The effect of inoculating endophytic N2-fixing bacteria on ...

Plant and Soil 242: 205–215, 2002. © 2002 Kluwer Academic Publishers. Printed in the Netherlands.

205

The effect of inoculating endophytic N2 -fixing bacteria on micropropagated sugarcane plants A.L.M. Oliveira1, S. Urquiaga2, J. Döbereiner2,† & J.I. Baldani2,3 1 Biotechnology, Universidade

Federal do Rio de Janeiro, Ilha do Fundão, Rio de Janeiro, Brazil Agrobiologia, BR 465, Antiga Estrada Rio-Sao Paulo, Km 47, Serop´edica, Rio de Janeiro, CEP 23851970, Brazil 3 Corresponding author∗ . Current address: Embrapa Agrobiologia BR465, Antiga Estrada Rio-São Paulo, CEP: 23851-970, Serop´edica, Rio de Janeiro, Brazil 2 Embrapa

Key words: biological nitrogen fixation, endophytic bacteria, plant–bacteria interactions, plant inoculation, sugarcane

Abstract The aim of this work was to evaluate the effect of the inoculation of endophytic N2 -fixing bacteria on the development of micropropagated sugarcane plants. The endophytic population of each inoculated species was monitored during the growth period, and biological nitrogen fixation (BNF) contribution of each inoculation treatment was assessed using the 15 N-isotope dilution technique. Seven different combinations of inoculum were used, using five endophytic diazotrophic species (Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae, Herbaspirillum rubrisubalbicans, Azospirillum amazonense and Burkholderia sp.), originally isolated from sugarcane plants. The results showed a clear physiological effect on the development of the inoculated plants, resulting in alteration of the dry matter-partitioning pattern and increase on root dry matter as compared to uninoculated plants. Indeed, all inoculated diazotrophic species could be reisolated in high numbers from the rhizomes of the inoculated plants, even 400 days after inoculation (DAI), suggesting the establishment of the inoculated bacteria. However, a negative effect of the mixture of all five species on the survival of plantlets was observed 45 days after inoculation, just after acclimatization. The analysis of the BNF contribution using the 15 N-isotope dilution technique showed that inoculation promoted some increase in the BNF contribution to the plant tissues. The best treatment was the mixture of all five strains, followed by the treatment with a mixture of Herbaspirillum spp. The contribution was much lower when the plants were inoculated with a mixture of G. diazotrophicus with A. amazonense and Burkholderia sp. A BNF contribution around 30% of total nitrogen accumulated was observed in micropropagated plants inoculated with the mixture of strains, suggesting that the combination of species in the inocula is the best strategy to improve sugarcane crops dependent on the biological nitrogen fixation process. Introduction Sugarcane is one of the most important crops in Brazil with a cropping area of 4.6 million ha. In general low inputs of nitrogen fertilizer to sugarcane are routine in Brazil because of the low yield response to N application (Azeredo et al., 1986). Actually, the N fertilization recommended by COPERSUCAR (the largest Brazilian cooperative of sugar farmers and industri∗ FAX No: +50-21-21-2682-1230.

E-mail: [email protected] † Deceased.

alists) is 50 kg N/ha/year in the plant cane, and a maximum of 100 kg N/ha/year to subsequent ratoon crops (COPERSUCAR, 2000). Several authors observed low sugarcane response to N fertilization in Brazil (Lima et al., 1987; Ruschel et al., 1975; Trivelin et al., 1995; Urquiaga et al., 1992) and found evidences that the biological nitrogen fixation (BNF) is one of the processes involved with the observed effect. Urquiaga et al. (1992) observed variations on BNF in the range of 12.8% (S. barberi var. Chunee) to 34% (Saccharum sp. var. SP-70 1143) in the first harvest. The Saccharum sp. commercial

206 hybrids var. SP-70 1143 and CB 45-3 were the most promising genotypes, with the nitrogen derived from BNF reaching a potential of 70 and 68%, respectively (mean of three years). The role of BNF in graminaceous plants begun to be elucidated with the discovery of the semi-solid N-free media by Dra. Johanna Döbereiner group’s in the 1970s (Döbereiner et al., 1972). Several diazotrophic species were isolated includding the endophytes isolated from sugarcane plants growing in Brazil. These include Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae and Herbaspirillum rubrisubalbicans, Azospirillum amazonense and a Burkholderia species provisionally named ‘B. tropicalis’ (Baldani et al., 1997; Döbereiner et al., 1995; James, 2000). These bacteria have been detected in very high numbers (ranging from 104 up to 107 cells per gram of fresh weight) and it has been postulated that they are effective in supplying N to the plant host. The role of such endophytic diazotrophic species on sugarcane growth, individually or in a complex endophytic community is not yet known. Few specific inoculation experiments are available on sugarcane (James and Olivares, 1998; Moraes and TaukTornisiello, 1997; Muthukumarasamy et al., 1999; Paula et al., 1991; Sevilla et al., 1998). Sugarcane micropropagation began in Brazil in the 1970s but it was commercially introduced about 10 years ago. The most convincing advantages presented by the micropropagation process is the clonal cleaning from pathogenic endophytes such as Leifsonia xilii (ratoon stunting disease pathogen). However, besides eliminating the pathogenic micro-organisms, the process virtually eliminates all the other beneficial organisms that could promote plant growth. The use of micropropagated sugarcane to reintroduce selected strains of diazotrophic endophytic bacteria by the inoculation procedure opens new avenues to any selected strain to colonise such axenic plants, diminishing the competition by the native community during the initial stage of root colonization. The aim of this work was to evaluate the effect of endophytic diazotrophic bacteria on the development of a micropropagated Brazilian sugarcane variety. The endophytic population of each inoculated species was monitored during the growth period, and BNF contribution of each inoculation treatment was accessed using the 15 N isotopic dilution technique.

