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TITLE: Sugarcane growth promotion by the endophytic bacterium Pantoea agglomerans 33.1 1
RUNNING TITLE: Sugarcane growth promotion endophyte 2
3
Quecine, M.C.1*, Araújo, W.L. 2*, Rossetto, P.B.3; Ferreira. A. 4; Tsui, S. 1; Lacava, P.T.5, 4
Mondin, M.1; Azevedo, J.L. 1; Pizzirani-Kleiner, A.A. 1 5
1. Department of Genetics, Escola Superior de Agricultura “Luiz de Queiroz”, University of São 6
Paulo, Av. Pádua Dias 11, P.O. BOX 83, 13400-970, Piracicaba,SP, Brazil. 7
2. Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, Av. 8
Prof. Lineu Prestes, 1374 - Ed. Biomédicas II, Cidade Universitária, 05508-900, São Paulo, SP, 9
Brasil 10
3. CENA – University of São Paulo, Av. Centenário 303, 13400-970 Piracicaba SP, Brazil, 11
4. Brazilian Agricultural Research Corporation, Embrapa Agrosilvopasture, Av. das Itaúbas, 12
3257, CEP 78550-194, Sinop, MT, Brazil. 13
5. Institute of Natural Sciences, Federal University of Alfenas, Rua Gabriel Monteiro da Silva, 14
700, Alfenas 37130-000, MG, Brazil 15
*Corresponding authors. 16
Mailing address: 17
Department of Genetics, Escola Superior de Agricultura “Luiz de Queiroz”, University of São 18
Paulo, Av. Pádua Dias 11, P.O. BOX 83, 13400-970, Piracicaba, SP, Brazil. E-mail: 19
[email protected], Tel. (55-19) 3429 4248 20
Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, Av. Prof. 21
Lineu Prestes, 1374 - Ed. Biomédicas II, Cidade Universitária, 05508-900, São Paulo, SP, 22
Brasil. E-mail: [email protected], Tel. (55-11) 3091 8346 23
Copyright © 2012, American Society for Microbiology. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.00836-12 AEM Accepts, published online ahead of print on 3 August 2012
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Abstract 24
The promotion of sugarcane growth by the endophytic Pantoea agglomerans strain 33.1 was 25
studied under gnotobiotic and greenhouse conditions. The GFP-tagged strain P. agglomerans 26
33.1:pNKGFP was monitored in vitro in sugarcane plants by microscopy, re-isolation and 27
quantitative PCR (qPCR). Using qPCR and re-isolation 4 and 15 days after inoculation, we 28
observed that GFP-tagged strains reach similar density levels both in the rhizosphere and inside 29
the roots and aerial plant tissues. Microscopic analysis was performed at 5, 10 and 18 days after 30
inoculation. Under greenhouse conditions, P. agglomerans 33.1-inoculated sugarcane plants 31
presented more dry mass 30 days after inoculation. Cross-colonization was confirmed by re-32
isolation of the GFP-tagged strain. These data demonstrated that 33.1:pNKGFP is a superior 33
colonizer of sugarcane due to its ability to colonize a number of different plant parts. The growth 34
promotion observed in colonized plants may be related to the ability of P. agglomerans 33.1 to 35
synthesize IAA and solubilize phosphate. Additionally, this strain may trigger chitinase and 36
cellulase production by plant roots, suggesting the induction of a plant defense system. However, 37
levels of indigenous bacterial colonization did not vary between inoculated and non-inoculated 38
sugarcane plants under greenhouse conditions, suggesting that the presence of P. agglomerans 39
33.1 has no effect on these communities. In this study, different techniques were used to monitor 40
33.1:pNKGFP during sugarcane cross-colonization, and our results suggested that this plant 41
growth promoter could be used with other crops. The interaction between sugarcane and P. 42
agglomerans 33.1 has important benefits that promote the plant’s growth and fitness. 43
44
45
46
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Introduction 47
The existence of endophytic bacterial communities has been recognized for over a 48
hundred years (32). Initially, these microorganisms were considered to be neutral with regard to 49
their effects on host plants; more recently, however, their positive impact has been verified in a 50
broad range of crops (67), in which they may contribute directly to plant growth by promoting 51
nutrient availability, biological nitrogen fixation and the production of phytohormones (38, 69). 52
Indirectly, they may also reduce microbial populations that are harmful to the plant, acting as 53
