Influence of organic fertilization on the number of culturable diazotrophic endophytic bacteria isolated from sugarcane

European Journal of Soil Biology 46 (2010) 387e393

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European Journal of Soil Biology journal homepage: http://www.elsevier.com/locate/ejsobi

Original article

Influence of organic fertilization on the number of culturable diazotrophic endophytic bacteria isolated from sugarcane Ricardo Pariona-Llanos, Felipe Ibañez de Santi Ferrara*, Hebert Hernán Soto Gonzales, Heloiza Ramos Barbosa Departamento de Microbiologia, Universidade de São Paulo, Av. Prof. Lineu Prestes, 1374, 05508-900 São Paulo, SP, Brazil

a r t i c l e i n f o

a b s t r a c t

Article history: Received 24 February 2010 Received in revised form 20 July 2010 Accepted 16 August 2010 Available online 9 September 2010 Handling editor: Kristina Lindström

The numbers of culturable diazotrophic endophytic bacteria (CDEB) from roots, stems and leaves of sugarcane submitted to organic, inorganic or no fertilization were compared. In order to determine the size of the N2 fixing populations, the Most Probable Number technique (MPN) was used. The quantification of diazotrophic bacteria by using the acetylene reduction assay (ARA) was more accurate than observing the bacterial growth in the vials; to confirm N2 fixing capability, the detection of gene nifH was performed on a sample of 105 isolated bacteria. The production of extracellular enzymes involved in the penetration of the plants by the bacteria was also studied. The results showed that organic fertilization enhances the number of CDEB when compared with conventional fertilization used throughout the growing season. The maximum number of bacteria was detected in the roots. Roots and stems presented the greatest number of CDEB in the middle of the cropping season and in leaves numbers varied according to the treatment. Using two pairs of primers and two different methods, the nifH gene was found in 104 of the 105 tested isolates. Larger amounts of pectinase were released by isolates from sugarcane treated with conventional fertilizers (66%), whereas larger amounts of cellulase were released by strains isolated from sugarcane treated with organic fertilizers (80%). Ó 2010 Elsevier Masson SAS. All rights reserved.

Keywords: Most probable number Endophytic diazotrophic bacteria Sugarcane Organic fertilization

1. Introduction Human activity is causing major increases in the amount of nitrogen cycling between living organisms and the environment. The consequences of such activity have already caused the rate of nitrogen entering the land-based nitrogen-cycle to double, and that rate is continuing to climb. Excessive nitrogen additions can pollute ecosystems and alter both their ecological functioning and the living communities they support [58]. Organic agricultural methods are believed to be more environmentally sound than conventional agriculture, which is dependent on the routine use of herbicides, pesticides and inorganic nutrient application. Organic agriculture results in less leaching of nutrients and higher carbon storage [14], less erosion [41] and lower levels of pesticides in water systems [24,27]. Organic manures are not only sources of major nutrients, but they also provide other micronutrients and plant growth-promoting molecules, which together lead to increased crop yields [27]. * Corresponding author. Universidade de São Paulo, Instituto de Ciências Biomédicas, Departamento de Microbiologia, Av. Prof. Lineu Prestes, 1374; CEP: 05508-000 São Paulo, SP, Brazil. Tel.: þ55 11 30917346; fax: þ55 11 30917354. E-mail address: [email protected] (F. Ibañez de Santi Ferrara). 1164-5563/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejsobi.2010.08.003

In natural ecosystems, plants represent a huge ecological niche, in which a great diversity of microorganisms is found. The microbial population is spread all over the host, continually interacting with the plant: on the surface of roots, stems and leaves or colonizing its inner tissues [46]. Some of these microbes are deleterious and others are beneficial to plants. Among the latter are plant growth promoting bacteria, able to synthesize substances that enhance plant growth, stimulate plant defenses or promote biocontrol [38]. Sugarcane (Saccharum officinarum L.) is grown in more than 120 countries, mainly in Brazil and India [3]. For centuries, Brazilian sugarcane has been cultivated with low N inputs, suggesting a possible interaction between the plant and diazotrophic bacteria [11]. Several genera of diazotrophic, endophytic bacteria were isolated from roots, stems and leaves of sugarcane: Enterobacter, Pantoea, Klebsiella, Pseudomonas, Herbaspirillum, Gluconacetobacter, Azospirillum [4,5,28,53]. Biological Nitrogen Fixation (BNF), the reduction of N2 to ammonium, causes great transformations in the Nitrogen cycle. BNF is carried out by prokaryotes only, bacteria and archaea, which include most of the bacterial phylogenetic groups [9]. BNF is extremely important because it plays an effective role in the natural interactions between organisms and is a powerful

