Journal of Experimental Botany, Vol. 51, No. 349, pp. 1443-1448,
August 2000
© 2000 Oxford University Press
Original Papers |
Hydrolytic enzymes and ability of arbuscular mycorrhizal fungi to colonize roots
Departamento de Microbiología del Suelo y Sistemas Simbioticos, Estación Experimental del Zaidín, CSIC, Prof. Albareda 1, Apdo. 419, E-18008 Granada, Spain
Received 16 February 2000; Accepted 5 April 2000
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Abstract |
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The production of hydrolytic enzymes from external mycelia associated with roots and colonized soybean roots (Glycine max L.) inoculated with different arbuscular-mycorrhizal (AM) fungi of the genus Glomus, and the possible relationship between these activities and the capacity of the AM fungi to colonize plant roots was studied. There were differences in root colonization and plant growth between the Glomus strains, and also between two isolates of G. mosseae. Hydrolytic activities in the root and external mycelia associated with roots differed in the AM fungi tested. Correlations were only found between the endoxyloglucanase activity of the external mycelia associated with roots of the AM fungi tested and the percentage root colonization or plant growth. However, hydrolytic activities of roots colonized by the different endophytes correlated with those of external mycelia. The hydrolytic activities were not qualitatively different because the endoxyloglucanase from AM colonized roots and the external mycelia did not show a high degree of polymorphism in the different species of fungus tested. The possible role of the hydrolytic activity of external hyphae of AM fungi was discussed as a factor affecting fungal ability to colonize the root and influence plant growth.
Key words: Arbuscular mycorrhiza, Glomus sp., Glycine max, hydrolytic enzymes.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Although arbuscular mycorrhizal fungi (AM) show no specific variations in their ability to colonize a great range of host plants, they vary considerably in their population biology, ecological specificity and symbiotic activity (Giovannetti and Gianinazzi-Pearson, 1994
). There are many reports of inter- and intraspecific differences in the efficiency of AM fungi in terms of plant growth and protection (Harley and Smith, 1983; Sieverding, 1991; Ruiz-Lozano and Azcón, 1995; Ruiz-Lozano et al., 1995). The physiological basis for these variations is poorly known. The ability of mycorrhizas to increase plant growth can in most cases be explained by an increased phosphorus uptake. The hyphal spread of AM fungi is an important factor influencing the phosphorus supply to the host plant (Jakobsen et al., 1992). Mycorrhizal fungi may differ in their capacity to develop an external hyphal system regardless of their capacity to colonize the root cortex (Graham et al., 1982). The morphological properties and spatial distribution of the external hyphae in soil and differences in hyphal uptake, translocation capacities and metabolic activity seem to play an important role in the efficiency of AM fungi (Kothari et al., 1991; Jakobsen et al., 1992).
Differences in the ability of mycorrhizal fungi to enhance phosphorus uptake and growth of the host plant, even between species for which the extent of root colonization is similar, may be due to functional differences in the host–fungal interface. Different band patterns of enzymatic activities in AM fungi belonging to the same genus have been observed (Hepper et al., 1988). It has been suggested that enzymatic polymorphism in different strains of AM fungus may be related to their efficiency in root colonization and influence on plant growth (Hepper et al., 1988; Rosendahl and Sen, 1992).
Hydrolytic enzymes seem to be involved in the penetration and development of AM fungi in plant roots. Cellulase, pectinase and xyloglucanase activities have been found in colonized roots and in the external mycelium of AM fungi (García-Romera et al., 1991a; García-Garrido et al., 1992b; Rejón-Palomares et al., 1996b). Differences in cellulase and pectinase activities between some Glomus isotypes have been observed (García-Garrido, 1991; García-Romera et al., 1991b). It is therefore possible that variations in colonization capacities of host tissues may be related to the ability of the fungi to produce hydrolytic enzymes (Gianinazzi-Pearson, 1994). Of the different hydrolytic enzymes, xyloglucanases are the least well known; however, they seem to play an important role in plant cell wall extension (Hoson et al., 1995).
This study tests the possibility that differences in the ability of the fungus to produce hydrolytic enzymes are related to variations in the level of root colonization.