Materials and methods Plants Sugarcane micropropagated plants of SP 70-1143, a Brazilian variety with high BNF potential (Urquiaga et al., 1992), was used as a host plant to evaluate the effect of the bacterial endophytic diazotrophs as inoculant. The plants were micropropagated according to James et al. (1994), inoculated and acclimatized at the CTC, (Centro de Tecnologia Copersucar, Piracicaba, SP, Brazil). After the plantlets had been rooted, the clumps were split and four to five individualised plants were transferred to flasks with 50 mL of MS media modified by Reis et al. (1999), were N and all the other salts were reduced to one-tenth of the original concentration and no plant hormones were present. Forty-eight hours after plant transfer, only those flasks with no apparent contamination were used for inoculation with the endophytic diazotrophic bacteria. A microbiological analysis was carried out in random samples to be sure that no diazotrophic endophytes were initially present on such plants. The effect of the nitrogen residues present on the MS modified media (1/10 of the original ionic strength) was not taken in account since the plants remained 1 week on this medium, and the metabolic activity is not so high at this stage because of the limitations of light and nutrients. Therefore the N assimilated by these plants did not influence the plant growth. In addition, the plants were acclimatized in a substrate and transferred to soil, both containing N. Inoculation Plant inoculation was performed as described by Reis et al. (1999). All bacterial strains were grown overnight in Dyg’s liquid media (Weber et al., 1999). A 100-µL bacterial suspension was inoculated into flasks containing micropropagated plants as previously described, providing a initial bacterial populations in the MS medium of approximately 2.0 × 105 cells per mL. Three flasks for each inoculation treatment were used. The number of cells reach up to 3.4 × 108 cells per ml of inoculation media 7 days after inoculation (unpublished data). The very small ammount of rich media used as inoculum (100 µL) and considering the long term experiment (12 months) let us consider that these factors did not influence the plant development since they remained on these flasks only for a week. The endophytic diazotrophic bacteria used in this study were originally isolated from

207 Table 1. Endophytic diazotrophic strains used and source of isolation∗ Name

Strain

Tissue associated

Sugarcane variety

Gluconacetobacter diazotrophicus (Gd) Herbaspirillum seropedicae (Hs) Herbaspirillum rubrisubalbicans (Hr) Azospirillum amazonense (Aa) Burkholderia sp. (Bt)

BR 11281 BR 11335 BR 11504 BR 11115 BR 11366

Roots Roots Stems Roots Germinated buds

Saccharum sp. hybrid SP 701143 SP 701284 SP 775181 SP 711406

∗ From Embrapa Agrobiologia Culture Collection.

sugarcane, and belongs to the Embrapa Agrobiologia Culture Collection (Table 1). Several combinations of strains were used to inoculate the micropropagated sugarcane plants, and were based on their frequency of occurrence in field grown sugarcane plants, priorizing the combination of G. diazotrophicus and Herbaspirillum spp. Uninoculated plants were used as a control. Previous work showed that the small ammount of inocula (100 µL) and short time period of the plant in this medium (5 days) did not influence the plant growth. Additional information can be found in James et al. (2001). Acclimatization Five days after incubation of the inoculated sugarcane plants, they were transferred to individual plastic cells filled with commercial seedling non-sterile substrate (PLANTMAX from EUCATEX , São Paulo, Brazil) with a total of 72 plants for each treatment. The plants were acclimatized for 45 days after inoculation (DAI) in a greenhouse with controlled humidity, temperature and water supply (COPERSUCAR, 1989), and then transferred to pots containing 15 N-labelled soil. The process applied here is routinously followed by sugarcane farmers in many countries of the world working with micropropagated sugarcane plants. Culture media The inoculated strains were reisolated in semi-specific semi-solid N-free and solid selective media. LGI-P with sugarcane extract was used to reisolate G. diazotrophicus, LGI to reisolate A. amazonense, JMV to reisolate Burkholderia sp. and JNFB to reisolate H. seropedicae e H. rubrisubalbicans (Döbereiner, 1992; Döbereiner et al., 1995b).

