In planta histochemical staining of fungal alkaline phosphatase activity for analysis of efficient arbuscular mycorrhizal infections

Mycol. Res. 97 (2): 245-250 (1993)

245

Printed in Great Britain

In planta histochemical staining of fungal alkaline phosphatase activity for analysis of efficient arbuscular mycorrhizal infections

B. TISSERANT, V. GIANINAZZI-PEARSON, S. GIANINAZZI AND A. GOLLOTTE Laboratoire de Phytoparasiiologie, INRA-CNRS, Station de Ginetique ei d'Amelioration des Plantes, INRA, BV 1540, 21034 Dijon cedex, France

A histochemical procedure was developed to visualize and estimate the proportion of arbuscular mycorrhizal infections showing fungal alkaline phosphatase activity, as compared to the total amount of fungal tissue (trypan blue staining) and of living mycelium, indicated by succinate dehydrogenase activity. In roots of Allium porrum and Platanus acerifolia, only a small proportion of living intraradical mycelium showed alkaline phosphatase activity during early infection but this increased greatly just before the mycorrhizal growth response of the host plant. Infection revealed by all three stains reached a maximum at 6 weeks after inoculation, after which the level of trypan blue stained infection remained constant but the proportion showing succinate dehydrogenase and alkaline phosphatase activity declined as the infection aged. Alkaline phosphatase activity was absent from virtually all abortive entry point hyphae formed on roots of a resistant myc-, nod- mutant of Pisum sativum, although succinate dehydrogenase activity was detected. Observations suggest that the alkaline phosphatase activity is induced by colonization of host roots and that this fungal enzyme could proVide a useful marker for analyzing the symbiotic efficiency of arbuscular mycorrhizal infections.

Staining techniques using trypan blue, chlorazol E or fuchsin (Phillips & Hayman, 1970; Brundrett et ai., 1982; Grace & Stribley, 1991; Merryweather & Fitter, 1991) to reveal fungal structures in arbuscular mycorrhiza do not distinguish living from dead fungus, and therefore give no indication of the metabolic state of the fungal tissue. Succinate dehydrogenase, a mitochondrial enzyme, has been used histochemically to detect the proportion of infected roots in which the fungus is 'alive' (Ocampo & Barea, 1985; Kough & Gianinazzi-Pearson, 1986; Smith & Gianinazzi-Pearson, 1990) and has shown that the fractional infection with this enzyme activity declines as plants age (Kough, Gianinazzi-Pearson & Gianinazzi, 1987; Hamel, Fyles & Smith, 1990; McGee & Smith, 1990). This method does not, however, appear to be indicative of the efficiency of the fungus in terms of plant growth (Vierheilig & Ocampo, 1989). The positive role of arbuscular mycorrhizae in the growth of many herbaceous and woody plant species generally results from a greater efficiency of mycorrhizal roots to take up phosphate, mainly due to the capacity of actively absorbing external hyphae to better explOit the labile pool of available soil phosphate (Smith & Gianinazzi-Pearson, 1988). Studies of the physiological basis of phosphate absorption, accumulation and translocation by arbuscular mycorrhizal fungi indicate that all these processes are metabolically dependent (Gianinazzi-Pearson & Gianinazzi, 1986; Smith & GianinazziPearson, 1988; Thomson, Clarkson & Brain, 1990). Amongst enzymes that could be involved in the phosphate nutrition of arbuscular mycorrhizal fungi, alkaline phosphatase activity has been localized within the phosphate-accumulating

