Acid phosphomonoesterase activity of ectomycorrhizal roots in norway spruce pure stands exposed to pollution

sdi B&i. mochem. Vol. 23, No. 7. pp. 667-631, 1991 Printed in Great Britain. All rights reserved

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ACID PHOSPHOMONOESTE~SE ACTIVITY OF ECTOMYCORRHIZAL ROOTS IN NORWAY SPRUCE PURE STANDS EXPOSED TO POLLUTION Uepartment of Pedology and Geology, University of Agriculture,Zemedelska Bmo 613 00, Czechoslovakia

3,

(Accepted 20 January 15%) Sammary-The release of orthophosphate ions from organic compounds is essential for continuous phosphorus cycling in forest ecosystem. An important stage of this process in coniferous forests of the temperate zone is the production of acid phosphomonoesterase (PME) by ectomycorrhizaj fungi. The effect of artificial and natural poihttant inputs during repeated &tort periods of high concentration on the activity of the specific enzyme was studied. The acid PME activity of spruce mycorrhisas was used as an indicator of anthropogenic pressure on forest soils. The seasonal dynamics of the activity of acid PME was monitored from February 1989 to January 1990. The results have showed a sign&ant decmase of acid PME activity in ectomycorrhizal spruce roots as affected by pollutant input. The amount of acid PME activity may become one of the characteristic of the changing biochemical procuses in soils under the effects of air pollution. The method presented is simple enough to be included in an integrated system of ecological analysis routinely used in field research to monitor forest decline.

MATERIALS

INTitODUCMON

The aim of ecological monitoring is to determine, interpret and forecast changes in the environment under the ef&ts of natural and anthro~~~c factors. On the basis of measuring a response to the stress factors objective information can be obtained on changes in ecological properties. Changes in the quantitative and qualitative structure of soil biocoenoses in response to pollution contamination of the environment markedly affect ~torn~~~~l symbiosis (cf. Jansen ef al., 1988). For this reason theories have appeared indicating that forest decline in air-polluted areas starts from the root system (Mayer, 1988) because ectomycorrhizal roots die off and do not regenerate. The main benefit of ectomycorrhizal symbiosis for tree species is the ability of the mycosymbiont to take up poorly availabk nu~ents and transfer them, together with vitamins and growth substances, to the host body. Mechanisms of uptake, storage and transfer of nutrients to the host plant by the ectomycorrhixal fungi has been unambiguously proved for phosphorus (Bartlett and Lewis, 1973; Williamson and Alexander, 1975; Ho and Zak, 1979, Alexander and Hardy, 1981; Doumas et al., 1984; Pang and Kolenko, 1986; Berjaud et al., 1987; Mejstfik, 1988; Cudlin and Mejstfik, 1989). Ectomycorrhixal fungi have a perfect enzymatic apparatus to ensure the transport of P into tissues of the host plant. Furthermore, 70-90% of P in forest soils may be tied up in organic P compounds (Halstead and McKerchner, 1975; Chaxiev, 1982; Harrison, 1983, 1987). Consequently ectomycorrhixal interrelationships are a fundamental precondition for the proper growth of coniferous forests on soils with a primary P deficiency where organically-bound phosphates are the only available source of P for tree species.

Artificially polluted area

Damage simulations and studies of seasonal fluctuation of acid PME activity were assayed on three types of experimental plots in a mature Norway spruce [Piceu abies (L.) Karst.] monoculture on the MAB Research Station “Rtijec nad Svitavou” (the Drahany Uplands, Czechoslovakia). The sampling sites were located at 630m alt. on mylonitixed acid granodiorite; the soil type was classi%d as Dystric Cambisol (Bd). Two types of plots were treated with elemental sulphur (Lettl ef al., 1981; Kennedy et al., 1985; Maynard et al., 1986): (i) S lOO-medium-polluted plots (100 kg S ha-’ yr-‘) and (ii) S 300-heavily-polluted plots (300 kg S ha-’ yr-I). The third type of plots was left untreated and served as control (C). Each of the experimental plots were represented by three independent sub-plots. The ~rnu~tjons were carried out by the personnel of the MAB Research Station during autumn in repeated short periods (20-29 November 1986, 29 October-12 November 1987, 26 October-16 November 1988 and 15-30 November 1989). Sampling was conducted midmonth between February 1989 to January 1990. Samples of spruce mycorrhizas were coUected from each of the subplots. The samples were mixed together and stored in PVC bottles at 4°C (at this temperature the samples were considered as fresh). Four replicates were produced from each mixed sample for analysis. Ectomycorrhizal acid PME activity was monitored in mature Norway spruce pure stands under the influence of repeated short periods of air pollution at high concentrations. Investigation was conducted at four localities at the southeastern outskirts of the

667

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K. RrusEK

Tabk 1. Addp~~m~~~~activity(~gp-Np5omg~‘myc. roots h-‘) of Norway qntcc [F&o &es (L.) Rats.] myawrh&s under high S-loadingto tree nut&ion; see for legendto the text. Means of 4 mplicptcs Time of sampling

