The role of rhizobacteria in rice plants: Growth and mitigation of toxicity

Marcela C F Rêgo, Aline F Cardoso, Thayná da C Ferreira, Marta C C de Filippi, Telma F V Batista,Rafael G Viana, Gisele B da Silva

1 Plant Protection Laboratory, Institute of Agrarian Sciences, Federal Rural University of Amazon, Belém 066.077-830, Brazil

2 Phytopathology Laboratory, Brazilian Enterprise for Agricultural Research-Rice and Beans, Goiania 75375-000, Brazil

Abstract Allelopathic compounds reduce the growth and productivity of upland rice plants, especially in consecutive plantations.The rhizobacteria Pseudomonas fluorescens BRM-32111 and Burkholderia pyrrocinia BRM-32113 have been recorded as growth promoters in rice. This study was developed to understand the effect of the application of rhizobacteria on upland rice plants in consecutive plantations. Experiments were conducted in a completely randomized design with four replications of four treatments: rice seed inoculated with P. fluorescens BRM-32111, rice seed inoculated with B. pyrrocinia BRM-32113(both sown on soil with rice residue), non-inoculated plants sown on soil with rice residue (control with residue (WR)), and non-inoculated plants on soil with no residue (NR). Roots and seedling growth were adversely affected by allelopathic compounds in control WR plants. Plants inoculated with rhizobacteria P. fluorescens BRM-32111 or B. pyrrocinia BRM-32113 induced an increase of 88% in biomass, 3% in the leaf area, 40% in length, 67% in root biomass, 21% in chlorophyll a,53% in chlorophyll (a+b), 50% in rate of carbon assimilation (A), 227% in A/rubisco carboxylation efflciency (Ci) and 63%in water use efflciency (WUE) compared to control WR plants. These results indicate that rhizobacteria P. fluorescens BRM-32111 and B. pyrrocinia BRM-32113 increase the tolerance of rice plants to stress from allelochemicals. There are possible practical agricultural applications of these results for mitigating the effects of environmental allelochemistry on upland rice.

Keywords: allelopathy, B. pyrrocinia, P. fluorescens, rhizobacteria, rice

1. Introduction

Consecutive plantations of upland rice experience a reduction in growth and productivity of up to 65% in flve years of cultivation (Fageria and Baligar 2003; Pinheiroet al.2006). The same allelopathic effect is observed in maize and sorghum plantations (Amb and Ahluwalia 2016). This effect is probably due to the degradation of plant residues that release allelochemicals and the accumulation of metabolites from root exudates present in the soil. Among the metabolites that produce allelopathic effects in rice are terpenoids, steroids, phenols, coumarins, flavonoids,tannins, alkaloids, cyanogenic glycosides, and other compounds (Rice 1984; Putnam 1988).

Allelopathy is deflned as any process involving secondary metabolites produced by plants, microorganisms, viruses,and fungi that influence the growth and development of agricultural production and biological systems (Torreset al. 1996). These compounds can act by inhibiting germination, growth, and reduction of plant root depth. The deleterious effect may vary according to the cultivar and the concentration and combination of metabolites (McPhersonet al. 1971; Chouet al. 1991).

The development of technologies to mitigate the damage of allelochemicals in consecutive rice plantations is an important topic for research in upland conditions.Approaches to mitigation include the selection of rice cultures tolerant to allelochemicals (Wathugala 2015); the rotation of crops with Fabaceae family species asVigna unguiculata(L.) Walp,Crotalaria paulinaSchrank, andMucuna aterrimaPiper and Tracy (Marenco and Santos 1999); and the use of microorganisms, such as arbuscular mycorrhizal fungi (AMF) and plant growth promoting rhizobacteria (PGPR) (Khalil 1999; Major 2010).