agents of biological control through competition, antibiosis or systemic resistance induction (61, 54
72). 55
Members of the Enterobacteriaceae family (Gammaproteobacteria) are frequently 56
described as rhizosphere colonizers of sugarcane and other grasses (84). This class includes 57
Enterobacter sp. (8, 49), Klebsiella (49), Enterobacter cloacae and P. agglomerans (formerly 58
Erwinia herbicola) (63). Many studies have reported the endophytic presence of 59
Enterobacteriaceae members in various crop species (77). P. agglomerans has been described as 60
an important corn and wheat endophyte (65), and it has also been isolated from potato stems (2), 61
rice seeds (65, 66) and citrus leaves (1). Many studies have shown the potential of Pantoea spp. 62
for systemic resistance induction (37, 44, 56) and protection against pests and plant-pathogenic 63
microorganisms (4, 9, 28, 59). Additionally, these bacteria may induce plant growth by 64
increasing the nitrogen supply in non-symbiotic associations (2, 7, 70, 81), solubilizing 65
phosphorus (45, 46, 73) and stimulating phytohormone production (79, 85). 66
Much of the current research in the field focuses on the capacity of endophytes to 67
colonize several different agronomically important hosts. The term for this phenomenon in 68
endophytes, cross-colonization, is adopted more commonly from the plant-pathogen literature 69
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(23). Zakria et al. (84) demonstrated that strains of Pantoea sp. (18-2) from sweet potato and 70
Enterobacter sp. (35-1) from sugarcane are able to endophytically colonize rice and promote its 71
growth. K. pneumoniae 342 (Kp342), isolated from corn and subsequently labeled with the green 72
fluorescence protein (GFP), was able to colonize C. roseus and C. sinensis (42). 73
The introduction of a potentially beneficial bacterium with cross-colonization capacity 74
requires careful monitoring. One of the oldest techniques for monitoring microorganisms in an 75
environment is to plate the organism on solid media in a laboratory; this technique has several 76
limitations, especially the considerable time required to obtain results. Recently, molecular 77
approaches have been developed to monitor bacterial species in host environments. For example, 78
tagging with related gene markers, such as GFP, has been particularly useful for following 79
bacterial infection pathways and for characterizing tissue and organ colonization (14, 27, 75, 76). 80
The qPCR technique, commonly employed to quantify and study clinical (5, 17, 57), 81
phytopathogenic (54, 82, 15, 47, 6) and endophytic (41) bacteria, has also been used for these 82
purposes. 83
Sugarcane tissue culture has been used to remove pathogens from desirable material (34), 84
and it has been used in breeding programs to reduce cloning time and rapidly obtain large 85
amounts of material with the desired genotype. For this reason, we studied the possibility of 86
plant growth promotion during sugarcane seedling acclimation. We aimed to reduce the time of 87
seedling development under greenhouse conditions, therefore reducing the costs of seedling 88
production. A plant-growth promoting strain of P. agglomerans 33.1, previously isolated from 89
Eucalyptus grandis, was evaluated during its interactions with sugarcane, with a focus on its 90
potential for cross-colonization and promotion of sugarcane growth under nursery conditions 91
during seedling acclimation. 92
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Materials and Methods 94
95
Microorganisms and growth conditions 96
Pantoea agglomerans 33.1 isolated from Eucalyptus grandis plants (59) and its 97
derivative tagged strain 33.1:pNKGFP (25) as well as Escherichia coli derivatives were routinely 98
grown at 28°C and 37°C, respectively, on Luria Bertani (LB) medium (68). The tagged strain 99
33.1:pNKBOR was obtained according to the methodology described by Ferreira et al. (25). All 100
of the strains (Table 1) were stored in 20% glycerol at -80°C. The plasmids were propagated and 101
isolated from E. coli DH5-α pir or Top 10 and purified with the Plasmid Miniprep Kit (Mobio) 102
according to the manufacturer’s recommendations. 103
104
Plant material 105
