Inoculation of plants with a flavobacterium species results in altered rhizosphere enzyme activities

Soil Biol. Biorhem. Vol. 26, No. 7, pp. 871-882, 1994 Copyright c, 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0038-0717(!24)EOOO3-I 0038-0717/94 $7.00 + 0.00

INOCULATION OF PLANTS WITH A I;LA~0&4CTERZUA4 SPECIES RESULTS IN ALTERED RHIZOSPHERE ENZYME ACTIVITIES JANE L. MAWDSLEY* Research

School

of Biosciences,

and RICHARD G. BURNS?

Biological Laboratory, University Kent CT2 7NJ, England

of Kent, Canterbury,

(Accepted I7 December 1993) Summary-The inoculation of wheat seedlings with Flavobacferium P25 (1 x lO’/seedling) resulted in increased activity of four hydrolases: a-galactosidase (+ 40%) /I-galactosidase ( + 23%), a-glucosidase ( + 18%) and b-glucosidase ( + 28%) in the endorhizosphere (rhizoplane and root) by day 14. Neither the release of enzymes from dead P25 cells nor altered enzyme activities expressed by starved P25 cells could account for the changes in activity. Studies to investigate if the changes were due to P25 affecting enzyme production by the indigenous bacterial species, showed that there was no correlation between the increases in activity following inoculation and the numbers of indigenous bacteria expressing the relevant activities. However, whilst the total number of bacteria expressing the four different enzymes were present in approximately equal numbers at the start of the study, this changed as the plant developed so that by the time the plant was 35 days old 60-70% of indigenous isolates expressed a-glu and /?-glu but only l&20% expressed a-gal and /l-gal. Inoculation also affected plant root protease activity with inoculated plants expressing only 45% of the activity measured in non-inoculated plants. This may explain the increase in carbohydrase activity in the rhizosphere of inoculated plants.

Nonetheless, it is generally reported that enzyme activity is greater in the rhizosphere than in bulk soil (Jandera et al., 1989) because of the greater substrate availability and increased numbers of microorganisms, even though few experiments have quantified this relationship (Burns et al., 1989; Lynch, 1990). Jandera et al. (1989) demonstrated protease activity to be significantly higher in the wheat rhizosphere than in bulk soil. They also showed activity to change with plant age and suggested that this was due to amino acids, released by the root, regulating enzyme synthesis or activity. Much debate has focused on the origin of enzymes in soil although, of the three main sources (plant roots, soil animals and microorganisms) the last named are considered to be the most important (Tabatabai and Fu, 1992). Microorganisms contribute to soil enzyme activity both through the synthesis and externalization of enzymes by living cells and the release of intracellular enzymes from lysing dead cells. Enzymes released into the soil are often adsorbed onto clay or humic particles (Burns, 1982) resulting in the soil having a background activity not directly associated with the extant microbial biomass. In addition, a close spatial relationship between microorganisms and plant roots may result in activities that are greater than the sum of the two components. Such an effect was recorded by Dodd et al. (1987) who showed phosphatase activity to be significantly greater in the rhizospheres of rape, wheat and onion in the presence of the

INTRODUCTION Before a potentially beneficial microbial species is released into the environment it is necessary to understand the factors affecting its survival and growth. However, perhaps even more important, is a knowledge of the inoculant’s metabolic activity in soil and how it may affect indigenous soil activities (Young and Burns, 1993). The study of enzyme activity in soil has been the subject of much research for many years (Skujins, 1967; Ladd, 1978; Burns, 1982; Tabatabai and Fu, 1992). However, the activity of enzymes in the rhizosphere has not been studied to such an extent (Burns, 1985). Similarly, the production of extracellular enzymes by inoculants is a factor undoubtedly affecting rhizosphere competence but has received little attention (O’Sullivan et al., 1991). The production of extracellular enzymes by an inoculant, enabling it to degrade some or all of the substrates released into soil from plant roots, will affect the survival and growth of both the inoculant and the indigenous population. However, O’Sullivan et al. (1991) showed extracellular protease production was not important in the survival of a Pseudomonas sp. in the sugarbeet rhizosphere.

*Present address: AFRC

Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, Dyfed SY23 3EB, Wales. TAuthor for correspondence. 871

JANE L. MAWDSLEY

872

and

mycorrhizal fungus Glomus sp. than in non-inoculated plants. The increases were shown to be due to a stimulation of root phosphatase activity rather than a direct consequence of the phosphatase-positive fungus. Four carbohydrate enzymes, cc-galactosidase (c(gal), fi-galactosidase (p-gal), t( -glucosidase (cI-glu) and fi-glucosidase @-glu), were selected as indicators of metabolic activity. All these enzymes play a significant part in the degradation of common polymers (e.g. cellobiose and maltose) in soil, the monomeric products (i.e. glucose and galactose) being important as carbon and energy sources for soil microorganisms (Eivazi and Tabatabai, 1988). The aim of our research was to investigate enzyme activity, its origins and its location in the rhizosphere of wheat during the early stages of growth and monitor the effect of bacterial inoculation with a Flauobacterium species on this activity. MATERIALS

