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1. INTRODUCTION
The drastic losses of original native forests in the Northwest region of Parana state in Southern Brazil occurred along the last century during the process of colonization. From 100% of forest cover in the 19th century, only 5% of the Atlantic Forest was remnants and many reforestation projects in degraded areas were carried with native trees (IPARDES, 2010).
Leguminous plants have great potential to restore degraded areas, and in addition increase soil quality and fertility (ALVINO-RAYOL et al., 2011). Schizolobium parahyba (Vell.) S.F. Blake var. parahyba (LEWIS, 2010), commonly known as guapuruvu, is a leguminous with fast grow that is used in a reforestation project (PIETROBOM; OLIVEIRA, 2004; SEREDA et al., 2008; CALLADO; GUIMARÃES, 2010). S. parahyba is a pioneer tree that occurs naturally in Atlantic Forest (LORENZI, 1992) in the states of Bahia, Espírito Santo, Paraná, Rio Grande do Sul, Santa Catarina and São Paulo (CARVALHO, 2005). This is considered an ecologically and economically important species due to its significant wood yield potential (BORTOLETTO JÚNIOR; BELINI, 2002).
The use of growth-promoters microorganism inoculum should be considered as a technological improvement of intensive forest cultivation using N-fixing bacteria and arbuscular mycorrhizal fungi (AMF) to increase wood production (SCHIAVO; MARTINS, 2003; SIVIERO et al., 2008). In the rhizosphere soil there are several species of microorganisms composing the microbial community that act in biogeochemical cycles with an important role on soil fertility and plantgrowth (ANDRADE, 2004; HERNANDÉZ-ORTEGA et al., 2011).
Pioneer leguminous woody plants establish symbiosis with AM fungi, which result in benefit for both, transferring P for plant and carbohydrates to fungi (ZANGARO et al., 2003). Many bacteria from rhizosphere soil can promote plant growth and named plant growth promoting rhizobacteria (PGPR) (ARTUSOON et al., 2006). In many cases, Rhizobium sp. strains, a symbiotic bacteria, can act as PGPR, mainly in non-nodule formation plant as S. parahyba (SIVIERO et al., 2008). The inoculation of legume plants with PGPR and AM fungi can increase plant growth (ABDALLA et al., 2000; MARIN et al., 2010), and there are several reports that show a beneficial effects in the interaction of AMF and diazotrophic bacteria (BAREA et al., 2005; RAIMAM et al., 2007; SALA et al., 2007; MIYAUCHI et al., 2008). The aim of this work was to evaluate the contribution of the microbial inoculants Glomus clarum and Rhizobium sp. in promote plant growth of S. parahyba under field conditions.
2. MATERIAL AND METHODS
2.1. Experimental Design
The experiment was carried out at Xambrê, PR, in southern Brazil (23° 47'27''S and long. 53° 35'45''W) from March to November 2011. The climate is humid subtropical, the mean of rainfall around 1.4 m year-1 (IAPAR, 2000) and the soil was a Rhodic Ferralsol (FAO-UNESCO, 1989).The study area (120 m X 60 m) previously was covered with Brachiaria decumbensand 60 days before planting was sprayed glyphosate to desiccate the grass.
The experimental design was a randomized complete block arranged in a factorial treatment combination with five replications and three factors: N-fixing bacteria (Rhizobium sp.), AM fungi (G. clarum) and fertilize NPK (20:5:20), resulting in the following treatments: 1. Control; 2. Rhizobium sp.; 3. G. clarum; 4. Fertilizer; 5. Rhizobium sp. + fertilizer; 6. Rhizobium sp.+ G. clarum; 7. G. clarum + fertilizer; and 8. Rhizobium sp.+ G. clarum + fertilizer). Each block was composed by eight plots corresponding to the eight treatments and each plot had twenty plants arranged in spacemen of 3 m X 3 m.
2.2. Soil and plant
Soil chemical characterization was made from a composite sample collected before experiment installation at 0-20 cmof depth. The chemical soil analysis was: pH (CaCl2) 4.5; Al3+ 0.26 cmol cdm-3; H + Al 4.60 cmolcdm-3; Ca2+ 0.86 cmol cdm-3; Mg2+ 0.35 cmol cdm-3; K+ 0.26 cmol cdm-3; P 24.0 mg dm-3; C 7.37 g dm-3. Seeds of S. parahyba were collected at the campus of the State University of Londrina, Londrina, PR, Brazil, selected, mechanically scarified and sown in tubettes in substrate Rhodic Ferralsol (FAO-UNESCO, 1989) mixed with vermiculite 4:1. After 30 days, the seedlings were taken to the experimental area and planted.
