Low recovery frequency of Gluconacetobacter ... - Springer Link

Symbiosis (2011) 54:131–138 DOI 10.1007/s13199-011-0133-3

Low recovery frequency of Gluconacetobacter diazotrophicus from plants and associated mealybugs in Cuban sugarcane fields Patricia Ortega-Rodés & Eduardo Ortega & Diethelm Kleiner & F. G. Loiret & Rosa Rodés & Jesús Caballero-Mellado

Received: 21 April 2011 / Accepted: 10 October 2011 / Published online: 29 October 2011 # Springer Science+Business Media B.V. 2011

Abstract This study was aimed to isolate and identify the N2-fixing bacterium Gluconacetobacter diazotrophicus from 11 sugarcane varieties, grown under field conditions in four Cuban provinces, and from their associated mealybugs Saccharicoccus sacchari. Identification was based on morphological and biochemical tests and PCRamplification of 16S rRNA genes using species-specific primers. From all sugarcane varieties and numerous mealybug colonies sampled, G. diazotrophicus isolates were recovered from inside sugarcane stems of only three varieties, and one from S. sacchari colony. These four isolates showed acetylene reduction activity in nitrogen-free media and contained nifH genes which were PCR-amplified using specific primers. ERIC-PCR fingerprinting was used to compare the Cuban G. diazotrophicus isolates with type and reference strains of N2-fixing Gluconacetobacteria. The very low frequency of G. diazotrophicus isolates recovered Jesús Caballero-Mellado: In memoriam Electronic supplementary material The online version of this article (doi:10.1007/s13199-011-0133-3) contains supplementary material, which is available to authorized users. P. Ortega-Rodés : E. Ortega (*) : F. G. Loiret : R. Rodés Lab. Fisiología Vegetal, Dpto. Biología Vegetal, Facultad de Biología, Universidad de La Habana, La Habana, Cuba e-mail: [email protected] D. Kleiner University of Bayreuth, Bayreuth, Germany J. Caballero-Mellado Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Ap. Postal 565-A, Cuernavaca, Morelos, Mexico

is probably related with the high doses of nitrogen fertilizers applied to the sugarcane in the Cuban fields for almost 30 years. Some genetic differences, using ERICPCR, were detected among G. diazotrophicus strains, which could be related with its source. Keywords ERIC-PCR . Frequency of appearance . Gluconacetobacter diazotrophicus . Saccharicoccus sacchari . Sugarcane

1 Introduction Gluconacetobacter genus was proposed by Yamada et al. (1997) as one of the four genera for acetic acid bacteria. In the last few years novel acetobacteria species have been reported, and some other reclassified in the genus Gluconacetobacter. Species such as G. diazotrophicus, G. johannae and G. azotocaptans are able to fix nitrogen (Gillis et al. 1989; Fuentes-Ramírez et al. 2001). G. diazotrophicus, the best known nitrogen fixing bacterium of this genus, has been isolated from many different sources, and is one of the most common bacteria isolated from sugarcane tissues (Boddey et al. 2003). In addition, G. diazotrophicus has been isolated from the pink mealybug Saccharicoccus sacchari (Ashbolt and Inkerman 1990), an insect that lives associated to sugarcane as part of its life cycle (Inkermann et al. 1986). Thus, S. sacchari has been proposed as a vector of G. diazotrophicus to colonize the sugarcane internal tissues (Ashbolt and Inkerman 1990; Caballero-Mellado et al. 1995). Despite many efforts to isolate G. diazotrophicus from sugarcane fields in Cuba, very few reports have described its presence and a very limited number of isolates has been

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recovered in those studies (Coego et al. 1992; Dong et al. 1994, 1995; Loiret et al. 2004). For almost 30 years in Cuba high doses of nitrogen mineral fertilizers (100–150 kg N. ha−1) were applied for sugarcane production (Cuéllar et al. 2002), which could explain the very low isolation frequency of G. diazotrophicus from sugarcane. Furthermore, to our knowledge the isolation of G. diazotrophicus from mealybugs associated with sugarcanes cultivated in Cuba has not been reported. The aim of this study was to assess the presence of G. diazotrophicus in tissues of 11 varieties of sugarcane and of the mealybugs associated in four main sugarcaneproducing Cuban provinces.