vacuoles of hyphae, and particularly along the fungal tonoplast (Gianinazzi, Gianinazzi-Pearson & Dexheimer, 1979; Dexheimer, Gianinazzi-Pearson & Gianinazzi, 1982; JacquelinetJeanmougin, Gianinazzi-Pearson & Gianinazzi, 1987). The corresponding enzyme has been separated by gel electrophoresis of extracts of arbuscular mycorrhizal roots infected by different fungi (Gianinazzi-Pearson & Gianinazzi, 1976, 1978,1983; Gianinazzi-Pearson et al., 1978; Vielhauer, 1989) and the activity has been shown to increase during the early stage of infection development which coincides with plant growth stimulation (Gianinazzi-Pearson & Gianinazzi, 1978). Detectable alkaline phosphatase activity decreases as the infection ages and with reductions in mycorrhizal effects on plant growth associated with increased phosphate additions to soil (Gianinazzi-Pearson & Gianinazzi, 1983). These observations have led to the hypothesis that this enzyme activity could indicate the existence of a functional arbuscular mycorrhizal infection in terms of phosphate nutrition. Fungal alkaline phosphatase activity has been localized at the light microscope level in cross sections of mycorrhizal roots (Smith & Gianinazzi-Pearson, 1990) but this does not permit an evaluation of the physiological state of the infection in the whole root system. In the present study, we have developed a method to visualize and estimate the proportion of arbuscular mycorrhizal root systems showing fungal alkaline phosphatase activity and we have investigated whether this enzyme could provide a useful physiological marker of an efficient symbiosis in terms of plant growth improvement (Smith, McGee & Smith, 1990).

In planta staining of fungal phosphatase

MATERIALS AND METHODS In a first experiment (Expt I), seeds of Platanus x acerifolia (At.) Willd. were germinated on moist filter paper at 20°C. One week after germination, 30 homogeneous seedlings were individually transplanted into plastic pots containing 400 g (dry weight) of a mixture (3: I : I, by vol.) of a disinfected clay loam soil (pH 7'9, 24 ppm Olsen P), gravel and Terra-green (Oil Dry SA R). Half the plants were inoculated with Glomus spp. (isolate LPA7) by introducing 0'5-1'0 g of chopped mycorrhizal leek roots into the planting hole. Uninoculated plants were used as controls. Plants were maintained in a growth chamber (20°, 70% humidity, 16 h, 300 j.lmol s-1 m- 2 ). Plants received weekly 16 ml Long Ashton nutrient solution without P (Hewitt, 1966). Plants were watered daily and were harvested 3, 4, 5, 6 and 7 weeks after transplanting. In a second experiment (Expt 2) the effect of rooting medium and host plant were investigated. The same protocol was used as in Expt I except that Allium porrum L. and P. acerifolia were planted in the soil mix with Terra-green replaced by perlite, and plants were harvested at 2, 4, 6 and 12 weeks after transplanting. A third experiment (Expt 3) was deSigned to see whether a successful endomycorrhizal infection was necessary for activation of fungal alkaline phosphatase. For this, Pisum sativum L. cv. Frisson and an isogenic myc- nod- mutant (P53, Gianinazzi-Pearson et al., 1991) were inoculated with Glomus intraradices Schenck & Smith (isolate LPA 8) in the same clay loam soil as in Expt 1 but mixed with 25 % sterile gravel. In all experiments, the efficiency of the mycorrhizal infection was assessed in terms of plant growth (dry mass of shoots) and the mycorrhizal infection determined by non-vital or vital staining procedures. Root systems were cut into I em lengths and samples were taken to visualize the mycorrhizal infection by staining with trypan blue in lactophenol (Phillips & Hayman, 1970), after clearing root samples in 10% KOH, or by staining for succinate dehydrogenase and alkaline phosphatase activities. Succinate dehydrogenase (SOH) activity was revealed by deposition of purple formazan following reduction of nitroblue tetrazolium in the presence of succinate using the method described by Smith & Gianinazzi-Pearson (1990), but modified for clearing root samples after the reaction. Visualization of fungal structures was improved by clearing roots in sodium hypochlorite solution (3 % active chlorine) at room temperature, instead of using boiling chloral hydrate. Alkaline phosphatase (ALP) activity was determined by a modified azo dye method using Fast Blue RR salt in the presence of a-naphthyl acid phosphate (Pearse, 1968). Root pieces were incubated overnight in a reaction medium containing 0'05 M Trisjcitric acid buffer (pH 9'2), 1 mg ml- 1 Fast Blue RR salt, 1 mg ml- 1 a-naphthyl acid phosphate, 0'5 mg ml- 1 MgC1 2 and 0'8 mg ml- 1 MnC1 2 4H 2 0. For P. acerifolia it was necessary, prior to this, to incubate root pieces 2 h at room temperature in a digestion medium containing 0'05 M Trisjcitric acid buffer (pH 9'2), 0'05 % sorbitol, 15 units ml- 1 cellulase and 15 units ml- 1 pectinase units in order to ensure penetration of the reagents. Background staining