PGPRs are beneflcial bacteria capable of inducing growth and increasing plant tolerance to biotic and abiotic stresses (Kloepperet al. 1980; Kremer 2006; Mishra and Nautiyal 2012). The use of microorganisms to increase tolerance of allelochemicals is a sustainable option that allows cultivation of soil without excess fertilizers. One of the actions of the allelochemicals on plants is the alteration of the mitotic activity of young cells, with a decrease in cell numbers, cell stretching, or both, an action that results in inhibition of metabolic activities and lower growth rates in plants (Rice 1984). The inhibition in growth may also be due to a decrease in germination, which can be attributed to a change in enzymatic activity that affects the mobilization of storage compounds during germination which, in turn,causes a swelling response and increasing the peroxidase level in rice plants.

The application of AMF and PGPRs increase plant tolerance to allelochemical compounds (Khalil 1999; Major 2010). AMF acts on plants by altering absorption of water(Egerton-Warburtonet al. 2007), nitrogen, phosphorus,and metals (Bartoet al. 2011) and inhibits the effects of allelochemicals present in the soil on plants (Bartoet al.2012).

Technology that modifles allelochemicals by transforming them into plant growth regulators in sorghum enables efflcient management of agriculture, due to the degradation of the allelochemicals in the soil (Bhadoria 2011; Uddinet al.2014; Ihsanet al. 2015).Sorghum bicolor(L.) releases a radicular exudate called sorgoleone that can inhibit weed growth, but the sorghum plant becomes tolerant to allelochemicals when treated with microorganisms capable of using sorgoleone as a carbon source and mineralize it through complete degradation of CO2in the soil (Gimsinget al. 2009).

However, it has been hypothesized that PGPRs, besides inducing growth in rice plants (Rêgoet al. 2014), also induce increased tolerance to the allelochemicals left in the soil by previous crops. This mechanism had not been demonstrated on upland rice. Therefore, our objective for this study was to investigate the application of PGPRs(Pseudomonas fluorescensBRM-32111 orBurkholderia pyrrociniaBRM-32113) on rice plants grown on soil containing rice plant residues.

2. Materials and methods

2.1. Plant materials and microorganisms

In fleld conditions, soil preparation for upland rice cultivation is done by plowing and leveling the soil. When the soil is compacted, subsoiling must be performed. Immediately after the rainy period, acidity is corrected by liming, and recommended fertilization, after which the planting is done,which is done after the end of the rainy season, however,the soil must be moist during the growth period, the control of invasive plants are required by both the competition for spatum and nutrients and by the allelopathic compounds that can be released by them, just as the rice plants themselves can leave in the fleld and interfere with future plantings,necessitating the opening of new planting areas.

In this job, the planting soil was from an area of commercial cultivation. The experiment used a completely randomized design with four replications (one replication was one pot with flve plants) of four treatments. The two PGPR treatments were rhizobacteriaPseudomonas fluorescensBRM-32111 andBurkholderia pyrrociniaBRM-32113, each inoculated in seeds in soil with plant residue. There were two controls: seeds with water (noninoculated seeds) in soil with residue (control WR), and noninoculated seeds in soil without residue (control NR). Spring rice seeds Primavera were inoculated with a suspension ofPseudomonas fluorescensBRM-32111 orBurkholderia pyrrociniaBRM-32113, made according to the methodology described by Fillipiet al. (2011). Control seeds were submerged in sterilized distilled water. For all treatments except control NR, 23 mg of rice roots were added to the soil of each pot, as described by Ranagalageet al. (2014).Content of allelopathic compounds in Table 1. The soil was collected from a non-cultivated area of the Amazonian ecosystem in Pará, Brazil, and was characterized as follows:H2O pH 5.4, 589 g clay kg–1, 66 g silt kg–1, 144 g sand kg–1,63 mg K+dm–3, 4 mg P dm–3, 0.4 mg Ca2+dm–3, 0.2 cmolc Mg2+dm–3, 0.1 cmolc Al3+dm–3, and 3 mg Si kg–1.