Bacterial counting (MPN) The most probable number method was used to monitor the bacterial endophytic population. The plants were washed in tap water, and then surface sterilised with 1% chloramine T solution for 5–10 min, according to the plant age, and then rinsed three times with sterile distilled water for 10 min each time. This treatment has been shown to eliminate the surfacecolonizing bacteria in different gramineous plants (Barraquio et al. 1997; Biró et al., 2000; Stolzfus et al., 1997). Unattached and/or loosely attached target bacteria were not considered although it is that part of the inoculated bacteria could be at the surface of the roots. Reports on survival of inoculated endophytic species have showed a poor survival of these bacteria in the soil environment (Baldani et al., 1997). One to 10 g of fresh weight (according to the plant age) was macerated in isotonic sterile solution (NFB medium salts, in 20% of ionic strength) with pestle and mortar or blender, depending on material resistance, diluted in the same solution in a 10% volume each dilution (until reach the 10−6 dilution). Aliquots of 100 µL of each dilution were inoculated in semi-solid selective media into 5-mL vials (LGI-P, JNFB, JMV or LGI), to reisolate the inoculated species G. diazotrophicus, Herbaspirillum spp., Burkholderia sp. and A. amazonense, respectively. There were three replicates for each dilution. Ten days after inoculation the vials were analysed for the presence or absence of a characteristic veil pellicle formed on semi-solid N-free media. The characterization of bacteria species isolated on semisolid N-free media was also confirmed based on the characteristic of the colony morphology formed on solid medium and also by microscopic analysis. The number of cells present in samples was calculated using McCrady tables (Döbereiner et al., 1995b, Tarrand et al, 1978).

208 Experimental design There were two sizes of the pots: pots of 20-L capacity to evaluate the plant growth and bacterial establishment at an earlier stage (200 days). For the long term analysis (400 days) pots of 60-L size were used. The 60 litter’s pots were filled with 50 kg of a B-horizon of a red-yellow podzolic soil Itaguai series (typic Hapludult) from the Embrapa-Agrobiologia experimental field (Seropédica-RJ) while pots of 20-L were filled with 20 kg of the same soil. After chemical analysis, the soil was amended with all major (P and K) and minor sugarcane nutrients (Mg, Ca, Zn, Mo, Cu, etc., Urquiaga et al., 1992). 15 N-enriched ammonium sulphate (10-at m % 15 N) was added at a concentration of 10 ppm (about 20 kg of nitrogen per hectare) plus 120 g of Brachiaria trash (powdered aerial plant tissues) to immobilise the N and limit the N availability only for 60-L pots. The smaller pots also received the same amount of nitrogen (ammonium sulphate) but not labelled. There were eight treatments, two harvest times and three replicates totalling 48 pots. The pots were arranged in a completely random design outside the greenhouse. Filtered tap water was used to irrigate pots when necessary. The inoculated and acclimatized plants were harvested 200 and 400 days after planting and sampled for microbiological analysis and 15 N enrichment. Nitrogen derived from air (Ndfa) Nitrogen fixation by the plants in the pots was measured by first determining the weight of the dry matter by drying the stems, trash (dead leaf material), green leaves, rhizomes and root samples for 1 week in an oven at 60◦C. The samples were powdered in two stages. Firstly the plant samples were grinder in a wiley mill and then powdered in a ball mill. Two to four milligrams of each sample were used to determine the 15 N enrichment in a mass spectrometer (Delta Plus, Finnigan, U.K.). The contribution of nitrogen derived from air (Ndfa) was estimate by the equation (Urquiaga et al., 1992): Ndfa = 100 × [1 − (at m % 15 N excess inoculated plant/at m % 15 N excess control plant)] Statistical analysis The experiment was designed to evaluate the effects of mixtures of endophytic diazotrophic bacteria as inocula, in supplying N to micropropagated sugarcane

plants. The unexpected interactions among sugarcane plants and endophytic diazotrophic bacteria, and interactions of endophytic diazotrophic inocula and natural colonizing endophytes pushes the error out of a normal distribution, as confirmed by Lilliefors test applied for normality. Since the micropropagated sugarcane plants have high genetic uniformity, it becomes difficult to evaluate the variation on growth parameters using parametric statistics. Therefore, the statistical analyses were carried out using tests analogous to F-test for confidence intervals, (Kruskal-Wallis test; Sprent, 1989). The confidence intervals were always 95% (α = 0.05). Nitrogen derived from air, fresh and dry weights, plant colonization by diazotrophic endophitic species, and the dry matter partitioning between roots and aerial parts, sucrose content and number of plants per pot were evaluated.