stem protected by overlapping leaf sheath, were considered as potential host of G. diazotrophicus. 2.2 Growth media Isolation and nitrogenase activity assays were performed in LGI-P medium (Döbereiner 1993). DYGS medium (Rodrigues-Neto et al. 1986) modified without malic acid and modified potato agar medium (Cavalcante and Döbereiner 1988) were used as rich media for purification purposes. The isolates were alternatively streaked on LGI-P and potato agar modified or DYGS modified media to obtain pure cultures. 2.3 Isolation of bacteria

2 Materials and methods 2.1 Sample sources Seventy stems were collected by sampling five apical internodes of sugarcane plants of 8–12 months (11 varieties) from 14 combinations of varieties and localities. Locations, sugarcane varieties and fertilization levels are described in Table 1. Five colonies of mealybugs, S. sacchari, feeding from sugarcane stalks were sampled from every field and plant variety. The individual internodes, and female mealybug colonies feeding attached to the

Internode isolates were obtained from the sap of apical internodes of the canes. Pieces of stems were washed with tap water. The rind was removed with a sterile sharp knife, and the cylinder of parenchyma tissue rinsed with 70% ethanol and flamed. Sap from the internodes was collected by centrifugation following the method of Dong et al. (1994). Aliquots of the sap (100 μl) were spread onto Petri dishes containing 1.5% agar LGI-P medium (Reis et al. 1994). The centrifugation method (Dong et al. 1994) has been useful for the isolation of different species of endophytes,

Table 1 Locality, soil type, sugarcane variety and presence (+) or absence (−) of Gluconacetobacter-like bacteria in plant or insects (S. sacchaari) sampled Locality

Samples per locality

Soil type

Fertilization (kg N. ha-1) / Precipitation seasona

Mayabeque Sugarcane Research Station (western country)

25

Ferralic Rhodic Cambisol

100–150 / Wet

Jovellanos Sugarcane Research Station (central country)

15

Ferralic Rhodic Cambisol

Not fertilized for 5 yearsb / Wet

Camagüey Sugarcane Research Station (eastern country)

25

Chromic Cambisol

100–150 / Dry

Fontanar, Havana Ecological Farm(western country)