2.2. Seed germination and seedling vigor

In a plastic box(11 cm×11 cm×3.5 cm) containing 100 g of soil and moistened with 50 mL of sterile distilled water,we sowed 25 seeds per replicate, totaling 100 seeds per treatment. Seven days after sowing, germination (%) and fresh seedling mass were measured. This assay was repeated twice.

2.3. Promotion of growth

The experiment was conducted under greenhouse conditions at Universidade Federal Rural da Amaz?nia(UFRA), Belém, PA, Brazil. Pots contained 750 g dystrophic oxisol, fertilized with 1.17 g N-P-K (nitrogen, phosphorus,and potassium), 0.35 g of urea, and 0.7 g of micronutrients(sulfur 3.9%, boron 1.8%, copper 0.85%, manganese 2.0%, and zinc 9.0%) each. Ten seeds were sown per pot.Plants were thinned to flve plants per pot at 11 days after germination. Twenty-one days after sowing, we evaluated total biomass (mg), plant height (cm), leaf area (cm2), relative chlorophyll content (SPAD), root biomass (mg), root length(cm), and gas exchanges, and quantifled photosynthetic pigments. This assay was repeated four times.

2.4. Anatomical study of the leaf

The size of the stomata was calculated as equivalent to the area of the ellipsoid representing the area of the stomatal pores (ASP), using the formula ASP=(π×Length×Width)/4,according to methodology described by Minnocciet al.(1999) and Bartoliniet al. (1997). The stomatal density (SD,stomata mm–2) (n=20 plants per treatment) of the inferior lamina was calculated according to Sacket al. (2003). The density of foliar tissues (DFT, mg cm–3) was calculated as the ratio of leaf mass per unit area of leaf (Mg cm2) and leaf thickness (Wright and Westoby 2002). Measurements were made using a 0.5 cm×0.5 cm piece of the leaf blade with the aid of a Motic microscope (BA400, China Group Co.).

2.5. Quantification of pigments

Using another leaf, 15 mg of tissue was removed, macerated in 240 μL of 98% EtOH, incubated at 80°C for 20 min,and centrifuged at 4°C at 14 000 r min–1for 5 min. The supernatant was collected, and the pellet was subjected to two more extractions, in 80 and 50% EtOH, respectively.The supernatants were collected and homogenized.Pigment extraction steps were performed under an ice bath and in the absence of light, according to the methodology deflned by Porra (1989). Subsequently, an aliquot of 20 μL of the ethylic extract of each sample was added to a reaction medium with 120 μL of 98% EtOH and 40 μL of the ethyl mix.Absorbance of this sample was estimated at wavelengths(λ) of 645 and 665 nm (OD645and OD665, respectively).Concentrations of chlorophylla,b,a/bratio, and chlorophyll(a+b) were calculated using eqs. (1) and (2) below. They were then normalized using the fresh mass of each sample.

Table 1 Allelopathic compounds, root content of flve plants (one pot) of rice obtained from microbiolized seeds with bioagents,21 days after sowing

2.6. Gas exchange

The rice plants were evaluated for gas exchange with a portable photosynthesis system (LI-6400XT; LI-COR,Lincoln, NE, USA). The net rate of assimilation of CO2(A),stomatal conductance (Gs), transpiration (E), intercellular concentration of CO2(Ci), estimated water use efflciency(WUE) (A/E), andcarboxylation efflciency of rubisco (A/Ci)were evaluated at a 400 μmol mol–1concentration of CO2,50% relative humidity, 28°C temperature, 300 μmol s–1air flow, and a photon flux density of 1 200 μmol m2s–1. The instrument was used according to the manufacturer’s instructions.

2.7. Statistical analysis

The data obtained in the experiments were subjected to analysis of variance (ANOVA). Means were compared using Duncan’s Multiple Range Test (P＜0.05) and standard error(P＜0.05) calculated in the Software SPSS 21.0. The linear correlation coefflcient (P＜0.05) between the growth variables of rice plants at 21 days after sowing was calculated in the Software Past 2.0.

