Applied Soil Ecology 14 (2000) 249–255
Isolation and symbiotic characteristics of Mexican Frankia strains associated with Casuarina Luis Vásquez, Néstor-Octavio Pérez, Mar´ıa Valdés∗ Escuela Nacional de Ciencias Biológicas, del Instituto Politécnico Nacional, Apartado Postal 264-CON, 06400 Mexico, DF, Mexico Accepted 27 January 2000
Abstract In the absence of available symbiotic nitrogen-fixing Frankia strains associated with Casuarina trees in Mexico for reforestation purposes, isolation was undertaken using root nodules from trees growing in different habitats in Mexico, from the coast of the Gulf of Mexico up to 2550 m above the sea level. A total of 24 strains were isolated and clonal cultures were obtained from one filament of each strain. The use of acetate as the sole carbon source was essential for the isolation of the endosymbiont from the nodules due to the fact that other contaminant actinomycetes utilize propionate. Clonal cultures were obtained, and cultural and symbiotic characteristics of pure cultures were assessed. All strains grew well in stirred DPM (defined propionate medium) with no mineral nitrogen. Isolates showed hyphae, multilocular sporangia and characteristic vesicles. The presence of the gene nifH was also demonstrated, with all strains being able to nodulate Casuarina equisetifolia. Nitrogenase activity (acetylene reduction) of the formed root nodules varied among the different associations depending on the isolate used to inoculate the plants. Several of the isolates can be used as inoculants for the propagation of Casuarina trees. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Frankia; Nitrogen fixation; Carbon source; Infectivity; Effectivity
1. Introduction The actinomycetal endophyte Frankia infects the roots of a wide range of angiosperms culminating in the development of root nodules and the establishment of a nitrogen-fixing symbiosis. Among these plants are the nitrogen-fixing trees, Casuarina equisetifolia Forst & Forst and C. cunninghamiana Miq., which were introduced in Mexico at the beginning of the century. They are valued as windbreaks, for land stabilization and soil improvement. C. equisetifolia is not only able to grow in dunes and soil containing little or no nitrogen, but grows successfully along the ∗ Corresponding author. Tel.: +52-53-41-32-35. E-mail address:
[email protected] (M. Vald´es)
highly polluted avenues of Mexico City. Symbiotic nitrogen fixation contributes to the success of these plants in marginal sites. However, the inoculation practices of this fast growing tree are not known in Mexico, and there are no selected strains of Frankia available for commercial use. Mexico is not the exception among the developing countries which need to propagate and inoculate Casuarina trees to ensure better performance in the afforestation areas. Relative success has been achieved with the isolation and culture of Frankia strains from root nodules of many actinorhizal plants including Casuarina. There are still several actinorhizal plants whose microsymbionts have not been isolated. Up to now, no selective isolation techniques have been developed and only a small percentage of isolation attempts have
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been successful. Some of the difficulties in isolating the symbiotic Frankia could also be its slow growth and the preponderance of faster growing contaminating eubacteria and actinomycetes associated with the soil-borne nodule. Another difficulty is that most of the soil bacteria remain unculturable (Torsvik et al., 1990; Bakken, 1997) and many Frankia strains seems to be part of this recalcitrant soil population or part of the less saprophytic bacteria (Rouvier et al., 1996). Most isolation procedures utilize liquid culture medium, which may lead to the development of cocultures (Benson and Hanna, 1983) or other contaminant actinomycetes (Niner et al., 1996). The way to purify a filamentous bacterium forming sporangia is through monosporal cultures (Lumini and Bosco, 1996) or through cultures from a single filament. Pure cultures are an essential prerequisite to characterize a bacterium or to characterize its interaction with plants. The lack of knowledge of the microsymbiont has lead to the misunderstanding of the physiology of nitrogen fixation in actinorhizal nodules. This study presents a simple modification of the isolation technique to obtain Frankia strains from C. equisetifolia as well as their clonal cultures. Their growth characteristics in stirred culture, the occurrence of the gene nifH and their effectivity in several plants were also included in this study.