5

Ferric Rhodic Ferralsol

Not fertilized for 10 yearsb / Wet

a

Total rain three months before sampling: Wet, 400–600 mm; Dry, 50–150 mm

b

Relative to date of sampling

Variety

ML 3–18 My 5514 Ja 60-5 C 323-68 C 1051-73 ML 3–18 B 42231 CP 34-79 C85-371 C88-393 C123-94 C8612 C1051-73 ML 3–18

Source of microorganism Stem

Insect

− − − − − + + + − − − − − −

− − − − − − − − − − − − − +

Low recovery of G. diazotrophicus from sugarcane and mealybugs

G. diazotrophicus among them (Palus et al. 1996; Ortega et al. 2001; Loiret et al. 2004). Apoplastic sap contain sugars, other nutrients and conditions which permit the biological activity of the endophyte G. diazotrophicus and harbor it (Dong et al. 1994; Tejera et al. 2006) Female specimens of sugarcane pink mealybugs, S. sacchari, associated to the stems of sugarcanes were collected and surface sterilized as follows: washed with sterile distilled water, 5% chloramine T in H2O (5 min), 70% ethanol (2 min); and finally rinsed three times with sterile sucrose solution (5%). Insects were then squashed in 40 μl of sterile sucrose solution (5%) and centrifuged 10 min at 60 g to remove heavy debris maintaining the bacteria in the supernatant (Tejera et al. 2004). Aliquots of the supernatants (100 μl) were inoculated onto agar LGI-P medium plates. Only colonies able to growth in LGI-P medium after 7 days and with morphology similar to G. diazotrophicus were selected. Isolates were subcultured from each colony to obtain pure cultures. The isolates were compared to reference N-fixing strains of Gluconacetobacter (Table 2) using different physiological and biochemical tests (Gram stain, nitrate reductase, as Harrigan and McCance 1968; catalase, as Eising and Gerhart 1987; grow in 30% of sucrose, as Cavalcante and Döbereiner 1988; antibiotic resistance, as Ureta et al. 1995). Only one colony from each source with similar characteristics to G. diazotrophicus was selected for further work. 2.4 DNA extraction and electrophoresis conditions The isolates were grown on liquid DYGS medium for 12 h; cells were collected and resuspended on 350 μl TEN buffer. The protocol of Ausubel et al. (1987) was followed for DNA isolation. Agarose gels (0.8%) were prepared in electrophoresis buffer (66.88 mM Tris, 22.54 mM boric acid, 1.27 mM

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Na2EDTA x 2 H2O). After electrophoresis (80 V), DNA was stained in aqueous ethidium bromide solution (0.5 μg ml−1) and gels photographed. 2.5 Oligonucleotide primers and PCR conditions nifH gene amplification was done by using primers NifH-F and NifH-R (Rösch et al. 2002), with the following cycling parameters: 30 s at 94°C, 30 s at 60°C and 1.5 min at 72°C and a final extension at 68°C for 7 min; 35 cycles. 23S rRNA gene amplification was carried out using primers AD (as universal) and 1440 (as species specific) (Kirchhof et al. 1998), after 25 cycles as follows: 30 s at 94°C, 30 s at 52°C and 3 min at 68°C and a final extension at 68°C for 7 min. In addition, the species specific primers 5′-TGAGTAACGCGTAGGGATCTG-3′, and 5′-GGAAA CAGCCATCTCTGACTG-3′, which specifically amplify 16S rRNA genes from G. diazotrophicus, were used following the conditions previously described (FrankeWhittle et al. 2005). For comparison among G. diazotrophicus strains, ERIC1R and ERIC2 primers described by Versalovic et al. (1991) were used. ERIC-PCR amplifications were carried under these cycling conditions: 30 cycles of 30 s at 94°C, 30 s at 52°C and 3 min at 68°C and a final extension at 68°C for 7 min. All PCR reactions started at 95°C for 15 min, for activation of Hot Start Taq polymerase. The PCR reaction mixtures contained 400 ng of total DNA, 8 nmol L−1 of primer, 0.01 mmol L−1 nucleotides, 1 μL buffer 10X, and 5 units Hot Start Taq polymerase from Qiagen Taq PCR Master kit. 2.6 Nitrogenase activity Nitrogenase activity was estimated by the acetylene reduction assay (ARA) (Hardy et al. 1968) in 5 days

Table 2 Bacterial strains used in this study Bacterial strains

Host species

Source

PAl5T (G. diazotrophicus) CFNE 550 (G. diazotrophicus) CFNE 501 (G. diazotrophicus) UAP-5560 (G. diazotrophicus) T2 (G. diazotrophicus) CFN-Ca54T (G. azotocaptans) CFN-Cf55T (G. johannae) K-12 (Escherichia coli) FV-ML-1 FV-B42-1 FV-CP34-1 FV-MBF-1

Roots of sugarcane Mealybugs-CB 45 3 Stem of sugarcane Var. Z Mex 55 32 Stem of sugarcane Var. Z Mex 55 32 Nodal roots of sugarcane var. ML 3–18 (F) Rhizosphere of coffee plant Rhizosphere of coffee plant

Cavalcante and Döbereiner 1988 Caballero-Mellado et al. 1995 Caballero-Mellado et al. 1995 Fuentes-Ramírez et al. 1993 Loiret et al. 2004 Fuentes-Ramírez et al. 2001 Fuentes-Ramírez et al. 2001