Results Establishment of the inoculated endophytic diazotrophic bacteria It was assumed that the surface sterilization procedure was able to eliminate the non-endophytic bacteria present at the surface of the sugarcane tissues. MPN counting carried out just after the inoculation period (7 days after inoculation) on surface sterilized plant tissues, resulted in populations up to 107 cells per gram of fresh weight of inoculated bacteria and no bacteria on the control (uninoculated) plants (Table 4). Diazotrophic bacteria were successfully reisolated from both roots and aerial parts of the inoculated plants (Tables 2 and 3). The only exception was at the last harvest (12 months) when no bacteria could be reisolated from the aerial parts (Table 3). The endophytic population of the root tissues reached approximately the same number of cells by the end of the experiment in all inoculated treatments. This suggests that the endophytic population was not influenced by the initial inoculum content, and probably was controlled by the host plant. The uninoculated plants showed an increase of the naturally colonised endophytic population, such as Azospirillum-like, Herbaspirillum-like, Burkholderialike and in a smaller frequency of G. diazotrophicuslike species. Those species were probably originated from soil, water supply, air or neighbour inoculated sugarcane plants. The major bacteria present in the control plants were Azospirillum and Burkholderialike bacteria. Soil associated tissues, such as roots and

209 Table 2. Endophytic population present in roots of micropropagated sugarcane plants inoculated with a mixture of different diazotrophic bacteria (values represent mean of three replicates) Inoculation treatment1

Species recovered (log number of cells/g−1 fresh weight) G. diazotrophicus Herbaspirillum spp. A. amazonense Burkholderia sp. (Lgi-P) (JNFB) (LGI) (JMV) Days after inoculation Days after inoculation Days after inoculation Days after inoculation 45 200 400 45 200 400 45 200 400 45 200 400

Control plant (not inoculated) Gd Gd+Hs+Hr Gd+Bt+Aa Mixture of all five species Hs+Hr Hs+Hr+Bt+Aa Bt+Aa

BD∗ 3.68 5.93 6.40 4.44 ND∗∗ ND ND

1.28 5.13 5.81 6.03 5.98 ND ND ND

1.95 5.40 5.48 6.95 5.60 ND ND ND

3.43 ND 6.15 ND 4.43 6.15 5.52 ND

3.92 ND 6.15 ND 6.86 6.15 5.93 ND

4.25 ND 5.85 ND 5.95 4.60 6.18 ND

2.73 ND ND 4.73 3.32 ND 4.10 2.73

5.03 ND ND 6.08 6.23 ND 5.78 6.32

4.60 ND ND 7.65 5.60 ND 5.48 3.95

1.80 ND ND 3.97 2.13 ND 2.53 4.60

3.46 ND ND 4.02 4.92 ND 3.04 4.04

5.60 ND ND 6.66 6.18 ND 5.48 7.65

1 See Table 1 for strains identification. ∗ BD, Number of cells below the minimum level of detection. ∗∗ ND, not determined. Countings were carried out on semi-solid media and varied according to the inoculated bacteria, except for the

control plants where all media were used. Table 3. Endophytic population present in aerial parts of micropropagated sugarcane plants inoculated with a mixture of different diazotrophic bacteria (values represent mean of three replicates) Inoculation treatment1

Species recovered (log number of cells/g−1 fresh weight) G. diazotrophicus Herbaspirillum spp. A. amazonense Burkholderia sp. (Lgi-P) (JNFB) (LGI) (JMV) Days after inoculation Days after inoculation Days after inoculation Days after inoculation 45 200 400 45 200 400 45 200 400 45 200 400

Control plant Gd Gd+Hs+Hr Gd+Bt+Aa Mixture of all five species Hs+Hr Hs+Hr+Bt+Aa Bt+Aa

BD∗ 1.39 5.51 5.12 5.77 ND∗∗ ND ND

2.38 3.50 3.68 5.70 6.59 ND ND ND

BD BD BD BD BD ND ND ND

1.66 ND 5.48 ND 6.14 4.86 5.83 ND

5.96 ND 6.40 ND 6.69 5.32 5.74 ND

BD ND BD ND BD BD BD ND

1.10 ND ND 4.15 3.57 ND 4.79 4.26

5.55 ND ND 6.00 6.61 ND 5.59 6.58

BD ND ND BD BD ND BD BD

1.13 ND ND 3.81 3.85 ND 3.75 1.99

1.99 ND ND 5.74 5.59 ND 4.75 5.94

BD ND ND BD BD ND BD BD

1 See Table 1 for strains identification. ∗ BD, Number of cells below the minimum level of detection. ∗∗ ND, not determined. See explanation in Table 1.

rhizomes presented an earlier colonization of naturally occurring diazotrophic endophytes, as compared to other plant parts (Tables 2 and 3). It is important to note that the mixture of all endophytic diazotrophic species as inoculum had a negative effect on plant survival rate after acclimatization (Table 4). Indeed, the inoculum mixtures, which presented the lowest survival of cane plantlets, were always associated with Herbaspirillum. However no symptoms of pathogenicity were observed on any inoculation treatment.

