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 refor-estation 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 establish-ment of a nitrogen-fixing symbiosis. Among these plants are the nitrogen-fixing trees, Casuarina equi-setifolia 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 ex-ception 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 isola-tion and culture of Frankia strains from root nodules of many actinorhizal plants including Casuarina. There are still several actinorhizal plants whose mi-crosymbionts have not been isolated. Up to now, no selective isolation techniques have been developed and only a small percentage of isolation attempts have
been successful. Some of the difficulties in isolating the symbiotic Frankia could also be its slow growth and the preponderance of faster growing contaminat-ing 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 con-taminant 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 occur-rence 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 H2O2(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 incu-bated 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 sepa-rately broken into filament fragments by using a hy-podermic 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 un-der stirred conditions (100 rpm) at 30◦C (Schwencke, 1991). Growth was evaluated through protein deter-mination 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).
PCR technique. The PCR mixture contained 50 ng of DNA, the manufacturer’s buffer (Gibco BRL), 3 mM MgCl2, 250mM of each dNTPs, 0.4mM of each primer and 2.5 U of Taq polymerase (Gibco BRL) in a 50ml volume. In order to amplify an internal part of the nifH gene of the tested isolates, the primer FGPH256 (5′-GAG TCC GGT GGC CCG GAG CC-3′) (Maggia et al., 1992) and the complementary sequences of the primer FGPH750 (5′-GAA GAC GAT CCC GAC CCC GA-3′) were utilized (Simonet et al., 1991). The first primer is used to amplify part of the nifH region and the second is a Frankia spe-cific 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 elec-trophoresis in 2% agarose gels, using the 123 bp lad-der (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 ni-trogenase activity in vitro. Activity was measured un-der 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 in-cubated 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 homog-enized. The innoculum used was a washed Frankia culture equivalent to 10mg of protein which was ap-plied 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 condi-tions, because they often show contamination. Plants
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 acti-nomycetes 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 acti-nomycetes or containing fluffy white colonies. The colonies were located at the bottom of the tubes, of-ten adhering to the walls. Under the microscope these colonies exhibited structures previously described for the genus Frankia: septate hyphae 1.0–1.5mm 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 fila-ments, 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 100–200mm in diameter showing a star fish shape. Twelve different clonal cultures were obtained. Strain characteristics are discussed in the following paragraph.
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 C. equisetifolia 2110
Calpulalpan, Tlaxcala C. equisetifolia 2550
Cd. Valles-Tamasopo, Mpio. Valles, San Luis Potos´ıa C. equisetifolia 200
Ciudad Madero, Tamaulipasa C. cunninghamiana 5
Ciudad Valles, San Luis Potos´ı C. cunninghamiana 90
2 km al E deOrizaba, Veracruza C. equisetifolia 1125
Cerro Gordo, Veracruza C. equisetifolia 640
Chachalacas, Veracruza C. equisetifolia 0
Chununtzent, Mpio. Huehuetl´an, San Luis Potos´ıa C. equisetifolia 80
El Juke, Carretera Poza Rica-Tuxpan, Veracruza C. cunninghamiana 100
El Mangal, Medellin, Veracruz C. equisetifolia 130
Epifanio Navarro, Mpio. Pueblo Viejo, Veracruza C. cunninghamiana 100
Escanelilla, Mpio. de Pinal de Amoles, Queretaroa C. equisetifolia 1150
Flores Mag´on, Veracruza C. equisetifolia 0
2.5 km al E deOrizaba, Veracruza C. equisetifolia 1100
Guti´errez Zamora, Veracruz C. equisetifolia 10
Isla del Amor, Mpio. de Alvarado, Veracruza C. cunninghamiana 0
Ixtaczoquitl´an, Vercaruza C. equisetifolia 1020
Jalapa de Enriquez, Veracruza C. equisetifolia 1420
Las Amapolas, Veracruz C. cunninghamiana 110
Miradores del Mar, Veracruz C. equisetifolia 880
Miraflores, Tlalmanalco, Edo. de M´exicoa C. equisetifolia 2260
Playa Paraiso, Veracruza C. equisetifolia 0
Playa Norte, Veracruz C. cunninghamiana 0
Poza Rica, Veracruza C. equisetifolia 100
Puente Tam´os, Mpio. de P´anuco, Veracruza C. equisetifolia 220
Punta El Morro, Veracruz, Veracruz C. cunninghamiana 0
Rancho San Fern´ando y Manuelita, San Luis Potos´ıa C. cunninghamiana 60
Rancho Los Manantiales, Mpio. de Alamo, Veracruza C. cunninghamiana 250
San Ciro de Acosta, San Luis Potos´ıa C. equisetifolia 960
Tampico Alto, Veracruza C. equisetifolia 100
Tecolutla-Gutierr´ez Zamora, Veracruz C. equisetifolia 0
Ursulo Galv´an, Veracruza C. cunninghamiana 10
Xochimilco, M´exico, DFa C. equisetifolia 2200
Zacapoaxtla, Puebla C. equisetifolia 1975
Zaragocita, Mpio. de Tancoco, Veracruza C. cunninghamiana 300
aIndicates that nodules from these sites were propagated on C. equisetifolia seedlings.
carbon source compared to the BAP and BAPpcm media.
3.3. Amplification of the nifH gene
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
the same DNA preparation of the native isolates with Frankia BR and Streptomyces ssp. as controls.
3.4. Infectivity and effectivity
Fig. 1. PCR amplification of the nifH gene from the Frankia strains using the primers FGPH256 and FGPH750′
. 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 occur-rence 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 Casua-rina 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
Table 2
Infectivity and nitrogenase activity (ARA) in vitro and in planta of native Frankia strains isolated from Casuarina equisetifolia
Frankia strain Nodulation (%)a ARA in vitro nMol C
2H2/mg/h ARA in vivo nMol C2H2/g/h
IPNCe1 100 1006 51130
IPNCe2 75 1091 36030
IPNCe4 100 64 70470
IPNCe5 100 470 44180
IPNCe6 100 846 14360
IPNCe14 100 1320 16140
IPNCe15 100 1346 22570
IPNCe16 87.5 337 18630
IPNCe17 87.5 619 109600
IPNCe18 100 675 26770
IPNCe2O 75 462 18800
IPNCe22 87.5 1166 32800
BR (reference strain) 75 535 44620
aPercent of nodulated plants of the total inoculated.
the inoculated plants. Nitrogenase activity of the cul-tures 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 reduc-tion activity by the different Frankia strains in planta were different and higher for several strains in com-parison to the reference strain. However, effectivity in terms of growth of the plants during the test period showed no differences among the different combina-tions of microorganism–plant (data not shown).
4. Discussion
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 nurs-eries. 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.
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 capabil-ities, and belong to a single genetic group, the No. 1, of seven molecular phylogenetic groups of the Aus-tralian 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. How-ever, 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 actino-mycete developed. This actinoactino-mycete exhibits acety-lene 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 prerequi-sites 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 cocul-ture. 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 microorgan-isms 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 vesi-cles, 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 dif-ferentiation 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. Prote-olytic 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 (acety-lene 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 estab-lishment 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 re-search was supported by CONACYT (1370-N9206) and the I.P.N. (Polytechnical Institute, DEPI 93630 and DEP1970430).
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