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Embrapa Agrobiologia, Caixa Postal 74505, CEP 23851-970, Seropédica, RJ, Brazil Department of Plant Microorganism Interaction
Stéphane Declerck
Université catholique de Louvain, Mycothèque de l'Université catholique de Louvain2, Unité de microbiologie, 3 Place Croix du Sud, 1348 Louvain-la-Neuve, Belgium
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ABSTRACT |
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Mycelium development and architecture and spore production were studied in Scutellospora reticulata from single-spore isolates grown with Ri T-DNA transformed carrot root-organ culture in monoxenic system. Culture establishment, anastomosis occurrence and auxiliary cell development also were examined. Seventy percent of the pregerminated disinfected spores colonized the transformed carrot roots. After 8 mo, the average spore production was 56 (24–130) per 30 cm3 of medium. Of the spores produced, 75% germinated and produced new generations in monoxenic culture. The mycelium network was formed by thick light-brown hyphae, which exhibit two major architecture patterns related to either root colonization or resource exploitation, and lower-order hyphae, bearing auxiliary cells, branched absorbing structures (BAS), hyphal swellings (HS) and forming anastomoses. BAS were formed abundantly in extramatrical mycelium and frequently had HS resembling vesicles, a feature not previously reported in the Gigasporaceae, to the best of our knowledge. Few anastomosis were observed within the mycelium and most often corresponded to a healing mechanism that form hypha bridges to reconnect broken hyphae or overcoming obstructed areas within a hypha. Numerous auxiliary cells were produced during culture development and their role was inferred.
Key words: anastomosis, arbuscular mycorrhizal fungi, auxiliary cell, branched absorbing structures, Gigasporaceae, Glomeromycota, hyphal swelling, monoxenic culture
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Among the seven AM fungi genera, Scutellospora contains approximately 17% of the described species (de Souza 2000). This genus was erected following dichotomy of the genus Gigaspora sensu Gedermann & Trappe (Walker and Sanders 1986). Despite the high diversity and occurrence of viable spores of Scutellospora species in natural ecosystems, some species are difficult to isolate and maintain in culture collections. For example, Scutellospora crenulata Herrera-Peraza; Cuenca & Walker is reluctant to grow in pot cultures (Herrera-Peraza et al 2001) while S. projecturata Kramadibrata & Walker and S. spinosissima Walker & Cuenca can be grown in mixed-species pot cultures but not as single species (Kramadibrata et al 2000, Walker et al 1998).
Monoxenic culture (MC) consists of an explant of Ri T-DNA transformed carrot root associated with AM fungal propagules on a synthetic nutrient-agar media. MC has been successful as a cultivation system for more than 25 AM species (see Fortin et al 2002 for a review) and is proving useful for studies of the fungal symbiont (Harrison et al 1999). However, most data generated under MC conditions were obtained with Glomus and Gigaspora species while in situ observations on in vitro-produced cultures of Scutellospora species were seldom reported. Our main objectives were: (i) to establish and describe the long-term culture of Scutellospora reticulata (Koske, Miller & Walker) Walker & Sanders under MC in association with RiT-DNA transformed carrot roots; and (ii) to describe the fungal development, i.e., spore germination, root colonization, extraradical mycelium development and architecture, and spore production.
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MATERIAL AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The strain was isolated from eroded soil in Brazil (see Santos et al 2000 for site characteristics) and cultured from a single spore on Brachiaria decumbens Stapf & Prain. The plants were grown in plastic pots containing 1L of a mixture of clay soil and sand (1:1 v/v), pH 4.8–5.2, amended with 5.5 g/Kg of rock phosphate and fertilized intermittently with 1/10 strength nutrient solution (Hoagland and Arnon 1938) without P. After 8 mo of growth, pot contents were dried in situ. Roots were removed, chopped and homogenized again with the remaining substrate. The pot culture presented an average of 2 spores per cm3 having 343 µm diam (mean 203–476 µm). The pot culture was stored at room temperature (under tropical conditions) until required. After 2 yr, spores were extracted by wet sieving and centrifugation, selected under a stereomicroscope as described by de Souza and Berbara (1999). After cleaning, the spores were re-examined under a stereomicroscope, and those appearing clean and in good condition were transferred to a blood-collect glass tube with a micropipette.
Establishment of monoxenic cultures –
Spores were surface sterilized at room temperature by a procedure adapted from Bécard and Piché (1992). After surface sterilization, spores were transferred individually with a micropipette to Petri plates containing water agar 0.8% (Agar bacteriological No. 1, OXOID Ltd., Hampshire, U.K.) pH 6.0 and incubated in an inverted position in the dark at 27 C until germination.