AND METHODS

Microorganism A triple antibiotic resistant (streptomycin, 250 /lg ml-‘; rifampicin, 100 pg ml-‘; kanamycin, 50 pg ml-‘) Flavobacterium species (designated FIavobacterium P25) was used in all experiments. This bacterium, which most resembles Flavobacterium balustinum, was isolated originally by Thompson et al. (1990). Cultures for soil inoculation were prepared by inoculating nutrient broth with P25 picked from a 2-day culture on P25 selective agar (nutrient agar supplemented with P25 selective antibiotics) and kept at 25°C for 16 h on an orbital shaker at 180 rev min-‘. The cells were harvested (by centrifuging at 3500 rev min’ for 15 min), washed twice in sterile phosphate buffer (0.2 M, pH 7.2) and resuspended to give an optical density (600 nm) of Iequivalent to a cell density of 1 x 109cfu ml-‘. Non -viable cells Dead entire P25 cells were prepared by resuspending a washed 16 h culture of P25 in 2% (w/v) paraformaldehyde for 15 min. This procedure, whilst killing the cells, does not disrupt the cell membranes (unlike autoclaving or treatment with sodium azide) and hence does not result in the release of intracellular enzymes. Intact dead cells had the same activity as intact live cells. This enabled us to mimic the effect of inoculant cells dying in soil and to monitor any subsequent release of enzymes as the cells were degraded. Following treatment with paraformaldehyde, cells were recentrifuged, washed twice in buffer and used in an identical manner to viable P25. A sample was plated on P25 selective agar to check non-viability. Starved cells The carbon- and nitrogen-limited environment of soil is very different from the nutrient rich medium

RICHARD G. BURNS

used in laboratory culture of P25. Therefore, a preparation of cells was starved in the laboratory so as to mimic the physiological state likely to be induced following inoculation. Starved P25 cells were prepared by resuspending cultures (as previously) in 20 ml sterile phosphate buffer and maintaining them at 4°C for 21 d prior to inoculation. A sample was plated on P25 selective agar to confirm viability. Soil and wheat plants A calcareous grassland soil (pH 6.9; 12% sand, 55% silt, 33% clay; organic matter 2.1%) of the Coombe series, with no history of cultivation or agricultural treatment, and which had been the original source of P25, was used in all studies. Soil was collected from a depth of 15-30 cm and large stones and organic debris removed before the soil was transported back to the laboratory in sealed polythene bags. Prior to use the soil was sieved (2.88 mm) to remove the majority of remaining stones. Subsamples of soil were used to determine field moisture content (0.22 ml g-l), field matric potential ( - 206 kPa) and moisture holding capacity (0.60 ml gg’). Wheat (Triticum aestivum var. Avalon) seeds were surface sterilized using the method of Maplestone and Campbell (1989) and germinated on I/ 10 strength tryptone soya agar at 25°C for 72 h. Wheat rhizosphere experiments The simple apparatus used to monitor the effect of P25 inoculation on ectorhizosphere (the volume of soil around plant roots under the influence of root exudates and secretions) and endorhizosphere (macerated roots containing both root-bound and intracellular enzymes) enzyme activity consisted of a 50 cm3 boiling tube containing soil (10 g dry wt equivalent) packed to a bulk density of 1.4 g cmm3. Soil was planted (at 1 cm depth) with a single surface sterilized seedling and 1 ml of washed P25 cells (1 x lo9 cfu g-r) was applied directly to the seedling, immediately after planting and before covering with soil. In unplanted tubes 1 ml of the inoculum was added directly to the surface of the soil. The soil was maintained at 60% MHC (0.36 ml gg’; -20.6 kPa) by regular additions of distilled water. All tubes were kept in a plant growth chamber with a light intensity of 42001x, a light-dark cycle of 16-8 h and a constant temperature of 20°C. After initial measurements of the effect of P25 inoculation on soil and rhizosphere enzyme activity, further investigations were undertaken in order to study the factors responsible for the changes in these activities with time, plant age and physiological state of the cells. These included: (i) inoculating plants with either starved or dead P25; (ii) monitoring the effect of P25 inoculation on the glucosidase and galactosidase activity of the indigenous microflora; and (iii) investigating the effect of inoculation on soil protease activity.