246 reported to occur with the azo dye method by Pearse (1968) was removed after ALP staining, without affecting enzyme activity localization, by incubating root samples 5 min in a sodium hypochlorite solution (1 % active chlorine). Controls consisted of incubation in the reaction medium in the presence of 20 mM KCN or NaF (inhibitors of alkaline and acid phosphatase, respectively), without substrate, or in 0'1 M acetate buffer at pH 5. Fractional infection was estimated microscopically as the intensity of infection of the root cortex (M%) (Trouvelor. Kough & Gianinazzi-Pearson, 1986) revealed by each stain and for P. sativum in Expt 3, number of entry point hyphae cm- 1 root was also determined. Data for three replicate plants at each harvest were analyzed by variance analysis (ANOVA) and Duncan's test.

RESULTS Expt 1 Values for fractional infection of roots (M%) by Glomus sp. and estimated by non vital staining (trypan blue) were similar to those obtained by SOH staining up to 6 weeks after

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Weeks after transplanting Figs 1-2. Expt I, P. acerifolia-Glomus sp. Fig. 1. Fractional infection (M%) of root cortex revealed by trypan blue (-e-), succinate dehydrogenase ( - 0 - ) and alkaline phosphatase staining (-.-). Fig. 2. Shoot dry mass of non-mycorrhizal ( - 6 - ) and mycorrhizal ( - .- ) plants. Bars indicate standard error of values; data points bearing different letters are Significantly different at the 95 % confidence level at each harvest.

247

B. Tisserant and others transplanting (Fig. 1). SDH activity indicated that 80 to 90% of mycelium present in roots was alive throughout the experiment. Alkaline phosphatase activity was indicated by a black staining of hyphal contents which could be observed in whole root pieces, entry point hyphae, intercellular hyphae and arbuscule branches (Figs 3--6). Values for fractional infection using the alkaline phosphatase activity stain were generally significantly lower than those for trypan blue or SDH staining. At three weeks, only a very small part of the living Glomus sp. mycelium presented an alkaline phosphatase activity (M % = 4), but this increased sharply at 4 weeks (M % = 45), indicating a stimulation in fungal metabolism (Fig. 1). Furthermore, this increase in alkaline phosphatase activity between 3 and 4 weeks occurred just before the plant growth response to the mycorrhizal infection, seen as a significant increase in shoot dry mass at 5 weeks after transplanting (Fig. 2). Control mycorrhizal root samples incubated with 20 mM KCN, or at pH 5, did not stain for alkaline phosphatase activity, whilst this persisted in root samples incubated in 20 mM NaF. No infection was detected with any staining procedure in root samples of uninoculated plants.

Expt 2 For P. acerifolia, succinate dehydrogenase and trypan blue staining gave a similar infection curve up to 6 weeks as in Expt 1, but differences in M % values at 2 weeks between these and alkaline phosphatase staining were less important in Expt 2 (Fig. 7). However, the growth stimulation in mycorrhizal plants (as compared with non mycorrhizal plants) occurred earlier and was already significant at 4 weeks after transplanting (Fig. 8). Fractional infection estimated by trypan blue staining increased to 6 weeks and then remained constant to 12 weeks, whereas the infection curves with vital staining (SDH and ALP) increased then significantly decreased at the last harvest (P = 0'05 for difference in values at 6 and 12 weeks). Differences appeared between M % values for SDH and ALP activity with a greater decrease in ALP stained roots (Fig. 7). For A. porrum, the fractional infection in mycorrhizal roots revealed by ALP activity was very low at 2 weeks after transplanting but increased greatly up to 6 weeks (Fig. 9), which coincided with a rapid growth response in the mycorrhizal plants (Fig. 10). As in P. acerifolia, fractional infection revealed by trypan blue staining was similar to that with SDH activity at 2 weeks, increased to 6 weeks then slightly to 12 weeks whilst the proportion of infection showing SDH and ALP activity decreased after 6 weeks, with the latter always being lower.