2.3. Bacteria inoculum
The bacterial strain used as inoculum was Rhizobium sp. isolated from nodules of Cassia sp. provided by our own collection. To prepare the inocula, the strain was cultivated in Petri dishes with YMAmedia (VINCENT, 1970) plus Congo red (0.25%) and incubated at 28 °C 48 hÉ1. The cells were suspended in sterile saline solution (NaCl 0.85%) at 107 colony forming unit (CFU mLÉ1) according to CaCO3 solution standard and 1 mL of bacterial suspension was dropped around the seedling when the first pair of leaves appeared.
2.4. AM Fungi inoculum
The inoculum of G. clarum was from our own collection and is keeping in pots with Brachiaria decumbens. Ten grams of crude inocula (spores, colonized root and mycelia) containing 53 spores g-1 of soil was added in the tubettes, after inoculation a thin layer of soil was covered (around 2 cm) and then the seed was sowed. The number of AMF spores and root colonization of B. decumbens which was present in the experimental area before the establishment of the experiment was determined and the number of spores and root colonization of S. parahyba were evaluated at the end of experiment. Treatment with chemical fertilization occurred with addition of 100 g of NPK fertilizer 20-5-20 per plant.The percentage of plants roots infected with AM fungi was estimated on stained samples (PHILLIPS; HAYMAN, 1970) by the grid-line intersect method (GIOVANETTI; MOSSE, 1980) by microscopic examination.
2.5. Data analysis
The variables evaluated were total height and survival at 30, 60, 120,180 and 240 days, and stem diameter (10 cm above the soil) at 180 and 240 days after planting seedlings. Data sets were tested for normality and homogeneity of variance and were evaluated by analyses of variance (ANOVA). The Tukey's Honest significant difference test was performed at p d” 0.05.
3. RESULTS
After 30 days no significative differences were observed in the variables analyzed. After 60 days and during the all experiment the plants treated with fertilizer showed higher height when compared with others treatments (Table 1).
Table 1 Total height (cm) of Schizolobium parahyba pv. parahyba treated with AM fungi (Glomus clarum), PGPR Rhizobium sp., and fertilizer NPK 20-5-20 at 30, 60, 120, 180 and 240 days after seedling planting. (Gc) Glomus clarum; (Rhi) Rhizobium sp.; and (Fert) Fertilizer. Means in the column with the same letter are not significantly different according to Tukey test (p< 0.05).
Tabela 1 Altura total (cm) do Schizolobium parahyba var. parahyba tratada com fungo MA (Glomusclarum), PGPR Rhizobium sp. e fertilizante NPK 20-5-20 aos 30, 60, 120, 180 e 240 dias após o plantio das mudas. (Gc) Glomusclarum; (Rhi)Rhizobium sp.; e (Fert) Fertilizante. Médias nas colunas com a mesma letra não apresentam diferenças significativas pelo teste de Tukey (p<0,05).
| Treatment | 30 days | 60 days | 120 days | 180 days | 240 days |
|---|---|---|---|---|---|
| Gc | 23.94 a | 31.15 a | 37.32 a | 44.51 a | 78.73 a |
| Gc control | 25.21 a | 32.79 a | 39.03 a | 46.04 a | 75.99 a |
| Rhi | 24.20 a | 32.05 a | 38.21 a | 42.94 a | 75.27 a |
| Rhi control | 24.96 a | 31.89 a | 38.14 a | 47.61 a | 80.01 a |
| Fertilizer | 24.78 a | 33.97 b | 41.82 b | 50.06 b | 84.60 b |
| Fert. control | 24.38 a | 29.97 a | 34.53 a | 39.51 a | 68.65 a |
| ANOVA (p values) | |||||
| Gc | 0.0567 | 0.1183 | 0.1986 | 0.8616 | 0.3166 |
| Rhi | 0.2442 | 0.8804 | 0.9605 | 0.2159 | 0.5997 |
| Fertilizer | 0.5363 | 0.0005 | 0.0001 | 0.0053 | 0.0049 |
| Gc*Rhi | 0.5465 | 0.5201 | 0.9303 | 0.6658 | 0.8595 |
| Gc*Fert. | 0.6758 | 0.6316 | 0.9061 | 0.4208 | 0.1484 |
| Rhi*Fert. | 0.2098 | 0.3429 | 0.0825 | 0.1650 | 0.5934 |
| Gc*Rhi*Fert. | 0.6758 | 0.5078 | 0.2063 | 0.7645 | 0.8865 |
The same results were observed in stem diameter (10 cm above the soil) after 180 and 240 days, plants treated with fertilizer showed larger stem diameter when compared with others treatments (Table 2).