Two to 3 d after germination, a block of agar containing a single germinated spore was transferred to another Petri plate close to a transformed carrot (Daucus carota L.) root approximately 70 mm long (Dalpé and Declerck 2002). The modified Strullu-Romand (MSR) medium (see Declerck et al 1998, modified from Strullu and Romand 1986) was solidified with 4 g/L Gel-GroTM (ICN Biomedicals Inc, Irvine, California, U.S.A.) and used as growing medium. Each experimental unit consisted of a successful single spore-carrot root culture in a Petri plate (90 mm diam) containing 30 mL MSR medium. Cultures were incubated in an inverted position in the dark at 27 C for up to 8 mo.
Data collection and harvest – The germination rate of surface-sterilized spores was assessed for three sets of 100 spores each during a 4 wk period. Ten Petri plates each containing 10 spores composed a set. The development of fungal extraradical mycelia and the differentiation of hyphal swelling tips, auxiliary cells and spores were observed for up to 8 mo in 10 plates arranged randomly. Spore dimensions were assessed using 12 randomly chosen, mature spores in each experimental unit. Mature and juvenile spores were differentiated by color, septa formation in the subtending hyphae and by the absence of cytoplasm activity in the sporogenous bulbous subtending hyphae. Germination of monoxenic propagated spores was assessed at the end of the experiments. After 8 mo, spores were removed from the growth medium with forceps, germinated as described above and re-associated with transformed carrot roots on the MSR medium to test their ability to form new colonies and spores. The occurrence of anastomoses was evaluated in 100 intersection points per experimental unit. The intersections were observed in primary as well as secondary and lower-order hyphae. Observations were made through a dissecting microscope and an inverted compound microscope. Digital pictures were captured with a Sony DXC-950 P Power HAD 3-CCD digital camera, and the spore and mycelia measurements were taken with Leica Qwin V 1.0 software (Leica Imaging Systems Ltd., Cambridge, U.K.).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Monoxenic cultures establishment, sporulation and subculture – Seventy percent of the pregerminated spores colonized transformed carrot roots and produced spores. For 30% of noninfective spores, failure was attributed to damage to the germination tube after transfer to a Petri plate or failure of the germ tube to contact actively growing roots.
The extramatrical mycelium development was extensive (Figs. 1, 2) and numerous infection units were formed in active growing roots (Fig. 3). The first daughter spores were formed approximately 10–12 wk after colonization (Figs. 1, 2) and sporulation was observed thereafter for 5–8 mo. After 8 mo, the average spore production per Petri plate was 56 (24–130) per 30 cm3 of MSR medium. The spores were globose (Fig. 4) in all the cultures, with an average diam of 376 µm (280–500 coefficient of variation of 10.67%, n = 120). In Petri plates with extensive colonization, spores were distributed over the entire plate, mostly deep in the growth media, close to the roots, but also in the external cells layers of older roots (data not shown) and on the plate lid. The spores produced in the MC germinated within 2–3 d, averaged 76% germination in water and 75% in sugar-free MSR medium after 2 wk incubation at 27 C. These germinated spores were able to re-associate with Ri T-DNA transformed carrot roots on the MSR medium and produced new spores (data not shown).
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and Bago et al (1998a) (Figs. 1, 2) forming the main branching network of the culture. Thinner lower-order hyphae branched from this network and formed auxiliary cells, sporogenic hyphae and spores (Figs. 1, 2), hyphal swellings (HS) and branched absorbing structures-like (BAS-L) (Bago et al 1998b). The RH presented two architectural patterns occurring concomitantly in the same colony (Figs. 1, 2). In one pattern, RH formed open circles or spirals. The number of hyphal branches always was higher on the convex side of the partial circle or spiral (Fig. 1). This pattern facilitated the radial spreading of the hyphae and was associated with primary root colonization. It was formed during the nonsymbiotic (after spore germination) and symbiotic (after root colonization) phases. The other pattern was characterized by RH that grew straight or were only slightly curved (Fig. 2), expanding for several centimeters and branching in a radial pattern with irregular distances between branches. These RH mostly were formed by extraradical hyphae during root colonization. They exhibited intense cytoplasm flow in contrast with lower-order hyphae, were active throughout the 8 mo of observations and seldom formed septa.