3. Results

3.1. Seed germination, seedling vigor, and growth promotion in plants

Control WR seeds had a 2% reduction in germination when compared to control NR seeds (Fig. 1), but the seeds inoculated withP.fluorescensBRM-32111 had a 2% increase in germination compared to control WR seeds(Fig. 2-A). The length of the seedlings in all treatments with residue in the soil had an average reduction of 22%(Fig. 2-B) compared to control NR seedlings. The biomass of seedlings inoculated withP.fluorescensBRM-32111 was 27% higher than that of the control WR seedlings(Fig. 2-C).

The control WR plants had a reduction in all variables related to growth promotion (Fig. 2). The biomass of the plants inoculated with the rhizobacteria increased 88%compared to control WR (Fig. 2-D). The length of plants withB.pyrrociniaBRM-32113 increased by 25 and 22%in relation to control NR and control WR, respectively(Fig. 2-E). Plants inoculated with rhizobacteria showed an increase of 3% in leaf area, 30% in relative chlorophyll content (SPAD), 40% in root biomass, and 67% in root length in relation to control WR (Fig. 2-H).

There were 19 and 91% increases in stomatal pore length in plants inoculated withP.fluorescensBRM-32111 in relation to control NR and control WR, respectively (Figs. 3-A and 4). The stomatal pore area and stomatal density were 102 and 83% higher, respectively, in plants treated withP.fluorescensBRM-32111 than those in control WR(Fig. 3-B). DFT was 18 and 8% higher in plants inoculated withP.fluorescensBRM-32111 in relation to the control NR and control WR plants, respectively (Figs. 3-D and 4).

3.2. Quantification of pigments

The concentration of pigments was reduced in control WR plants when compared to control NR. In plants inoculated with rhizobacteria, there were increases of 21%in chlorophyllaand 53% in the sum of chlorophyll (a+b)(Fig. 5). For chlorophyllband the chlorophylla/bratio,there were increases of 22 and 25%, respectively, in plants inoculated withP.fluorescensBRM-32111 in relation to the control NR, and 21 and 28%, respectively, in relation to the control WR (Fig. 5-B–D).

Fig. 4 Electromicrographs of the abaxial face of leaves of rice obtained from seeds treated with plant growth promoting rhizobacteria (PGPR) at 21 days following sowing in soil without residue (NR) and with residue (WR). A, control NR. B, BRM-32111 WR. C, BRM-32113 WR. D, control WR. Arrows,stomata.

3.3. Gas exchange

All gas exchange parameters were different in control WR plants than in control NR and rhizobacteria treatment plants.The plants inoculated with the rhizobacteria showed an increase of 37 and 50% inAin relation to the control NR and control WR, respectively (Fig. 6-A). For WUE, the increases were 128 and 63% compared to the control NR and control WR, respectively (Fig. 6-E). However, there was a 227% increase in rubisco carboxylation efflciency in plants with rhizobacteria and control WR compared to the control NR (Fig. 6-F). However, control NR plants showed a 47% increase inGs, 42% inCi, and 60% inEcompared to plants inoculated with rhizobacteria (Fig. 6-B–D).There is a correlation between the parameters related to photosynthetic pigments and gaseous exchanges (Table 2).

4. Discussion

4.1. Seed germination, seedling vigor, and growth promotion in plants

Growth promotion can take place directly (inducing changes physiological, biochemical, molecular and morphological,anatomical) or indirectly (through induction of resistance to biotic and abiotic stresses) defined by Bachet al. (2016).Studies carried out by Filippiet al. (2011), Rêgoet al. (2014),Nascenteet al. (2016), and Buenoet al. (2017), indicate that isolates BRM-32111 and BRM-32113, present action to promote growth in plants of both forms (direct and indirect).