Sugarcane stem var. ML 3–18 (Jv) Sugarcane stem var. B 42231 (Jv) Sugarcane stem var. CP 34–79 (Jv) Mealybug associated to sugarcane var. ML3-18 (F)

This This This This

study study study study

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2.7 Statistical analysis Statistical analysis was based on the use of the variance and Duncan test for multiple comparison with α=0.05 and α= 0.01. A dendrogram was made using NTSYSpc 2.1 program. Similarity between isolates was calculated using Dice Coefficient using phenotypic and genotypic characteristics (Dice 1945).

3 Results 3.1 Isolation and purification of nitrogen-fixing bacteria from sugarcane and mealybug We obtained 34 isolates displaying different colony morphologies and able to grow in LGI-P medium, of which only 13 showed similar colony morphology to G. diazotrophicus reference strains; however only four isolates showed nitrogenase activity and produced brown colonies on potato agar; the nine remaining isolates were not included in the next experiments. These isolates were named FV-ML-1, FV-B42-1, and FV-CP34-1 isolated from sugarcane stems, and FV-MBF-1 from sugarcane mealybug (Table 2). No nitrogen-fixing isolates were recovered from Havana or Camagüey Research Stations, neither from sugarcane stems nor from the colonies of mealybugs associated to sugarcanes. Microorganisms with the target characteristics were recovered from the stems of sugarcane of all varieties from the fields at the Jovellanos Research Station (103–104 CFU per mL of apoplastic sap); however, none isolate similar to G. diazotrophicus was recovered from the mealybugs sampled from the sugarcane plants growing at that Station. Despite all attempts to isolate G. diazotrophicus-like bacteria from mealybugs associated to sugarcane, only one isolate was obtained from the field in Fontanar (Table 2). 3.2 Characterization of the isolates Nitrogenase activity quantification of the isolates is shown in Fig. 1. FV-ML-1 strain showed the highest nitrogenase

700 a

nmol ethylene mg protein-1 h-1

cultures growing in semisolid LGI-P medium. Column filled with Porapak was used for gas chromatography. The samples were incubated in 10% acetylene for 2 h. Nitrogenase activity was expressed as nanomoles of ethylene produced per time unit and total protein content. Protein content of bacterial biomass, collected by centrifugation, was determined according to Bradford (1976) in cell suspensions heated for 3 min at 100°C in 1 M NaOH according to Cohjo et al. (1993). Bovine serum albumin was used as standard.


600 500 400

b b

300 b

200

b

100 0 FV-B42-1

FV-MBF-1

FV-ML-1 FV-CP-34-1

T2

Fig. 1 Acetylene reduction activity in G. diazotrophicus isolates after growth during 5 days at 30°C in semisolid LGI-P medium. Cultures were incubated 2 h in 10% acetylene for determinations. Data with different letters are significantly different at a P value of 0.05. n=5, SEx=85.33

activity among the new isolates, which was in the order of the ones described by Stephan et al. (1991) and FloresEncarnación et al. (1999). The nitrogenase activity in the bacteria isolated from the insect (FV-MBF-1) was also similar to the majority of the isolates obtained from the plants in this study. Supporting their nitrogen-fixing capacity, the five Cuban isolates (four new and T2) also amplified a 500 bp nifH gene fragment (Online Resource 1). These primers amplify the target nifH gene segment in a wide range of N2-fixing bacteria (Rösch et al. 2002). The Cuban isolates FV-ML-1, FV-B42-1, FV-CP34-1, FVMBF-1, T2 and the type strain PAl5 of G. diazotrophicus showed similar results in: Gram stain (−), NO3- reductase (−), motile (+), catalase (+), nitrogenase activity as ARA (+) and growth in 30% sucrose (+); however some differences were observed in antibiotic resistance (Table 3). A fragment of 23S rRNA gene with the expected size (411 bp) (Kirchhof et al. 1998) was amplified from the four isolates recovered in this study, from T2 and from the G. diazotrophicus reference strains (Online Resource 2). These isolates also produced a PCR-amplified product of the correct size (913-bp; Online resource 3) with G. diazotrophicus 16S rRNA gene specific primers (Franke-Whittle et al. 2005). Altogether, these evidences strongly supported that the isolates FV-ML-1, FV-B42-1, FV-CP34-1 and FV-MBF-1 belong to the species G. diazotrophicus. The majority of the strains produced PCR products using ERIC primers (Fig. 2). ERIC-PCR fingerprinting yielded relatively simple electrophoresis patterns, combining distinct DNA amplification products ranging approximately from 200 to 2500 bp. An 800 bp band was amplified in all the Cuban isolates, with FV-ML-1, FV-B42-1 and FV-CP34-1 (isolated from sugarcanes) showing identical profiles and sharing at least two amplification products with isolates FV-MBF-1 (isolated from mealybugs) and strain T2