Branched absorbing structures and hyphal swellings –
Structures resembling BAS-L reported by Bago et al (1998b) were formed as ramifications of primary, secondary or higher-order hyphae and also as ramifications of sporogenous hyphae (Fig. 5–7). These structures were formed by numerous thin (<1–5.3 µm), generally straight but sometimes contorted, hyphae extending radially in the medium in a volume frequently larger than 2 mm3 (Fig. 5). These structures were transient and their cytoplasm content retracted after septation. This process occurred 1–2 wk after the BAS-L formation (Fig. 6). During cytoplasm contraction cytoplasm frequently was arrested inside small, thin single-wall hyphal swellings (HS), which typically developed at the hyphal extremity of the BAS-L (Fig. 6). These HS were hyaline, globose to ovoid with dimensions of 13–18 µm by 26–36 µm. These structures resembled extraradical vesicles or immature spores because they frequently contained dense cytoplasmic material typically found in fully expanded spores and auxiliary cells (Fig. 7). The number of these HS varied from just a few to several hundred for a single BAS-L. In a single culture, HS could be 20 times as abundant as differentiated S. reticulata spores.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). For example, Morton (1995) reported contamination of a putative Microdochium species in a culture of S. gregaria INVAM CL756 and relates this contamination to the low production of spores, while Hijri et al (2002) reported the contamination by ascomycetes in apparently healthy spores of S. castanea BEG1. The use of S. castanea spores contaminated with ascomycetes caused problems for interpreting DNA analysis (Schussler 1999). The MC of S. reticulata provides a new perspective in the cultivation of this genus and extends the range of AM fungi genera maintained on excised roots (Fortin et al 2002). This system allows nondestructive observations of extraradical mycelium (Bago et al 1998a) and spore developmental sequence (de Souza and Berbara 1999, Dalpé and Declerck 2002). Culture and subculture of S. reticulata – The spore germination rate was high for both 2 yr dried pot culture (59%) and ROC-produced (75%) spores, demonstrating the long-term survival potential of this accession and the viability of the MC produced spores.
The composition of the MSR medium with pH adjusted to 5.5 before autoclaving (Declerck et al 1998) appeared adequate for this isolate, since the fungal life cycle was properly completed. In addition, a high number of successful single spore MC (70%) was obtained and could be increased by manipulation of the Petri plate, such as vertical incubation (Diop et al 1992) or by a better placement of the germinated spore and the root organ culture explant. Spore production within this system varied between 24–130 per Petri plate with 30 mL medium, which is comparable to the production in single-spore open-pot culture (mean two spores per mL dry soil).
The sporulation dynamics of S. reticulata was similar to that of Gigaspora margarita Becker & Hall (CNPAB 1 and 16), Gi. albida Schenck & Smith (INVAM 927 and BR607A, de Souza unpublished results) and Gi. rosea Nicolson & Schenck DAOM194757 (Diop et al 1992), which formerly was identified as Gi. margarita (Bago et al 1998c) or in competition with Glomus intraradices Schenck & Smith (Tiwari and Adholeya 2001). Compared with G. intraradices Schenck & Smith, G. versiforme (Karsten) Berch, G. proliferum Dalpé & Declerck (Declerck et al 2000, 2001), which start to produce spores in less than 1 mo in MC, these Gigasporaceae species exhibit a long vegetative phase before sporulation (2–3 mo). The sporulation dynamics of S. reticulata and Gi. margarita, as compared with G. intraradices, G. versiforme and G. proliferum under similar growing conditions, are consistent with "K"-like strategists for these three Gigasporaceae species, as compared to these three Glomus species, which behave like "r" strategists.
Mycelium architecture and development –
MC technique let us study the development of the external mycelium and its structures for lengthy periods and detect structures and patterns of mycelium growth previously reported. Bago et al (1998a) divided the extraradical spreading of the Glomus intraradices in monoxenic system into three stages: (i) proliferation of runner hyphae acting as conducting channels, which divide dichotomously and extend the fungal colony radially; (ii) development of arbuscule-like structures, which are formed at regular intervals along the runner hyphae and which might play a preferential role in nutrient uptake; and (iii) formation of spores in zones already colonized by runner hyphae and arbuscule-like structures. Those three stages also occur in S. reticulate. However, the pattern of mycelium branching is distinct. The Glomus mycelium branches more profusely and with lower angles than S. reticulata. In addition, S. reticulata forms the circular or spiraled mycelium that has not been reported to occur in Glomus.
The RH observed here are nearly identical to what Friese and Allen (1991) defined as runner hyphae. However, we observed that these hyphae also play a role in the allocation of resources within the fungal network. This was supported by the dense bidirectional cytoplasmic/protoplasmic streaming observed from roots to the surrounding environment, i.e., the various structures of lower-order hyphae and from lower-order hyphae to roots. In that sense, RH works as a "circulatory system".
