The influence of root-knot nematodes (Meloidogyne javanica) on yield and on activity of sucrose synthase and invertase in roots of eggplants (Solanum melongena)

Physiological

Plant Pathology

(1984)

‘25, 209-217

The influence of root-knot nematodes (Meloidogyne javanica) on yield and on activity of sucrose synthase and invertase in roots of eggplants (Solarium melongena) W.

CLAUSSEN

Institutfuer

Obsfbau und Gemuescbau, der Universitaet

Bonn, Auf dem Huegel 6,053

Bonn, Federal Republic of Germany

and A. F. BIRD CSIRO Institute of Biological Resowces, Division South Australia 5001, Australia (Accepted for publication

of Horticultural

Research,

G.P.O.

Box 350, Adelaide,

July 1984)

Infection of eggplants (Solarium melongena) with root-knot nematodes (Meloidogyne javanica) decreased total plant and fruit dry weight, increased sucrcee synthase activity in galls and in the whole root system and also increased invertase activity in galls hut not in the whole root system. No sucrose synthase activity was detected in homogenates of infective larvae of M. javanica but sucrose hydrolysing activity was detected in these nematodes. Thus sucrose synthase activity appears to be associated only with plant tissue whereas sucrose hydrolysing activity may be associated with both plant and nematode. The starch and sucrose content of galls of infected plants were much lower than that of adjacent roots or of the roots of uninfected plants.

INTRODUCTION

Root-knot nematodes have a wide host range which includes many species of crop plants. Members of this genus have a world-wide distribution and Meloidogyne javanica is one of the most cosmopolitan of its species [4]. Most estimates of yield loss caused by nematodes are based on comparisons between yields with or without the addition of nematicides. These comparisons might over emphasize the effect of nematodes on crop loss, since nematicides may inhibit other parasitic organisms attacking the plant or, indeed, at least in some cases, actually stimulate plant growth. However, considerable loss of yield can be readily demonstrated under glasshouse conditions using host plants grown both in the presence and absence of root-knot nematodes [4]. Studies on the plant’s physiological changes associated with parasitism by these nematodes have not received a great deal of attention perhaps because of the added complexities associated with the study of two interacting organisms. It has been shown, using radio tracer techniques, that there is an accumulation of translocated 0048-4059/84/050209+09

$03.00/O

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1984 Academic

Press Inc.

(London)

Limited

W. Claussen

210

and A. F. Bird

photosynthates in tomato root galls induced by M. javanica [.5]. Also it seems that these nematodes may interfere in some way with the production of assimilates in the leaves

[W Sucrose synthase (UDP-glucose : n-fructose 2-gluccsyltransferase, E.C. 2.4.1.13) is considered to be a good marker for the distribution of assimilates in eggplants [7, 81. In this paper we have used it to test whether or not the reported higher demand for assimilates in nematode infected roots is accompanied by an increase in the activity of this enzyme. Sucrose synthase catalyses the readily reversible reaction UDP-glucose + n-fructose = sucrose + UDP [6]. Its main physiological importance appears to lie in its ability to cleave sucrose [I]. UDP-glucose, released by this cleavage, and its derivatives, can act as substrates for the synthesis of cell walls and storage compounds [IO]. A demand for these substrates during the growth of nematode galls should result in an increased sucrose synthase level in roots. Both acid and alkaline invertases are also thought to be involved in plant growth and the storage of assimilates [I] and in this paper we have included studies on the effects of M. javanica on the activity of these enzymes in eggplants and compared them with those of sucrose synthase mentioned above.

MATERIALS

AND

METHODS

Eggplants (Solunum melongena L. cv. Mission Bell) were grown from seed in an air conditioned glasshouse (18”-28 “C) under normal sunlight and in similar, but shaded, conditions throughout the year. Shade was achieved using shade cloth which only allowed 30% of the normal (glasshouse) sunlight to be transmitted. In all other respects the two sets of conditions were the same. The plants were grown in quartz sand (0.2-1.0 mm in diameter) in 10 litre black plastic pots. Nutrient solution [ 71 was supplied once a day ( 100-500 ml, depending on plant size) and additional water was supplied if necessary. The eggplants were each infected three times, at 2 weekly intervals during their flowering stage, with approximately 30000 freshly hatched larvae of M. javanica. These nematodes were obtained from egg masses that had been dissected from infected tomato roots and placed in shallow distilled water in Petri dishes. Larvae to be homogenized for enzyme analysis were centrifuged repeatedly in sterile distilled water, the supernatant being removed in each instance, until aliquots examined under the microscope showed them to be free of bacterial contamination. The plants were infected by pouring the nematodes in 5 ml of water on to the sand surface and this was then covered with aluminium foil to reduce evaporation. No water nor nutrient solution was added for the next two days in order to prevent the leaching of nematodes. Control plants were treated in the same way but with 5 ml of water instead of the suspension of infective larvae. The first samples were taken 14 days after the last infection. Enzymes were extracted from roots and galls immediately after harvesting. The extraction medium, adjusted to pH 7.4, was made up of 0.1 M Tris-HCI, O-1 M cysteine and 10 mM MgSO,. The fresh sample was homogenized in 15 ml of this medium in 50 ml centrifuge tubes using a Polytron homogenizer on ice for 1 min. Extracts were squeezed through “mira cloth” (Calbiochem), centrifuged in a refrigerated centrifuge (Sorval