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Table 3 Growth of the isolates in media with selected antibiotics according to Ureta et al. 1995 Antibiotica resistance

PAl5T

T2

FV-ML-1

FV-B42-1

FV-CP34-1

FV-MBF-1

Ch Amp Tc Str Km Cb Rif

− − − − − + +

− + − − − + +

− + − − − + +

− − − − − + +

− − − − − − +

− + − − − + +

Ch Chloramphenicol 150 μg. mL−1 ; Amp Ampicillin 75 μg. mL−1 ; Tc Tetracycline 10 μg. mL−1 ; Str Streptomycin 100 μg. mL−1 ; Km Kanamycin 100 μg. mL−1 ; Cb Carbenicillin 250 μg. mL−1 ; Rif Rifampicin 10 μg. mL−1

a

(isolated from sugarcane roots, Loiret et al. 2004). Isolate FV-MBF-1 and strain CFNE550 showed simpler but very similar ERIC patterns (Fig. 2). A cluster analysis that included all the above characteristics further supported that the Cuban isolates belong to the G. diazotrophicus species (Fig. 3), which was clearly differentiated from other species like G. johannae and G. azotocaptans. Isolates FV-ML-1, FV-B42-1 and FV-CP34-1, all collected within sugarcane stems, formed a separated subgroup, whereas FV-MBF-1 (isolated from mealybug) clustered with strain CFNE 550, also isolated from mealybug (Caballero-Mellado et al. 1995) and the type strain UAP 5560. The two strains isolated from S. sacchari were therefore included in this last sub-group, indicating certain similarity among them. However, CFN550 and UAP-5560 were clearly differentiated. This fact, together with data indicating that strains UAP 5560 and CFNE 550 belong to different ETs group (Caballero-Mellado et al. 1995), support

Fig. 2 DNA electrophoresis of PCR amplification products obtained with primers ERIC1R and ERIC2. Lane 1, 100 pb Ladder Plus; lane 2, FV-MBF-1; lane 3, FV-ML-1; lane 4, G. diazotrophicus T2; lane 5, FV-B42-1; lane 6, FV-CP34-1; lane 7, G. diazotrophicus PAl5T; lane 8, G. azotocaptans CFN-Ca54T; lane 9, G. johannae CFN-Cf 55T; lane 10, G . diazotrophicus CFNE 550; lane 11, G . diazotrophicus UAP 5560; lane 12, 100 pb Ladder Plus

the suitability of our methodology to compare strains of G. diazotrophicus.