Bacterial inoculation and rhizosphere enzyme activities The effect of P25 inoculation on the number of glucosidaseand galactosidase-positive indigenous microorganisms was measured concurrently with the effect of P25 inoculation on soil and rhizosphere enzyme activities. In addition to performing enzyme assays, a dilution series was prepared and soil, ectorhizosphere or endorhizosphere samples were plated in triplicate onto l/l0 strength tryptone soya agar supplemented with 50 pg ml-’ cycloheximide (TSAC). Plates were kept at 25°C for 72 h prior to being replica plated onto TSAC, TSAC +pnitrophenyl tl -p-galactopyranoside, TSAC +pnitrophenyl P-D-gafactopyranoside, TSAC fp-nitrophenyl a-D-glycopyranoside and TSAC +pnitropenyl /?-D-glucopyranoside (20 ml of 25 mM pyranoside solutions being added to 980 ml of media in all cases giving a final concentration of 0.5 mM). The plates were kept at 25°C for 24 h and the total number of colonies and the number showing enzyme activity (indicated by a bright yellow halo around the colony) counted. Enzyme assays Galactosidase and glucosidase. The assays used for or-galactosidase (E.C. 3.2.1.2), fi-galactosidase (E.C. 3.2.1.23), cc-glucosidase (E.C. 3.2.1.20) and /?-glucosidase (E.C. 3.2.1.21) were those described by Eivazi and Tabatabai (1988). After initial studies to determine detection sensitivities and optimal conditions, all samples were incubated statically with 1 ml 25 mM p-nitrophenyl substrate at pH 6.5, 37°C for 1 h in the absence of microbial inhibitors. These conditions gave results which were well within the accurate detection limits governed by the sensitivity of the spectrophotometer (Philips PU8720 u.v./vis. scanning spectrophotometer). Treatment samples were assayed in triplicate and the control samples in duplicate. Figures show the means of such replicates with bars representing the standard error of the mean. Student’s t-test was used to assess the difference between two treatment means. Protease. The protease assay was modified from that of Ladd and Butler (1972) and Speir and Ross (1975). The sample of soil, rhizosphere soil or root (prepared in an identical manner to that used for glucosidase assays) was incubated in a 25 ml Erlenmyer flask with 10ml 0.1 M Tris buffer (pH 8.1) containing 1% (w/v) sodium caseinate for 2 h at 30°C. Controls contained no sodium caseinate. Trichloroacetic acid, 17.5%, 4 ml, was added and the sample mixed before being transferred to a 15 cm3 centrifuge tube and spun at 2,OOOrevmin’ for 1Omin (MSE Centaur 2 Bench Centrifuge). The supernatant fraction, 2 ml, was removed to a second centrifuge tube and treated with 3 ml 1.5 M Na,CO, and 1 ml 33% (v/v) Folin-Ciocalteau reagent (Sigma Chemical Co.). The tubes were vortexed and left 30 min before being recentrifuged (3 min 2000 rev min-‘). Supernatant, 2 ml, was removed to a cuvette and the colour read at 700 nm (Philips

873

PU8720 u.v./vis scanning spectrophotometer). A standard curve (range (M.5 mM) was prepared by diluting a 1 mM stock solution of tyrosine. All samples were assayed in triplicate. The effect of protease on the glucosidase and galactosidase activity of P25 was measured using a commercial preparation of protease (Streptomyces griseus protease, Sigma Chemical Co., U.K.). 1 ml of a 1 x lo9 cfu ml-’ P25 suspension f 4.8 units protease (1 mg ml-‘) was used to carry out a standard glucosidase or galactosidase assay. Preparation of samples for enzyme assays P25 cells. Tests were made to discover the cellular location and activity of the galactosidases and glucosidases in P25. 500 ml of a 16 h P25 culture was centrifuged (9820 g, 15 mitt, SC). The supernatant fraction was removed, the pellet resuspended in 20 ml phosphate buffer (0.2 M, pH 7.2) and the centrifugation step repeated. After a second wash the supernatant fraction was again removed and the pooled supernatant, the extracellular enzymefraction, stored. The pellet was suspended in 10 ml buffer and the cells were disrupted by passing twice through a French Pressure Cell (9000 PSIG, SLM Aminco SLM Instruments Inc.). Any unbroken whole cells and the cell debris were removed by a slow spin (5000 rev min-’ 10min MSE Centaur 2 Bench Centrifuge) and discarded. The supernatant fraction was transferred to a clean centrifuge tube and the cell walls and membranes were pelleted by centrifugation (3 1,000 g, 40min, 5°C) and were designated as the membrane bound enzyme fraction. The supernatant from this step was designated as the intracellular enzyme fraction. In order to determine specific enzyme activities the amount of protein present in each sample was calculated using a modified Bradford assay (Bradford, 1976). The appropriately diluted protein solution (1 ml) was added to 1 ml of colour reagent and the absorbance read at 595 nm (Philips PU8720 u.v./vis. scanning spectrophotometer) after 2 min. Bovine serum albumin, covering the range O-20 pg ml-‘, was used to prepare a standard curve. Soil and rhizosphere. Soil and rhizosphere samples were prepared by pooling the contents of three replicate soil tubes in a 150ml beaker. In planted soils, the roots were shaken free of adhering soil by hand and transferred to a sterile universal containing 20 ml modified universal buffer. The roots were vortexed (1 min) to remove any remaining ectorhizosphere soil, transferred to a separate universal containing 20 ml modified universal buffer, and macerated using a Polytron homogenizer (speed 5, 2 x 30 s, Polytron Kinematica AG, Littau, Lucerne, Switzerland). Non-planted soil (of the same period of incubation as planted soil) (1 g), planted soil (1 g) plus 1 ml root washings, or 1 ml homogenized root suspension were used as soil, ectorhizosphere and endorhizosphere

JANE

874

I.