Inoculation effects on plant development Analysis carried out at 45 days showed that G. diazotrophicus promoted a significant increase in root dry matter as compared with the other inoculant treatment (Table 5). On the other hand, A. amazonense and Burkholderia sp. induced a significant decrease on root dry matter as compared to the other treatments, including uninoculated control plants. Similar effect was observed when inoculated in a mixture containing

210 Table 4. Effect of inoculation with different endophytic bacteria on the sugarcane plantlet survival Treatment1

Survival rate2

Control plant Gd Hs+Hr Gd+Hs+Hr Gd+Bt+Aa Hs+Hr+Bt+Aa Bt+Aa Mixture of all five species

88.88 A5 93.05 A 73.61 AB 83.33 A 88.88 A 69.44 BC 97.22 A 58.33 D

Total bacterial counts (log number of cells) 45 Days4 7 Days3 BD∗ 7.14 7.14 7.45 7.62 7.62 7.45 7.75

3.52 3.68 6.15 6.35 6.41 5.54 4.61 4.75

1 See Table 1 for strains identification. 2 Percentage of live plants out of 72 after the acclimatization period (45 days after inoculation). 3 Means of four surface sterilized plantlets at 7 days. 4 Means of three samples of surface sterilized roots at 45 days. 5 Values followed by same letter

did not differ significantly from Kruskal–Wallis test at 5% of significance.

∗ BD, Number of cells below the minimum level of detection.

Table 5. Total dry matter accumulation and shoot-to-root (S/R) ratio of micropropagated sugarcane plants inoculated with a mixture of different bacteria combination (values represent for mean of three replicates) Inoculation treatment1 45 DM2

S/R3


2.72 B4 3.70 A 2.72 B 2.57 B 2.32 B 1.97 C 1.91 C 2.75 B

1.65 B 1.15 C 2.20 A 2.07 A 1.23 C 1.45 B 1.77 B 1.47 B

CV%

29.8

Days after inoculation 200 DM S/R 34.48 B 47.99 A 43.66 A 52.07 A 47.82 A 36.76 B 47.29 A 43.51 A

2.21 A 0.39 B 1.40 A 0.82 A 0.90 A 1.00 A 0.95 A 0.81 A

27.2

400 DM

S/R

476.7 B 438.8 C 645.8 A 448.6 C 563.3 B 549.5 B 474.7 B 599.2 A

3.6 D 2.9 C 2.2 B 2.4 B 1.9 A 2.9 C 2.5 B 1.7 A

27.4

1 See Table 1 for strains identification. 2 DM, Total dry matter accumulation in grams per plant. 3 S/R, shoot-to-root ratio. 4 Values followed by same letter did not differ significantly from

Kruskal–Wallis test at 5% of significance. CV%, coefficient of variance.

Herbaspirillum spp. but not with G. diazotrophicus (Table 5). A significant increase in dry matter production due to the inoculation was observed, mainly with G. diazotrophicus plus Herbaspirillum spp., as compared to uninoculated plants at 200 DAI. However, plants inoculated with a mixture of Herbaspirillum spp. plus Burkholderia sp. plus A. amazonense did not differ from uninoculated plants (Table 5). On the other hand, the inoculation with only G. diazotrophicus species promoted a slight decrease on dry matter accumulation of aerial parts (200 DAI), but it stimulated an increase

in the dry matter production of roots as compared to uninoculated plants (data not shown). The shoot-toroot ratio of plants at the beginning of the development (200 DAI) confirmed the decrease in dry matter of aerial parts when G. diazotrophicus was used as inoculant (Table 5). Statistical significant differences between aerial and rooted parts at 400 days after inoculation were observed for all inoculum mixture, as compared to uninoculated plants (Table 5). There was a generalized tendency of transferring carbon supply to roots in all inoculum mixture tested. The plants inoculated

211 Table 6. Effect of inoculation with different endophytic diazotrophic bacteria mixture on the stalk number and stalk fresh weight per pot in grams, 400 days after inoculation (values represent for mean of three replicates) Treatment1

Stalk number per pot

Stalk fresh weight


7.3 5.3 5.7 5.7 5.0 2.3 4.3 4.7

803.9 D2 651.6 G 974.7 A 696.9 F 871.8 BC 844.2 C 730.2 E 893.3 B

CV%

48

23.5

1 See Table 1 for strains identification. 2 Values followed by

same letter did not differ significantly from Kruskal–Wallis test at 5% of significance.

