Factors affecting appressorium development in the vesicular-arbuscular mycorrhizal fungus Glomus mosseae (Nicol. & Gerd.) Gerd.

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1 New Phytol. (1993), 123, Factors affecting appressorium development in the vesicular-arbuscular mycorrhizal fungus Glomus mosseae (Nicol. & Gerd.) Gerd. & Trappe BY MANUELA GIOVANNETTP, LUCIANO AVIO\ CRISTIANA SBRANA^ AND ANNA SILVIA CITERNESI' ^ Istituto di Microhiologia Agraria, Centro di Studio per la Microbiologia del Suolo, Via del Borghetto 80, Pisa, Italy ^ Scuola Superiore di Studi Universitari e Perfezionamento, Via G. Carducci 40, 56100, Pisa, Italy {Received 27 May 1992; accepted 29 July 1992) SUMMARY Investigations on the lack of appressorium formation in the roots of the non-host plant Lupinus albus L. showed that root exudates do not inhibit mycelial growth of the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus mosseae (Nicol. & Gerd.) Gerd. & Trappe, but that they hinder hyphal attachment and fungal recognition of roots. Exudates do not stimulate hyphal proliferation in the rhizosphere. G. mosseae hyphae were able to recognize and attach to excised roots of both lupin and host plants, forming swellings resembling appressoria. No growth of G. mosseae hyphae was observed around simulated roots consisting of nylon, silk, polyamide and glass threads, whereas appressoria were formed on heterologous hyphae of VAM fungal species. The hypothesis that a purely thigmotropic stimulus could trigger hyphal attachment and the further differentiation of appressoria was excluded. Key words: Appressorium, vesicular arbuscular mycorrhizas, recognition, pre-infection events. Reports varv concerning earlv interactions be- INTRODUCTION u i f A 'u * 1» c tween mycorrhizal tungi and non-host plants, some Vesicular-arbuscular (VA) mycorrhizal fungi are authors have found that species of the genus Glomus obligate symbionts and their survival depends on the are able to invade senescing roots of non-host plant ability of their hyphae to infect a host plant rapidly species (Hirrel, Mehravaran & Gerdemann, 1978; and efficiently. Spore germination has been reported Glenn, Chew & Williams, 1985; Gianinazzi-Pearson to be independent of the presence of a host plant & Gianinazzi, 1989; Giovannetti & Lioi, 1990). (Hepper & Smith, 1976; Daniels & Trappe, 1980), Other authors have reported that Glomus caledonium though recent reports have shown that both spore and the species of G/omws denominated E3 are able to germination and hyphal growth, up to the formation form appressoria on roots of different species of the of infection structures, could be increased by the non-host genus, Brassica (Ocampo, Martin & Haypresence of a host plant or of its root exudates man, 1980; Tommerup, 1984). In contrast, recent (Becard & Piche, 1989; Gianinazzi-Pearson, Bran- results have shown that two species of VA mycorzanti & Gianinazzi, 1989; Nair, Safir & Siqueira, rhizal fungi were not capable of invading Brassica 1991). Mycorrhizal infection is established by a roots, although they did form swellings resembling peculiar structure, the appressorium, whose form- appressoria on the root surface (Glenn, Chew & ation is the first and most important indication of Williams, 1988). fungal recognition of a potential host plant (Staples The genus Lupinus is generally considered to be & Macko, 1980). The study of appressorium in- non-host to VA mycorrhizal fungi, though some itiation and development in VA mycorrhizal host authors have reported sparse VA mycorrhizal inplants has been neglected and only a few studies have fection in some species (Trinick, 1977; Bedmar & recently been reported (Lackie et al, 1987; Garriock, Ocampo, 1986). In a previous paper (Avio, Sbrana & Peterson & Ackerley 1989). Giovannetti, 1990), we described the absence of 8-2

2 116 M. Giovannetti and others infection and appressorium formation by three VA mycorrhizal fungi in different Lupinus species, and we refuted the hypothesis of Morley & Mosse (1976) that the cause of such an absence could be seed-coat exudates. Recently, Gianinazzi-Pearson & Gianinazzi (1992) reported that intergeneric grafts between lupin and pea induced the failure of a true VA mycorrhizal symbiosis in pea roots, suggesting the presence of an inhibitory shoot factor in lupins. In this study we performed experiments with the aim of: (i) ascertaining whether a chemotropic factor associated with non-host roots could hinder fungal recognition of roots; (ii) establishing whether appressorium formation could be elicited by a purely thigmotropic stimulus. sterile sand and, 6 d after germination, were transplanted into pots containing sterile, acid-washed quartz grit. Each root system was gently placed between two millipore membranes (0-45 //rn diam. pores). Another membrane, inoculated with 10 sporocarps of G. mosseae, each containing about 12 spores, was placed over the millipore sandwich