Control

Febnuuy March April M&Y June J&Y AUgW sep-b=

14.5 13.1 13.1 16.9 to.1 18.1 14.6 25.9 36.9 36.2 12.9 16.2 2.39 i&ii 19.0 2.44

OClObCt

Nowmher Delxmber JMUUy SEM JUtIC SEM

TN!attttCtlt s 100 10.6 10.5 10.6 10.6 15.6 12.8 10.7 15.9 17.1 21.7 10.6 9.7 1.12 Saiajka 16.5 1.07

10.6 10.2 10.2 10.2 13.9 to.7 6.2 10.7 11.8 12.4 4.5 6.2 0.86 Stnrfiny Albin 12.7 I .03

Ostravsko-Karvinsky Coalfield (the Moravian-SileSian Beskids, Czechoslovakia). Flysch bedrock, mesoclimatic regime, altitude and local orography determined the diversity of soil types: Dystric Cambisol (Bd)-Be&e and Albmovo nam&sti,and Orthic Podxol (PO)-Salajka and SmrEiny. The monitoring sites were selected for their different degrees of ~~o~~~c pollution: (i) a control locality, &%e (the least damaged, corresponding to C), (ii) a medium-polluted locality, Salajka (ccrresponding to S 100) and (iii) heavily-polluted locality, Sm&ny and Albinovo nami?sti (corresponding to S 300). Samples from each locality were obtained from independent sub-plots in June 1989 and stored in PVC bottles at 4OC. METHODS Reagents (1) Succinatc-Borate Buffer Solution (SBB Solution), pH 4.8. Prepare 11 of the S-B buffer solution by mixing 350 ml of 50 mm011-l sodium tetraborate

4Or

m

Pl0r

comol s loo

s300

cf

Tabk 2, IntIuart of simuktcd hi& S-loadingon the acid PME activity in Norway rpnwr [P&w &es (tf Kant] pure stands

COfltrOl

;g S300

SbUldStd dcviatioab

Mepnb 19.86 13.01 19.86 9.79 13.01 9.79

8.61 3.72 8.61 2.78 3.72 2.78

*5.47 f2.37 * 5.47 f 1.76 f 2.37 +1.76

f 7.72. f3.34 f 7.72,’ f 2.49 k3.34. f 2.49

‘Refer to the text for explanation of plot symbols. “fig p-NP SOmg-’ cctomycorrhizal roots h-l. *Di~Efctlccs k1wen plots significant at the level of 01to.95 (P = 0.05). **Diffemnccs tctwm plots significent at the level of 0 -0.99 (P -0.01).

(A Solution) and 650 ml of 50 mm01I-’ succinic acid (B Solution). A Solution: Dissolve 19.1g of Na,B,O, 10 H,O in I 1 of distilled water. B Solution: Dissolve 5.9 g C&O, in 11 of distilled water. (2) p-Nitrophenyl Phosphate Solution (p-NPP Solution), 500 pmol l- *. Dissolve 180mg NazC6H.,N02P0, (Lachema o.p., Brno, Czechoslovakia) in 11 of the SBB Solution. (3) Potassium Hydroxide, 1 mol I-‘. Dissolve 5.6 g KOH in lOOm1of distilled water. (4) ~-Nitrophenol Standard Solution (p-NP Solution), initial dilution. Dissolve 0.1 g C,H,OH NO, (Lachema o.p., Bmo, Czechoslovakia) in 1OOmlof the SBB Solution. To provide precise and accurate measurement it is recommended to use freshly prepared solutions. Procedure (1) Analysis is made of 50 mg of washed mycorrhixal roots. The sample is placed into a 100.ml Erlenmeyer flask and then 12ml of the p-NPP Solution is added. (2) The reaction mixture is shaken and then kept for 60min at 25°C.

February

a

August

March

m

September

April

Coniidenccinterval OI- 0.95 4 10.99

m

October

a

November

m

December

m

January

Fig. 1.The relationshipbetweenacid PME activity of Norway spruce [ Piceu &es

(L.) Karst] myoorrfiizas

and diffmnt anthropogcnic load; treatment identitication from the text.

Pho~homon~~~

Fob

March

April

June

May

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activity under acid deposition

July

Sept

Aug

Ott

Nov

Dee

Jan

Fig. 2. Fluctuation in activity of ectomycorrhizal acid PME in Norway spruce [Picea abies (L.) Karst] pure stands during the season 1989-W; set for legend to the text. (3) The readion mixture is filtered into a test tube and 8 ml of 1 mol KOH 1-l is immediately added to the filtrate. Due to strong alkalization the yellow colour of the p-M! ions develops. (4) To construct the calibration curve it is necessary to determine the absorbance of the standard solutions. 1 ml of p-NP Solution is diluted to 100 ml with the SBB Solution. 1,2,3. . . 10 ml of this solution is filled to 12 ml by the SBB Solution. The final standard solutions contain 10,20,30. . . 100 l(g p-NP. The solutions are filtered and 8 ml of 1 ma1 ICOH 1-I is added to the filtrate. A mixture of 12 ml of the filtered SBB Solution and 8 ml of 1 mol KOH 1-l is used as a blank. The optical density of all the filtrates is measured against the blank at 410 rnp. The amount of p-NP released is determined after the hydrolysis of the substrate and is a measure of acid PME activity. The results are given as ;_~,p-Np released from Mmg of mycorrhizal roots