The sugarcane seedlings (var. SP80 1842 and SP80 3240) were initially cultivated under 106
in vitro conditions. The seedlings were supplied by Dr. Sabrina Moutinho Chabregas (CTC, 107
Piracicaba, SP, Brazil). 108
109
Cross-colonization under gnotobiotic assays 110
For plant inoculation, the bacterial cells were transferred to 50 ml of LB medium 111
supplemented with kanamycin (100 μg/ml) and incubated at 28°C under shaking conditions (150 112
rpm) until the late log phase. The cells were harvested (at 4500 g for 15 min) and washed with 113
phosphate-buffered saline (PBS) at pH 6.5, and the cell density was adjusted to 105 CFU/ml. The 114
SP80 1842 sugarcane seedlings were cultured in 50-ml tubes containing 7 ml of agar-free MS 115
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medium (51) with the endophytic bacteria at 28º C under 16-h photoperiods. In the control 116
plants, PBS was used in place of the bacterial suspension. Eighteen days after bacterial 117
inoculation (DAI), three plants per treatment were sampled for fluorescence microscopy (FO) 118
and scanning electronic microscopy (SEM) analysis. The re-isolation and qPCR procedures were 119
performed on 5 plants at 5 and 14 DAI. 120
121
Fluorescence microscopy 122
Roots and aerial fresh tissues were cut and immediately observed with visible light and 123
fluorescence microscopy (Zeiss, Axiophot II, Germany) under a green (FITC, 510 nm) 124
fluorescence filter. Both images were combined, using the overlay module in the MetaVue 125
program (Universal Imaging Corporation, USA), to identify the bacterial cells. 126
127
Scanning electron microscopy (SEM) 128
Plant tissue were cut (approximately 2 cm2), fixed in buffered Karnovsky solution (2% 129
glutaraldehyde, 0.001 M CaCl2, 2.5% paraformaldehyde) at 4°C and rinsed three times (10 min 130
for each rinsing) with a 0.05 M cacodylate buffer at pH 7.2. The samples were infiltrated with a 131
30% glycerol solution for 30 min and treated with an osmium tetroxide solution in a 1% 132
cacodylate buffer for 2 h. The samples were then washed three times in distilled water, 133
dehydrated with acetone (in a series of 30, 50, 70, 90 and 100% acetone dilutions), dried to the 134
critical point using a dryer (BALZERS-CPD 030), mounted on aluminum stubs and coated with 135
gold (BALZERS-MED 010). The metal-coated samples were examined using SEM (LEO-136
ZEISS). 137
138
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Southern blot analysis 139
The number of insertions of the pNKGFP fragment in the 33.1:pNKGFP genome was 140
confirmed using Southern blotting. For this technique, probes were generated by PCR, using 141
primers (PPNKF: 5’ - CCT TCA TTA CAG AAA CGG C - 3’ and PPNKRII: 5’ - GGT GAT 142
GCG TGA TCT GAT CC - 3’) designed by the OligoPerfect Designer program (Invitrogen) 143
based on the pNKBOR sequence. 144
These primers amplify a 362 bp sequence located between the insertion sites (IS) that 145
correspond to a portion of the kanr gene and a conserved plasmid fragment. The probe specificity 146
was evaluated using DNA from the strains, plasmids (Table 1) and plants. The PCR reaction 147
contained 1 μL of the samples (10 ng), 0.2 M of primers PPNKF and PPNKRII, 3.75 mM of 148
MgCl2, 0.2 mM of a dNTP mixture and 2.5 U of Taq DNA polymerase (Fermentas) combined 149
with buffer 1X for a final volume of 25 μl. The PCR program consisted of an initial denaturation 150
step at 94ºC for 4 min, followed by 35 cycles of denaturation at 94ºC for 30 s, annealing at 58ºC 151
for 45 s and extension at 72ºC for 30 s. The final step was a 10 min extension at 72ºC. The 152
amplified products were separated by electrophoresis with a 1.2 % agarose gel and stained with 153
ethidium bromide. 154
The probes were labeled using the random-primed DNA labeling method, and the 155
hybridization was confirmed with the Gene ImagesTM AlkPhos DirectTM labeling and detection 156
system (GE Healthcare) according to the manufacturer’s instructions. 157
158
Quantitative PCR (qPCR) 159
Sugarcane seedlings inoculated either with or without 33.1:pNKGFP were sampled, and 160
the roots and aerial parts of these plants were separated. To separate the bacterial cells from the 161