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Materials and methods |
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Growth of plants and inoculation procedures
Inocula of the AM fungi used were two isolates of G. mosseae (Nicol. and Gerd.) Gerd. and Trappe (BEG 12) from Rothamsted Experimental Station (UK) and from the University of Kiel (Germany); G. fasciculatum (Thax. sensu Gerd.) Gerd. and Trappe (BEG 58) from Dijon (INRA); G. clarum (Nicol. and Schenck) and G. intraradices (Schenck and Smith) from the Instituto Venezolano de Investigaciones Científicas (IVIC); and G. deserticola (Trappe, Bloss and Menge) from the Instituto de Investigaciones Agrobiológicas de Galicia (CSIC). All mycorrhizal inocula consisted of soil, spores, mycelium, and infected root fragments from an open pot culture of soybean plant (Glycine max L.). Ten grams of inoculum with similar characteristics (an average of 30 spores g-1 and 75% of roots infected) of one of the six Glomus isolates was added to each pot at sowing just below the soybean seeds. This saturating amount of the soil inoculum was enough to result in optimal root colonization (Ruiz-Lozano, 1995
). Soybean plants were obtained from surface-sterilized seeds (5 min in 0.75% NaOCl). Seeds were sown in moistened sand, and after 2 weeks, ten seedlings were transplanted to each pot, with three replicate pots per treatment. Plants were inoculated with 10 g of inoculum, and uninoculated plants were given filtered leaching (Whatman No. 1 filter paper) from the inoculum soil (20 ml pot-1, 1 : 2 w:v, soil : water). Plants were grown in 300 ml capacity open pots of soil collected from the province of Granada, Spain. The pots were filled with a gray loam soil obtained from the garden of the Estación Experimental del Zaidín (Granada, Spain). The soil (pH 8.1, 1 : 1, soil : water method) contained (mg kg-1): 6.2 P (NaHCO3-extractable), 0.3 N, and 132 K, and consisted of: 35.8% sand, 43.6% silt, 20.5% clay, and 1.8% organic matter. It was steam-sterilized and mixed with sterilized quartz sand at a proportion of 2 : 3 (v : v).
The plants were kept in a controlled-climate glasshouse, and were watered regularly and given 10 ml Hewitt's nutrient solution per week (Hewitt, 1952). The solution used for AM-inoculated plants lacked phosphate. Natural light was supplemented by Sylvania incandescent and cold-white lamps, 400 µmol m-2 s-1, 400–700 nm; with a 16/8 h light/dark cycle. Air temperature was 25–19 °C, and relative humidity was 50%.
Plants were harvested after 30 d. The root system was separated from the shoot, and dry weight of the shoot was determined. The root system was washed and rinsed several times with sterilized distilled water and parts of the root system were cleared and stained (Phillips and Hayman, 1970). The percentage of total root length that was colonized by AM fungi was measured as described previously (Giovannetti and Mosse, 1980).
External mycelia associated with the roots were isolated from roots of 30-d-old soybean plants colonized with the different G. mosseae isotypes by rubbing the roots while submerged in sterile water and passing this water through a 50 µm mesh sieve (Benabdellah et al., 1998). The external mycelium was collected with forceps under a dissecting microscope and mycelium dry weight was determined. Viability of the harvested hyphae was assessed by determining succinate dehydrogenase activity (Hamel et al., 1990).
Preparation of extracts for enzyme assays
Roots (10 g fresh weight) were pulverized in a mortar under liquid nitrogen. The resulting powder was homogenized in 30 ml of 100 mM TRIS-HCl buffer (pH 7) plus 0.02 g polyvinyl-polypyrrolidone (PVPP), 10 mM MgCl2, 10 mM NaHCO3, 10 mM ß-mercaptoethanol, 0.15 mM phenylmethyl sulphonyl fluoride (PMSF) and 0.3% (w : v) X-100 Triton. Sodium azide (0.03%) was added to all solutions. The liquid was filtered through several layers of cheesecloth and centrifuged at 20 000 gfor 20 min.
The supernatant was dialysed against several hundred volumes of the same diluted extractant solutions (1 : 9, v : v) for 16 h at 4 °C. The samples were then frozen until used.
External mycelia were frozen in liquid nitrogen and finely pulverized in a mortar. The resulting powder was suspended (30 mg ml-1) in the same extractant solution as for roots. The suspension was briefly sonicated (1 min, 5 times at 80 W) and centrifuged at 20 000 g for 20 min; the pellet resuspended and sonicated again, and washed by centrifugation with the same buffer three times. The supernatant was used as a crude enzyme extract.
Enzyme assays
The extracts were assayed to determine the activities of endoxyloglucanase (endo-XG), endoglucanase (endo-GN) (EC 3.2.1.4), endopolymethylgalacturonase (PMG), and endopolygalacturonase (endo-PG) (EC 3.2.1.15).