Table wheat

Glucosidase

roots,

L. MAWDSLEY and RICHARD G. BURNS

and galactosidase

non-inoculated

soil

activities

associated

Fhobacrerium

and

replicates

with

P25.

sterilized Values

wheat

are

the

seeds, sterilized mean

of

three

+_ SEM Specific

activity

(mg .’ protein)

Sample Seeds-non-germinated Seeds-germinated Roots-sterile

(72 h) (5 d)

8&l 140 *

I3

80 T 8

I59 f

I3

73 f

226 + 20

Soil-fresh P25 (I6

41 k4 1224+62 38+

htiintact

cells

P25 (I 6 h~xtracellular P25 (I6

htmembrane

P25 (I6

htintracellular

bound

I

24 +_ I

0

0

0

0

28 k I 8

35 F 2 2333 2 I61 27+

I

0

0

2288 A I I4

220

0

249 + 36

samples, respectively. Dry weight equivalents were determined for each sample by oven drying aliquots at 105°C. Each study was conducted over a period of 35 days with samples being assayed at days 1, 7, 14, 21, 28 and 35. The data presented is from day 14 at which time there were high populations of viable inoculant bacteria present and a distinct rhizosphere had developed. In general, the results at day 14 were representative of those obtained throughout the study; any differences are discussed in the text.

RESULTS Glucosidase and galactosidase activities of Flavobacterium P25

The results of experiments carried out to investigate the initial glucosidase and galactosidase activities of Flavobacterium P25, soil, gnotobiotic 5-day old wheat roots and germinated and non-germinated wheat seeds are shown in Table 1. cc-Glu was the

F = 5 L Tm 9

7*1

480 & 32 1516+99 2OOi_I4 0 0 224 k I4 41*3

predominant enzyme in P25 and was detected in the membrane bound, intracellular and the extracellular enzyme fractions. Low amounts of /?-Glu and cc-gal activity were detected in the intracellular fraction and p-glu activity was also detected in the membranebound fraction. j-gal was not produced by Flavobacterium P25. Eflect of plant age and inoculation with viable Flavobacterium P2.5 on soil and rhizosphere enzyme activity

The activity of the four enzymes in non-rhizosphere soil, ectorhizosphere soil and the endorhizosphere was measured over a 35-day period in non-inoculated non-sterile soil. In non-rhizosphere soil there were no significant changes in activity, but in the ectorhizosphere activity of all four enzymes increased during plant development so that by day 35 activity was significantly (P < 0.05) greater (mean = + 18%) than at the start of the study. In the endorhizosphere (Fig. 1) cc-gal and cc-glu activity

6500 5500 4500 3500 2500 1500

-0 0, 3 1400 z 1200 _zi 1000 s 800 5 600 4 400 f 200 n. 0 Plant

age (days)

Fig. 1. Effect of plant age on enzyme activity in the endorhizosphere of wheat grown in non-inoculated soil; a-gal (m), p-gal (A). cl-glu (m) and p-glu (+). Bars represent standard errors.

Bacterial inoculation and rhizosphere enzyme activities

‘-

875

2500

u

z 1z x s 5 g I

800 600 400 200

a-gal

B-gal

a-glu

Fig. 2. Activity of enzymes in the endorhizosphere of wheat grown in non-inoculated (0) and inoculated ) soil 14 days after inoculation. Bars represent standard errors.

doubled over the 35 d study whilst a-gal activity also increased significantly (P < 0.05) by 37%. In contrast, p-glu activity decreased by 50%. Inoculation of planted soil produced consistent increases (+ 1740%) in the activity of all four enzymes in the endorhizosphere. These increases were measured each week during the 35 d study and a typical result is shown in Fig. 2. In the ectorhizosphere increases in activity were also recorded although these were less than in the endorhizosphere ( + 518%). In non-planted soil there were no significant changes in enzyme activities. The factors determining the increase in endorhizosphere activities were studied in greater detail. Effect of dead Flavobacterium P25 on endorhizosphere enzyme activity

There was a possibility that the increases in activity in the endorhizosphere recorded when seedlings were inoculated were due to the release of intracellular enzymes from dead and lysing P25 cells rather than enzyme secretion by viable, actively metabolizing cells. This was investigated by ‘inoculating’ seedlings with intact P25 cells which had been killed by treating with 2% w/v paraformaldehyde (Fig. 3). If all the intracellular enzymes were released from lysing P25 cells (calculated from the activities of P25 grown in nutrient broth) and had remained active in soil this would account for all of the increase in a-glu activity but < 1% of the increase in activity of the other three enzymes in soil inoculated with viable cells. Over the first 14 days of the study the presence of viable P25 led to an increase in activity of all four enzymes. In contrast, the inoculation of soil or seedlings with non-viable P25 led to significant