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sugarcane surface, the root was incubated in 2 ml of PBS buffer at 28ºC for 2 h, and the bacterial 162
cells were harvested by centrifugation. Total bacterial DNA was extracted according to Araújo et 163
al. (1). After this surface sterilization, the total DNA of the root and aerial plant parts was 164
extracted by the CTAB method, according to Doyle and Doyle (22). 165
The qPCR analysis was performed in a 25-μl final volume, which contained 12.5 μl of 166
the master mix of Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen) and the PPNKF 167
and PPNKRII primers (10 μM each). Aliquots of the master mix (20 μl) were dispensed in the 168
wells, and 5 μg of DNA (50 ng/μl) were added as a PCR template. The qPCR reaction cycles 169
consisted of a denaturation step at 94ºC for 5 min, 40 cycles at 94ºC for 30 s and a final step at 170
61ºC for 15 s. The plasmidial fragment quantification was performed using an iCycler iQ Real-171
Time PCR instrument (Bio-Rad Laboratories Inc., USA). Four replicates in duplicate were used, 172
and a standard curve was obtained for every run using a known copy number (105 to 108) of the 173
linearized plasmid pNKGFP. 174
175
Sugarcane-growth promotion and defense-protein production 176
The micro-propagated sugarcane seedlings (var. SP80-1842 and SP80-3240) were 177
transferred to plastic pots containing the organic substrate Eucatex PlantMax® Horticultura 178
(Eucatex). The transferred plants were first acclimated in a humid chamber at 28ºC for seven 179
days. The bacterial wild type 33.1 and tagged 33.1:pNKGFP were inoculated into the substrate 180
(108 CFU/plant), and the pots were then transferred to a greenhouse at 28ºC. The substrate of 181
control pots was inoculated with PBS buffer that did not contain any bacterial cells. The dry 182
mass, related defense protein production and bacterial cross-colonization of the plants were 183
evaluated at 30 DAI. 184
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Forty plants from each treatment were sampled, washed and divided into root and aerial 185
parts. The vegetative tissues were weighed and dried at 55ºC for 5 days to reach a constant 186
weight. 187
Enzymatic activity was assessed using separate total protein extractions from the leaves 188
and roots according to Ferreira-Filho et al. (26). The amount of protein present in the supernatant 189
was measured using the Bradford procedure (10), with bovine serum albumin (BSA) as the 190
standard. Four replicates were used for the measurements of enzymatic activity. 191
The endoglucanase and chitinase activity from the crude plant extract was assayed using 192
CMC-RBB and CM-Chitin RBV (Lower). The enzyme activity was measured as the absorbance 193
per milliliter of the substrate reaction per hour (29). 194
195
Bacterial re-isolation 196
The densities of P. agglomerans in the rhizosphere and inside the sugarcane tissues were 197
evaluated at 4 and 15 DAI in the gnotobiotic assay and at 30 DAI in the greenhouse assay. To 198
promote the detachment of bacteria from the roots, the plant samples were placed in a new sterile 199
tube containing 2 ml of PBS and agitated at 120 rpm for 1 h. The cell suspension was diluted and 200
plated on 5% TSB for bacterial quantification. The seedlings were washed in running tap water 201
and surface-disinfected using serial rinsing in 70% ethanol and 2% hypochlorite according to 202
Araújo et al. (1). To confirm the efficiency of the disinfection process, aliquots of the sterile 203
distilled water used in the last washing were spread onto 5% TS agar medium and examined for 204
surface contaminants after a 3-day incubation at 28°C. The surface-disinfected samples were 205
macerated in a PBS buffer, and the appropriate dilutions were plated onto 5% TS agar 206
supplemented with kanamycin (100 μg/ml) and benomyl (50 μg/ml). The cultivable bacterial 207
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density associated with sugarcane was also evaluated under greenhouse conditions. For this 208
process, the macerated samples were plated on TS agar without the selective antibiotic, and the 209
number of tagged strains among the indigenous community was measured by exposing the plates 210
to UV light. 211
212