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agricultural weapon. Cereals grown with N supplies much below their need, associated with diazotrophic bacteria can obtain up to 30% of their N from BNF when fertilized with ample PK and minor elements. The largest effect in this group was obtained with sugarcane, which can obtain up to 150 kg N ha1 from BNF [11]. A considerable fraction of such exchanges involves endophytic N2fixing bacteria [50], which interact with the host plant cells and tissues according to different degrees of dependence [22]. Due to its characteristics, the use of organic manures together with BNF may enhance the benefits to the environment. To obtain information about the bacteria-host-environment interaction, it is important to consider the number of microorganisms involved in the interaction and to try to learn which mechanisms promote such interaction. Within certain limits, the larger the size of the bacterial population, the greater its contribution to the interaction. Persello-Cartieaux and co-workers [37] demonstrated for Pseudomonas thivervalensis that an inoculum of ideal number (105 CFU ml1) produced favorable morphological changes to colonized plantlets whereas inocula above 106 CFU ml1 caused irreversible damage to plants. Continuous environmental changes have increased the interest in studies about the consequences of such changes for living organisms. Microorganisms that play an important role in nature can be used to evaluate these consequences. The size of a given bacterial population may indicate if certain human practices which impact the environment have positive or negative consequences. One way to estimate viable microbial population sizes in different environments, such as water, plant and animal tissues [21,47] is the Most Probable Number (MPN) [36,39,48,51]. In order to function, endophytic bacteria need to colonize the host plant and their penetration depends on appropriate entry mechanisms. One such mechanism allowing for the active penetration of endophytic bacteria into plant tissues involves the use of hydrolytic enzymes such as pectinase and cellulase [43]. Pectinolytic and cellulolytic enzymes are produced by a number of endophytic bacteria such as Azoarcus sp. [19] and Klebsiella oxytoca [26]. The aim of this study was to quantitatively compare the culturable endophytic nitrogen-fixing bacteria populations in sugarcane submitted to organic, conventional or no fertilization, to detect the presence of the nifH gene in those bacteria and to asses their production of the enzymes used to penetrate the plant.

Organic Agriculture Movements e by ECOCERT International, certify agency Franc-German, accredited by European Community (1999); and by the International Certification Services of Japan (2000). Sugarcane organic treatment consists of: the filter cake (residues from sugar), vinasse (residues from the alcohol distillery), phosphate rock and basaltic rock. 2.2. Microorganisms Culturable Diazotrophic Endophytic Bacteria (CDEB) from sugarcane (Saccharum sp.). Root, stem and leaves of sugarcane plants were analyzed in order to determine the number of diazotrophic endophytic bacteria. 2.3. Culture media Two synthetic semisolid media, deprived of combined nitrogen source, were tested to grow diazotrophic endophytic bacteria: LGI-P [44] and NFb [10]. 2.4. Plant disinfection and CDEB enumeration Samples of roots, stems and leaves weighing 3 g were washed in tap water. Using a sterilized punch, the inner section of the stem was withdrawn to avoid contamination. Roots and leaves were disinfected externally [2]. To perform the control of disinfection, samples of leaves and roots with cut tips were closed with paraffin to isolate the internal organ content, sealing the endophytic bacteria inside the organ. These materials were incubated for 24 h at 30  C, in a nutrient broth. In case of microbial growth, all the study material was discarded. After the disinfection, leaves and roots were floated in distilled sterile water (1:10 p/v) and processed for 1 min in a Warring blender. Samples of stem removed from the plant were macerated in distilled sterile water (dilution 1:10) with sterile mortar and pestle. The extracts of organs were submitted to decimal dilutions to obtain the suspensions with decreased concentrations of bacteria. Preliminary tests were performed to determine the most adequate incubation period and to determine if MPN should be obtained through culture growth (turbidity) or through ARA. The cultures were incubated during 2, 3, 4, 5, 7, 14 and 21 days. In the 21 days incubation test, fresh medium was added to the cultures, according to the Villemin and co-workers method [57].