2. Materials and methods 2.1. Sampling To increase the probability of finding diversity, root nodules were collected from C. equisetifolia and C. cunninghamiana trees growing in different ecosystems going from the dunes of the Gulf of Mexico up to an altitude of 2550 m. A survey was completed within an area of around 117 000 km2 . The Atlantic seaboard was selected because the genus was introduced for the first time in this region and has been continuously propagated there. 2.2. Isolation in liquid nutrient medium Seedlings of C. equisetifolia were grown in plastic pots filled with sterile sand and watered with N-free
nutrient solution (Hoagland and Arnon, 1950). Plants were grown in a greenhouse at 28/14◦ C and a 14/10 h photoperiod. When seedlings were 6 weeks old they were inoculated with the suspension of disinfected and crushed nodules collected in the field and stored in polyethylene bags, and refrigerated. A dark green color, indicative of the presence of nodulation, of the plants occurred within 16 weeks. Nodules were cleaned thoroughly with tap water and the individual lobes surface sterilized with sodium hypochlorite (3:10) and H2 O2 (30%) for 1 min in each solution, and rinsed several times with sterile distilled water. The epidermis of the lobes was removed, lobes were dissected into small pieces and transferred to test tubes containing liquid defined propionate medium (DPM) (Baker and O’Keefe, 1984) supplemented with acetate instead of propionate. Test tubes were incubated at 28–30◦ C for 5–9 weeks. 2.3. Purification of colonies To obtain clonal cultures from the isolates, colonies showing the phenotypic traits of Frankia were separately broken into filament fragments by using a hypodermic syringe and diluted 1:100 (v/v) with sterile distilled water. One millilitre of this suspension was transferred to a Petri plate containing melted modified DPM agar. The plate was agitated, allowed to solidify and incubated at 28◦ C in an anaerobic jar with no reducing agents. Plates with a single filament were examined until a colony had formed. Colonies were removed, homogenized, transferred to nitrogen-free liquid DPM medium, and subcultured every week under stirred conditions (100 rpm) at 30◦ C (Schwencke, 1991). Growth was evaluated through protein determination using the Bradford assay (Bradford, 1976) with cells that were centrifugated and sonicated. 2.4. Amplification of nifH genes Genomic DNA was obtained through a CTAB (cetyltrimethylammonium bromide) protocol. This protocol was described originally for mycobacteria (Van Soolingen et al., 1991) and was adapted for Frankia in our laboratory (Pérez et al., 1999). The DNA of each of the isolates was used as a template for the amplification of the nifH gene using the
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PCR technique. The PCR mixture contained 50 ng of DNA, the manufacturer’s buffer (Gibco BRL), 3 mM MgCl2 , 250 M of each dNTPs, 0.4 M of each primer and 2.5 U of Taq polymerase (Gibco BRL) in a 50 l volume. In order to amplify an internal part of the nifH gene of the tested isolates, the primer FGPH256 (50 -GAG TCC GGT GGC CCG GAG CC-30 ) (Maggia et al., 1992) and the complementary sequences of the primer FGPH750 (50 -GAA GAC GAT CCC GAC CCC GA-30 ) were utilized (Simonet et al., 1991). The first primer is used to amplify part of the nifH region and the second is a Frankia specific nifH primer. The reaction was carried out at 35 cycles of 95◦ C for 1 min, 50◦ C for 1 min, and 72◦ C for 2 min on an Ericop Easy-Cycler Thermal Cycler. The primers were synthesized by Gibco BRL. Five microlitres of PCR products were subjected to electrophoresis in 2% agarose gels, using the 123 bp ladder (Gibco, BRL) as a molecular marker. The gel was stained with ethidium bromide and observed under a UV light source. 2.5. Infectivity and effectivity tests All Frankia strains obtained were tested for their nitrogenase activity in vitro. Activity was measured under air with the acetylene reduction assay in 6–10 ml serum vials. Samples were removed aseptically from cultures growing in the nitrogen-free medium and incubated with 10% acetylene (v/v) for 24 h. Ethylene production was measured using a Pye Unicam gas chromatograph fitted with a 80–100 mesh Porapak N column, injector and column temperatures were 100◦ C with the detector at 150◦ C, following the Postgate method (Postgate, 1971). Frankia strains were tested for their ability to infect actinorhizal plants belonging to other host specificity groups as well as their original host plant, validating Koch’s postulates. The strains were grown in DPM, washed in distilled water, centrifugated and homogenized. The innoculum used was a washed Frankia culture equivalent to 10 g of protein which was applied to the roots of each 5-week old C. equisetifolia, Alnus acuminata spp. glabrata, Elaeagnus angustifolia and Myrica cerifera seedlings. Uninoculated seedlings were used as controls. Alder plants were obtained from aseptic germination and grown under the same conditions, because they often show contamination. Plants
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were grown for 6 weeks in the greenhouse under a photoperiod of 14/10 h and temperatures of 28/14◦ C. Acetylene reduction assays were conducted at the end of each experiment with the nodules of each plant.