Bacterial plant-growth promotion mechanisms 213
Indole acetic acid (IAA) production 214
The indole acetic acid (IAA) production of the 33.1 strain was assessed using the 215
quantitative method developed by Bric et al. (12). The absorbance (OD520 nm) values obtained 216
were interpolated from a standard curve to determine the IAA concentration. Two independent 217
experiments were performed in triplicate. 218
219
Phosphate solubilization 220
The ability of strain 33.1 to solubilize inorganic phosphate was evidenced by a halo 221
obtained after cultivation of the bacteria on culture media supplemented with two inorganic 222
phosphate sources, Ca3(PO4)2 and Al(PO4), at 28°C for 72 h, according to Verma et al. (81) and 223
Hara and Oliveira (31), respectively. An estimation of the halo size (cm) divided by the colony 224
size (cm) generated a solubilization index (SI) that was used to quantify the phosphate 225
solubilization. 226
227
Biological nitrogen fixation (BNF) 228
The ability of the strain to fix atmospheric nitrogen was assessed according to Dobereiner 229
et al. (19). To confirm the ability of the strain to fix atmospheric nitrogen, seven consecutive 230
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streaks were performed in the Nfb medium. The nitrogenase activity was measured after 5 days 231
of incubation using an acetylene reduction assay (33). The chromatography gas analysis was 232
completed at the Post-Harvest Laboratory, ESALQ-USP. The samples were incubated with 10% 233
acetylene for 2 h at 28°C. The nitrogenase activity was expressed in nanomoles of ethylene per 234
hour. 235
236
Statistical analysis 237
The statistical analyses of the data were conducted using the SAS program (SAS Institute 238
Inc., Cary, NC, USA) and were designed to account for the random design and sub-factorial 239
nature of the qPCR and re-isolation data. To quantify the bacteria, the obtained data were log-240
transformed to stabilize the variance. The bars represent the means +/- standard errors of four 241
replicates. The asterisks (* and **) indicate significant differences (α = 0.05 and 0.01, 242
respectively) according to Student's t test. 243
244
Results 245
Microscopy analysis of sugarcane interactions with the 33.1 and 33.1:pNKGFP strains 246
The behavior of the P. agglomerans 33.1 and 33.1:pNKGFP strains during their 247
interactions with micro-propagated sugarcane plants was evaluated using SEM. No bacteria or 248
fungi were observed in the control treatments on any of the observed samples (Fig. 1A). The 249
33.1 and 33.1:pNKGFP cells aggregated on the root surface (Fig. 1B, C and D). 250
The formation of a biofilm by 33.1:pNKGFP on the sugarcane root surfaces was observed 251
at all of the sampled points, as described above (Fig. 2A, C and E), but bacterial fluorescence 252
was observed only at 5 DAI (Fig. 2B). We observed a fluorescence reduction after this period but 253
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not a reduction in the bacterial biofilm aggregate (Fig. 2D and F). The sugarcane samples also 254
showed auto-fluorescence; however, the presence of a few 33.1:pNKGFP cells fluorescing in the 255
bacterial aggregation is clearly visible. Otherwise, in the MS suspension, bacterial fluorescence 256
was not affected in the aggregated cells (data not shown). We did not observe fluorescent 257
bacterial cells inside of the sugarcane tissues, probably due to the loss of bacterial fluorescence 258
observed in the biofilm aggregation. 259
260
Monitoring of the 33.1:pNKGFP sugarcane cross-colonization using qPCR and re-isolation 261
To develop a qPCR technique to monitor P. agglomerans 33.1:pNKGFP in sugarcane, 262
the specificity of the designed set of primers was first tested using conventional PCR. The 263
primers PNKF and PNKRII were tested against plasmids and bacterial strains (Table 1) that were 264
specific to the tagged strain (Fig. 3A). No amplification was observed for the DNA of sugarcane 265
without 33.1:pNKGFP inoculation (data not shown). The 33.1:pNKGFP strain presented only 266
one insert of the plasmid (Fig. 3B); therefore, it was used for the quantification of P. 267
agglomerans in the sugarcane seedlings. 268
Using this qPCR technique under gnotobiotic conditions, we observed a higher number 269