with G. diazotrophicus plus A. amazonense plus Burkholderia sp. or with a mixture of all five endophytic diazotrophic bacteria species, promoted the highest differences on dry matter partitioning as compared to control plants (Table 5). The inoculation with Herbaspirillum spp. induced the greatest increase in total dry matter as compared to all others inoculum combinations, while inoculation with G. diazotrophicus strain promoted a significant decrease on dry matter of stems as compared to uninoculated control plants (Table 5). A relatively high difference between the highest and the lowest value for number of plants per pot was observed (Table 6). However, no statistical difference was observed probably due to the high coefficient of variance. The stalk fresh weight measured after 400 days of inoculation shows both positive and negative effects due inoculation treatment (Table 6). The inoculation treatments with Herbaspirillum spp., followed by mixture of all five species, and the inoculation with A. amazonense and B. tropicalis plus G. diazotrophicus or plus Herbaspirillum spp. presented significative increases on stalk fresh weight as compared to uninoculated control plants. On the other hand, inoculation with A. amazonense and B. tropicalis without the presence of G. diazotrophicus or Herbaspirillum spp., inoculation with G. diazotrophicus plus Herbaspirillum spp. and the inoculation of sugarcane plantlets with G. diazotrophicus alone presented decreases on stalk fresh weight as compared to uninoculated control plants. The plants inoculated with Herbaspirillum

spp. and with a mixture of all five species promoted the highest increase on total dry matter and stalk fresh weight among all inoculum treatments (Tables 5 and 6). The cane yield presented is apparently low because the harvest of plants was made before the end of normal growth period, and the pots limited growth. BNF contribution The biological nitrogen fixation (BNF) contribution of the inoculated diazotrophic bacteria was measured by the 15 N isotopic dilution technique. There was a certain consistence on the values of BNF contribution in all plant tissues sampled, except to the roots and trashes where quite variable results were observed (data not shown). Therefore, BNF contribution was calculated based on the indicator leaf (leaf +3) and the rhizomes of plants grown 12 months in the pots (plus 45 days of acclimatization). The results showed that all the inoculated bacterium, either as a single or in a mixture, contributed to the amount of nitrogen accumulated in the plant (Table 7). The BNF contribution varied from 3 to almost 24% when the rhizome material was analysed, and these values varied from 3.6 to almost 34% when the leaf +3 tissues were used to calculate the BNF. However, the best inocula treatment was the same using either rhizome or leaf as sample (Table 7). The highest BNF contribution was obtained when a mixture of all five endophytic diazotrophic species was inoculated (23.7% for the rhizome and 33.7% for the leaf +3). The second best treatment was the inoculation with Herbaspirillum spp. (17 and 11.9%). On the other hand, the lower BNF contribution was observed when the plants were inoculated with a mixture of G. diazotrophicus, plus Burkholderia sp. plus A. amazonense (3–3.6%). The other inoculant treatments did not show the same pattern when both tissues were compared. On an average, the G. diazotrophicus followed by G. diazotrophicus plus Herbaspirillum presented a BNF contribution of 9.4 and 9.7% of total N accumulated in the plant. The inoculum contained Burkholderia sp. plus A. amazonense present the lowest BNF contribution.

Discussion The MPN counting showed that all inoculated species were able to colonize the sugarcane plant tissues 7 days after inoculation. Usually, this period is enough for bacterial penetration through wounds and cracks

212 Table 7. Biological nitrogen fixation contribution of different endophytic diazotrophic bacteria to micropropagated sugarcane plants SP 70-1143 variety grown in pots containing 15 N labeled soil for 12 months (values represent mean of three replicates) Treatment1

at m % 15N BNF (Ndfa)2 excess Rhizome

at m % 15N BNF (% Ndfa) excess Leave +3

BNF (% Ndfa) (mean3 )


0.490 B4 0.433 B 0.402 B 0.446 B 0.476 B 0.412 B 0.428 B 0.374 A

0.590 B 0.548 B 0.520 B 0.529 B 0.569 B 0.567 B 0.565 B 0.391 A

0 9.2 14.6 9.7 3.2 9.4 8.1 29.2

0 11.7 16.9 9.1 2.9 11.9 12.7 23.7

0 7.11 11.9 10.3 3.6 3.9 4.2 33.7

1 See Table 1 for strain identification. 2 Ndfa, nitrogen derived from air, referred as transfer to plant tissues by BNF processes and based on comparison with isotopic enrichment of uninoculated sugarcane plants, 3 Mean, BNF calculated using the arithmetic mean of rhizome and leave +3 isotopic enrichment, 4 Values followed by same letter

did not differ significantly from Kruskal–Wallis test at 5% of significance.

