Differential hyphal morphogenesis in arbuscular mycorrhizal fungi during pre infection stages

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1 New Phytol. (1993), 125, Differential hyphal morphogenesis in arbuscular mycorrhizal fungi during pre infection stages BY M. GIOVANNETTP, C. SBRANA\ L. AVIO\ A. S. CITERNESP AND C. LOGP ^ Istituto di Microbiologia Agraria, Centro di Studio per la Microbiologia del Suolo, C.N.R., Via del Borghetto 8, Pisa, Italy ^Scuola Superiore di Studi Universitari e Perfezionamento 'S. Anna', Via Carducci 4, 561 Pisa, Italy {Received 9 March 1993; accepted 22 June 1993) SUMMARY Roots of host plants elicit a local change in morphology in the hyphae of arbuscular mycorrhizal fungi, prior to the formation of appressoria. The elicited hyphae switch from their original branching pattern and apical dominance to differentiate in a new irregular, septate branching pattern with reduced inter-hyphal spacing. The extensive hyphal development associated with roots of host plants was shown to be due to the differential growth pattern described, and to precede the further cascade of events leading to appressorium formation and the development of a functional symbiosis. Key words: Recognition, pre-infection events, chemotropism, hyphal morphogenesis, arbuscular mycorrhizas. INTRODUCTION Arbuscular mycorrhizal (AM) endophytes are biotrophic fungi which obtain nutrients from the root cells of host plants. After spore germination, signals from the roots elicit the differentiation of appressoria, which are specialized infection structures formed by many fungi in order to penetrate surfaces of host plants (Staples & Macko, 198; Giovannetti et al., 1993). Previous studies have shown that hyphal growth of AM fungi in the rhizosphere was increased by root exudates of host plants (Elias & Safir 1987; Becard & Piche, 1989 a; Gianinazzi-Pearson, Branzanti & Gianinazzi, 1989; Nair, Safir & Siqueira, 1991; Giovannetti et al., 1993). On the contrary, non-host plants like Brassica and Lupinus lacked some factors eliciting hyphal proliferation in the rhizosphere (Glenn, Chew & Williams, 1988; Giovannetti et al., 1993). Some authors found that root exudates of host plants not only stimulated hyphal growth, but also exerted some morphogenetical effects on the fungus (Mosse & Hepper, 1974; Powell, 1976; Glenn, Chew & Williams, 1985; Gemma & Koske, 1988; Mosse, 1988). Recently, some fiavonoids like quercetin have been shown to promote spore germination, hyphal elongation and hyphal branching in Glomus etunicatum (Tsai & Phillips, 1991). The experimental system devised previously for study of the early stages of AM infection (Giovannetti et al., 1993) was used here to investigate the morphological events elicited in the hyphae of AM fungi by the roots of host plants. MATERIALS AND METHODS Fungal cultures The following AM fungi, maintained in Medicago sativa L. pot-cultures, were used: Glomus mosseae (Nicol. & Gerd.) Gerd. & Trappe (Rothamsted isolate), Glomus intraradices Schenck & Smith (isolated from a local serpentine soil), an undescribed species of Glomus, named A6, isolated from a nursery soil. Plant material The following host and non-host plant species were used: (a) hosts: Fragaria vesca L., HeUanthus annuus L., Octmum basilicutn L., Lycopersicon esculentum Mill., Triticum aestivum L.; (b) non-hosts: Brassica

2 588 M. Giovannetti and others Figures 1, 4-7. For legend see opposite.