3. Results Table 1 shows the location and altitude of the sites of the isolation trials. Seedlings of C. equisetifolia inoculated individually with nodule samples coming from 36 different sites in the field, were nodulated by nodule samples from 25 sites only. 3.1. Isolation on liquid medium After 4–12 weeks, tubes were examined for the occurrence of Frankia colonies. Contaminating actinomycetes other than Frankia developed in the tubes supplemented with glucose or pyruvate or propionate. Media supplemented with acetate as the sole carbon source remained clear, devoid of contaminating actinomycetes or containing fluffy white colonies. The colonies were located at the bottom of the tubes, often adhering to the walls. Under the microscope these colonies exhibited structures previously described for the genus Frankia: septate hyphae 1.0–1.5 m in diameter, polymorphic multilocular sporangia, and spherical refringent vesicles. A total of 24 Frankia isolates were obtained. 3.2. Purity of cultures Petri dishes with modified DPM medium and no mineral nitrogen, inoculated with fragmented filaments, were carefully observed under a dissecting microscope at the beginning of the incubation and during the 4–6 weeks. At that time, the fluffy colonies were 100–200 m in diameter showing a star fish shape. Twelve different clonal cultures were obtained. Strain characteristics are discussed in the following paragraph. The growth of clonal cultures is expressed as g protein/ml in BAP (Murry et al., 1984), BAPpcm (Schwencke, 1991) and DPM (Baker and O’Keefe, 1984). Better growth was obtained with the DPM medium supplemented with propionate as the sole
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Table 1 Survey locations and origin of samples of root nodules of Casuarina introduced in different regions in Mexico, from the dunes of the Gulf of Mexico to the highlands Location
Host plant
Altitude (m)
Atotonilco El Grande, Hidalgo Calpulalpan, Tlaxcala Cd. Valles-Tamasopo, Mpio. Valles, San Luis Potos´ıa Ciudad Madero, Tamaulipasa Ciudad Valles, San Luis Potos´ı 2 km al E deOrizaba, Veracruza Cerro Gordo, Veracruza Chachalacas, Veracruza Chununtzent, Mpio. Huehuetl´an, San Luis Potos´ıa El Juke, Carretera Poza Rica-Tuxpan, Veracruza El Mangal, Medellin, Veracruz Epifanio Navarro, Mpio. Pueblo Viejo, Veracruza Escanelilla, Mpio. de Pinal de Amoles, Queretaroa Flores Mag´on, Veracruza 2.5 km al E deOrizaba, Veracruza Guti´errez Zamora, Veracruz Isla del Amor, Mpio. de Alvarado, Veracruza Ixtaczoquitl´an, Vercaruza Jalapa de Enriquez, Veracruza Las Amapolas, Veracruz Miradores del Mar, Veracruz Miraflores, Tlalmanalco, Edo. de M´exicoa Playa Paraiso, Veracruza Playa Norte, Veracruz Poza Rica, Veracruza Puente Tam´os, Mpio. de P´anuco, Veracruza Punta El Morro, Veracruz, Veracruz Rancho San Fern´ando y Manuelita, San Luis Potos´ıa Rancho Los Manantiales, Mpio. de Alamo, Veracruza San Ciro de Acosta, San Luis Potos´ıa Tampico Alto, Veracruza Tecolutla-Gutierr´ez Zamora, Veracruz Ursulo Galv´an, Veracruza Xochimilco, M´exico, DFa Zacapoaxtla, Puebla Zaragocita, Mpio. de Tancoco, Veracruza
C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C.