containing the roots, so that only root exudates could diffuse through the membrane. Controls were set up in the same way, without plants. Five replicate plants for each trial were used. After 10, 15 and 20 d, the plants were removed from the pots, the millipore membranes with the sporocarps were opened and the germinated sporocarps were examined under a dissecting microscope, after staining with a few drops of 0-05 % trypan blue in lactic acid. Sporocarp germination was checked and hyphal growth was MATERIALS AND METHODS assessed by the gridline intersect method, after Fungal cultures positioning a gridline on the millipore membrane The following VA mycorrhizal fungi, maintained in (Hepper, 1979). Using this method, the growth of lucerne pot-cultures, were used: Glomus coronatum hyphae, in the absence of root colonization, could be Giovannetti, Glomus mosseae (Nicol. & Gerd.) Gerd. examined. A similar experiment was performed and Trappe; Glomus sp. strain A6. using P. sativum var. Frisson and the corresponding isogenic mutant P2 [susceptible and resistant to mycorrhizal infection respectively (Due et al., 1989)] Plant material harvested 20 d after transplanting. The following plant species were used: Lupinus albus L. (lupin), Medicago sativa L. (lucerne), Pisum sativum L. var. Frisson (pea), isogenic mutant P2 Experiment 3. Pre-infection events on excised roots of host and non-host plants were examined by (Due et al., 1989). decapitating plants. Seeds of lupin, pea var. Frisson, the corresponding pea mutant, P2, and lucerne were Experimental design germinated in sterile sand. Ten-day-old seedlings Experiment 1. This experiment investigated the lack were transplanted into pots and inoculated with 10 of appressorium formation on roots of intact plants germinated sporocarps of G. mosseae placed between of the non-host lupin. Twenty seeds of L. albus were millipore membranes as described in expt 2. The surface-sterilized in 1 % sodium hypochlorite for shoots of the four plant species were excised, leaving 20 min, rinsed in sterile water, sown in pot cultures the root systems. Controls consisted of sporocarps of the host plant M. sativa infected with G. mosseae, alone, placed between millipore membranes. Each and grown for 4 wk. The seedlings were then trial consisted of five replicates. The root systems harvested and their roots checked for appressorium and controls were watered daily. After 10 d the roots formation, after clearing in 10% KOH and staining were cleared and stained as described in expt 1, and with trypan blue (Phillips & Hayman, 1970). At the carefully examined under a light microscope to same time, 10 lucerne and 10 lupin seedlings, grown check for the presence of appressoria. This exin sterile sand from sterilized seeds, were placed periment was repeated twice. together between millipore membranes (0-45 fim After cutting, viability of the decapitated root diam. pores), each containing 30 pre-germinated systems was checked daily for 6 d using 10//g ml"' sporocarps of G. mosseae and checked for appres- fluorescein diacetate (FDA) (Sigma, F7378) in sorium formation after 14 d. Another five sterile phosphate buffer (0-1 M, ph 7-4) (Widholm, 1972). seedlings of these two plant species were trans- The roots were stained with the FDA solution for planted into Petri dishes containing 1 o water agar 10 min at 21 C, rinsed in the same buffer and supplemented with 60 mg T' bromocresol purple, observed using a Reichert Polyvar fluorescence to visualize variations in ph by the change in colour microscope equipped with the followingfiltercomof the dye indicator (Dinkelaker, Romheld & bination: excitation filter BP , barrier filter Marschner, 1989). LP 520, and dichroic mirror DS 510. Experiment 2. Spore germination and hyphal extension of G. mosseae were studied in the presence of root exudates of host and non-host plants. Sterilized seeds of L. albus and M. sativa were germinated in Experiment 4. This experiment was designed to test whether a purely thigmotropic stimulus might be involved in attachment and appressorium formation. For this purpose, we utilized different kinds of

3 Appressorium development in Glomus mosseae 117 physical structures simulating roots. Double millipore filter sandwiches were set up, as described in expt 2, each containing, on one side, the root system of the host plant M. sativa and, on the other side, nylon threads of different diameters (65, 120, 180, 350 fim) or threads of silk (30/^m diam.), cellulose {10 fim diam.), polyamide (20/^m diam.) and glass {100 fim diam.), with 10 sporocarps of G. mosseae. Three replicates were included in each trial. After 2 wk the membranes were carefully opened and the mycelium was stained and examined as described in expt 3. This experiment was also carried out omitting the host plant. Five millipore membrane sandwiches, containing six sporocarps of G. mosseae and six spore clusters of Glomus A6 each were