of 33.1:pNKGFP cells in the rhizosphere and in sugarcane tissues at 15 DAI. The strain had 270
multiplied and moved from sugarcane roots to aerial parts by 15 DAI, increasing bacterial cell 271
density in aerial tissues and in root tissues (Fig. 4A). The number of bacterial cells detectable 272
with qPCR is similar to that detectable with re-isolation, and both techniques showed an increase 273
in the number of bacterial cells in the rhizosphere and aerial plant parts (Fig. 4B). 274
In the greenhouse assay, 33.1:pNKGFP was also able to colonize acclimated sugarcane 275
when added to the substrate. A greater number of tagged bacterial cells were observed in the 276
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rhizosphere than in the aerial parts (Table 2). The density of the indigenous bacterial community 277
was not affected by the addition of 33.1:pNKGFP. A similar number of indigenous bacterial cells 278
were observed in the rhizosphere in all treatments, either with or without the bacterial addition of 279
strain 33.1 or 33.1:pNKGFP. 280
281
Plant-growth promotion 282
After substrate inoculation, P. agglomerans 33.1 significantly promoted the biomass 283
accumulation of the sugarcane plants (Fig. 5A, B). In var. SP80-1842, this result was observed in 284
only aerial plant tissues, whereas for var. SP80-3240, increased biomass accumulation was 285
observed in root tissues as well (Fig. 5B). For both sugarcane varieties, we observed that 286
bacterial inoculation induced chitinase and cellulase production in root tissues (Fig. 5D). 287
The 33.1 strain was able to solubilize two different phosphate sources. Calcium 288
phosphate was hydrolyzed to a greater extent by the 33.1 strain than was aluminum phosphate, 289
with SI values of 3.4 and 1.72, respectively. Even during phosphate solubilization, 33.1 showed 290
the capacity to produce IAA, generating approximately 100 µg/ml in the assessed conditions. 291
Nitrogen fixation by 33.1 was confirmed in all seven consecutive inoculations in the Nfb 292
medium. All of the incubations developed a halo under the medium surface; however, very little 293
nitrogenase activity (0.03 nmol/h ethylene) was detected by acetylene reduction. 294
295
Discussion 296
P. agglomerans (Erwinia herbicola) is a cosmopolitan bacterium that lives in diverse 297
environments, including soil (55, 83), water (50), insects (18) and humans (16). This species has 298
been found endophytically in many important crops (34), acting as a plant-growth promoter (2, 299
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45, 46, 70, 79, 81, 85), biocontrol agent (4, 9, 59) and even a systemic resistance inducer (37, 44, 300
56). In this study, we evaluated the interactions of P. agglomerans with sugarcane, aiming to 301
confirm the bacterium’s capacity to cross-colonize this crop and to increase the growth and 302
fitness of sugarcane seedlings. The strain 33.1 was selected due to its identification as a growth 303
promoter in its original host, E. grandis (60). The mechanisms associated with plant-growth 304
promotion and cross-colonization in this bacterium had, however, not been previously described. 305
The 33.1 and 33.1:pNKGFP strains both interacted similarly with sugarcane, forming 306
aggregates around the roots and infecting their hosts through radicular fissures. Frequently, 307
endophytic bacteria live in the soil, and prior to systemic colonization, the bacterial cells attach 308
to the roots close to the fissures caused by lateral root emergence (13). Compant et al. (14) also 309
observed the presence of Burkholderia sp. strain PsJN at lateral root emergence sites, suggesting 310
that crack entry colonization occurred in grapevine plantlets in a similar manner to the 311
phenomenon previously observed with the same strain in potato plants (62). These results 312
suggest that endophytic bacteria, which are able to colonize different host plants, could use the 313
same approach in their first colonization step. Many endophytic species have a broad range of 314
hosts; Herbaspirillum seropedicae, for example, has been found in a wide variety of crops, 315
including sorghum, sugarcane, corn and other grasses (3, 53). An endophyte isolated from a 316