4 Discussion Sugarcane has been cultivated in Cuba for more than five centuries as a main crop from western to eastern part of the island, however reports of finding G. diazotrophicus associated with the plant are very scarce and null with mealybug. We hypothesized that it could be possible to recuperate the strain from the plant and the insect with relative abundance. Seventy plants growing in 14 fields of sugarcane and their associated mealybugs (20 colonies) in four locations of Cuba were explored for the presence of G. diazotrophicus, however only the plants from three fields (21%) and the mealybugs from one field (7%) harbored G. diazotrophicus, evidencing a low frequency of recovery (Table 1). According to the morphology of the colonies, growth on LGI-P medium, to form brown colonies on Potato-agar medium (Cavalcante and Döbereiner 1988), biochemical, and molecular features, isolates FV-ML-1, FV-B42-1, FV-CP34-1 and FV-MBF-1 are strains of G. diazotrophicus. González et al. (2004) used ERIC-technique to differentiate acetic acid bacteria at the genus level. Using ERIC-PCR we were able to obtain differences among strains of G. diazotrophicus, what shows the usefulness of this technique to detect differences between strains of G. diazotrophicus (Fig. 2). G. diazotrophicus, Herbaspirillum spp. Azospirillum spp. Pantoea sp. and Burkholderia spp. have been isolated from sugarcane cultivars (Baldani et al. 1997; 2002; Boddey et al. 2003; James 2000; Loiret et al. 2004; Castro et al. 2011; Omarjee et al. 2008). Von Dohlen et al. (2001) and Thao et al. (2002) reported primary and secondary endosymbionts in mealybug but they did not work with pink sugarcane mealybug. Ashbolt and Inkerman (1990), Caballero-Mellado et al. (1995) and Franke et al. (2000) isolated acetic acid bacteria, α-proteobacteria from pink

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Fig. 3 Dendrogram showing relationships among G. diazotrophicus isolated from S. sacchari (FV-MBF-1), from plants of Cuban (T2; FV-ML-1; FV-B42-1; FV-CP34-1), Mexican (UAP-5560; CFNE-550) and Brazilian (PAl5T) varieties of sugarcane, and G. azotocaptans (CFN-Ca54T) and G. johannae (CFN-Cf 55T ) isolated from coffee rhizosphere in Mexico. Similarity coefficient Dice were obtained using genotypic and phenotypic data

sugarcane mealybugs; this is the first report of G. diazotrophicus isolated from pink mealybug associated with sugarcane in Cuban conditions. Because mealybugs live in close association with sugarcane stems during part of their life cycle (Inkermann et al. 1986), it has been proposed that S. sacchari could be a vector for G. diazotrophicus spreading in sugarcane cultivars (Ashbolt and Inkerman 1990). The insects might become infected during sap feeding and inoculate the bacteria to other stems. Caballero-Mellado et al. (1995) suggested that G. diazotrophicus is sucked from sugarcane by S. sacchari and the population within insect would be a subset of the sugarcane population. In contrast Ashbolt and Inkerman (1990) suggested that G. diazotrophicus may be autochthonous microbiota of mealybugs associated with sugarcane. In this study we recovered different strains isolated from cane internodes and insects. Since the mealybug isolate originated from a different field than the three sugarcane isolates, it is not possible to speculate about their primary origin, plant or insect. However, the fact that G. diazotrophicus was more frequently isolated from the plant than from the insects suggests that the primary host would be the plant and the insect would be a transmission vector, and that perhaps the insects do not provide the optimal conditions for multiplication or survival of the bacteria. If the strains differ due to isolation from the plant or insect, the function as transmission vector suggested by Ashbolt and Inkerman (1990) would not exist. We were able to recover G. diazotrophicus from mealybugs in 1 out of 20 colonies associated with plants from 11 varieties growing in 4 localities; if G. diazotrophicus were an autochthonous microbiota of insect as suggested, it should be possible to recover strains of G. diazotrophicus more frequently from the insect. We extend the results of Versalovic et al. (1991), who demonstrated that ERIC-like sequences are present in many diverse eubacteria, and can be an efficient target for primer