All hydrolytic activities were assayed by the viscosity method (Rejón-Palomares et al., 1996a) using xyloglucan from nasturtium seed (Tropaeolum majus L.) extracted as described previously (McDougall and Fry, 1989), carboxymethylcellulose (CMC), citrus pectin, and Na polygalacturonase as substrates. The reduction in viscosity was determined at 0–30 min intervals. Approximately 0.5 ml of the reaction mixture was sucked into a 1 ml syringe and the time taken for the meniscus to flow from the 0.70 ml to 0.20 ml mark was recorded. The reaction mixture contained 1 ml of 0.5% substrate in 50 mM citrate-phosphate buffer (pH 5) and 0.2 ml enzyme. Viscosity reduction was determined at 37 °C. One unit of enzyme activity was expressed as specific activity (U mg-1 protein) (U reciprocal of time in hours for 50% viscosity lossx103) (Rejón-Palomares et al., 1996b).
Polyacrylamide gel electrophoresis
Xyloglucanase enzymes were separated by non-denaturing electrophoresis on 8% polyacrylamide slab minigels (MiniProtean II, Bio-Rad) amended with 0.1% xyloglucan in 50 mM TRIS–0.1 M glycine buffer (pH 8.8) (García-Garrido et al., 1992a). The electrode tank contained the same TRIS-glycine buffer (pH 8.8) as used in the gel. The wells were filled with 30 µl of either root or fungus extract (175 µg protein) and 3 µl 0.05% bromophenol blue. Electrophoresis was done at 4 °C and a constant current of 20 mA per gel for 4 h.
The gels were incubated with 50 mM citrate-phosphate buffer (pH 5) at 37 °C for 16 h, after which they were stained with 0.1% Congo red for 15 min. Washing in 1 M NaCl followed this until the bands became visible.
Protein determination
Total proteins were determined by the method of Bradford (Bradford, 1976) using a Bio-Rad kit with BSA as the standard.
Statistical treatments
Data were subjected to one way ANOVA and Tukey test (P=0.05) evaluated differences in treatment means. Percentage data were subjected to arcsine transformation before analysis by linear regression.
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Results |
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Microscopic observations of stained roots showed no presence of AM fungi in uninoculated controls. The percentage of root length colonization of soybean root ranged from 22% by G. mosseae (BEG 12) to 50% by G. deserticola. Higher dry weights of the external mycelia associated with the roots were observed in roots colonized either with G. intraradices or G. deserticola. The differences in shoot dry weight between soybean plants inoculated with G. mosseae (BEG 12), G. clarum or G. intraradices and uninoculated controls were not significant. Shoot dry weights of soybean inoculated with G. fasciculatum and G. deserticola were higher than with the other treatments (Table 1
).
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As Table 2
shows, endo-XG, endo-PMG and endo-PG activities were higher in mycorrhizal plant roots than in uninoculated controls. Mycorrhizal plants showed higher endo-GN activity than uninoculated controls, except in roots colonized by G. mosseae (BEG 12). Plants colonized with G. deserticola showed the highest endo-XG and endo-PMG activities. Endo-GN activity was higher in roots colonized by G. fasciculatum, G. intraradices and G. deserticola than in roots colonized with the other Glomus strains. However, endo-PG activity was lower in roots colonized by either G. mosseae isotypes (Table 2).
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Comparison of hydrolytic activities from the external mycelia associated to roots colonized with different Glomus strains shows that the external mycelia of G. deserticola exhibited the highest endo-XG and endo-PG activities. Endo-GN activity was lowest in the external mycelia of G. mosseae (BEG 12). Endo-PMG activity was variable in the external mycelia of the different endophytes tested; however, G. deserticola showed the highest and the two G. mosseae isotypes the lowest activity (Table 3
).
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No correlation was observed between shoot dry weight, percentage of root length colonization and dry weight of external mycelia of the different endophytes tested. However, endo-XG activity in the external mycelia correlated with the dry weight of their external mycelia, with the shoot dry weight of colonized plants and the percentage of root colonization. The hydrolytic activities of roots colonized by the different endophytes correlated with the homonymous enzymatic activity of the external mycelia associated to roots (Table 4
).