The isolates BRM-32111 and BRM-32113 previously tested in upland rice plants inoculated with rhizobacteria,and not inoculated (control). In plant inoculated, there was an increase in siderophore production and enzymatic activity(peroxidase, chitinase, and β-1,3-glucanase) (Filippiet al.2011), increase in plant length and biomass and anatomical adaptations (Rêgoet al. 2014), the highest absorption of nutrients, nitrogen, phosphorus, calcium, silicon, iron, and manganese (Nascenteet al. 2016; Buenoet al. 2017), and change in the synthesis of indoleacetic acid (AIA) (Nascenteet al. 2016), these changes induced by the rhizobacteria evidenced their effect of growth promoter.

In the present study, rice residues added to soil reduced germination, length, and biomass of upland rice seedlings.However, in rice seedlings inoculated with rhizobacteriaP.fluorescensBRM-32111, the damage to germination,length, and biomass was mitigated when compared to noninoculated plants grown in control WR, indicating that the rhizobacteria induced protection against the harmful effects of the allelochemicals at the seedling stage. In continuous upland rice plantations, there is a reduction of up to 65%in productivity in subsequent crops, which is attributed to several factors including the release of allelochemicals from rice residues in soil (Chou 1991). The major allelochemicals include phenolic acids, p-coumaric acid, ferulic acid, benzoic acid, syringic acid and salicylic acid (Chou and Lin 1976;Rice 1984; Putnam 1988). Soil rice residue compounds reduce germination, plant growth, leaf expansion, and root elongation, as shown in the present study (Figs. 1 and 2).These negative effects have also been recorded for corn cultivation onSorghum bicolorand rice residues (Amb and Ahluwalia 2016). These damages have been attributed to a chain of signals perceived by radicles that absorb the allelopathic substances released into the soil after rice roots decompose. When allelopathic substances come in contact with the cellular membrane of the root, they cause depolarization, a change in the efflux of ions, and a reduction in hydrolytic conductivity, resulting in lower absorption of water and nutrients by the roots and thus interfering in the development of the plant (Baziramakengaet al. 1995;Lehman and Blum 1999).

Fig. 5 Treament control (not inoculated), and plants inoculated with Burkholderia pyrrocinia BRM-32113 and Pseudomonas fluorescens BRM-32111. Chlorophyll a (mg g–1 from fresh paste (FP)) (A), chlorophyll b (mg g–1 from fresh mass (MF)) (B), chlorophyll(a+b) (mg g–1 from MF) (C), chlorophyll a/b (mg g–1 from MF) (D), at 21 days after germination, in soil without residue (NR) and soil with residue (WR) rice plants. Bars (SE) followed by the same lowercase letter do not differ signiflcantly (Duncan, P＜0.05), n=20.

Fig. 6 Treament control (not inoculated), and plants inoculated with Burkholderia pyrrocinia-BRM-32113 and Pseudomonas fluorescens BRM-32111. Net assimilation rate of carbon (A) (mmol CO2 m–2 S–1) (A), stomatal conductance to water vapor (Gs)(mmol H2O m–2 S–1) (B), intercellular carbon (Ci) (mmol H2O m–2 S–1) (C), sweat rate (E) (mmol H2O m–2 S–1) (D), efflcient use of water (WUE) (A/E) (E), carboxylation efflciency of rubisco (A/Ci), at 21 days after germination, in soil without residue (NR) and soil with residue (WR) rice plants. Bars (SE) followed by the same lowercase letter do not differ signiflcantly (Duncan, P＜0.05), n=20.