2. Material and methods 2.5. Most probable number technique (MPN) 2.1. Sugarcane Saccharum sp (variety SP801816) grown under the following conditions: organic fertilization (ORG-F), conventional fertilization (CONV-F) and no fertilization, or control (NO-F), hereinafter designated by the letters indicated in parentheses. Sugarcane was grown in Fazenda São Francisco, in Sertãozinho, São Paulo State (latitude 21100 ; longitude 48 070 ). Three adjacent areas, one for each type of fertilization, measuring five square kilometers of purple latossoil were chosen as the collection site. Soil pH was close to neutral, between 6.0 and 7.0. The plants were 10 months old and 2.5 m high at the beginning of the experiment. Three individual plants, having been submitted to each type of fertilization, were collected randomly from each experiment area every month, from March to October, 2006. Sugarcane was fertilized once, immediately after cutting the preceding crop. For organic fertilization (ORG-F) 100 m3 ha1 of vinasse containing 80 kg of N e 300 kg of K were used. For conventional fertilization (CONV-F) 465 kg ha1 of NPK fertilizer 20-0-20 (93 kg ha1 of N and K). Organic fertilizer is certified by international agencies as: Farm Verified Organic, Inc, North Dakota, USA, accredited for IFOAM (1997); International Federation of

Thirty five 10 ml vials, containing four milliliters of LGI-P and NFb media, were inoculated with 250 ml of sequenced dilutions (101 to 107) of triturated organs. The vials were incubated for 5 days at 30  C. The MPN was estimated by consulting the McCrady table [29]. The MPN of CDEB was estimated during eight months in different plant organs submitted to organic, conventional and no fertilization, during the growing season. 2.6. Acetylene reduction assay (ARA) Nitrogenase activity by ARA was performed following Anderson and co-workers [1]. The bacterial cultures with acetylene 10% in the air phase were incubated without shaking at 30  C, for 72 h. Samples of vials presenting positive ARA were spread on LGI-P solid medium. Colonies with different characteristics (e.g. color, size, mucosity, rough or smooth edge) were isolated and again tested for their ability to reduce acetylene and grown in medium LGI-P liquid for DNA extraction in order to look for the presence of the nifH gene and to test for pectinase and one type of cellulase, the endoglucanase.

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performed according to the ECL System (Amersham Pharmacia) recommended protocol, and hybridization was carried out at 60  C for 16 h, without formamide. Samples that turned out labeled were considered to be positive. Escherichia coli DNA was used as a negative control and Azospirillum brasilense DNA as positive control.

Table 1 Primers used for the detection of the nifH gene. Primer

Sequence (50 / 30 )

Goal

Pol-F Forward Pol-R Reverse PPf Forward

TGC-GAY-CCS-AAR-GCB-GAC-TC ATS-GCC-ATC-ATY-TCR-CCG-GA GCA-AGT-CCA-CCA-CCT-CC

PPr Reverse

TCG-CGT-GGA-CCT-TGT-TG

-

nifH nifH nifH nifH nifH nifH

Source PCR PCR PCR Dot Blot PCR Dot Blot

389

[40] [40] [42]

2.12. Pectinolytic and endoglucanase activities assay

[42]

2.7. Genomic DNA extraction DNAs were extracted according to the Wizard Genomic DNA Purification Kit, Promega catalog number #A1120, and following the manufacturer’s instructions. The quality of the DNA extraction was verified through 1% agarose gel electrophoresis dyed with ethidium bromide. 2.8. PCR reaction and sequencing Amplification of the nifH gene was performed in 1x the enzyme buffer, 25 mM dNTP’s, 10 mM primers Pol-F and Pol-R or PPf and PPr, respectively (Table 1) described for the nifH gene of Azotobacter vinelandii (av) [40] and Azospirillum brasilense (ab) [42], 1.5 ml MgCl2, 2 U Taq polimerase plus water in order to adjust the final reaction volume and the DNA concentration to 50 ng ml1. Temperatures were 91  C per 20 s; 55  C per 15 s and 72  C per one minute. Cycles were repeated 30 times.