(P < 0.01) increases in a-glu (+ 246%) and /I-glu

( + 37%) but resulted in no change in a-gal and /?-gal activities. However, at subsequent sampling dates any differences gradually decreased so that by day 35 (Fig. 4) the activities of all four enzymes in the dead P25 treatments were the same as in non-inoculated controls and significantly less than in seedlings grown in soil inoculated with viable cells. Effect of starved Flavobacterium P25 on endorhizosphere enzyme activity Flavobacterium P25 cells, which had been starved at 4°C in phosphate buffer for 21 days, and significantly (P < 0.001) lower ( - 57%) amounts of a-glu and higher (+ 208%) amounts of /I-glu than cells grown in nutrient excess medium. Therefore, studies were undertaken to establish if the increase in endorhizosphere /I-glu activity following inoculation was a result of the nutrient limiting conditions in soil (Newman, 1985). The results (Fig. 5) demonstrate that, although starvation does affect P25 enzyme production, it is not responsible for the changes in enzyme activity that occur following inoculation. This was shown by the fact that whilst inoculated seedlings showed the significant increases in activity of all four enzymes, there were no differences between plants inoculated with starved or non-starved cells. E#ect of viable Flavobacterium P25 on the enzyme activities of indigenous bacteria

The a- and /I-glucosidase and a- and /I-galactosidase activities of P25 inoculated and non-inoculated soil, ectorhizospheres and endorhizospheres were measured for 35 days and compared to the numbers

876

JANE

z : % $ I P

L. MAWDSLEY and RICHARDG. BURNS

1000 800 600 400 200 0

a-gal

B-gal

a-glu

) P25 on endorhizosphere activity compared to Fig. 3. Effect of inoculation with viable ( that measured in non-inoculated controls (0) 14 days after inoculation. Bars represent standard errors.

of indigenous bacteria expressing one or more of the four enzymes. With the exception of a-glu activity, for which increases in endorhizosphere activity corresponded to increases in the numbers of cc-glu positive bacteria for the first 21 days (Table 2), there was no relationship between increases in activity and the numbers of relevant bacterial isolates. Changes in the percentage of indigenous endorhizosphere bacteria expressing enzyme activity in non-

F

inoculated plants are shown in Fig. 5. At day 0, the numbers of bacteria expressing the different enzymes were present in approximately equal numbers (ca 7 x 10’ cfu g-‘). However, as the root developed, the numbers of bacteria with different activities changed dramatically. Thus by the time the plant was 35 days old, 60-70% of bacterial isolates were cr-glu and /I-glu positive but only lO-20% were a-gal and p-gal positive. The changes in the proportions of enzymepositive indigenous bacteria were similar in inocu-

2800

A= 2600 5 2400 i

L 2200 OI 2000

2 1800 1600

;

:: 1400 4

1200

2 1000 z

800

600 : 0” 400 L 7 a

200 0 a-gal

B-gal

aiglu

) P2S on endorhizosphere activity compared to Fig. 4. Effect of inoculation with viable ( that measured in non-inoculated controls (0) 35 days after inoculation. Bars represent standard errors.

1900 1800 c 1700 ; 1800 2 1500 i

CI,

z

800

3 4

700 600

f

500

5

400

5

300

a-gal

a-glu

B-gal

Fig. 5. Effect of inoculation with ‘starves (a) or non-starved ( ) P25 on endorhizosphere activity compared to that measured in non-inoculated controls (0) 14 days after inoculation. Bars represent standard errors.

lated soils, with the exception of cl-glu-positive bacteria over the first 21 days of the study which were 2040% greater than in the non-inoculated soils.

compared to the activity measured in an untreated culture.

Effect of viable Flavobacterium P25 on soil and rhizo sphere protease activity

DISCUSSION

There was no significant effect of P25 inoculation on soil, ectorhizosphere or endorhizosphere protease activity up to day 14. However, by day 21 there was a 29% decrease in endorhizosphere protease activity which became more pronounced with time so that by day 35 plants inoculated with P25 had only 55% of the activity measured in non-inoculated controls (Fig. 7). Flavobacterium P25 is protease positive and a suspension of 1 x lo9 cells ml-’ expressed an activity of 4p~ tyrosine released h-‘. However, at no stage did gnotobiotic wheat roots or their rhizospheres express any protease activity. Commercial preparations of protease (4.8 units activity) when incubated for 2 h with P25 cells decreased cc-glu ( - 29%) and /I-glu ( - 38%) activity (significant at P < 0.01)