Rice plants inoculated withP.fluorescensBRM-32111 orB.pyrrociniaBRM-32113 increased germination, length,and biomass in relation to non-inoculated plants grown in soil with residue (control WR) (Fig. 2). Studies have shown that there is a relationship between plant growth and soil microorganisms in the presence of allelopathic compounds.This relationship is affected by multiple mechanisms. In the present study, PGPRsP.fluorescensBRM-32111 andB.pyrrociniaBRM-32113 induced increases in growth and root biomass, which may be due to an improvement in the soil-plant-microorganism crosstalk process. Root exudates stimulate formation of quorum-sensing bacteria with an increase in the release of polysaccharides that combined with particles of clay, induce the formation of mucigel (Guckertet al. 1975), a substance that facilitates root growth and protects PGPRs in the rhizosphere (Kiers and Denison 2008; Van Dam and Bouwmeester 2016;Venturi and Keel 2016). Rhizosphere bacteria can also degrade allelochemicals through enzymatic action, which may alter the toxicity of the allelochemical compounds by turning them into a non-toxic form (Baiset al. 2006; Bhadoria 2011; Inderjitet al. 2011; Mishraet al. 2013; Zouet al. 2014;Wuet al. 2015). The allelochemical compounds released by grasses (phytotoxins, 2,4-diacetyl phloroglucinol (2,4- DAPG), phenazine, hydrogen cyanide (HCN), antibiotics and cell wall degradation enzymes from plant roots) may have been degraded by PGPRsP.fluorescensBRM-32111 andB.pyrrociniaBRM-32113 in the present study. This may have favored the growth of rice plants on soil with rice residues.

Table 2 Estimation of linear correlation coefflcients between growth variables of rice plants at 21 days after sowing1)

In addition to growth promotion, these microorganisms also act on the modulation of the stress response gene, in the inhibition of antioxidant enzymes, and in the secretion of allelochemicals in soil (Barazani and Friedman 1999; Baiset al. 2006; Mishraet al. 2013). The root system, aerial part,biomass, stomatal density, foliar and leaf mass, leaf area and stomatal pore were all the largest in rice plants inoculated withP.fluorescensBRM-32111. These morphological adaptations of the plants inoculated withP.fluorescensBRM-32111 favored the acquisition of CO2, which helps the plant to develop its potential for growth (Galméset al.2007; Millaret al. 2011).

4.2. Quantification of pigments

The non-inoculated rice plants in control WR had a reduction in the concentration of photosynthetic pigments, whereas,there was an increase in chlorophyll concentration in plants inoculated withP.fluorescensBRM-32111. In control WR,plants showed a drastic reduction of chlorophylla, which may be related to the loss of the ability of cells to synthesize chlorophyll when exposed to an allelochemical compound,as occurs in green algae (Chlorella vulgaris) exposed to the allelochemical N-fenil-2-naftilamina (Bornman and Vogelmann 1991; Haifenget al. 2009).

As for chlorophyllbanda/bratio, there was an increase in plants inoculated withP.fluorescensBRM-32111,indicating an increase in the efflciency at the center of the photosynthetic reaction complexes of antenna systems PSII and PSI. These systems can be divided into complexes of inner antennas containing chlorophyllband the complexes of capture, transmission and dissipation of excess light (NPQ,qP,Fv/Fm) (Haifenget al. 2009). The allelopathic compound N-phenyl-2-naphthylamine, in green algae, alters the transport function of photosynthetic electrons, with a current from PSII (photosystem II) to PSI (photosystem I), forming donor-receptor electron complexes with phylloquinone and naphthoquinone (Dwivedi and Rao 1972; Haifenget al.2009). The rhizobacteriumP.fluorescensBRM-32111 may have stimulated plants to tolerate stress by maintaining the antenna complex, electron transport, or both.