Pectinolytic activity was determined using citric pectin (Sigma) at pH 5.0 (for the detection of polygalacturonase) and pH 7.0 (for the detection of pectate lyase). Bacteria were incubated in Petri dishes containing solid MP medium and incubated at 30  C, for 4 days [17]. After colony development, the medium was covered with a lugol solution for one to two minutes. For endoglucanase activity isolates were inoculated on solid medium containing 1% carboxymethyl cellulose (CMC), and incubated at 30  C for 4 days. Following bacterial growth, the medium was covered with congo red dye during 15 min and subsequently covered by a 1 M solution of NaCl [52]. Pectinolytic and endogluconase activity became evident through the development of a discoloured area surrounding the colonies. 2.13. Data analysis Data analysis was performed using the program STATgraphicsÒ and the data were analyzed by the one-way analysis of variance ANOVA, by the Tukey test (P < 0.05) and comparison test for proportions (P < 0.05). 3. Results

2.9. Purification of PCR fragments The amplified products were purified with GFXTM PCR DNA and Gel Band Purification Kit, GE Healthcare catalog n 27-9602-01, according to the manufacturer’s instructions. 2.10. Sequencing reaction Sequencing reactions were performed according to the protocol prescribed by the DYEnamicTM ET dye terminator kit (MegaBACETM), Amersham Biosciences/GE Healthcare catalog n US81090 with 5 pmoles of primer and 4 ml de DYE ET. The concentrations of amplified products used in the sequencing reactions varied according to the size of the fragments under analysis. 2.11. Dot blot hybridization DNAs were spotted onto Hybond Nþ membranes (ECL System; Amersham Pharmacia, Piscataway, NJ, USA), as recommended by the manufacturer’s protocol. Dot-blot hybridizations were performed using a 705-bp probe of Azospirillum brasilense Sp7T nifH gene (Genbank accession number M64344), amplified by PCR using primers PPf and PPr [42]. Probe labeling and hybridizations were

Experiments performed with different culture media (data not shown) revealed that LGI-P and NFb were the most efficient media for bacterial growth once they were allowed to reach a higher number of bacteria. Data presented in Table 2 indicate that the most adequate incubation-time to estimate the highest MPN was 5 days because in that case there has been growth in the highest dilution that, according to the McCrady table [29], was equivalent to the highest population. The enumeration of diazotrophic bacteria was based on the number of ARA positive vials, despite the fact that a higher MPN was obtained by visualization the level of growth. This pattern was observed in all three plant organs analyzed (Table 3). A greater number of CDEB was observed in roots of ORG-F plants. There have been no significant differences between the values of MPN of CDEB present in roots and stems of CONV-F and NO-F plants. In all three types considered, leaves presented the lowest values of MPN (Table 4). Sugarcane roots of ORG-F plants always yielded greater numbers of bacteria than those of CONV-F or NO-F plants, the last sampling point being the exception (Fig. 1a). CONV-F and NO-F plants did not show significant differences. In the stem, the number of bacteria did not significantly vary between March and June, irrespective of the fertilizing conditions. In July and August, where the MPN peaks were observed, NO-F sugarcane stems have shown

Table 2 Preliminary test to determinate the optimal incubation period of CDEB cultures with acetylene. “Highest ARA positive dilution” indicates the highest bacterial dilution where acetylene reduction was detected; “MPN” corresponds to values indicated on McCrady’s table listing the different incubation periods. The five days incubation period allowed for the detection of the highest MPN of CDEB in the sugarcane plant. Incubation period (days)

2

3

4

5

7

14

21

Highest ARA positive dilutiona

102 1.7  101  0.5  101

103 2.8  103  1.9  103

105 1.7  104  1.55  104

106 2.8  105  3.4  104

104 1.4  104  3.0  103

103 2.2  102  4.5  101

102 3.3  101  1.6  101

MPN (Bacteria g1)b a b

Qualitative ARA. Means of three replicates with standard deviations.