cance of the effect of high S-loading on acid PME activity (Table 2). The continuous monitoring for 12 months showed a statistically significant negative correlation between the simulated load of sulphur compounds on the activity of acid PME (being above the limit between random and true deviations in all combinations). Similar results were found for naturally polluted and art&ally polluted localities (sampled in June 1989). These findings tend to confirm the above-mentioned conclusion for forest soils under the influence of indust~al pollutants. Furthermore, the health of mycorrhizal roots was assessed in situ showing a visible disparity in turgidity and richness with increasing acid deposition. Throughout 1 yr a seasonal fluctuation of acid PME activity in an artificially polluted area was monitored. A two-peak seasonal pattern with a sharp autumn m~imum was observed (Fig. 2). DISCUSSION

RESULTS Monitoring of acid PME activity in both naturally and artificially polluted plots demonstrated a decrease in enzyme activity (Table 1 and Fig. 1).

oNO2

Methods for determination of soil phosphoesterase activity are based on the hydrolysis of a specific organic compound of phosphorus. In the majority of such studies, ~-ni~ophenyl phosphate (Tabatabai and Bremner, 1969) is used as the substrate:

0 Na I OH

P-O \

+ Na2HP04

-

NOz

0 Na

Na2G~H~N02PO~

-

Enzyme

Gf,H40H

NO2 + Na2HP04

H20

The values from art&ally-polluted experimental plots were tested to determine the statistical signifi-

Substrate specifity and selectivity result in the identification of a particular enzyme. p-Nitrophenyl

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K. r&&K

phosphate is a monoester of o~hophospho~c acid. The catalyst of its decomposition to p-nitrophenol and monophosphate is a phosphomonoesterase. The measurement used was a modification of the method by Woolhouse (1969). The Woolhouse method is widely used (Bartlett and Lewis, 1973; Williamson and Alexander, 1975; Malcolm and Vaunt, 1979; Alexander and Hardy, 1981) because of its sensitivity and accuracy. The modification we used owing to its simplicity is also suitable for routine monitoring in basic field research. It is an universal procedure for a variety of samples with a standard comparative level for soils under the changing influence of anthropogenic pressures. Correctly interpreted, the results appear to be a sensitive indicator of soil enxymatic procemes. However, phosphatases are continually synthesixed, accumulated and ~rn~~eo~ly inactivated and decayed. The values recorded refer to the instantaneous relationship between these counteracting processes which are dependent on external ecological conditions in both soil and plant. Therefore, large-scale random sampling is necessary. The enzyme is more active in soils of pH close to the pH optimum of the enzyme. Phosphorus ~~~labi~ty depends on the pH of soils as well. The absorption of H,PO, itself would be ~~~ti~lly prevalent in soils having pH values less than 2.8 and plant uptake of its bivalent and trivalent anions HPO:- and PQ3- is dominant in soils with pH values higher than 7.2 (Walker, 1972; Corbridge, 1977). In the majority of soils in the temperature zone, that is in the pH range from 3 to 7, the monophosphate HrPOi ion is most readily absorbed by plants (Hayman, 1975; Barber, 1984). Organic ph~pho~ compounds are principally esters of orthophosphoric acid. The catalysts releasing monophosphate ions from organic phosphate esters are phosphomonoesterases (EC 3.1.3.). Acid phosphoesterases play a crucial role in the hydrolysis of organically-bound phosphorus in acid soils (Hoffmann, 1968; Eivazi and Tabatabai, 1977; Juma and Tabatabai, 1978; Dick and Tabatabai, 1978; Ho, 1979; Chaxiev, 1982; Malcolm, 1983; Rojo et al., 1990). Thus, acid phosphomon~te~~s (EC 3.1.3.2.) should play a important role in the natural supply of inorganic phosphate ions to tree species in temperate forest soils. Large-area symptoms of severe damage by air pollutants in the Moravian-Silesian Beskids were first reported after an extreme meteorological event in the night preceding 1 January 1979. Meteorological conditions of air pollutant dispersion in the orographitally complicated region lead to an unfavourable vertical composition of the atmospheric layers. The situation causes extremely high concentration of industrial pollutants in a thin layer. The die-back of spruce stands in this region is obviously affected by repeated short periods of lethal concentrations of industrial pollutants (Forchtgott, 1988; Kratochvilovi et al., 1989). Since the circumstances described above have been documented widely (prinze and Krause, 1989; Rak, 1990), the use of this bioindicative test in the continuous integrated monitoring could be relevant.

Adinow&dg-ts-The author is indebtedto w&agues of his from the Institute of Forest Ecology, Bmo-gob&ice (Czohoslovakia) for their assistance in field activities.

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A Mech-

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activity under acid deposition

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