4.3. Gas exchange

One of the mechanisms that characterizes phytotoxicity induced by allelochemicals is the inhibition of photosynthesis and evolution of oxygen through interaction with components of photosystem II (PSII) (Einhelling 1996). In the present study, the photosynthetic apparatus of the control WR rice plants was adversely affected by the rice residue in the soil, but the damages were mitigated when the plants were inoculated with the rhizobacteriaP.fluorescensBRM-32111 orB.pyrrociniaBRM-32113. Allelochemicals reduce photosynthesis by causing a reduction in electron transfer or by reducing and inhibiting the synthesis of adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate (NADPH), reducing stomatal conductance and transpiration (Wuet al. 1999; Yuet al.2003). As observed in this study, rice plants grown in WR had a drastic reduction inGsandE, as already observed in other plants. This effect was attributed to the allelochemicals of the phenol groups, which are allelopathic compounds due to the decomposition of grass and which cause disturbances in the membrane cells and compromise the plant’s ability to regulate water transport as occurs in stomatal guard cells,which may lead to the reduction ofGsandE(Einhellig 1996;Weiret al. 2004).

The increases in the gas exchange parametersA,WUE, andA/Ciin rice plants inoculated withP.fluorescensBRM-32111 orB.pyrrociniaBRM-32113 and seeded in soil with residue resulted from the stimulus to plant growth. These plants had greater leaf area and photosynthetic pigments,which are directly linked to an increase in the photosynthetic rate, which aids in the biomass accumulation in the rice plants inoculated with the rhizobacteria (Galméset al. 2007; Millaret al. 2011) (Table 2). The estimation of linear correlation coefflcient (P＜0.05) between growth variables of rice plants,all variables more related, correlation obtained in root biomass and length in relation as variables of plant length, leaf area,water efflciency, and rubisco carboxylation efflciency.

The characteristics of control NR and control WR were similar to those of corn and soybean plants exposed to allelochemical compounds released by black walnut (Juglas nigraL.), which led to a reduction in the net assimilation rate of CO2(Shibu and Gillespie 1998). However, the rice plants inoculated withP.fluorescensBRM-32111 orB.pyrrociniaBRM-32113 had an increase in the rate of CO2assimilation and greaterGsandCicompared to control WR plants,indicating that these rhizobacteria alter the physiological behavior of the plants, increasing plant tolerance to the allelochemicals. Strains ofPseudomonas fluorescens,Bacillus subtilis, and other species that associate with the roots of plants were able to provide tolerance to allelopathy and to degrade the allelochemicals released fromGmelina arborea(Hauser 1993; Barazani and Friedman 2001).The plants inoculated withP.fluorescensBRM-32111 had characteristics of plants considered tolerant to the effects of allelochemicals, with greater leaf area, DTF, and root biomass, which indicates the occurrence of thickening of cell walls that contributes to increase gas exchange efflciency and WUE (Niinemets 2001).

Rotation of crops has been suggested as a way to decrease the allelopathic effects in plants. In crop rotation studies with upland rice and wheat in Pakistan, the crops had reduced production in successive plantations, even with the adoption of good agricultural practices. These losses may be mitigated by the use of microorganisms(P.fluorescensBRM-32111 orB.pyrrociniaBRM-32113)that induce increased tolerance of upland rice plants to allelopathic residues.

The rhizobacteriaP.fluorescensBRM-32111 andB.pyrrociniaBRM-32113 have been recorded as growth promoters and blast suppressants in upland rice (Filippiet al.2011; Rêgoet al. 2014), induced increased plant tolerance to allelopathic compounds and should be evaluated in the fleld to conflrm this positive effect on upland rice. If conflrmed, this effect could facilitate management for increased productivity in the same area and consecutive years of planting and reduce the need to open new areas for rice planting.

5. Conclusion

The rhizobacteriaPseudomonas fluorescensBRM-32111 andBurkholderia pyrrociniaBRM-32113 mitigate physiological damage, induce plant growth, and induce tolerance to allelochemical compounds in rice plants and should be investigated in fleld experiments for their potential to be inserted into upland crop management.

Acknowledgements

The authors thank the National Council for Scientiflc and Technological Development, Brazil, the Amazon Research Foundation, Brazil, and the Rural Federal University of Amazon, Brazil for the research funding and the Brazilian Federal Agency for the Support and Evaluation of Graduate Education for the grant of a doctorate scholarship.