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Table 3 Preliminary test to compare MPN of CDEB measured either through observation of the growth (turbidity) or nitrogenase activity (ARA) in roots, stems and leaves. Different letters indicate significant differences among populations measured in the same plant organ. Organ

MPNa ARAb

Turbidity Root Stem Leaf a b

6

5

9.9  10  5.6  10 (a) 9.3  106  5.4  105 (a) 4.0  104  9.7  103 (a)

5.1  105  5.5  104 (b) 5.8  104  2.1  103 (b) 1.7  104  3.8  103 (b)

Means of three replicates with standard deviation. Qualitative ARA.

to harbor the smallest bacterial population. In September and October, at the end of the cropping season, the number of bacteria was significantly larger in the stems of ORG-F sugarcane than CONV-F sugarcane but neither of the two significantly differed from that of the control (Fig. 1b). The bacterial population hosted in the leaves of ORG-F plants was significantly larger than that found in leaves of CONV-F and NO-F plants, during the first four months of the crop period (Fig. 1c). Thereafter there were no significant differences in the number of microorganisms in the leaves of plants grown under any of the three conditions. The values of MPN measured during the crop period show that in roots and stems bacteria were found in greater numbers during the months of July and August, for all three conditions under which the plants were grown (Fig. 1a and b). In both organs the bacterial population was smaller in March and April and, after reaching the peak, it reverted to levels similar to those. The size of the bacterial population in leaves differed according to the conditions the plants were grown under (Fig 1c). In CONV-F plants there has been no significant change in the number of bacteria in the leaves. Conversely, in ORG-F and NO-F plants, the largest populations of diazotrophic endophytes occurred early, in March and April. Starting in May, the number of bacteria fell for both ORG-F and CONV-F plants, and remained stable through October. Of the 105 strains tested through Dot Blot hybridization, 63 (60%) were positive for the nifH gene. Fig. 2 shows the results of the test for the nifH gene in 52 isolated bacteria. Fragments of approximately 705 and 360 bp were obtained with PCR amplification reactions. Of the 105 PCR tested strains, using primers PPf and PPr, 41 (39%) turned out positive; when using primers Pol-F and Pol-R nifH was detected in 48 (46%) samples (Table 5). The nifH gene was detected in all but one strain isolated from sugarcane (in this case grown under NO-F conditions); in 61.2% of isolates, the nifH gene was identified by either PCR or Dot-Blot alone; in all other cases the gene was detected by both techniques. No significant differences were found among fertilization conditions ORG-F, CONV-F and NO-F when Dot-Blot or PCR analyses were performed with Azospirillum brasilense primers. However, when PCR analysis was performed with primers described for Azotobacter vinelandii, the nifH gene was detected in a significant smaller number of NO-F samples. Table 6 shows that 66% of the 35 strains isolated from CONV-F sugarcane produced pectinase, a significantly larger number than the percentage of pectinase-producing strains isolated from ORG-F or NO-F plants (P ¼ 0.003), which amounted to 38% e 29%,

Fig. 1. MPN of CDEB obtained throughout the cropping period from roots (A), stems (B) and leaves (C) of sugar cane grown under different fertilization conditions: conventional (CONV-F), organic (ORG-F) or none (NO-F) (n ¼ 3).

respectively. The study of the production of endoglucanase yielded opposite results. The number of isolates with endoglucanase activity remained at 66% in CONV-F sugarcane. However, the percentage of endoglucanase-producing bacteria in ORG-F plants was 80% and only 47% in strains from NO-F plants. Such are significant differences (Table 6). The proportion of strains isolated from CONV-F or ORG-F plants did not differ significantly in terms of their ability to excrete at least one of the enzymes studied (Table 6). The number of positives among isolates from those two conditions (80% for ORG-F and 70% for CONV-F) was significantly higher than those from NO-F plants (52%). 4. Discussion This study assesses the effects of the addition of organic fertilizer to a sugarcane plantation on the numbers of CDEB and on the production of endoglucanase and pectinase by those organisms, compared to the addition of conventional or no fertilizer.

Table 4 Mean MPN and standard deviation of endophytic bacteria obtained from roots, stems and leaves of sugarcane grown under ORG-F, CONV-F or NO-F (n ¼ 3). Different lowercase letters indicate significant differences among populations subjected to the same treatment and upper-case letters indicate significant differences among populations measured in the same plant organ. Organ

ORG-F

CONV-F

NO-F

Root Stem Leaf

3.76  1.92  105 (a,A) 4.27  2.24  104 (b,A) 2.74  1.56  103 (c,A)

9.66  5.46  103 (a,B) 2.02  1.32  104 (a,A) 7.19  2.26  101 (b,B)

1.85  0.97  104 (a,B) 3.64  1.59  104 (a,A) 8.03  2.32  101 (b,B)

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Fig. 2. Dot Blot Hybridization of the nifH gene from a sample of strains isolated from ORG-F, CONV-F and NO-F sugarcane plants. (þ) Azospirillum brasilense DNA () Escherichia coli DNA.