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Because pectinase (endo-PMG, endo-PG) and cellulase activities (endo-GN) of AM roots are difficult to detect (García-Romera et al., 1991b
; García-Garrido et al., 1992a) and since endo-XG is the only hydrolytic enzyme whose activity correlates with root colonization and plant growth, xyloglucanase activity was selected for electrophoretic analysis. Several electrophoretic bands of endo-XG activities were observed in 30-d-old soybean plants. The bands in root extracts differed between the endophytes tested. Only one band of endo-XG activity was detected in the external mycelia of each of the different endophytes tested. The electrophoretic band of the external mycelia of G. mosseae (BEG), G. fasciculatum, G. clarum G. intraradices, and G. deserticola showed the same electrophoretic mobility (line 4). Only G. mosseae (KIEL) produced a xyloglucanase band with different electrophoretic mobility (line 2). Several electrophoretic bands present in non-inoculated plants were absent in some of the mycorrhizal plants (Fig. 1).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The inter- and intraspecific differences in the effectiveness of AM fungi for root colonization and plant growth are well known (Harley and Smith, 1983
; Sieverding, 1991). Differences were found in root colonization and plant growth not only between the different Glomus strains, but also between the isolates of G. mosseae BEG-12 and KIEL. However, as has been observed elsewhere, AM fungi differ in their ability to enhance growth of the host plant, regardless of the extent of root colonization (Graham et al., 1982). One of the most important factors that influences the efficiency of different AM fungal strains seems to be their external mycelium. The production of external hyphae may vary considerably between AM fungi (Sanders et al., 1977; Graham et al., 1982; Abbott and Robson, 1985; Kothari et al., 1991). No clear relationship seems to exist between the amount of external hyphae in soil and the growth responses observed in colonized plants (Jakobsen et al., 1992; Frey and Schuepps, 1993). Other factors such as the difference in rate of appresorium formation, in hyphal uptake and translocation capacities of nutrients, and in the metabolic activity of the external hyphae, seem to have more influence on the efficiency of AM fungi (Jakobsen et al., 1992; Frey and Schuepps, 1993; Giovannetti and Citernesi, 1993). One of the factors involved in the penetration of root by AM fungi is the production of hydrolytic enzymes (García-Garrido et al., 1992a; García-Romera et al., 1991a). The activities of the hydrolytic enzymes tested were higher in AM colonized than in non-AM colonized root (García-Romera et al., 1991a). Quantitative differences in hydrolytic activities of different AM fungi in the root and the external mycelia associated to roots were observed. No correlations were found between most of the hydrolytic activities of the AM fungi and percentage root colonization or plant growth. The low and localized production of hydrolytic enzymes does not allow the establishment of a close relationship between their production and AM root colonization (García-Garrido et al., 1996; García-Romera et al., 1991b). However, a correlation between endo-XG activity of the external mycelia and root colonization and plant growth was noted. This result indicates that the activity of this enzyme is an important factor influencing fungal colonization and plant growth (Rejón-Palomares et al., 1996a).
It is clear that the external hyphae play an important role in colonization capacity and efficiency of plant growth. These studies shows that the enzymatic activity of the external hyphae associated with roots colonized by AM fungi varies greatly depending on the fungal isolate used. These findings also indicate a correlation between the hydrolytic activities of these external mycelia with those of their colonized roots. Relationships between the metabolic activity of colonized roots and external hyphae have been found (Kothari et al., 1991; Jakobsen et al., 1992; Frey and Schuepps, 1993). The production of hydrolytic enzymes by external hyphae may therefore be a relevant variable that should be considered since colonization of roots requires efficient penetration mechanisms by the external hyphae (García-Romera et al., 1997).
Different isozyme activities in species of the genus Glomus show clear variations between species and geographically different isolates (Rosendahl and Sen, 1992). However, the hydrolytic enzyme endo-XG from AM colonized root and external mycelium does not show a high degree of polymorphism between the different species tested. A similar absence of polymorphism was found for other enzymes such as alkaline phosphatase and cellulase (García-Garrido, 1991; Gianinazzi et al., 1992). The presence of one band of xyloglucanase activity in non-mycorrhizal roots which was absent in mycorrhizal roots, suggest qualitative inhibition by the fungus of some enzymatic activity of the plant. Inhibition of plant protein synthesis by AM fungi has been observed in several plant–AM fungi associations (García-Garrido et al., 1993; Dumas-Gaudot et al., 1994).
A time-course study underway in this laboratory will shed further light on the role of hydrolytic enzymes from external mycelia in the mycorrhization of roots.
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Acknowledgments |
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Financial support for this study was provided by the DGICYT, Spain.
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Notes |
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1 To whom correspondence should be addressed. Fax: +34 58 129600. E-mail: igarcia{at}eez.csic.es
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