Glucosidases and galactosidases have been detected in fresh and marine waters, sediments, soil, microorganisms, plants and animals (Hayano and Tubaki, 1985; Eivazi and Tabatabai, 1988; Boon, 1991; Chrbst, 1991). These enzymes are considered to be important in the soil because they degrade carbohydrates and provide growth and energy substrates for soil heterotrophs (Eivazi and Tabatabai, 1988). The initial experiments described here provide information about the activity of the four enzymes in: the inoculant Flavobacterium P25; non-inoculated, nonsterile soil; and gnotobiotic wheat seeds and roots. Flavobacterium P25 expressed intracellular cc-gal, a-glu and j?-glu activities. cc-G111was shown to be intracellular, membrane bound and extracellular (i.e. secreted into the surrounding environment). a-Glu was the most abundant enzyme in all of the three

Table 2. The effect of P25 inoculation on the percentage of a-glucosidase-positive bacteria and the total a-glucosidase activity of wheat roots. Values are the mean of three replicates f SE. PNP = p-nitrophenol Inoculated Plant age (days) 7 14 21 28 35

PNP released (rg g-’ h-‘) 190 260 386 684 368

f f + + f

6.01 26.2 10.0 26.3 34.4

Non-inoculated Isolates with activity (%) 62.9 53.0 69.3 42.8 64.1

f f f + f

3.14 4.08 5.61 3.34 5.45

PNP released (pg g-’ h--l) 140 145 295 631 342

f f + + f

20.2 6.06 32.3 48.3 32.3

Isolates with activity (%) 54.4 36.7 53.1 10.4 69.3

+ f f + f

2.17 5.01 5.22 5.24 3.10

JANE L. MAWDSLEY and REHARD G. BURNS

878

b

E

70-

‘g

60 -

5

50-

! = .z

40-

z

20-

30 -

10 ”

114

i

2’1

2t3

35

Time (days) Fig. 6. Effect of plant age on the expression of a-gal (m), P-gal (A), a-glu (a) and /3-glu (+) by the indigenous endorhizosphere bacteria of non-inoculated wheat. Bars represent standard errors.

fractions whilst b-gal was not detected in any of the fractions. All four enzymes were detected in the soil: /?glu >> a-glu > a-gal > b-gal. This is consistent with the report that fi-glu activity is more active in soil than a-glu, a-gal or p-gal (Tabatabai, 1982). The relative activities of the three other enzymes appears

to vary with soil type, as shown by Eivazi and Tabatabai (1988) who investigated activity in five different soils. This is not surprising as many factors, such as soil composition, cropping history and soil amendments can affect enzyme activity (Tabatabai, 1982) as well as the composition of the indigenous microflora.

T L

r soil Fig. 7. Effect of P25 ( in non-inoculated

rhizosphere

root

) on soil, ectorhizosphere and endorhizosphere protease activity in relation to that controls (0) 35 days after inoculation. Bars represent standard errors.

Bacterial

inoculation

and rhizosphere

Sterile 5-day old wheat roots showed activity of all four enzymes: /?-glu >> a-gal > B-gal > a-glu. Assays were performed using root homogenates and were undoubtedly a mixture of both intracellular and root bound (or rhizoplane) enzymes. The actual externalization of enzymes from roots was confirmed by detecting all four enzymes at low activities ( < 20 pg PNP gg’) in agar on which the seeds were germinated for 72 h prior to planting and in sand in which axenic seedlings were grown (data not shown). Enzymes have been recorded frequently in rhizosphere soil although their origin is not clear (Rovira and McDougall, 1967). However, one study by Chang and Bandurski, 1964), demonstrated that enzymes attached to sterile corn (Zea map) roots hydrolysed sucrose, cellobiose, ATP, DNA, RNA and pyrophosphate, whilst invertase and nuclease were secreted as soluble exoenzymes into the surrounding liquid growth medium. Their study, and ours show that seedlings and young roots release hydrolytic enzymes into the soil and that this takes place in the absence of microorganisms. Germinated seeds (t = 72 h) expressed significantly higher (11.4-29.8x) activities of ail four enzymes compared with non-germinated dry seeds. This is not surprising because during germination carbon stores within the seed are metabolzied by endogenous enzymes to provide the energy required for the early stages of plant development (Greulach, 1973); the enzymes responsible for the breakdown of the carbon would be expected to increase in activity (Milthorpe and Moorby, 1974). Of the four enzymes assayed, a-gal was the most active in both germinated and non-germinated seeds. Enzyme activity in the rhizosphere increased as the wheat plants developed, probably paralleling increases in root exudates and secretions and microbial biomass (Rovira and McDougall, 1967). We have shown that all four enzymes are passed from the root into the soil and it is likely that a proportion of these plant enzymes will become complexed to clay and humic colloids (Stotzky and Burns, 1982; Burns, 1986). It is known that such immobilized enzymes may survive as active catalysts in soil (Burns, 1982). One of the major sources of carbon input to soil is obviously plants via their roots (Fogel, 1985), and increased enzyme activities have been demonstrated in the rhizosphere in response to rhizodeposition. For example, SotolvP et al. (1989) showed B-l,3 glucanase activity to be IO-50 times greater in the rhizosphere of 3-6 day old barley (Hordeutn vulgare) than in bulk soil, whilst Burns et al. (1989) measured lower but significant increases in phosphatase activity in the rhizosphere of 26-day old maize (Zea map) ranging from 1.3 (alkaline phosphatase) to 2.7 times (acid phosphatase). Similarly, other organic matter inputs such as plant litter (Sinsabaugh et al., 1991) or amendments with green manures or composts (Tate et al., 1991) have been shown to increase soil enzyme activities.