set up, placed in moistened sterile sand and checked for appressorium formation on the hyphae of the different fungal species after 2 wk. Fungal mycelium growing on the membranes was treated with the fluorescent brightener Uvitex CFI (stilbene sulphonic acid derivative, obtained from Ciba-Geigy, Varese, Italy), which binds to fungal cell walls, and observed under u.v. light as previously described (Giovannetti, Avio & Salutini, 1991). The same trial was performed using G. coronatum instead of Glomus A6. exudates of the host plants, lucerne, pea var. Frisson and the pea mutant P2, the hyphal lengths of the germinated sporocarps were, respectively, 3-2 x, 3-1 X, and 2-5 x the length of the controls (Table 1). Similarly, the length of mycelium per germ tube developing from each sporocarp was, respectively, 2-7 X, 2-8 X, and 2-2 x the length of the controls (Table 1). The data shown in Table 1 illustrate the validity of the method used for determining hyphal length in G. mosseae (i.e. using sporocarps instead of spores): the 400 RESULTS Lack of appressorium formation on intact roots of the non-host plant lupin {Experiment 1) L. albus grown within pot cultures of mycorrhizal M. sativa did not become infected; after 4 wk no appressoria were formed and no hyphal growth around lupin roots was observed. The occurrence of lucerne roots, growing inside the same sandwich and sometimes attached to lupin roots, did not elicit any appressorium formation on the lupin root surface. After staining, no hyphae of G. mosseae were observed attached to lupin roots. When roots of sterile seedlings of lupin were placed in agar medium containing bromocresol purple, they caused a rapid colour change from purple to yellow in less than 24 h, indicating a decrease in ph to below 5-2, whereas no colour change occurred in the substratum in the presence of lucerne roots. Hyphal extension of G. mosseae in the presence of host and non-host plant root exudates {Experiment 2) The length of G. mosseae mycelium increased from the first to the third harvest in the presence of lucerne root exudates, whereas it did not change in the presence of lupin exudates or in the controls (Fig. 1). Lupin root exudates did not inhibit hyphal elongation of G. mosseae. At the last harvest the length of mycelium per germinated sporocarp in the presence of lupins ( mm) was not significantly different from that of the controls ( mm) (Table 1). In the presence of the root 0 Days Figure 1. Hyphal growth of Glomus mosseae in the presence of root exudates of host {Medicago sativa, # ) and non-host {Lupinus albus, ^ ) plants and in controls ( ). Asterisks represent values significantly different from controls at P < Table 1. Hyphal growth of Glomus mosseae in the presence of root exudates from host and non-host plants Plants Control Lupin Lucerne Pea, var. Frisson Pea mutant, P2 Hyphal length/germinated sporocarp (mm) lll-3±13-3a 108-0±7-6a 355-2±36-7b 341-6±48-9b b Hyphal length/germ tube (mm) 17-6±2-8a 18-0 ±0-7 a 47-2±7-7b 48-7±9-4b 39-l+4-5b Means followed by the same letter within the same column are not significantly different {P < 0-05; Tukey's test).

4 118 M. Giovannetti and others Figures 2-7. Light micrographs of interactions between Glomus mosseae and excised roots of the non-host plant lupin. Scale bars, 16^ Figure 2. Swellings resembling appressoria, formed on a single root cell. Figure 3. Swelling developing a penetration hypha, showing the empty hyphal tip. Figure 4. Large swelling developing three penetration hyphae. Figure 5. Hyphal swelling showing retraction septa (arrows). Figure 6. Hyphal swelling formed on the edge of two contiguous cells. Figure 7. Hypha originating from a swelling, which retracts its cytoplasm and forms consecutive septa, isolating the empty hyphal tip (arrows). slightly different number of spores inside each sporocarp did not alter variability in the data obtained. Moreover, the accuracy of this method is demonstrated by the fact that the hyphal length that could be measured in the presence of each host plant remained the same. Pre-infection events on excised host and non-host roots {Experiment 3) On the first and second day after excision of the shoots, the root systems of both lupin and lucerne were still viable. On the third day, 60 % of lupin