The MPN method is the most appropriate one to estimate the number of same-type bacteria including different genera (e.g. CDEB) [32]. When compared to others methods, MPN showed similar results: a significant positive correlation coefficient of 0.9 between the direct plating method and the MPN technique was obtained by Scherer et al. [48]. Experimental data showed that to correctly employ the MPN method to a defined group of microorganisms, it is necessary to adapt the conditions (media, temperature, O2 concentration, etc) to maximize the growth of the microorganisms under analysis [18]. In the present study the best growing conditions for CDEB were five days of culture in LGI-P or NFb media and analysis of the nitrogenase activity by ARA after 72 h of incubation. The verification of the proper incubation time was performed because various genera with different metabolic features were involved. Villemin and co-workers [57] adapted the MPN method to both aerobic and anaerobic free living diazotrophic bacteria by adding fresh medium to the cultures after 21 days of incubation, followed by a new period of incubation to favor the bacterial growth. This method was tested in this study but the numbers of bacteria (Table 2) were 104 times lower than those obtained with five days of incubation. A decrease of pH values from 6.0 to 4.0, approximately, after the fifth day probably made the environment inhospitable to some genera. Even the addition of fresh medium was not sufficient to rehabilitate the culture. To confirm these results, a culture was prepared in semisolid LGI-P medium and, at different times, samples taken from such culture were spread in solid LGI-P medium. The isolated colonies showed that the diversity decreased after the fifth day (data not shown). That happened because not all organisms survive for the same amount of time under the same conditions; it also showed it was necessary to change the method and, as a result, the five days incubation period was chosen and the addition of fresh medium was abandoned. Table 5 Number of nifH positive strains for each fertilization condition using three different methods: Dot Blot Hybridization (DB), PCR with primers PPf and PPr (ab) and PCR with primers Pol-F and Pol-R (av). Condition

Total

DB

PCR (ab)

PCR (av)

nifH positive independently of method

ORG-F CONV-F NO-F

30 33 42

23 (76.7%) 18 (54.5%) 22 (52.4%)

18 (60.0%) 16 (48.5%) 7 (16.7%)

10 (33.3%) 16 (48.5%) 22 (52.4%)

30 33 41

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Villemin and co-workers [57] recommended that the quantification of diazotrophic bacteria by using the ARA was more accurate than visually observing the bacterial growth in the vials. Therefore, once the N2-fixing capacity of the studied bacteria was established, the assessment of the MPN by measuring nitrogenase activity was the quantifying parameter. Unlike the data observed by Villemin and co-workers [57] for free living diazotrophics, in this study, the estimation of the MPN exclusively by the observation of the turbidity/ halo of the cultures resulted in higher values (Table 3). These data can be explained by the growth of non-N2 fixing bacteria that either could have some kind of nitrogen reserve [49] or could possibly have used very low quantities of combined nitrogen that always contaminate the ingredients used to elaborate culture media. To confirm the results obtained with the acetylene reduction assay, the presence of nifH genes was searched in 105 randomly obtained isolates. In the present study, the nifH gene was detected in all the isolated bacteria, with one exception. The Dot-Blot and PCR techniques, used for that purpose with primers of nifH genes from Azospirillum brasilense and Azotobacter vinelandii, complemented each other, since the application of the two techniques on every sample allowed for the detection of the nifH gene in practically all the microorganisms. In the single case where the gene was not detected, different primers could have been used [59]. If that bacterium is capable of reducing acetylene, it should carry a nifH, which was not amplified by the primers used in the study. The results obtained in the present study show that roots of sugarcane grown under organic fertilization harbor a larger number of diazotrophic bacteria than the leaves of the same plants; such data are consistent with those in the literatures. Other studies of sugarcane have shown that roots contained larger bacterial populations that were viable for longer periods of time than the aerial organs of the plants [13,35]. Absent pathological conditions, the plant defenses control the bacterial population, and its size varies according to the species in question; also, in rice, each plant organ sets an acceptable population density limit and the root contains the largest bacterial population [6,39]. Having direct contact with the soil makes the root the principal entryway for microorganisms into the plant and that organ becomes also a major transit area for bacteria in the plant. Moreover, the root system is a major drain of photosyntates produced by the aerial part of the plant, which are used as nutrients. Urquiaga and co-workers [55] showed that sugarcane roots contain an average of 25% more nitrogen than aerial plant tissues and attributed such data to the presence of a greater number of diazotrophic bacteria in that organ. Dong and co-workers [12] discussed the difficulties of the translocation of bacteria via the xylem vessels to the aerial part of the plant, a factor favoring the accumulation of microorganisms in the root. Besides, the smaller MPN of CDEB in the leaves of sugarcane may be explained by other reasons. Leaves, especially old and senescent ones, have less available nutrients, such as carbon. They also contain higher concentrations of O2 resulting from photosynthesis, and oxygen is a toxic gas as far as nitrogenase is concerned since it oxidizes the enzyme, irreversibly inhibiting it. Diazotrophic, endophytic bacteria may, or may not, posess mechanisms that protect the nitrogenase enzyme against irreversible inhibition by O2, which would prevent nitrogen fixation [8,20,54]. The lower MPN of CDEB found in leaves may be the result of the high O2 concentration and a low number of bacteria with an enzyme protective mechanism, since a few bacteria would find in those conditions a favorable environment for their development. Another possibility is that raised by the results of Olivares and co-workers [34]. After inoculation, they could not isolate H. seropedicae from the leaves and showed that artificial inoculation of sugarcane leaves by H. seropedicae elicits a hypersensitive response (HR) by the plant, which suggests sugar cane leaves are not a suitable

