-microbe interactions

could indeed be more than just that more N is gained than is lost through rhizode- posits. The volume of rhizosphere soil is important not only for N but also for other micro- and macronutrients. Moreover, the rhizodeposit investment might result in a better soil environment for root growth, as already hypothesized in the case of an increased root membrane permeability through the mineralization of fulvic acid-like organic compounds. On the other hand, any form of synzbiosis in nature implies a temporary reciprocal disadvantage in view of a near common benefit, which can be singly reached in alternative ways but only through much higher expenses of matter and energy. Last but not least, the loss of substrates and energy may result in a growth of nonpathogenic organisms, thus creating a more biologically safe environment ( 1 5,94).

Very recently, Blagodatsky and Richter (95) simulated the complex behav- ior of soil microrganisms after addition of soluble C (glucose, 2 mg C g" soil), with (100 pg N g" soil added as ammonium sulfate) or without addition of N.

Moreover, continuous root exudate flow mimicking root growth was simulated.

The mechanistic model set up for C and N transformations is quite simple as to number and type of different pools. ( l ) soluble C available for microbial con- sumption, (2) C and N held in the soil microbial biomass, (3) insoluble organic matter, (4) inorganic N, and ( 5 ) CO2 evolution. The new and central conceptual feature of the model is the introduction of a microbial activir~l sfnte ,function, depending on the amounts of C and N available for microorganisms and control- ling all living processes of soil microorganisms. This approach seems more prom- ising than fractionating soil organic matter in a number of distinct chemical pools of different recalcitrance, but it is quite unlikely to be determined experimentally.

Although the model is able to furnish satisfactory long-term simulations (up to 15 days) (96), the range of added substrates, both soluble and insoluble, and other soil processes-like nitrification, denitrification, and native organic matter solubilization-have to be included to prove its validity also for a very complex environment like that of the rhizosphere.

B. C-to-N Ratio in the Rhizosphere

Enhanced nutrient cycling in both the rhizosphere and bulk soil may depend

on the bacterial grazing by protozoa or nematodes with release of inorganic N.

Nematodes appear to be the primary consumers of bacteria in the rhizosphere, whereas protozoa are equally prevalent in rhizosphere and bulk soil (41,97). Esti- mated C-to-N ratios of bacterial-feeding nematodes range from 5 : l to I O : 1 (98,99) and are generally higher than those of their bacterial food source; thus the excess N is excreted as ammonia (100,101) by nematodes. The estimated