enzyme activities

879

Changes in activity of the four enzymes also occurred in the endorhizosphere. a-Gal and a-glu activity doubled over the 35 days of the study and P-gal also showed a significant increase (+ 37%). In contrast, /?-glu activity decreased by 50% by day 35. Sotolvl et al. (1989) reported that p- I,3 glucanase activity of barley (H. vulgare) roots decreased from day 4 to day 10 following an initial increase, whilst chitinase increased up to a maximum at day 7 and then decreased (Hanzlikovi et al., 1989). Inoculation of wheat seedlings led to increases in activity of all four enzymes, in both the ectorhizosphere and endorhizosphere, throughout the 35 days of the study. According to McLaren et al. (1960), plant roots actually take up enzymes from the environment and barley (H. vulgnre) roots were shown not only to adsorb quantities of lysozyme at their surface, but also to absorb the enzyme which could then be detected in the roots. This means that, although the increases in root activity could not have been caused by the uptake of enzymes from P25 (P25 does not produce /?-gal and has only low amounts of a-gal and /?-glu), they could have been a result of uptake of enzymes produced by the indigenous microflora many of which were positive for the enzymes in question. One explanation for the increases in enzyme activity following FIauobacterium P25 inoculation is that intracellular enzymes were released from dead P25 cells. The introduction of P25 to soil is followed by a rapid decline in cell numbers (Thompson et al., 1990) and we investigated the effect of introducing dead P25 cells to wheat seedlings. Dead P25 cells had no effect on the activity of a-gal and p-gal during the first 14 days (note that these enzymes are either not produced or produced in very low quantities by P25 grown in vitro) but resulted in increases in sc-glu ( + 246%) and p-glu (+ 37%) (both of which are located intracellularly, exclusively so in the case of p-glu). However, after day I4 there was no difference in enzyme activity between non-inoculated plants and those inoculated with dead P25 cells. This indicates that the dead cells are rapidly broken down in soil (Bohool and Schmidt, 1973, 1980) releasing intracellular enzymes which remain active for only a short time. It is clear that inoculation with live P25 cells is necessary for there to be long-term changes in activity. This suggests that prolonged enhancement of activity is due either directly to enzyme production in situ or indirectly to the influence of P25 on the root or the indigenous rhizosphere microorganisms. Viable Flavobacterium P25 cultures, which had been starved by storing in phosphate buffer, had lower ( - 57%) a-glu activity but higher ( + 208%) b-glu activity than non-starved cells assayed at late exponential growth phase. It was hypothesized that the increases in B-glu activity measured in the ectorhizosphere and endorhizosphere of inoculated plants was due to increased fi-glu production in the comparatively nutrient poor soil environment. The ability to

880

JANEL. MAWDSLEY and RICHARDG. BURNS

change metabolic activity is a major factor influencing survival of bacteria under conditions of starvation (Gray, 1976). If this was the case, initial concentrations of b-glu in soils inoculated with starved bacteria should be higher than those inoculated with non-starved bacteria but the distinction would decrease as the non-starved cells became nutrient limited. However, there were no significant differences between endorhizosphere enzyme activities from plants inoculated with starved or non-starved cells. Therefore changes in inoculant enzyme activities in response to nutrient limitation does not account for the increases in activity measured. Root exudates and secretions are affected by the presence of rhizosphere microorganisms (Barber and Lynch, 1977; Martin, 1978). It is possible that P25 inoculation, either directly or through a change in the composition or concentration of compounds produced by the root could lead to either: (i) a change in the microbial community structure such that certain species are enriched and associated enzymic activities increased; or (ii) a modification to the existing microbial population which is induced (or repressed) for the enzymes under study. Therefore, the a-gal, P-gal, a-glu and b-glu activities of inoculated and non-inoculated plants were measured and compared with the total numbers of indigenous (including P25) bacteria expressing these activities. In the majority of cases there was no positive relationship between the increases in activity and the numbers of relevant bacteria, i.e. the increased amounts of enzyme activity measured were not paralleled by increases in the numbers of bacteria expressing activity of the enzymes under investigation. The only exception was with total a-glupositive bacteria during the first 21 days of the study where increases in cc-glu activity measured could be matched to increases in the numbers of bacteria expressing a-glu activity. However, this can be accounted for by the large numbers of cc-glu producing P25 (> 1.3 x IO’cfu g-‘) root). Therefore, the recorded increases in enzyme activities were not caused by the inoculant altering the numbers of the appropriate indigenous microflora. Many investigators have attempted to relate enzyme activity in soil to total microbial numbers but the results have been inconsistent. Cochran et al. (1989) showed that whilst dehydrogenase (an enzyme that should be expressed in all viable microbial cells) activity in a subarctic forest soil was correlated with microbial biomass, there was no correlation with dehydrogenase activity in an agricultural soil. Perhaps less surprising was the lack of high correlation of biomass with phosphatase or urease (enzymes that will be associated with certain components of the microbial community) activity in either soil. Frankenberger and Dick (1983) investigated the relationship between activities of 11 soil enzymes and microbial respiration, biomass and plate counts. They showed that only phosphodiesterase and a-galactosidase ac-