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Table 6 Number of pectinase and endoglucanase producing CDEB isolated from sugarcane grown under ORG-F, CONV-F and NO-F. Different letters indicate significant differences. Fertilization

Number of tested strains

ORG-F CONV-F NO-F

31 35 48

Pectinase

Endoglucanase

Pectinase or endoglucanase producing strains

Producing strains

%

Producing strains

%

Producing strains

%

12 23 14

38(b) 66(a) 29(b)

26 23 23

80(a) 66(b) 47(c)

26 26 25

80(a) 70(a) 52(b)

location for the bacteria. Thus, only certain bacteria, which did not provoke that HR, would remain in the leaves. The larger number of bacteria in the leaves of ORG-F plants, when compared with CONVF or NO-F, may have been aided by a greater supply of nutrients to the leaves or by a mitigation of the HR in ORG-F sugarcane. The results of this study showed similar MPN between roots and stems of CONV-F or NO-F sugarcane. Most likely, conventional fertilization and the lower quantity of nutrients in the NO-F soil harmed the number of bacteria in the roots. No clear-cut data about the influence of organic fertilization on the number of CDEB were found in the literatures. Results of the present study (Fig. 1), on the other hand, offer a comparison of the number of CDEB under ORG-F, CONV-F or NO-F. Moreover, results comparing roots, stems and leaves show that the bacterial population was never lower in ORG-F plants than in CONV-F and NO-F plants. Taking into account ORG-F and CONV-F plants only, in the first case there was a stimulation of the number of CDEB in the roots (March through August) and in the leaves (March and April); the larger numbers reached in the stems (September and October) may be the result of a stimulus under ORG-F or of an inhibitory effect under CONV-F, since none of them differed significantly from the control. It becomes thus evident that, when compared to its conventional counterpart, organic fertilization had a positive effect on the number of CDEB throughout the sugarcane cropping season, stimulating the increase of the bacterial population within adequate limits and ensuring a beneficial association [16]. Leita and co-workers [25] have shown that organic fertilization increased microbial biomass and metabolism in soils. Conversely, studies show that CONV-F cultivation may harm the colonization of the plant by certain bacteria genera such as Gluconacetobacter diazotrophicus that may be eliminated from plants submitted to nitrogen fertilization in high concentration. However, the same effect was not verified with Herbaspirillum spp [31]. Given the fact that there were no significant differences among CDEB populations in CONV-F and NO-F plants, it could be suggested that better adapted bacteria replaced those bacteria more susceptible to conventional fertilizers. The occurrence and distribution of diazotrophic bacteria is influenced by the cultivar type [7] and by the type of plant fertilization. The type of fertilization may interfere with plant metabolism. Barley plants subjected to organic fertilization presented different concentrations of certain compounds when compared with plants treated with chemical fertilizers [33]. As a result, changes in metabolism alter the physiological state of the plant, and this subsequently affects its association with the diazotrophic bacterial population [30,45]. ORG-F contains 14% less N and vinasse and larger amounts of organic matter than CONV-F. Those facts might favor bacteria-plant interactions and lead to a greater colonization of the plant by bacteria under ORG-F cultivation. The number of CDEB changed during the cropping period (Fig. 1). In roots, stems and leaves there were two month periods when the microbial population was higher. Several factors may explain these differences, including plant age and tissue type [23]. Therefore, the maximum number of CDEB can only be obtained when the enumeration test is performed in the appropriate organs and period of time, and through the suitable method.