Expt 3 As in P. acerifolia and A. porrum, significant differences were observed in M % between non-vital and vital staining in mycorrhizal roots of 6-week-old P. sativum cv. Frisson infected with G. intraradices (Table 1), with SDH and ALP active infection being considerably lower. However, the overall values for M% were generally lower, ALP staining revealed

only 45% of total infection (compared to 66-79% in A. porrum and P. acerifolia) and no mycorrhizal growth response occurred in this less dependent plant species (results not shown). In the isogenic myc-, nod- pea mutant (P53) the values for M % were zero since in the incompatible interaction with G. intraradices, entry point hyphae were blocked at the appressorium stage. In P. sativum cv. Frisson, the number of trypan blue stained entry points cm- 1 root was significantly greater (P = 0'01) than in the isogenic P53 mutant (Table 1) and a high proportion of them showed SDH (70 %) or ALP (53 %) activity. Only 22 % of appressoria revealed by trypan blue staining showed an SDH activity on roots of the P53 mutant and alkaline phosphatase activity was detected in less than 1 % of the abortive entry points.

DISCUSSION The results reported here using a staining procedure for root pieces confirm the presence of an alkaline phosphatase activity in arbuscular mycorrhizal fungi, as previously shown by biochemical or ultrastructural analyses (Gianinazzi-Pearson & Gianinazzi 1978; Gianinazzi-Pearson et aI., 1978; Gianinazzi et al., 1979; Dexheimer et al., 1982) and, more recently, in cross sections of endomycorrhizal roots with increased P inflow rates (Smith & Gianinazzi-Pearson, 1990). This simple technique makes it possible to directly compare in mycorrhizal root systems the total amount of fungal tissue (trypan blue staining) and the proportion which is alive (SDH staining) with that likely to be associated with an active phosphate metabolism (ALP activity). The fact that an important increase in the proportion of intraradical mycelium showing alkaline phosphatase activity preceded the mycorrhizal growth response in A. porrum and P. acerifolia, and was considerably lower in non responsive P. sativum, does support the hypothesis that this enzyme is somehow involved in the processes of phosphate transfer from soil to roots by arbuscular mycorrhizal fungi. This has been previously suggested by its localization in the same cellular compartment as stored polyphosphate and by the inhibitory effects of high phosphate fertilization (Gianinazzi-Pearson & Gianinazzi, 1983). Several functions have been proposed for alkaline phosphatase in microorganisms, and in particular their role in hydrolysis of phosphate esters, in transphosphorylation as transferases and in transmembrane transport of inorganic phosphate (McComb, Bowers & Posen, 1979). There were significant differences in infection values between trypan blue and alkaline phosphatase staining throughout all experiments. The observed decrease in ALP active infection after six weeks agrees with results obtained in biochemical analyses where extractable mycorrhiza specific alkaline phosphatase activity declined in endomycorrhizal roots of ageing plants (Gianinazzi-Pearson & Gianinazzi, 1978; Vielhauer 1989). The decrease in the value of M% estimated by SDH staining, as compared with trypan blue staining, in relatively old plants (12 weeks) is similar to that previously reported and reflects senescence of the fungal mycelium as infection ages (Kough et aI., 1987; Hamel et al., 1990; Smith et al., 1990). The short digestion treatment of

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Figs 3-6. Fungal alkaline phosphatase activity in endomycorrhizal roots of P. acerifolia. Fig. 3. Intraradicular mycelium. Fig. 4. An entry point hypha (eh) on the root surface. Fig. 5. Intercellular hyphae (ih) and arbuscules (a). Fig. 6. Detail of an arbuscule. Bar = 200 11m for Fig. 3 and 20 11m for Figs 4-6.