binding sites to produce fingerprints of different bacterial genomes. Three main groups disjoin from the cluster analysis of strains (Fig. 3), stressing that the strains FV-ML-1, FV-B42-1, FV-CP34-1 and FV-MBF-1 collected from sugarcane stems and from the insect in Cuba belong to G. diazotrophicus, and differentiate from very related species like G. johannae and G. azotocaptans. The strain of G. diazotrophicus isolated from S. sacchari in Cuba shows certain but not complete similarity with the one isolate from the insect in Mexico. The fact that the sugarcane plants harbor nitrogen fixing microorganisms could be an excellent contribution to the plant nitrogen nutrition via BNF. In our work it was demonstrated the presence of G. diazotrohicus within the stem of sugarcane plants from 3 varieties (ML 3–18, B 42231 and CP 34–79) coming from fields in Jovellanos Research Station. These sugarcane genotypes have been used as commercial varieties for sugar production, as well as parents for obtaining new cultivars of sugarcane within the Cuban breeding programs (Pérez et al. 1997). Var. B 42231 and ML 3–18 are used as parents in breeding programs as they provide vigor and high sucrose content to the descendants. Other unknown characteristics (like the capacity to harbor nitrogen fixing bacteria) could be valuable to select for in the descendants. Boddey et al. (2003) considered that the plant genotype has an important role among the factors necessary to stimulate BNF in sugarcane. Three varieties from Jovellanos Research Station harbor natural G. diazotrophicus populations, and therefore could have certain genetic characteristics, which together with the environmental conditions, may facilitate the establishment of this beneficial plant-bacteria interaction. Different environmental factors have been suggested to influence the population of G. diazotrophicus within sugarcane plants. dos Reis et al. (2000) suggested that the decrease in the bacterial number of G. diazotrophicus was due to high levels of N fertilization and other environmental


factors, mainly rainfall; Muñoz-Rojas and CaballeroMellado (2003) further emphasized the negative effects of N fertilization. According to our results, it seems that the low frequency of isolation for G. diazotrophicus from sugarcane tissue or S. sacchari is more related to the high doses of nitrogen fertilization than with rainfall (Table 1). Some efforts have been made to isolate nitrogen-fixing bacteria from sugarcane plants in Cuban fields, but the presence of G. diazotrophicus has been confirmed in few studies and at very low frequencies (Coego et al. 1992; Dong et al. 1994, 1995; Loiret et al. 2004). It is noteworthy that the few isolates recovered from plants analyzed in three studies (Dong et al. 1994, 1995; Loiret et al. 2004) came from sugarcane growing in fields where low or no nitrogen fertilizers were applied for at least 10 years before sampling. No information is available concerning N fertilization levels applied in the fields where Coego et al. (1992) isolated only one strain (SRT4) of G. diazotrophicus. Likewise, we recovered G. diazotrophicus at very low frequencies from sugarcane cultivated in fields from four Cuban provinces with medium to high relevance for the crop. Thus, the low frequency of G. diazotrophicus found in this and other studies could be a consequence of the relatively high nitrogen fertilization (100–150 kg N.ha−1) used during long periods, almost 30 years, in Cuba (Cuéllar et al. 2002). In fact, the recovery from sugarcane occurred in field where N fertilizer had not been applied since 5 years before our sampling (Table 1). Considering the promoting effects of G. diazotrophicus on sugarcane plant grow (Muñoz-Rojas and CaballeroMellado 2003; Sevilla et al. 2001) it should be convenient to re-inoculate the bacteria into the plant, particularly in Cuba and some other countries where micropropagation of sugarcane is used for production purposes (Tian et al. 2009; Engelmann et al. 1999). Acknowledgements This research was supported by the grant for the joint Project Cub00/02 between the Cuban Ministry for Science and Environment (CITMA) and German Bundenministerium fur Bildung und Forschung (BMBF). The authors also appreciate the support of the grant C/4226-1 from the International Foundation for Science (IFS). The technical assistance of Milagros Diez-Cabezas and Helga Castorph is also acknowledged. Authors appreciate very much the critical review of Dr. Juan Sanjuan at CSIC. Authors dedicate this paper to the Memory of Jesús Caballero-Mellado, excellent scientist and wonderful person.

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