2110 2550 200 5 90 1125 640 0 80 100 130 100 1150 0 1100 10 0 1020 1420 110 880 2260 0 0 100 220 0 60 250 960 100 0 10 2200 1975 300
a
equisetifolia equisetifolia equisetifolia cunninghamiana cunninghamiana equisetifolia equisetifolia equisetifolia equisetifolia cunninghamiana equisetifolia cunninghamiana equisetifolia equisetifolia equisetifolia equisetifolia cunninghamiana equisetifolia equisetifolia cunninghamiana equisetifolia equisetifolia equisetifolia cunninghamiana equisetifolia equisetifolia cunninghamiana cunninghamiana cunninghamiana equisetifolia equisetifolia equisetifolia cunninghamiana equisetifolia equisetifolia cunninghamiana
Indicates that nodules from these sites were propagated on C. equisetifolia seedlings.
carbon source compared to the BAP and BAPpcm media.
the same DNA preparation of the native isolates with Frankia BR and Streptomyces ssp. as controls.
3.3. Amplification of the nifH gene
3.4. Infectivity and effectivity
The use of the primers mentioned above amplified a 500 bp band in all the isolates including the reference strain (Fig. 1). The reproducibility of the PCR reaction was examined by comparing the generated band of the PCR product in reactions repeated at least twice using
No one isolate was able to induce the formation of root nodules in plants belonging to other specificity groups; the less specific plant for its microbial partner, Myrica, was nodulated by all the Casuarina isolates. The results of the relative infectivity of all the strains
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the inoculated plants. Nitrogenase activity of the cultures was high, measured after 24 h of incubation with acetylene. Activity ranged from 64 to 1346 nMol of ethylene produced per mg of protein per hour. Under the test conditions, rates of acetylene reduction activity by the different Frankia strains in planta were different and higher for several strains in comparison to the reference strain. However, effectivity in terms of growth of the plants during the test period showed no differences among the different combinations of microorganism–plant (data not shown).
4. Discussion Fig. 1. PCR amplification of the nifH gene from the Frankia strains using the primers FGPH256 and FGPH7500 . All the strains displayed and band of about 500 bp 1 and 14: 123 bp marker (Gibco); Frankia strains 2: Br; 3: IPNCe1; 4: IPNCe2; 5: IPNCe5; 6: IPNCe6; 7: IPNCe16; 8: IPNCe17; 9: IPNCe18; 10: IPNCe20; 11: IPNCe22; 12: Streptomyces antibioticus; and 13: negative control without DNA.
in C. equisetifolia are included in Table 2. The occurrence in percent of infected plants is recorded together with the acetylene reduction activity in planta as well as the in vitro activity by the Frankia native strains. The Casuarina seedlings exhibited active nodules 4 weeks after inoculation. All cultures infected Casuarina seedlings roots. Sixty percent of the strains were able to nodulate all the inoculated seedlings; some of them induced nodule formation in only 75% of
Casuarina equisetifolia is one of the species of Casuarina most widely distributed in tropical and subtropical countries for multiple uses, it is also one of the most propagated in the Mexican nurseries. Its establishment becomes successful, mainly in marginal soil, when its roots are in an efficient nitrogen-fixing association with the soil filamentous bacterium Frankia. Thus, inoculation of seedlings is necessary using either crushed nodules or isolated and selected Frankia strains. Isolation and culture of Frankia strains from Casuarina root nodules have proven difficult (Diem and Dommergues, 1983; Zhang and Torrey, 1985). Currently, many Frankia strains nodulating Casuarina are available (although they are not characterized for effectivity) in several laboratories. Unfortunately, in
Table 2 Infectivity and nitrogenase activity (ARA) in vitro and in planta of native Frankia strains isolated from Casuarina equisetifolia Frankia strain IPNCe1 IPNCe2 IPNCe4 IPNCe5 IPNCe6 IPNCe14 IPNCe15 IPNCe16 IPNCe17 IPNCe18 IPNCe2O IPNCe22 BR (reference strain) a
Nodulation (%)a 100 75 100 100 100 100 100 87.5 87.5 100 75 87.5 75
Percent of nodulated plants of the total inoculated.