The production of hydrolytic enzymes is regulated in a way that they are excreted only at the onset of the colonization process and are repressed thereafter to prevent further damage to the plant [16,56]. Bacteria isolated from CONV-F sugarcane released larger amounts of pectinase, whereas those under ORG-F released more endoglucanase (Table 6). This finding may suggest there has been a selection of bacteria that are better adapted to the type of fertilization. The analysis of the results obtained with the 114 isolates, showed that there has been no significant difference, in general, among the numbers of bacteria able to release at least one of such enzymes. Since approximately 70% to 80% of the bacteria isolated from CONV-F and ORG-F sugarcane, respectively, released such enzymes, it may be suggested that this is an important characteristic for the establishment of the microorganisms inside the plant. Penetration can also take place at cracks, such as those occurring at root emergence sites or created by deleterious microorganisms, as well as by root tips [43]. Data from the literature show that organic fertilization provides for adequate growing conditions for sugarcane [15]. The results presented in this study reinforce that idea because they show organic fertilizers to have a positive effect on the numbers of CDEB, while conventional fertilizers do not have that effect. Moreover, as explained in greater detail in the first paragraph of the introduction, organic agricultural methods are believed to be environmentally sound for a number of reasons [14,24,27,41], while conventional agriculture may lead to excessive nitrogen additions and harm ecosystems [58]. For those reasons organic fertilization should be encouraged. Acknowledgements The authors wish to thank FAPESP, CNPq and CAPES for grants and fellowships, Fazenda São Francisco for kindly providing the sugarcane samples and Dr. Marcel Bouquet for volunteering his technical and linguistic skills to the English version of this paper. References [1] M.D. Anderson, R.W. Ruess, D.D. Uliass, J.S. Mitchell, Estimating N2 fixation in two species of Alnus in interior Alaska using acetylene reduction an 15N2 uptake, Ecoscience 11 (2004) 102e114. [2] W.L. Araújo, J. Marcon, W. Maccheroni Jr., J.D. Van Elsas, J.W.L. Van Vuurde, J.L. Azevedo, Diversity of endophytic bacterial populations and their interation with Xylella fastidiosa in citrus plants, Appl. Environ. Microbiol. 68 (2002) 4906e4914. [3] J.I. Baldani, V.M. Reis, V.L.D. Baldani, J. Döbereiner, A brief story of nitrogen fixation in sugarcane e reasons for success in Brazil, Funct. Plant Biol. 29 (2002) 417e423. [4] S.C. Bellone, C.H. Bellone, Presence of endophytic diazotrophs in sugarcane juice, World J. Microbiol. Biotechnol. 22 (2006) 1065e1068. [5] C.H. Bellone, S.C. Bellone, R. Pedraza, M.A. Monzon, Cell colonization and infection thread formation in sugar cane roots by Acetobacter diazotrophicus, Soil Biol. Biochem. 29 (1997) 965e967. [6] F. Chi, S.H. Shen, H.P. Cheng, Y.X. Jing, Y.G. Yanni, F.B. Dazzo, Ascending migration of endophytic rhizobia, from roots to leaves, inside rice plants and assessment of benefits to rice growth physiology, Appl. Environ. Microbiol. 71 (2005) 7271e7278. [7] L. Chiarini, A. Bevivino, C. Dalmastri, C. Nacamulli, S. Tabacchioni, Influence of plant development, cultivar and soil type on microbial colonization of maize roots, Appl. Soil Ecol. 8 (1998) 11e18.

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