3 Hyphal morphogenesis in arbuscular mycorrhizal fungi napus L., Dianthus caryophyllus L., Eruca sativa Lam., Lupinus albiis L. Experiment 1 The experiment assayed differential hyphal morphogenesis in AM fungi elicited by factors associated with host roots. Sterile seeds of the host and nonhost plants were germinated in sterile sand. Six days after germination, the root system of each seedling was sandwiched between two 47 mm diam millipore membranes (-45 fim diam pores). Another memhrane, containing 1 sporocarps of G. mosseae, was placed over the millipore sandwich containing the roots, using clips to hold the memhranes together. Each sandwiched root system was placed into a 1 cm diam pot, and then buried with sterile, acidwashed quartz grit (2-5 mm diam). Both sand and grit were autoclaved at 121 C for 3 min. Controls were prepared without roots, and five replicates were used for each trial. The experiment was performed under controlled conditions (2-24 C, 16 h photoperiod of irradiance 1/y.E m"^ s"', 6 % RH) and pots were watered daily. After 2 d the plants were removed from the pots, the sandwiches containing the sporocarps carefully opened, stained with -5 % trypan blue in lactic acid, and hyphal growth assessed with a Wild dissecting microscope at x 5 magnification, using the grid intersect line method (grid 47 mm diam, lines 3-66 mm apart) (Giovannetti & Mosse, 198). A second trial used the host plant O. basilicmn, chosen because in the initial experiment it had shown a marked adaptability to the 'sandwich' growing conditions. Plants were prepared as described above, then another millipore membrane containing 1 spores of either Glomus sp A6 or G. intraradices was placed on the millipore root sandwich. Replication and further treatments were as described above. 589 the sandwiches containing the sporocarps treated as in Expt. 1. Hyphal growth in five replicate plants was assessed at each time interval. The more extensive hyphal development and branching elicited in areas overlying the roots growing underneath the membranes was compared with hyphal growth in non-elicited areas. Hyphal density was assessed over five areas of 4 mm^ each, in plants harvested at 12, 14, 16, 18 and 2 d, using a microscope grid eyepiece under a Reichert Polyvar light microscope. For further quantitative and qualitative description of differential hyphal growth, the membranes from plants harvested at 2 d were cut into pieces, to give elicited and non-elicited areas. These were mounted in lactic acid on microscope slides and total hyphal length and the number of branches were measured over 2-81 mm^ areas, each 3 mm apart. A control trial, in which the plants were given 3 ml/pot of a half-strength Hoagland solution twice a week, was prepared to exclude the possibility that the differential hyphal morphogenesis could be due to nutritional stress caused by the ' sandwich' experimental conditions. Experiment 3 The time-course of hyphal differentiation was measured in the G. mosseae O. basilicum interaction. Plants were obtained as described in Expt. 1, and their root systems were sandwiched between millipore membranes, over which another sandwich, containing 1 germinated sporocarps of G. mosseae, was placed. Plants were transplanted into pots containing grit, harvested after 24, 36, 48 and 96 h, and the hyphae stained as in Expt. 1. Five plants were used for each period. Control plants were prepared without the intermediate membrane, to exclude the possibility that the differential hyphal morphogenesis could be due to the membrane itself. RESULTS Experiment 2 Experiment 1 The distinctive hyphal branching associated with host roots was measured in the interaction between G. mosseae and O. basilicum. Plants were germinated and grown as in Fxpt. 1. After 8, 12, 14, 16, 18, 2, 24 and 28 d, they were removed from the pots and Extensive hyphal development and branching was elicited in G. mosseae by roots of the host plants growing underneath the membranes. This was visible to the naked eye, after staining the membranes with trypan blue: a very densely branched hyphal Figure 1. Differential hyphal morphogenesis elicited in Glointis mosseae by roots of host plants growing underneath a millipore membrane: a densely branched hyphal network in close association with the roots delimits the outline of the entire root system. Scale bar, 2-4 mm. Figures 4-6. Light micrographs showing differential morphogenesis in hyphae of Glomus mosseae elicited by the roots of Ocimtim hasilictim, growing underneath a millipore membrane. Figure 4. Elicited (arrowhead) and non-elicited (arrow) hyphae. Scale bar, 57 /tm. Figure 5. Fan-like structures, with many hyphal branches arising laterally from elicited hyphae. Scale bar, 12-1 lira. Figure 6. Elicited hyphae, showing elkhorn-like structures, formed by many successive enlargements of hyphal tips, separated by frequent septa, strongly reminiscent of appressoria. Scale bar, 6-1 jim. Figure 7. Ean-like structures, where each hyphal br-anch forms an appressorium on the root of Ocimtim basilicum (arrowheads). Scale bar, 4/nn.