specific host family may colonize other plants, even plants in other families, suggesting a lack of 317
specificity to a particular host (84). For example, Klebsiella pneumoniae (Kp342) isolated from 318
corn was able to colonize wheat, rice, Arabidopsis thaliana and Medicago sativa (20). Azoarcus 319
indigens isolated from a grass pasture also colonized rice and sorghum (24, 62, 71), and a 320
Burkholderia sp. isolated from onion colonized grapevine (15) and potato (52). 321
Using qPCR and re-isolation techniques, we demonstrated that P. agglomerans 33.1, 322
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which was previously isolated from Eucalyptus plants, was able to grow in sugarcane seedlings 323
after systemic colonization. Endophytic bacteria are frequently found at a high concentration in 324
roots, and there is often a gradient of bacterial abundance from the roots to the stem and leaves 325
(43). These endophytes are hypothesized to colonize the root surface before colonizing the host 326
itself (30, 36, 64). Our data indicated that 33.1:pNKGFP has a preference for the rhizosphere 327
over the root and aerial parts of sugarcane, demonstrating typical endophytic behavior during 328
colonization. Under greenhouse conditions, this strain did not affect the density of the 329
endogenous bacterial community, but it was able to induce dry mass accumulation by IAA 330
production and phosphate solubilization. 331
Additionally, P. agglomerans 33.1 stimulated the production of chitinase and 332
endoglucanase by root tissues, with which the bacterial cells had more contact and stayed for a 333
longer time. During plant invasion, bacterial cells may produce hydrolytic enzymes that are 334
recognized by the plant, triggering the plant’s resistance system. Trotel-Aziz et al. (78) reported 335
an increase in the resistance of vines against Botrytis cinerea due to the augmentation of 336
chitinases and other compounds induced by the inoculation of the P. agglomerans strain PTA-337
AF1. 338
We demonstrated that 33.1, an endophytic bacterium that promotes the growth of E. 339
grandis, was able to cross-colonize and promote the growth of sugarcane. This bacterium also 340
induced the synthesis of chitinase and endoglucanase enzymes, which are associated with plant 341
protection against pathogens. Furthermore, new genes may be transferred to 33.1 to improve on 342
its wide range of benefits to host plants. Our results contribute to the understanding of the 343
interactions between plants and endophytes and demonstrate the viability of P. agglomerans 33.1 344
as an inoculant to improve sugarcane productivity. 345
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346
Acknowledgements 347
This work was supported by a grant from the Foundation for Research Assistance, São 348
Paulo State, Brazil (Grant 2002/14143-3). We thank FAPESP for the fellowship to M.C.Q. 349
(Proc. no. 2005/53748-6) and P.B.R. (Proc. no. 2003/01438-8). We also thank Dr. Elliot W. 350
Kitajima (NAP/MEA/ESALQ) for providing the scanning electron microscope and other 351
facilities, as well as Dr. Sabrina Moutinho Chabregas from CTC (Piracicaba, SP, Brazil) for 352
research protocols, valuable discussions and supplying the micro-propagated sugarcane plants. 353
354
References 355
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TABLE 1. Plasmids and strains used in this work. 619
620
621
622
623
624
625
626
627
628
629
630
631
632
633
634
635
636
637
638
639
640
641
Plasmids and Strains Description References
Plasmids
pNKBOR Knr miniTn10 Rossignol et al. (65)
pNKGFP Knr miniTn10, gfp Ferreira et al. (25)
pUC18 Amp r Perron et al. (58)
pCM88 Tet r , gfp Marx and Lindstrom (48)
pSMC21 Ap r , Kn r ,gfp Kuchma et al. (40)
pUC4K Kn r Taylor; Rose (74)
pJTT Kn r cry1Ac7 Downing et al. (21)
pGEMT-easy Amp r Promega
pWM1013 Kn r , desred Brandl and Mandrell (11)
Strains
DH5-α - λ pir recA1 endA1 hsdR1 relA1
λ::pir
Kolter et al. (39)
33.1 Strain from Eucalipto grandis. Procópio (60)
33.1;pNKBOR 33.1 harboring pNKBOR This work
33.1:pNKGFP 33.1 harboring pNKGFP Ferreira et al. (25)
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TABLE 2. Density of sugarcane-associated bacteria measured using re-isolation. 642