on the roots (James et al., 2001). The inoculation technique used to introduce endophytic bacteria in sugarcane micropropagated plants requires a short incubation period for the inoculated bacteria to colonize and establish inside the plant tissues (Reis et al., 1999). Here, all inoculated species were found colonizing endophytically the plant tissues in quite similar numbers (approximately 108 cells per plantlet). Nevertheless, a fluctuation in the population of the inoculated bacteria with time was observed. It has been shown that the MPN method usually under-estimate the population of nitrogen-fixing bacteria in sugarcane plant tissues (Boddey et al., 2001). These authors compared the ELISA and MPN method and found that the former is able to detect much higher populations of G. diazotrophicus and Herbaspirillum spp. It is also known that populations of these diazotrophs are also influenced by the nutritional state of the plant, including the nitrogen level (Reis et al., 2000), tissue utilized (roots, leaves and stems — surface sterilized or not) and age of the plants (Fuentes-Ramirez et al., 1999) as well as if the materials are from plants previously inoculated and grown either in sterilized or in the field conditions (Sevilla et al., 2001). Therefore, it does not mean that the bacteria were not present in the plant tissues such as that observed in the aerial parts of the sugarcane plants harvested at 400 days after inoculation. It could be just because the population is below the minimal level of detection of the method. A high mortality of plants inoculated with Herbaspirillum rubrisubalbicans could be partially ex-

plained by the pathogenicity caused by these species to some sugarcane varieties (Olivares et al., 1997), although no typical symptoms was observed in our experiment. It is known that G. diazotrophicus and Herbaspirillum spp. are able to colonise sugarcane tissues in high numbers (James, 2000). Therefore, it is plausible to assume that such heavy colonization could have blocked the xylem vessels at this earlier stage of the plant and consequently affecting survival of the plantlets. Bashan (1986) showed the same trend for eight different rhizosphere bacteria in influencing the plant roots inoculated in wheat, including two Azospirillum. However, the author found that low levels (102 – 104 cfu ml−1 ) had smaller positive effects besides high levels (up to 108 cfu ml−1 ) of inoculum decreased the root surface area. Indeed, the plant response was also affected by the inoculation timing, whereas late inoculations resulted in a decrease in plant response. In addition, metabolic misbalance caused by a high nutrient demand of both plant and the endophytic community introduced could also affected the plant survival. Differences on rhizosphere microbial composition among wheat (Triticum aestivum L.) genotypes starved on zinc and manganese (Zn and Mn) were observed by Rengel et al. (1998). This might help to explain the primary negative effect of the mixed inoculation with all five endophytic diazotrophic strains. If one assumes that nutritional deficiencies take place at the acclimatization period because of an excess of endophytic population, it is possible that such deficiencies may have influenced the total endophytic

213 populations, or even the plant survival. Analysis based on the dry matter accumulated at the end of acclimatization period (45 DAI) showed that plants inoculated with few strains (low inoculum size) were those which significantly increased the dry matter (inoculation with G. diazotrophicus or inoculation with Herbaspirillum spp.). The nutrient competition between host plant and high endophytic diazotrophic bacteria population probably limited plant growth and therefore the production of dry matter at this stage. Studies based on the dynamics of the endophytic populations are needed to improve and optimise such inoculation technology on micropropagated sugarcane plant. An analysis of 79 different experiments of graminaceous plant inoculated with Azospirillum (Bashan and Dubrovsky, 1996) suggested that the effect of Azospirillum should be considered as a whole plant effect because the high variability on the inoculation effects. Interestingly, the authors report that a common effect, on almost all inoculation studies, was the change on dry matter partitioning of the whole plant level, meaning that the proportion on root dry matter and aerial part dry matter could be changed on graminaceous plants due to plant growth-promoting bacteria (PGPB) effect. In our study, significant differences on shoot-to-root ratio were also reported, as compared to uninoculated control plants. Nevertheless, the inoculation with G. diazotrophicus plus A. amazonense plus Burkholderia sp. or with a mixture of all five strains, promoted the highest difference on dry matter production between roots and aerial parts (shoot/root ratio) at 200 DAI. Such differences was mainly due to an increase in dry matter accumulated on roots as compared to control plants. Interestingly, the inoculation of sugarcane plants with Herbaspirillum spp. increased the dry matter of roots and aerial parts, however the plant still produced relatively more root than aerial dry matter. Such effects observed here suggest other possible contributions of the endophytic diazotrophic inoculated species on micropropagated sugarcane plant development besides the BNF, including for example phytohormones. Sevilla et al. (2001) showed that G. diazotrophicus nif-minus mutant could also increase the total dry matter of sugarcane inoculated plants, suggesting an additional effect of G. diazotrophicus as plant growth promoting bacteria, confirming the results of Sevilla et al. (1998). Our results of the 15 N isotopic enrichment of micropropagated plants inoculated with G. diazotrophicus species plus A. amazonense plus Burkholderia sp. showed a low efficiency of transfer of