Table 2 Stem diameter of Schizolobium parahyba pv. parahyba after 180 and 240 days after seedling planting. (Gc) Glomus clarum; (Rhi) Rhizobium sp.; and (Fert) Fertilizer. Means in the column with the same letter are not significantly different according to Tukey test (p< 0.05).
Tabela 2 Diâmetro do caule de Schizolobium parahyba var. parahyba após 180 e 240 dias após o plantio das mudas. (Gc) Glomus clarum; (Rhi) Rhizobium sp.; e (Fert) Fertilizante. Médias nas colunas com a mesma letra não apresentam diferenças significativas pelo teste de Tukey (p< 0,05).
| Treatment | 180 days | 240 days |
|---|---|---|
| Gc | 1.22 a | 2.27 a |
| Gc control | 1.18 a | 2.30 a |
| Rhi | 1.16 a | 2.18 a |
| Rhi control | 1.24 a | 2.40 a |
| Fertilizer | 1.38 b | 2.60 b |
| Fert. Control | 1.00 a | 1.90 a |
| ANOVA (p values) | ||
| Gc | 0.5537 | 0.7631 |
| Rhi | 0.4850 | 0.3496 |
| Fertilizer | 0.0005 | 0.0011 |
| Gc*Rhi | 0.6919 | 0.6312 |
| Gc*Fert. | 0.8392 | 0.2757 |
| Rhi*Fert. | 0.3367 | 0.9691 |
| Gc*Rhi*Fert. | 0.6351 | 0.7519 |
Plants survival showed different response. After 30 to 120 days the survival of control plants was the same of more effective treatments, except for Rhi. After 120 days, all treatments showed differences when compared with control except for Rhi and Gc. The survival of control plant was very low after 240 days (16.7%) and plants inoculated with Rhizobium sp. and G. clarum presented 40 and 36.7% of plants survival, respectively. However, the treatment Fert showed 73.3% of survival, but differences were observed only for control, Rhi and Gc. In 180 and 240 days, the dual inoculation (Gc + Rhi) showed good response of survival against stress conditions (around 50%) as well as when the microorganisms were combined with chemical fertilizer as Rhizobium sp. and G. clarum with 50 to 60% of survival (Figure 1).
Figure 1 Effect of AM fungi (Glomus clarum), PGPR Rhizobium sp strain, and fertilizer NPK 20-5-20 on survival (%) on Schizolobium parahyba pv. parahyba at 30, 60, 120, 180 and 240 days after seedling planting. Bars represent the standard error of mean. Gc: Glomus clarum; Rhi: Rhizobium sp; and Fert.: Fertilizer.
Figura 1 Efeito do fungo MA (Glomus clarum), da cepa PGPR de Rhizobium sp, e fertilizante NPK 20- 5-20 na sobrevivência (%) de Schizolobium parahyba var. parahyba, durante 30, 60, 120, 180 e 240 dias após o plantio da muda. Barras representam o erro-padrão. Gc: Glomus clarum; Rhi: Rhizobium sp; e Fert.: Fertilizante.
During the experiment before 120 days the plants suffered a high stress conditions, first at all there was drought time for 60 days. After 120 days, in June occurred a frost for two days, in this conditions the tropical woody plant suffered an intensive stress with low temperature (Table 3).
Table 3 Monthly temperature and pluviometric index during the experimental time (March to November, 2011).
Table 3 Temperatura mensal e índice pluviométrico durante o experimento (março a novembro de 2011).
| Days | Month | Temperature | Precipitation |
|---|---|---|---|
| (°C) | (m) | ||
| Planting | Mar. | 20.5 | 1.031 |
| 30 | Apr. | 15.7 | 1.682 |
| 60 | May | 14.9 | 0.092 |
| 90 | Jun. | 11.0 | 1.389 |
| 120 | Jul. | 14.8 | 1.418 |
| 150 | Aug. | 15.1 | 1.081 |
| 180 | Sep. | 16.4 | 0.692 |
| 210 | Oct. | 18.4 | 192.0 |
| 240 | Nov. | 19.1 | 205.4 |
In the soil samples collected before establishment were found 28 spores g-1of rhizosphere soil, and root samples of B. decumbens showed 83% of AM root colonization. At the end of the experiment were found 28 spores of AM fungi g-1 of rhizosphere soil from inoculated plants of S. parahyba and roots showed 50% of AM colonization, in non-inoculated plants were found 19 spores g-1 of soil and 30% of AM root colonization.