4 59 M. Giovannetti and others Table 1. Effects of root factors of host (H) and non-host plants (NH) on differential hyphal morphogenesis in Glomus mosseae Plant species Sporocarps showing Confidence differential interval morphogenesis (%) (95%) Fragaria vesca (H) Helianthus annuus (H) Ocimum basilicum (H) Lycopersicon esculentum (H) Triticum aestivum (H) Brassica napus var. oleifera (NH) Eruca sativa (NH) Dianthus caryophyllus (NH) Lupinus albus (NH) Controls (18-47)* (36-77) (81-98) (52-82) (37-75) * Confidence intervals from the Geigy Scientific Tables. 4 r=-82 Table 2. Total hyphal development of Glomus mosseae in the presence of the root factors of the host plant Ocimum basilicum (H) or controls (C) Time after Total hyphal length (mm) CD O O o o. c 3 "5.c D. > 1 J Sporocarps showing elicited hyphae (%) Figure 2. Relationship between the number of germinated sporocarps showing differential hyphal morphogenesis and hyphal length. Slope, intercept and correlation coefficient calculated from linear regression analysis. net developed in close association with the roots, clearly delimiting the outline of the entire root system (Fig. 1). The proximity of roots of non-host species did not elicit any morphogenetical event (Table 1). Hyphal growth of the AM fungus on the membranes containing non-host plants was similar to that of the controls, i.e. in B. napus, D. caryophyllus, E. sativa, L. albus and control 111 9, 13-, 12-6, 19-2 and mm/germinated sporocarp respectively. It is (d) H * ** ** ** ** ** ** C *, ** Values statistically difterent from control at P < -5 and P < 1 respectively. interesting to note that hyphal length per germinated sporocarp was directly correlated with the number of germinated sporocarps showing differential hyphal morphogenesis (Fig. 2). The trial with O. basilicum and different AM fungi confirmed the results obtained with G. mosseae. Hyphae of the difterent species of AM fungi showed the same phenomenon of altered morphogenesis, giving profuse growth on the membrane surfaces directly overlying roots. Experiment 2 Hyphal development of G. mosseae in the presence of O. basilicum roots or in the controls is shown in Table 2. Eight days after inoculation, total hyphal length in the sandwiches containing plant roots was similar to that of the controls, whereas at d post-inoculation it was significantly greater than in the controls. The growth curve calculated for hyphal length of G. mosseae per germinated sporocarp showed a

5 E 6 r 5 ~ 4 Q. ro o O o Q. a en c x: Hyphal morphogenesis in arbuscular mycorrhizal fungi 591 *, i L _L J_ Days after inoculation Figure 3. Hyphal development of Glomtis mosseae in the presence of roots of Ocimtim basilictim (- -) and in the control (- -). Bars represent S.E.M. Asterisks represent values significantly different from controls at P < 1. Table 3. Density of Glomus mosseae hyphae growing on millipore membranes in areas overlying roots of O. basilicum (roots) and in areas remote from the roots (no roots) Time after inoculation (d) Hyphal length (mm mm Roots* l]-65± No roots O±O * Data in the two columns differ statistically at P < -1. Table 4. Time required by the AM fungus Glomus mosseae to initiate differential hyphal morphogenesis after inoculation on the host plant Ocimum basilicum Time (h) Elicited sporocarps (%)* a a 35-87±8-2b b * Values followed by different letters differ significantly at P< -5. phase and a plateau (Fig. 3). The plateau was reached in the controls on day 12, whereas in the host plant treatment it was reached on day 2. The growth rate of germinated hyphae, calculated from the measurements taken between day 8 and 12, was 1-65 //m/min in the controls and 3-43 /(.m/min in the presence of O. basilicum roots. Hyphal density in areas of the membranes overlying roots of O. basilicum was 3-5 times greater than in areas remote from the roots (differences significant at P<-1) (Table 3). Where differential growth was elicited, the number of branches per unit of hyphal length was much larger than where such growth was not elicited, i.e and branches per mm respectively, 2 d after inoculation. Interestingly, the zones of elicited fungal growth on the membranes were about mm wide, which is less than the diameter of the roots growing underneath. In these areas, the hyphae abandoned their original growth pattern of regular branching, spacing and apical dominance, and differentiated to give an irregular, septate, much-branched pattern (Fig. 4). Observation of the morphological changes shown by elicited mycelium at higher magnifications revealed a sequence of events. Firstly, the main hyphae altered direction to grow towards the roots. Once adjacent to roots, the long, straight directional hyphae differentiated into different shapes: for example, fan-like structures, consisting of many hyphal branches arising laterally or apically froni hyphae (Fig. 5); also, elkhorn-like structures, formed by many successive enlargements of hyphal tips, separated by frequent septa and strongly reminiscent of appressoria (Fig. 6). The same morphogenetical effects were obtained in controls with Hoagland's solution. Experiment 3 The hyphae of G. mosseae were able to change their elongation and branching pattern as early as 24 h after inoculation (Table 4), by which time % of the germinated sporocarps showed a differential growth pattern. In controls which had no intermediate membrane, the same kind of hyphal differentiation occurred when G. mosseae was directly in contact with the roots. Indeed, 48 h after the beginning of the interaction, the roots of O. basilicum elicited a detectable hyphal branching, which was immediately followed by appressorium formation (Fig. 7). DISCUSSION The most important result of this study is the detection of a distinct step in the early events of AM infection, which takes place before appressorium development. Evidence presented shows that: (1)