5 Appressorium development in Glomus mosseae roots were viable, whereas 70% of lucerne and pea roots were already dead. During the following days the root systems of the plants progressively died, up to the sixth day, when they appeared dead for the most part. Germ tubes originating from the germinated sporocarps grew along the surface of excised roots of the host plants lucerne and pea. Germ tubes were attached to roots, often branched, and formed hyphal swellings. Occasionally, true appressoria were formed on excised lucerne roots and the hyphae germinating from them penetrated adjacent epidermal cells, but failed to spread further. Although growth of G. mosseae hyphae was not elicited on the roots of intact lupin plants, they were able to grow around and to attach to the root surface of decapitated ones, leading to the formation of hyphal swellings (Figs 2-5). Such enlarged structures often originated from hyphae growing along the grooves between cells and were formed over clinal and anticlinal wall junctions between epidermal cells. Sometinies thin hyphae originated from the swellings, but these rapidly aborted, retracting their cytoplasm, forming consecutive retraction septa, which isolated the empty hyphal tips (Figs 6, 7). The total length of hyphae attached to excised lupin roots was significantly lower than that of hyphae attached to lucerne, pea var. Frisson, or pea mutant, P2, roots (Table 2). This was due to the shorter length of the lupin root system: the length of hyphae attached per cm of lupin root was not significantly diflferent from that on pea roots (0-50 and 0-99 mm respectively). The total number of 119 Table 2. Length of hyphae of Glomus mosseae attached to {AHL), and number of swellings {SN) formed on the excised roots of host and non-host plants Plants Lupin Lucerne Pea, var. Frisson Pea mutant. P2 SN AHL (mm cm~^ root) SN (mm"' AHL) 3-38a b 3O-15b 3-4a 100-6c 85-4bc 0-50a l-97b 0-99 a b 0-45 a 4-14b 2-71bc b 70-8 b l-04ab l-96ac AHL (mm) Values within columns followed by the same letter are not significantly different {P < 0-05; Tukey's test). hyphal swellings and the number of swellings per mm of attached hypha formed on excised lucerne roots were similar to those formed on pea, whereas hyphal swellings were less frequent on lupin roots (Table 2). Pre-infection events on simulated roots {Experiment 4) No growth or attachment of G. mosseae mycelium was observed around nylon threads of increasing diameter or on polyamide, silk, cellulose or glass threads. Interestingly, many structures similar to appressoria were formed by VA mycorrhizal fungi on the hyphae of different fungal species, i.e. G. mosseae Figures 8, 9. Light andfluorescencemicroscopy of interactions between homologous and heterologous hyphae of VA mycorrhizal fungi. Figure 8. Anastomosis occurring between hyphae of Glomus mosseae, stained with trypan blue. Scale bar, 12-5 //m. Figure 9. Appressorium-like structure formed by Glomus mosseae hypha on heterologous hypha of Glomus coronatum, stained with Uvitex and observed under u.v. light. Scale bar, 30 fim.

6 120 M. Giovannetti and others formed such structures on Glomus A6 and on G. coronatum hyphae, and vice versa. The observed structures were different frotn those formed by G. mosseae on its own hyphae, where anastomosis immediately occurred after an initial stage of attachment (Fig. 8). In contrast, G. mosseae tended to form attachment structures, very similar to appressoria, on Glomus A6 and G. coronatum hyphae (Fig. 9). DISCUSSION The results of our study show that: (1) root exudates of the intact non-host plant lupin hinder hyphal attachment and fungal recognition of roots by the VA mycorrhizal fungus G. mosseae \ (2) G. mosseae is able to recognize the roots of decapitated host and non-host plants but was unable to distinguish between them; (3) the factors leading to the production of appressoria in G. mosseae are not linked to purely thigmotropic responses. The VA mycorrhizal fungus G. mosseae is not able to penetrate, to form appressoria on, or to attach to roots of, intact lupin plants, even when infected roots of a lucerne (host) plant grow in close contact with them. This confirms previous results obtained by Avio et al. (1990), but contrasts with those of other authors, who reported some infection of this genus under field (Trinick, 1977) or laboratory conditions (Bedmar & Ocampo, 1986). The presence of lupin roots did not, however, influence hyphal growth of G. mosseae, as compared to controls, whilst root exudates of host plants (lucerne and pea) increased hyphal length, confirming results previously obtained in vitro by other authors (Becard & Piche, 1989; Gianinazzi-Pearson et al., 1989; Nair et al., 1991). Interestingly, root exudates of the VA mycorrhizal resistant pea mutant, P2, where mycorrhizal infection is inhibited but appressoria are formed (Due et al., 1989), had the same stimulatory effects as those from wholly compatible plants. In conclusion, our results on the non-host lupin are similar to those of Gianinazzi-Pearson et al. (1989), and are in agreement with those of Glenn et al. (1988) with the non-host Brassica. These authors proposed that non-host roots lack a factor which is present in the exudates of host roots and which elicits hyphal proliferation in the rhizosphere. The use of decapitated plants demonstrated that when lupin shoots were removed from the roots, G. mosseae hyphae could grow around and attach to the latter, giving rise to swellings and to some attempt at penetration of the outer root layer. The length of hyphae attached to excised