root samples necessary for ALP staining in P. acerifolia did not seem to affect enzyme activity, contrary to that reported for SDH activity in root samples submitted to a long digestion time (McGee & Smith, 1990). Furthermore. no differences in alkaline phosphatase activity of fungal mycelium have been observed with or without digestion of root samples in comparative studies of G. mosseae-infected soybean (unpublished data). However, when using ALP staining to reveal infection. it is necessary to determine whether prior enzyme digestion of roots is necessary for the penetration of the reagents. since this is dependent on the plant species. The properties of fungal alkaline phosphatase activity observed after staining of root pieces (low value at the beginning of infection. inhibition by cyanide. insensitivity to fluoride. decrease in activity in older plants) are similar to those described for the mycorrhizal specific alkaline phosphatase by Gianinazzi-Pearson & Gianinazzi (1978). Moreover. we have confirmed by ultracytochemistry that the enzyme is localized in vacuoles of intraradicular mycelium of Glomus sp. and Glomus infraradices, as previously reported for other arbuscular mycorrhizal fungi by Gianinazzi et al. (1979) and Dexheimer ef al. (1982). Several observations indicate that the alkaline phosphatase activity of arbuscular mycorrhizal fungi is induced by the host plant. A lag phase occurred in the

infection stained for alkaline phosphatase and only a small proportion of living intraradicaJ mycelium showed this enzyme activity during the earliest phase of infection. These observations. together with the fact that virtually none of the appressoria formed on roots of the isogenic myc-. nod- pea mutant showed an alkaline phosphatase activity, strongly suggest that the fungus must develop within root tissues before activation of the enzyme occurs. The inducible nature of this fungal alkaline phosphatase is also indicated by the fact that activity is only very weakly detectable in hyphae growing out from germinating spores and is limited to a very small region behind the actively growing hyphal apex (Kough & Gianinazzi-Pearson, 1985, and unpublished data). Amongst different factors that could affect the fungal alkaline phosphatase within host tissues, development of nutrient stress in the plant during early growth may be somehow associated with induction of the enzyme activity. In conclusion. the simple technique described in the present study for revealing the alkaline phosphatase activity of arbuscular mycorrhizal fungi in planta, coupled with that for detecting succinate dehydrogenase activity, improves the possibilities of studying the physiological activity of endomycorrhizal infections in situ. Although the role of the fungal alkaline phosphatase in the phosphate metabolism of ar-

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Figs 7-8. Expt 2, P. acerifolia-Glomus sp. Fig. 7. Fractional infection (M%) of root cortex revealed by trypan blue (-e-), succinate dehydrogenase ( - 0 - ) and alkaline phosphatase staining (-.-). Fig. 8. Shoot dry mass of non mycorrhizal ( - 6 - ) and mycorrhizal (-..t.- ) plants. Bars indicate standard error of values; data points bearing different letters are significantly different at the 95 % confidence level at each harvest.

Figs 9-10. Expt 2, A. porrum-Glomus /ntraradices. Fig. 9. Fractional infection (M%) of root cortex revealed by trypan blue ( - e - ) , succinate dehydrogenase ( - 0 - ) and alkaline phosphatase staining (-.-). Fig. 10. Shoot dry mass of non-mycorrhizal ( - 6 - ) and mycorrhizal (-..t.-) plants. Bars indicate standard errors of values; data points bearing different letters are significantly different at the 95 % confidence level at each harvest.

Table 1. Fractional infection (M%) and number of entry points cm- l root in 6-week-old P. sat/vum cv. Frisson and the isogenic myc, nod- (P53) mutant revealed by trypan blue (TB), succinate dehydrogenase (SOH) and alkaline phosphatase (ALP) staining

REFERENCES

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buscular endomycorrhiza is still a matter for speculation (Gianinazzi-Pearson & Gianinazzi, 1983; Smith & GianinazziPearson 1988), the present results suggest that the activity of this enzyme could provide a useful marker for analyzing the efficiency of an arbuscular mycorrhizal infection. This work was partly supported by the Ministere de l'Environnement et du Cadre de Vie and EPR-Bourgogne.

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In planta staining of fungal phosphatase

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(Accepted 17 June 1992)

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