biologically fixed nitrogen, when compared to others inoculation treatments. Nevertheless, the total dry matter production on those treatments was higher than the uninoculated control plants. Other inocula combinations contributed with less nitrogen to sugarcane plants, as observed with G. diazotrophicus plus Herbaspirillum inoculation. Probably other effects besides BNF of the endophytic diazotrophic species, were active on sugarcane inoculated plants, such as hormone production and/or plant dry matter partitioning influence (source and sink interference). PGPB effects have been mainly observed at the earlier plant development stage, as shown for G. diazotrophicus (Table 5). A very high dry matter production was observed at 45 and 200 days after inoculation in contrast to the dry matter accumulated at 400 days which was much lower than the control plants. It seems that the root mass production was stimulated (probably induced by bacterial phytohormones) instead aerial parts, reflecting in a low biomass production at 400 days. Several reports have shown the effect of plant growth promoting substances such as phytohormones produced by nitrogen fixing species (Bastian et al., 1998; Costacurta and Vanderleyden, 1995; FuentesRamirez et al., 1993). The analysis of bacterial gene expression in association with sugarcane plants, as well as the construction of others endophytic diazotrophic mutants could help to explain the observed effects on sugarcane growth. Recently, Muthukumarasamy et al., (1999) showed an increase on biomass production and leaf N-content of both micropropagated and sugarcane developed from buds when endophytic diazotrophic bacteria was inoculated together with arbuscular mycorrhizal fungi (AMF). Indeed, the authors found that the plants inoculated with mixed diazotrophs (G. diazotrophicus, Herbaspirillum spp., A. lipoferum and the AMF Glomus clarum) presented the highest biomass as compared to uninoculated plants provided with nitrogen supply (275 kg N ha−1 ). Interestingly, in another trial with several varieties, a mixed diazotroph inoculation amended either with 50% of the N recommended dose or without any N fertiliser, showed the highest yield as compared to uninoculated plants amended with 275 kg N ha−1 in two of eight sugarcane varieties. Higher BNF contribution was detected when based on rhizomes, green leaves (flag) and stems samples as compared to roots and trashes data. These results are in accordance to that published by Lima et al. (1987), who worked with four Brazilian sugarcane varieties

214 originated from sets. The authors found differential 15 N isotope enrichment among tissues from the same sugarcane plant. The discrepant values, found when trashes and roots were analysed, could be due to the low nitrogen content of such materials. Considering the high mobility of nitrogen inside plants, the differences observed on nitrogen content of different sugarcane tissues, seems to be due to differences in nitrogen management by the plant or differences in plant tissue maturity. Much higher BNF contributions than that found in our study were shown by Urquiaga et al. (1992) for the same sugarcane variety. The highest contribution observed here was approximately 30% of total nitrogen content, when five different endophytic diazotrophic species were used to inoculate micropropagated sugarcane plants as compared to 53% determined by the Urquiaga et al. (1992). However, those authors used the graminaceous species Bracchiaria arrecta as a negative control plant to quantify BNF in a three-year study. In this work, we used micropropagated sugarcane SP 70-1143 variety, with no native endophytic diazotrophic bacteria, to quantify the BNF contribution of the different inoculum combinations. We assume that no significant BNF occurred on uninoculated control plants, although microbiological analysis carried out on uninoculated control plants, just after acclimatization period, showed the presence of diazotrophic species naturally colonizing such plants. Therefore the BNF contribution with G. diazotrophicus species inoculated alone in micropropagated sugarcane plants could not be attributed to just this bacterium, because of the natural diazotrophic bacteria colonization. Interactions of the endophytic community are strongly suggested and in an experiment using fluorescent in situ hybridisation (FISH) with oligonucleotide probes (unpublished data), competition between different endophytic diazotrophic species were observed in vitro. Nevertheless, the use of selected endophytic diazotrophic species to inoculate micropropagated sugarcane plants contributed with around 30% of total plant nitrogen content as compared to natural endophytic diazotrophic species. Therefore, it seems that the BNF observed was underestimated. In addition, growth of the plants in pots also limits the complete development of the plant, and therefore could interfere on the BNF contribution of these plant/endophytic– diazotrophic bacteria associations. Indeed, the high BNF contribution observed for Saccharum spp. could not be attributed only to one or few species. It seems that the plant needs different endophytic species acting

during the different growth stages, or it is necessary an interaction between the host plant and a complex endophytic community to promote high transference of fixed nitrogen as well as the PGPB effects.

Acknowledgements The authors thank Copersucar for providing sugarcane micropropagated plants, and R. M. Boddey and V.M. Reis (Embrapa-Agrobiologia) for useful suggestions. This work was financially supported by the projects 03.2000.126 of Embrapa, PADCT III/FINEP (project 76.97.1138.00) and Pronex II (project 76.97.1051.00). This paper is a tribute to Dr. Johanna Döbereiner, who strongly stimulated the BNF research on Graminaceous plants around the World and was the main responsible for the development of this challenging area of research in Brazil.

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