tivities were significantly related to microbial numbers whilst alkaline phosphatase, amidase and catalase were highly correlated with microbial biomass. A study by Jha et a/. (1992) investigated the relationship between total bacterial and total fungal counts on one hand and activities of dehydrogenase, urease and phosphatase on the other. The authors chose four forest soils at two altitudes, the two soils at each altitude being at diferent stages of degradation (i.e. different organic matter contents and physical and chemical properties caused by slash and burn agriculture). Dehydrogenase activity was correlated to fungal counts in all four soil types but only to bacterial populations in the less degraded soils at the lower altitude. In the lower altitude soil urease activity was correlated to both fungal and bacterial populations separately, but at the higher altitude only to fungi. Phosphatase activity at the higher site showed no correlation to microbial numbers and at the lower site only to fungal numbers. In our study, the apparent lack of correlation between enzyme activities and numbers of bacteria expressing those activities (a more relevant measurement than total bacteria), suggests that the activity is largely extracellular and not closely associated with fungi or even soil fauna (Hayano and Tubaki, 1985; Kandeler et al., 1993). Initially the bacterial species expressing the four enzymes were shown to be present in equal numbers. However, as the plant developed, the proportions of bacteria with different enzyme activities changed so that by the time the plants were 35 days old 60-70% of isolates had a-glu and p-glu activity but only IO-20% had a-gal and a-gal activity. This demonstrates a microbial response to changes in the exudates produced by a plant as it develops and warrants further study. Another possible contribution to the changes in enzyme activity is that P25 influenced the activity of soil or rhizosphere proteases. Proteases would be expected to degrade a proportion of some or all of the four hydrolases unless they were protected by their association with clay and humic material (Burns, 1982). Over the first 14 days of the study there was no change in soil or rhizosphere protease activity following P25 inoculation. However, by day 21 rhizosphere protease activity had decreased by 29% and on day 35 by 45% in comparison with non-inoculated roots. This could account for the increases in glucosidase and galactosidase activities because, presumably less of the carbohydrases would be degraded. P25 cultures exposed to a commercial protease for 2 h at 37,‘C had reduced amounts of a-glu (- 29%) and /I-glu ( - 38%) activity. Sterile wheat roots did not possess any protease activity. This is consistent with the reports of Chang and Bandurski (1964) who failed to detect protease activity in sterile corn (Zea map) roots. Vagnerova and Macura (1974a) also reported that protease was

Bacterial

inoculation

and rhizosphere

not produced by sterile wheat roots, although, wheat grown in soil and significant protease activity indicating that rhizosphere microorganisms are responsible for the ‘root’ protease activity. This was confirmed by studies which showed that, while sterile wheat roots grown in a nutrient solution exhibited no protease activity, those inoculated with a suspension of three rhizobacterial isolates did (VBgnerovB and Macura, 1974b). The authors also demonstrated that the protease activity was present in the roots and not in the surrounding plant growth medium. This indicates that the microbial proteases are either adsorbed to the roots or adsorbed by the roots as had been demonstrated for other proteins (McLaren et aI., 1960). A third study by VBgnerova and Macura (I 974~) may provide an explanation to the decrease observed in root protease activity following P25 inoculation. These authors showed an inverse relationship between protease activity and the numbers of proteolytic bacteria present, with enzyme activity being greatest when numbers of proteolytic bacteria were lowest. P25 was shown to produce protease and hence inoculation of roots with proteolytic P25 may be directly responsible for the decreases in endorhizosphere protease activity. An alternative explanation is that a change in root exudates resulted in the enhancement of different bacteria which in turn affected protease activity. In conclusion, our study suggests that changes in carbohydrase

activities

Flavobacterium the inoculant

P25, and

following

are due

the plant.

inoculation

to interactions These

interactions

with between may

in protease activity although further studies are required to elucidate the role of changes in the quantity and quality of root exudates. Our work has shown clearly that microbial inoculation can affect the enzyme activity of both the indigenous microbial population and the plant and illustrates the importance of secondary (or indirect) effects of microbial inoculation on plant and microbial processes. include

alterations

Acknowledgement-This research was dentship from the Natural Environment

funded by a stuResearch Council.

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