643
a The 33.1:pNKGFP density was measured at 30 DAI. Values with the same letter within a 644
column are not significantly different (P > 0.005) according to Tukey’s test, which was 645
conducted with the SAS program (release 9.1). The results are the means of four replicates of 646
each sample. 647
b The bacterial density was measured at 30 DAI with 33.1 or 33.1:pNKGFP. The treatment 648
control contained an inoculation of PBS medium without bacterial cells. Values with the same 649
letter within a column are not significantly different (P > 0.005), according to the Tukey test 650
conducted with the SAS System (release 9.1). The results are the means of four replicates of each 651
sample. 652
653
Samples 33.1:pNKGFP density
Bacterial density
(CFU/gram of sample)b
(CFU/gram of sample)a Control 33.1 33.1:pNKGFP
Rhizosphere 2,9 x105 a 8,6x105A 12,4x105A 18,4x105 A
Root 4,3x102 b 7,1x105A 10,5x105A 4,5x105AB
Aerial part 2,2 x101 b 2.2x103B 1,4x103 B 1,5x103B
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FIG. 1. Sugarcane infection and root surface biofilm formation by 33.1 and 33.1:pNKGFP. 654
Control (A), 33.1 bacterial cells during root infection through rooting fissures, indicated by the 655
white arrow (B), and biofilm formation by 33.1 (C) and 33.1:pNKGFP (D) on the sugarcane root 656
surface. The samples were collected at three days after inoculation. The scanning electron 657
microscopy magnification was 500X in A, 1500X in B and D and 3000X in C. Scale bar: 20 µm 658
in A and D and 10 µm in B and C. 659
660
661
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FIG. 2. Visualization of 33.1:pNKGFP cells during biofilm formation on the sugarcane root 662
surface using light microscopy at 5 (A), 10 (C) and 18 DAI (E) and fluorescence microscopy 663
under a FITC (510 nm) filter to visualize the decrease in fluorescence at each respective time (B, 664
D and F).The arrows indicate the 33.1:pNKGFP bacterial cells that expressed GFP. The 665
microscopy magnification was 200X and the scale bar: 10 µm. 666
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FIG. 3. Specificity of conventional PCR with the PNKF and PNKRII primer set for the detection 669
of P. agglomerans 33.1:pNKGFP. (A) 1 - plasmid pNKBOR, 2 - plasmid pNKGFP, 3 - plasmid 670
pUC18, 4 – plasmid pCM88, 5 – plasmid pSMC21, 6 – plasmid pUC4K, 7- plasmid pJTT, 8 – 671
plasmid pGEMT-easy, 9 – plasmid pWM1013, 10 – 33.1:pNKBOR, 11 – 33.1:pNKGFP, 12 – 672
33.1, M – 100-bp DNA ladder (Fermentas). The PCR product, indicated by the arrow, is 360 bp. 673
(B) Integration of the pNKGFP fragment into the P. agglomerans 33.1 chromosome, confirmed 674
with Southern blot analysis of the plasmid and chromosomal DNAs cut with EcoRI and probed 675
with the 360 bp amplicon fragment obtained from a PCR reaction using the primers PNKF and 676
PNKRII. 1 - 33.1, 2 – plasmid pNKGFP and 3- 33.1:pNKGFP. 677
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FIG. 4. P. agglomerans 33.1:pNKGFP density during sugarcane cross-colonization, measured 678
using qPCR (A) and re-isolation (B) at 4 and 15 DAI. The abundance data, in CFU/g of tissue, 679
were log-transformed to stabilize the variance. The results are the means of the four replicates for 680
each sample. The bars represent the standard error of each treatment. 681
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FIG. 5. Sugarcane plant-growth promotion by P. agglomerans 33.1. The strain was added to the 683
substrate of acclimated (A) SP80-1842 and (B) SP80-3240 sugarcane. The data are the average 684
dried weights at 30 DAI. The bars represent the standard error of each treatment. The values with 685
asterisks (**) in the same tissue and varieties are significantly different (α = 0.01) from the 686
control according to Student’s t test. (C) Sugarcane variety SP80-1842 without (left) and with 687
33.1 inoculation (right). (D) Sugarcane resistance-protein production. a The index of enzymatic 688
activity was calculated based on the absorbance/ml of substrate/h. The values with asterisks (* or 689
**) are significantly different from the control treatment (α = 0.05 and 0.01, respectively) 690
according to Student’s t test. 691
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