ARA in vitro nMol C2 H2 /mg/h
ARA in vivo nMol C2 H2 /g/h
1006 1091 64 470 846 1320 1346 337 619 675 462 1166 535
51130 36030 70470 44180 14360 16140 22570 18630 109600 26770 18800 32800 44620
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developing countries there are no laboratories with strains that can be recommended. It appears that all the isolated and cultured strains coming from those countries into which the trees were introduced from Australia have more pronounced saprophytic capabilities, and belong to a single genetic group, the No. 1, of seven molecular phylogenetic groups of the Australian microsymbionts (Rouvier et al., 1996; Simonet et al., 1998). It also appears it is still not known what the media preferences are of the endosymbionts from the other phylogenetic groups. Nevertheless, several of our characterized strains should take their place among the available strains for inoculation studies in forestry plantations. Propionate is the most recommended carbon source for Frankia isolation from Casuarina nodules. However, the use of acetate as the sole carbon source for the isolation of Frankia from the Casuarina nodules was shown to be essential. It is important to mention that in all the isolation trials utilizing propionate with no nitrogen in the nutrient medium, a contaminant actinomycete developed. This actinomycete exhibits acetylene reduction activity (Niner et al., 1996; Villegas et al., 1997), and it seems that it is one of the rhizospheric microorganisms associated with soil-born nodules. Culture purity and identity are essential prerequisites when characterizing bacteria and when evaluating plant–bacteria interactions. Most of the isolation trials of Frankia are accomplished by submerging a piece of a disinfected nodule lobe into a liquid medium. This procedure may allow for the growth of a coculture. It may be possible for a slow-growing Frankia isolate to be masked by a faster contaminating one; physiological genotypic characters may be attributed to such a coisolate, while nodulation is induced by the slower-growing one (Lumini and Bosco, 1996). Also, nodules or individual lobes may host more than one Frankia strain (Benson and Hanna, 1983; Reddell and Bowen, 1985). The results show that the isolated microorganisms from C. equisetifolia nodules are actinomycetes belonging to Frankia according to the exhibited morphological and molecular features of the genus; procaryont septate hyphae, induced spherical vesicles, multilocular sporangia and the occurrence of sequences of the nifH gene. These actinomycetes fulfill Koch’s postulates by means of the original host infectivity. The Mexican isolates grew readily
in defined synthetic nutrient medium such as DPM. Lack of ammonium in the medium promotes the differentiation of vesicles by the microorganism fixing dinitrogen to sustain its growth. Growth decrease in BAP media, specially in BAPpcm , was attributable to autolysis of the mycelia in standing culture. Proteolytic activity (Horrière, 1984), aminopeptidades and different proteinases (Benoist and Schwencke, 1990; Benoist et al., 1992) have been reported and identified in Frankia strains from C. equisetifolia. The results of effectivity of the symbiosis (acetylene reduction rates) in young Casuarina plants showed considerable variation depending on the strain of Frankia used as inoculum, indicating the need to select the most efficient combination of plant–bacteria in field trials for plantation purposes for good establishment and growth. Several of our isolates (IPNCe1, IPNCe4 and IPNCe17) should take their place among the internationally available, recommended strains for Casuarina inoculations in nursery propagations. Acknowledgements We thank Evarista Fuentes, Margarita Juárez and Thelma Laguna for technical assistance. This research was supported by CONACYT (1370-N9206) and the I.P.N. (Polytechnical Institute, DEPI 93630 and DEP1970430). References Baker, D., O’Keefe, D., 1984. A modified sucrose fractionation procedure for the isolation of frankiae from actinorhizal root nodules and soil samples. Plant Soil 78, 23–28. Bakken, L.R., 1997. Culturable and nonculturable bacteria in soil. In: van Elsas, J.D., Trevors, J.T., Wellington, E.M.R. (Eds.), Modern Soil Microbiology. Marcel Dekker, New York, USA, pp. 47–61. Benoist, P., Schwencke, J., 1990. Native agarose–polyacrylamide gel electrophoresis allowing the detection of aminopeptidase, dehydrogenase and esterase activities at the nanogram level: enzymatic patterns in some Frankia strains. Anal. Biochem. 187, 337–344. Benoist, P., Muller, A., Diem, H.G., Schwencke, J., 1992. High-molecular-mass multicatalitic proteinase complexes produced by the nitrogen-fixing actinomycete Frankia strain BR. J. Bacteriol. 174, 1495–1504. Benson, D., Hanna, D., 1983. Frankia diversity in an alder stand as estimated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis of whole cell protein. Can. J. Bot. 61, 2919– 2923.
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