4. DISCUSSION
Plants from Brachiaria genus are largely used as AMF host, because grass as B. brizantha increase the potential of AM inocula in the soil due to its high level of colonization by AM fungi (SANTOS et al., 2000; CAPRONI et al., 2003; CORDEIRO et al., 2005; MELLO et al., 2006). The roots from B. decumbens showed high level of AM colonization before S. parahyba was planted in the experimental area.
The colonization rate of Brachiaria roots by native AM present in the experimental area did not influence the colonization root of S. parahyba by G. clarum, because the non-inoculated plants showed very low AM colonization when compared with inoculated plant with G. clarum. Is large known that our own inoculum of G. clarumshowed high infectivity and effectivity on plant host. The performance of introduced AMF depends of many factors to compete with native AMF such as fast infectivity and establishment and ability to maintain root colonization (WILSON; TOMMERUP, 1992).
On the other hand, Siviero et al. (2008) found that S. parahyba pv. amazonicum inoculated with G. clarum increased plant growth in Amazon area, and agree with Santos et al. (2000) where G. clarum showed high level of root colonization of Cryptomeria japonicum and success to compete with indigenous community of AM fungi.
The low effectiveness of G. clarum on plant growth probably was influenced by high level of P of soil (24.0 mg dm-3). Is large known that the high concentration P may influence negatively the AM fungi (BRESSAN; VASCONCELLOS, 2002; KIRIACHEK et al., 2009), also Costa et al. (2005) found that the inoculation of AMF in seedlings of Ancornia speciosa and Malpighia ermaginataincreases plant growth only in soil with low P around 3.0 and 4.0 mg dm-3.
G. clarumprefer pH up to 6.0 (SIQUEIRA; FRANCO, 1988), and the low pH 4.5 in the experimental soil may also influenced the establishment of the symbiosis between G. clarum and plant root. Also the interaction between Rhizobium sp. strain under field conditions is influenced by temperature, soil acidity, nutrient concentration and plant host. These factors can promote low response of inoculation, decreasing efficiency of plant to establish a symbiotic relationship with diazotrophic bacteria (MORAES et al., 2010). In spite of Schizolobium spp is non-nodule forming plant Siviero et al. (2008) found response of Rhizobiumsp inoculation acting as free living bacteria in the rhizosphere of S. parahyba pv amazonicum.
The presence of G. clarum and Rhizobium sp. did not promote S. parahyba growth but protected against stress conditions occurred during the experiment time. After planting the seedlings suffered with dry weeks and a freeze conditions for two days. The non-inoculated plants showed a high level of mortality when compared with inoculated plants with microorganisms or fertilized. Some studies demonstrated that S. parahyba did not support low temperatures and high decrease plant growth or death (CARVALHO, 2003; SOUZA et al., 2011). S. parahyba showed sensibility for temperature variation(CALLADO; GUIMARÃES, 2010) and low capacity of osmotic adaptation when compared with S. parahyba pv amazonicum during water stress (CARVALHO, 2005).
The high mortality observed in non-inoculated plants suggested that low temperatures affected endogenous mycorrhizal symbiosis (HEINEMEYER; FITTER, 2004), but not inoloculated AM fungi who might increase the tolerance of plants to drought keeping water potential gradient in the root protecting against oxidative stress increasing plant tolerance to drought (PORCEL; RUIZ-LOZANO, 2004). The effects of arbuscular mycorrhizal symbiosis on host plant tolerance against the water deficit caused by drought is related with the increase of stomatal conductance that consequently enhances the water use efficiency of plants (RUIZ-LOZANO; AROCA, 2010).
S. parahyba var. parahyba responded by chemical fertilize increased plant growth but not survival when compared with inoculated plants with G. clarum and/or Rhizobium sp. suggest that inoculation of microorganisms conferred climate stress resistance. The mortality occurred between 120 and 180 days especially in control plants, the results suggested that the nutrient deficiency associated with environmental factors caused plant stress and death.
5. CONCLUSIONS
The high level of P soil in the experimental area probably decreased the effectiveness of G. clarum and Rhizobium sp. on plant growth when compared with fertilized plants. Also the environmental conditions during winter time (dry season and freeze) increased plant mortality especially of control plants. Otherwise AM fungi and Rhizobium sp. protected plants against drought and freeze. Otherwise, the planting time must be between November to December to avoid the presence of young plants during winter time.