Enzyme activity

in M. javanica

infected

eggplants

211

RC 2B) at 50000g for 10 min and 5 ml of the supernatant were desalted at 2-4”C on a Sephadex G25 column (20 x 150 mm) equilibrated with 50 mM MOPS buffer at pH 7.4. The nematodes consisting of two batches of 50000 and two batches of 250 000 infective larvae were homogenized at 0 “C in 2 ml of 0.1 M MOPS buffer at pH 7.4 for 3 min using a Potter glass homogenizer. Under these conditions, microscopic examination revealed that very few nematodes remained intact.

I OO

I 4

I 8 Time

(weeks)

I 12

I 16 ‘

FIG. 1. Total dry weight of eggplants infected with the root-knot nematode (Meloidogyae jauunicu) compared with controls. Plants were grown under light and shade conditions, respectively. The first sample was taken 2 weeks after the last infection with nematodes. Values shown are means of two replicates. Vertical bars represent standard deviation. 0, Infected; 0, control; -, shade; ---, light.

Time

(weeks)

FIG. 2. Dry weight of fruit of eggplants infected with the root-knot nematode (Meloidogyae juvuaica) compared with controls. Plants were grown under light and shade conditions, respectively. Values shown are means of two replicates. Vertical bars represent standard. deviation. 0, Infected; 0, control; ---, shade; ---, light.

W. Ciaussen

212

and A. F. Bird

Enzyme assayswere made immediately after extraction. Sucrose synthase activity was assayed at 30 “C in a medium containing 30 pmol of Tris-HCl (pH 8.7)) 1 umol of UDP-glucose, 3 pmol of fructose, 5 pm01 of MgSO, in a total volume of 0.3 ml. The reaction was started by adding UDP-glucose to the medium and stopped by adding 0.2 ml of 0.2 M NaOH. The test tubes were covered with glass marbles and heated in a boiling water bath for 10 min, followed by the addition of O-2 ml Roe reagent and 1.7 ml concentrated HCl [16]. The covered test tubes were held at 80 “C for 10 min. Sucrose synthase activity was calculated from spectrophotometer readings at 520 nm. Invertase activity was measured by incubating the enzyme either at pH 4.8 in 100 pm01 of acetate buffer + 60 pmol of sucrose or at pH 7.5 in 100 pm01 of citratephosphate buffer + 60 ltrnol of sucrose. In each case the final volume was 1.1 ml. The reaction was started by adding the buffer-sucrose mixture to the enzyme preparation. The enzyme used in the blanks was boiled for 1 min before adding the buffered substrate. The concentration of reducing sugars released by hydrolysis was determined by the method described by Nelson [Z3]. Carbohydrates were determined enzymatically as follows-500 mg (f wt) of root were stored at - 20 “C immediately after sampling. Sugars were extracted from the samples with 10 ml of 80% ethanol in 50 ml centrifuge tubes using a Polytron homogenizer and centrifuged (Sorval RC 2B) at 10 000 g for 2 min at room temperature. The supematant was decanted and the pellet redissolved in 10 ml of 80% ethanol. After shaking for 5 min, the suspension was centrifuged again and the supematant added to the lirst one. Water (10 ml) was added to the pellet and the covered test tubes were heated in a boiling water bath for 1 h. Approximately 7 Units of amyloglucosidase (E.C. 3.2.1.3) in 5 ml 0.3 M acetate buffer (pH 4.8) were added to each

:tI+-LAd 4

8

Time

12

16

(weeks)

FIG. 3. Shoot :root ratio of eggplants infected with the root-knot nematode (Mclotig~ae juouaico) compared with controls. Plants were grown under light and shade conditions, respectively. Vahxs shown are means of two replicates. Vertical bars represent standard deviation. 0, Infected; 0, control; -, shade; ---, light.