Microscopic observations revealed that hyphal damage, caused by root growth, might have negatively affected spore formation in the MC. Some immature spores, located far from the disrupted hyphae, were arrested in their juvenile stage, probably due to the damage caused to the mycelia network. Similar damage and arrested spore development also might be expected in disturbed soils such as in tilled agricultural fields. This negative selection might help explain the low abundance of some Gigasporaceae species in arable fields (Daniell et al 2001, Helgason et al 1998, Jansa et al 2002), because spores are the main propagules for Gigasporaceae (Brundrett et al 1999 a, b, Klironomos and Hart 2002). Other factors observed in MC that might negatively affect fitness in agricultural soils include the long vegetative period before sporulation, the lengthy process of spore expansion and development.
Branching absorbing structures-like –
BSA-L reported here, resemble those described by Bago et al (1998b) for G. intraradices but were less ramified and frequently terminated in swollen tips, the HS. Bago et al (1998b) proposed that most BAS were involved in nutrient uptake capabilities and those appearing at the spore's subtending hyphae were implicated in spore formation. BAS-L longevity was ephemeral, and cytoplasm often was arrested inside the HS after septation of the subtending hyphae. For S. reticulata, these structures were localized near sites of sporulation, perhaps indicating a function related to sporulation, as demonstrated for the spore-BAS structures in G. intraradices (Bago et al 1998b). In some cases, i.e., G. clarum Nicolson & Schenck (de Souza and Berbara 1999), G. etunicatum Bercker & Gerdemann (Pawlowska et al 1999) and G. proliferum (Declerck et al 2000), HS were juvenile spores that differentiated into mature spores after successful culture establishment. In A. rehmii Sieverding & Toro, HS did not develop further, but they might help provide energy storage used to support further sporulation (Dalpé and Declerck 2002), thereby serving a similar role as suggested for auxiliary cells (see below).
Anastomoses –
Data on anastomoses formation mostly were reported for Glomus species (Giovanneti et al 1999, 2001, 2003). Anastomoses were observed to occur between hyphae originating from the same spore and from different spores of the same isolate (Giovannetti et al 1999), while no anastomoses were detected between hyphae belonging to different isolates (Giovannetti et al 2003). Concerning Scutellospora species, i.e., S. castanea Walker, neither interspecific nor intergeneric hyphal fusions were observed with germlings (Giovannetti et al 1999). In our experiment, we recorded anastomoses in intact arbuscular mycorrhizal networks of S. reticulata but in a low frequency as compared with the Glomus species studied (Giovanneti et al 1999, 2001, 2003). Bago et al (1999) also reported the existence of healing mechanisms in Glomus intraradices and Gigaspora rosea that restrict the damage induced by ageing or lytic events. These authors suggested that such mechanism could improve hyphal survival under adverse conditions. We also hypothesize that the healing mechanism reported in S. reticulata might decrease damage caused in the mycelium network that can negatively affect spore formation.
Auxiliary cells –
The biological function of auxiliary cells remains speculative (Bonfante and Bianciotto 1995). Jabaji-Hare (1988) observed high amounts of lipids within the auxiliary cells of a Gigaspora species, supporting the storage function of these structures. Roles in transitory storage (Pearson and Schweiger 1993) and reproduction (Pons and Gianinazzi-Pearson 1985) have been hypothesized, or they might represent vestiges of relict reproductive structures (Morton and Benny 1990). Our observations suggest a possible role in carbon storage, for use as energy for spore initiation and development and/or mycelial production and repair. Specific details supporting this supposition were (i) the abundance of auxiliary cells (approximately 600–700 per Petri plate, data not shown) for an average of 56 spores produced, (ii) the apparent changes in lipid content during the transition from young to older auxiliary cells and (iii) the high lipid content within these cells. The transitory role of auxiliary cells in spore production also was supported by previous work conducted with S. calospora (Nicol. & Gerd.) Walker & Sanders (Pearson and Schweiger 1994). These authors said that carbon stored in the auxiliary cells could be used during spore formation.
Final comments –
Although artificial, MC supports the life cycle of AMF, thereby generating viable propagules (spores). The major advantage of this system is that it allows for detailed, nondestructive observation of the entire fungal development over time. This is particularly useful in the study of extraradical mycelium development and function. As demonstrated here, the extramatrical mycelium play a major role in the S. reticulata life cycle and survival because spores are the main propagule in this family (Brundrett et al 1999a, b, Klironomos and Hart 2002). The observed properties of spore production also might dictate the ecology of this species to a large extent in the natural environment as well as in agricultural soils. However, further experiments are necessary to prove that MC can be used to predict AM species behavior in field soils. The use of MC should help us unravel some of the basic and applied aspects of the biology and ecology of the AMF symbiosis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Part of the Belgian Coordinated Collections of Micro-organisms (BCCM)
Accepted for publication May 11, 2003.
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LITERATURE CITED |
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