roots of lupin and the number of swellings formed per cm root were not significantly different from those observed on excised pea roots. These results show that inhibition of hyphal attachment in lupin is determined by a factor diffusing into the rhizosphere from the living, intact plants. Evidence for an inhibitor of mycorrhizal symbiosis produced in the shoot lupin has recently been provided from studies of intergeneric grafts between the mycorrhizal pea and the non-mycorrhizal lupin (Gianinazzi-Pearson & Gianinazzi, 1992). The proteoid roots of Lupinus albus L. have been shown to produce an acidic environment, rich in chelating agents such as citrate ions (Gardner, Parbery & Barber, 1982; Gardner, Barber & Parbery, 1983). Dinkelaker ef a/. (1989) demonstrated a strong acidification (ph 4-8) of the rhizosphere and excretion of citric acid by lupin proteoid roots, suggesting that this could be a mechanism of nutrient mobilization. We have shown that root exudates of lupin are also acidic (ph < 5-2) in very young root systems, where proteoid roots have not developed yet. An acidic environment inhibits both spore germination and mycorrhizal infection by G. mosseae (Mosse & Hepper, 1975 ; Green, Graham & Schenck, 1976). Thus, the factors involved in the lack of appressorium formation and hyphal attachment of VA mycorrhizal fungi to lupin roots could be: {a) the lack of a stimulus to hyphal proliferation in the rhizosphere; {b) shoot-produced inhibitory factors; {c) alterations in the rhizosphere ph, hindering the approach of hyphae to the roots, the first essential step for further development of the symbiotic event. However, the latter would not be active for fungi that tolerate acid phs. G. mosseae was able to form appressoria and penetrate excised roots of lucerne while the roots were viable. This, together with the fact that in lucerne excised roots both the length of attached hyphae and the number of swellings formed were much higher than in lupin or pea, suggests a higher degree of aflinity between G. mosseae and lucerne (Lioi & Giovannetti, 1987). The formation of hyphal swellings and of hyphae attempting penetration, previously observed by Glenn ei a/. (1985, 1988) in excised non-host.bra55?ca roots, were interpreted as initial stages of appressorium formation. Our observations of the production of swellings in excised host and non-host roots strengthen this hypothesis. The occurrence of swellings instead of appressoria on excised roots is very important as it suggests that VA mycorrhizal fungi are able to recognize the root surface, to attach to it, to produce the first morphological changes as appressoria, but that they probably require some chemical and/or physical factor associated with living roots to develop to further stages of the mycorrhizal infection. We never observed any appressorium formation in experiments with simulated roots, even in the presence of root exudates of a host plant. This suggests that both topographical and chemical signals are necessary to elicit appressorium differentiation by VA mycorrhizal fungi, as already found in

7 Appressorium development in Glomus mosseae some plant pathogenic fungi and in fungal mycoparasites (Kaminskyj & Day, 1984; Hoch et al, 1987; Manocha & Chen, 1990), and that a simple thigmotropic stimulus like a thread is not sufficient; in contrast, ectomycorrhizal fungi will form a fungal sheath around roots simulated from silicon (Read & Armstrong, 1972). Furthermore, the occurrence of structures similar to appressoria on heterologous hyphae of VA mycorrhizal fungi also suggests that attachment of hyphae, and the morphogenic events leading to the formation of appressoria, are the result of fungal recognition of signals associated with biological surfaces, rather than of purely physical structures resembling roots. Mycorrhizal infection is a multi-step process, during which many different signals cause a cascade of recognition events between host and symbiont (Tester, Smith & Smith, 1987; Gianinazzi-Pearson & Gianinazzi, 1989). These steps, morphologically distinct, consist of spore germination, hyphal growth around roots, hyphal attachment to the roots, appressorium formation, intraradical penetration and intraradical growth, up to the formation of arbuscules, the sign of the development of a compatible interaction (Harley & Smith, 1983). The mycorrhizal status, therefore, has to be conceived as the product of multifold signals of different nature, acting at various stages of the interaction, which eventually lead to the establishment of a functional symbiosis. ACKNOWLEDGEMENTS We thank Dr V. Gianinazzi-Pearson and Dr S. Gianinazzi for providing the seeds of the pea cultivar and isogenic mutant, and for access to unpublished results, Dr V. Gianinazzi-Pearson for critically reading the manuscript and Mr V. Gherarducci for his help in preparing the photographs. 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