Enzyme activity

in M. javanica

infected

213

eggplants

tube which was closed and kept overnight at 55 “C. After glucose released from starch, and glucose in ethanol extracts had been phosphorylated by hexokinase and oxidized by glucose-6-phosphate-dehydrogenase, reduced NADP was measured spectrophotometrically at 340 nm [3]. The glucose moiety of sucrose in the ethanol extracts was determined in the same way after hydrolysis catalysed by acid invertase [Z]. Fructose was determined after phosphorylation and conversion of fructose-6-phosphate to glucose-6-phosphate catalysed by glucose phosphate isomerase.

28-

Time

(weeks)

FIG. 4. Sucrose synthase activity in roots of eggplants infected with the root-knot nematode (Mcfoidogynejauunica) compared with controls. Plants were grown under light and shade conditions, respectively. The first sample was taken 2 weeks after the last nematode infection. Values shown are means of four replicates. Vertical bars represent standard deviation. 0, Infected; 0, control; -, shade; ---, light.

RESULTS

Nematode infected eggplants grew at a slower rate and yielded less fruit than did uninfected control plants (Figs 1 and 2). Similar results were obtained with plants grown in the shade although these differences were observed only in later harvests. Nevertheless, they were just as pronounced (Figs 1 and 2). Both infected and control plants grown in the shade grew more slowly and yielded much less fruit than their counterparts grown in sunlight. Infection with nematodes also delayed flowering and fruit set by several days. The shoot :root ratio (on a dry weight basis) was lower in plants infected with nematodes than in control plants under conditions of both sunlight and shade (Fig. 3). Sucrose synthase activity was higher in roots infected with nematodes (Fig. 4) in plants grown both under shade and in full sunlight than in similarly treated noninfected controls. In full sunlight these differences were most pronounced during the

214

W. Claussen TABLE

Sucrose syntha.~ activity, infected with Meloidogyne

invertase javanica

1

activity and starch and sucrose concentration in eggplant roots (galls and non gall tissues) sampledfrom the start of the experiment to sixteen week? Time

Treatment Gall f. wt ( y0 of total wt) Sucrose synthase activityb Galls Non-gall tissue Invertase activity (pH 4S)c Galls Non-gall tissue Invertase activity (pH 7.5)d Galls Non-gall tissue Sucrosee Galls Non-gall tissue Stared Galls Non-gall tissue

of harvest

(weeks)

0

5

9

13

16

6.8

9.8

18.1

19.6

41

14.8 f 1.2 6.4 + 0.4

12.8&1,4 2.8 * 0.1

16.1 & 1.9 16.6 * 1.1

12.1 5 1.1 13.6 & 1.5

10.4 + 1.1 16.4 + 0.9

2.4 & 0.02 0.24 f 0.03

2.3 f 0.03 0.5 + 0.02

4.0 f 0.05 0.4 * 0.05

6.1 * 0.05 1.4 + 0.09

10.6 & 1.2 3.3 * 0.5

2.6 k 0.3 1.9*0.2

3.6 f 0.2 0.6 & 0~05

5.6 f 0.6 0.4 f 0.05

8.9 5 0.8 0.6 & 0.07

12.6 * 1.3 3.1 If 0.4

-

0.4 + 0.02 0.6 * 0.07

0.5 * 0.04 0.6 + 0.05

-

0.4 f 0.03 1.9 * 0.2

0.5 * 0.05 1.8 *O.l

-

-

-

pPlants were grown under shade conditions. Values shown are means f SD. bpmol sucrose formed g-i f wt h-r. Cpmol sucrose hydrolised g-i f. wt h-l (acetate buffer). dpmol sucrose hydrolised g-r E wt h-r (citrate-phosphate buffer). cmg (100 mg) -i f. wt. fmg (100 mg)-r E wt.

TABLE Sucrose hydrolysing

activity

in

theinfective

of nematodes

homogenized

50 000 250 000 Walues

larvae of Meloidogyne

are the means

replicates,

javanica=

Sucrose hydrolysing sucrose hydrolysed

of two replicates

activity g-i f. wt h-r) pH

pH 4.8 0.09 * 0.01 0.47 * 0.06

shown

of four

2

(pmol Number

and A. F. Bird

7.5

0.08 i 0.01 0.51 10.05 + SD.

period of rapid fruit growth, 2-6 weeks after the first harvest. In plants grown in the shade the differences became pronounced whilst the fruit were of negligible size (Fig. 4). The activity of sucrose synthase was similar in galls of varying size. The activity of this enzyme in non-galled root tissue of infected plants was much less than in galls in the first two harvests (Table 1) but similar for harvests three and four and slightly higher for the final harvest.