6 592 M. Giovannetti and others factors associated with roots of host plants are the beginning of the interaction, and that such early signals recognized by AM fungi; (2) rec- differentiation is comparable to that occurring when ognition triggers reorientation of the direction of the fungus is in direct contact with the roots. The distinctive morphological structures formed hyphal elongation and the initiation of a different by G. mosseae on the membranes in contact with the branching pattern prior to the formation of roots can be divided into two types: (a) structures appressoria and the establishment of the symbiosis. originated by hyphal branching, with a strong Roots of all the host plant species tested were able directional growth; (b) enlarged structures, formed to elicit extensive hyphal proliferation in the three later, which resemble appressoria and whose origin species of AM fungi utilized, whereas roots of nonmight depend on the membrane surface and/or on host plants neither elicited nor inhibited hyphal the impeded contact with the root surface. growth. This confirms the hypothesis that non-host Interestingly, true appressoria have never been roots lack factors promoting hyphal growth in the observed on millipore membranes; this is contrary to rhizosphere (Glenn, Chew & Williams, 1988; what happens with many biotrophic fungi, which Giovannetti et al., 1993). Some authors have form appressoria in vitro on membranes of different suggested that fiavonoid compounds and COg structure and composition (Hoch et al., 1987; St. exuded by host roots might stimulate hyphal growth Leger et al., 1989; Kwon & Hoch, 1991). (Gianinazzi-Pearson et al., 1989; Nair et al., 1991; In conclusion, a chemodifferentiation, as defined Siqueira, Safir & Nair, 1991). The use of a physical barrier to impede formation by Staples et al. (1983), is the earliest detectable of the symbiosis made it possible to monitor hyphal morphological event indicating the successful recgrowth of G. mosseae in the presence of root factors ognition of a host plant by AM fungi. This step is a of the host plant O. basilicum. The rate of growth, pre-requisite for the further cascade of events leading after an initial large increase, became constant, to appressorium formation, root penetration and suggesting that the fungus responds to the stimulus colonization, which ultimately results in the decoming from the roots for a certain time and that it velopment of a functional symbiosis. stops growing if the successive events of the symbiosis (i.e. appressorium formation, root pen- ACKNOWLEDGEMENTS etration) do not take place (Becard & Piche, 1989a). This study demonstrates that the more extensive Research supported by National Research Council of hyphal growth found in the presence of host roots is Italy, Special Project RAISA, Sub-project N. 2, Paper N mainly due to a change in hyphal morphogenesis elicited by factors associated with roots. This suggests: (1) there is a concentration gradient of the REFERENCES chemical stimulus from the roots to the zones nearby, Becard G, Piche, Y o. New aspects on the acquisition of and such stimulus is perceived by the fungus only at biotrophic status by a vesicular-arbuscular mycorrhizal fungus, Giga.spora margarita. New Phytologist 112: the high concentrations occurring on the roots; (2) Becard, G, Piche, Y Fungal growth stimulation by CO^ the chemical stimulus is strongly associated with the and root exudates in vesicular-arbuscular mycorrhizal sym.. root mucilages and it is perceived by the fungus biosis. Applied and Environmental Microbiology 55: through the pores of the membranes; (3) a volatile Elias, KS, Safir, GR Hypbal elongation of Glomus fasiculatus in response to root exudates. Applied and Ensubstance elicits hyphal attraction to roots (Koske, vironmental Microbiology 53: ; Gemma & Koske, 1988). We could speculate Gemma JN, Koske RE Pre-infection interactions between roots and the mycorrhizal fungus Gigaspora gigantea: chemothat exudates from host plant roots, alone or in tropism of germ tubes and root growth response. Transactions association with volatile compounds (Becard & of the British Mycological Society 91: Piche, 19896), elicit a differential hyphal morpho- Gianinazzi-Pearson V, Branzanti B, Gianinazzi S In vitro enhancement of spore germination and early hyphal genesis which facilitates the search for suitable sites growth of a vesicular-arbuscular mycorrhizal fungus by host for adhesion and appressorium formation. The root exudates and plant Ravonoids. Symbiosis 7: specific elicitors remain to be determined, though Giovannetti M, Avio L, Sbrana C, Citernesi AS Factors affecting appressorium development in the vesicular-arbuscular other authors reported some stimulation of hyphal mycorrhizal fungus Glomus mosseae (Nicol. & Gerd.) Gerd. & branching in the presence of the fiavonoid quercetin Trappe. New Phytologist 123: (Tsai & Phillips, 1991). Giovannetti M, Mosse B An evaluation of techniques for measuring vesicular-arbuscular mycorrhizal infection in roots. Differential hyphal morphogenesis in AM fungal New Phytologist 84: hyphae in the vicinity of host roots has been Glenn MG, Chew FS, Williams PH Hyphal penetration mentioned by a few authors (Mosse & Hepper, 1975; of Brassica (Cruciferae) roots by a vesicular-arbuscular mycorrhizal fungus. New Phytologist 99: Powell, 1976; Glenn et al., 1985) but unfortunately, Glenn MG, Chew FS, Williams PH Influence of because of their methodology, they failed to dis- glucosinolate content of Brassica (Cruciferae) roots on growth tinguish between effects before and after mycorrhizal of vesicular-arbuscular mycorrhizal fungi. New Phytologist 11: infection. In contrast, the 'sandwich' experimental Hoch HC, Staples RC, Whitehead B, Comeau J, Wolf ED. system has shown that the stimulus to differentiation Signaling for growth orientation and cell difterentiation is perceived by the hyphae within 24 h from the by surface topography in Uromyces. Science 235:

7 Hyphal morphogenesis in arbuscular mycorrhizal fungi 593 Koske RE Evidence for a volatile atraetant from plant Endogone spores and infected root segments. Transactions of the roots affecting germ tubes of a VA mycorrhizal fungus. British Mycological Society 66: Transactions of the British Mycological Society 79; Siqueira JO, Safir GR, Nair MG Stimulation of vesiculararbuscular mycorrhiza formation and growth of white clover by Kwon YH, Hoch, HC Temporal and spatial dynamics of appressorium formation in Uromyces appcndiculatus. Experimental mycology IS: Staples RC, Macko V Formation of infection structures as flavonoid compounds. New Phytologist 118: Mosse, B Some studies relating to ' independent' growth of a recognition response in fungi. Experimental Mycology 4: vesicular-arbuscular endophytes. Canadian Journal of Botany Staples RC, Macko V, Wynn WK, Hoch HC Terminology to describe the differentiation response by 66: Mosse B, Hepper CM Vesieular-arbuscular mycorrhizal germlings of fungal spores. Phytopathology 73: 38. infections in root organ cultures. Physiological Plant Pathology St Leger RJ, Butt TM, Goettel MS, Staples RC, Roberts DW. 5: Production in vitro of appressoria by entomopathogenic Nair, MG, Safir GR, Siqueik-a JO Isolation and fungus Aletarhizium anisopliae. Experimental Mvcologv 13: identification of vesicular-arbuscular mycorrhiza-stimulatory compounds from clover {Trifolium repens) roots. Applied and Tsai SM, Phillips DA FUiyonoids released naturally from Environmental Microbiology 57: alfalfa promote development of symbiotic Glomus spores in Powell C LI Development of mycorrhizal infection from yitro. Applied and Environmental Microbiology 57:

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

New Phytol. (1993), 123, 115-122 Factors affecting appressorium development in the vesicular-arbuscular mycorrhizal fungus Glomus mosseae (Nicol. & Gerd.) Gerd. & Trappe BY MANUELA GIOVANNETTP, LUCIANO