Enzyme activity

in hf. javanica

infected

eggplants

215

1

Time

(weeks)

FIG. 5. Acid and alkaline

invertase activity in roots of nematode (Mc~sidogyacjuvan~ca) compared with controls. ditions. The first sample was taken 2 weeks after the last four replicates. Vertical bars represent standard deviation.

Time

eggplants infected with the root-knot Plants were grown under shade coninfection. Values shown are means of 0, Infected; 0, control.

(weeks)

FIG. 6. Starch and sucrose content of roots of eggplants infected with the root-knot nematode (Mcloidogyae jnoan&) compared with controls. Plants were grown under shade conditions. The first sample was taken 2 weeks after the last infection. Values shown are means of two replicates. Vertical bars represent standard deviation.

216

W. Claussen

and A. F. Bird

No sucrose synthase activity was detected in homogenized larval nematodes but sucrose hydrolysing enzymes with pH optima between 4.8 and 5.3, and 7*4 and 8.0, respectively, were detected in these organisms (Table 2). Both types of activity occurred in much higher concentrations in the roots of infected plants than in controls (Fig. 5), particularly in the last two harvests. However some activity was detected in the roots of non-infected control plants (Fig. 5) so that not all of the sucrose hydrolysing enzyme activity can result from this host-parasite relationship. The concentrations of starch and sucrose were low in the roots of infected plants grown in the shade compared with the roots of uninfected plants grown under similar conditions (Fig. 6). The concentrations of starch, in particular, were much higher in the roots of uninfected control plants at the last two harvests than in the equivalent infected roots (Fig. 6). Less starch was detected in galls compared with adjacent tissue (Table 1).

DISCUSSION

Eggplants grown under low light conditions showed more shoot growth and less root growth (Fig. 3) than those grown under high light conditions. In conjunction with this there is a lower sucrose synthase level in the roots (Fig. 4). On the other hand those plants grown under high light conditions were able to produce more assimilates which supported greater root growth (Fig. 3) and showed a higher sucrose synthase activity (Fig. 4). It has been shown that this enzyme is closely associated with the distribution of assimilates [7,8]. In these experiments we have used sucrose synthase as a marker and have shown that the demands of the parasitic nematode are reflected in increased levels of activity of the enzyme, particularly in plants grown in the shade (Fig. 4). These increased levels of sucrose synthase activity in infected plants, as compared with uninfected controls, were most pronounced during the phase of rapid fruit growth where the fruit competed with roots in obtaining photosynthetic products from the leaves. Thus, as may be expected, the growth of fruit took place much more slowly in nematode infected plants (Fig. 2). The sucrose synthase activities shown in Figure 4 are values for the whole root. Because galls only make up a small portion of the total root weight for the first two harvests (Table l), sucrose synthase activity for whole roots does not reflect the high activity in the galls of plants grown under low light conditions. The dramatic increase in sucrose synthase activity shown from harvest three onwards for non-galled roots in infected plants (Table 1) was responsible for the high activity of infected roots (Fig. 4). The high sucrose synthase activity measured at the final harvest for control plants grown in the shade (Fig. 4) might be due to the increase in carbohydrate levels demonstrated (Fig. 6). This is in agreement with earlier findings that changes in the sucrose and starch concentrations in roots are accompanied by similar changes of sucrose synthase activity [9]. The observations that nematode galls contain a lower carbohydrate concentration than roots ofuninfected controls (Table 1) confirm those of Owens & Novotny-Specht [1.5]. Starch, when present in galls is thought to be confined to the periphery [14]. Although no investigations on sucrose synthase activity have been carried out so far, invertase activity has been shown to occur in galls as well as in nematodes [II, 14, 271.

Enzyme activity

in M. javanica

infected

eggplants

217

It should be noted that the techniques used for the determination of the invertases in the nematode homogenates do not exclude the possibility of the presence of glycosidases. We have found that, in contrast to sucrose synthase activity, invertase activity remains low in non galled roots of infected plants for all harvests whilst activity in the galls is high (Table 1). Orion & Bronner [Z4] have shown, by histochemical means, that this invertase activity is concentrated mainly in the syncytia (giant cells). The findings that sucrose hydrolysing enzymes occur in the homogenates of infective larvae of M.javanica (Table 2) lead to the speculation that the endo-parasitic stages of this nematode may exude this enzyme into the syncytia (giant cells) that they induce and feed upon. Alternatively these nematodes may stimulate the production of this enzyme within the syncytia. We thank Miss S. D. Harris for expert technical assistance and the senior author gratefully acknowledges financial support from Deutsche Forschungsgemeinschaft.

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A photometric

of Biological

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