Effects of maize inoculation with Fusarium verticillioides and with two bacterial biocontrol agents on seedlings growth and antioxidative enzymatic activities

Applied Soil Ecology 51 (2011) 52–59

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Effects of maize inoculation with Fusarium verticillioides and with two bacterial biocontrol agents on seedlings growth and antioxidative enzymatic activities ˜ b , Elizabeth Agostini b , Miriam Etcheverry a,∗ Paola Pereira a , Sabrina G. Ibánez a Laboratorio de Ecología Microbiana, Departamento de Microbiología e Inmunología, Universidad Nacional de Río Cuarto, Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Ruta Nacional 36 Km 601 (5800) Río Cuarto, Córdoba, Argentina b Departamento de Biología Molecular, Universidad Nacional de Río Cuarto, Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Ruta Nacional 36 Km 601 (5800) Río Cuarto, Córdoba, Argentina

a r t i c l e

i n f o

Article history: Received 10 March 2011 Received in revised form 15 August 2011 Accepted 23 August 2011 Keywords: Maize Oxidative response Biological control Fusarium verticillioides Bacillus amyloliquefaciens Microbacterium oleovorans

a b s t r a c t Maize (Zea mays L.) is a staple food for the majority of the world’s population and different diseases may affect its emergence, growth and development. Fusarium verticillioides (Sacc.) Nirenberg (Teleomorph: Gibberella moniliformis Wineland) is the most commonly reported fungal species infecting this crop. The present work analyzes the bioprotective role of two bacterial agents, Bacillus amyloliquefaciens and Microbacterium oleovorans, against F. verticillioides in maize. Maize growth parameters, count and identification of Fusarium isolates as well as CAT, SOD, APX and POD activities were evaluated in 10 and 20 days old seedlings in relation to single inoculations and co-inoculations of the fungus and the bacterial agents. Endophytic count of F. verticillioides propagules was reduced after inoculation with B. amyloliquefaciens during both evaluated periods (10 and 20 days) while M. oleovorans presented a variable performance as an F. verticillioides antagonist. Inoculation with F. verticillioides promoted an increase in SOD activity in roots of 10 days old maize seedlings. S/R ratios of these seedlings were also significantly affected by inoculation with the fungus. Results suggest an early response of maize to infection, since such variations in enzyme activities and plant growth parameters were not observed 20 days after treatment. Moreover, no differences were observed in the number of POD isoforms between infected and non-infected plants. The overall results point out that maize infection by F. verticillioides may be attenuated after seed inoculation with B. amyloliquefaciens and this infection may represent a weak stressful factor, eliciting minor changes in the antioxidative response at early stages of plant growth. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Maize (Zea mays L.) is a key crop in terms of global cereal yields and represents one of the main crops grown in Argentina (SIIA, 2011). Maize cultivation allows Argentina to be among the world’s largest producer and exporter countries (NCGA, 2010). Fungi of the genera Fusarium, Aspergillus and Penicillium are frequently reported as colonizers of maize grains (Roigé et al., 2009; Makun et al., 2010). Fusarium verticillioides (Sacc.) Nirenberg is one of the most prolific toxin producers within Fusarium genus and causes ear rot of maize (Robertson-Hoyt et al., 2007; Bacon et al., 2008). This species can be found in association with maize as both a pathogen or as a symptomless intercellular endophyte, depending on diverse factors such as plant and fungal genotypes, environmental conditions, fungal inoculum size and presence of antagonists (Oren et al., 2003; Bacon et al., 2008).

∗ Corresponding author. Tel.: +54 0358 4676113; fax: +54 0358 4676231. E-mail address: [email protected] (M. Etcheverry). 0929-1393/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.apsoil.2011.08.007

Biological control of crops diseases and pests using microbial inoculants is being increasingly recognized as a viable, eco-friendly alternative that limits the massive use of synthetic chemical pesticides (Charan et al., 2011). Several groups of plant-endophytic bacteria occupy the same niches that many fungal pathogens. Consequently, they are ecological homologues for most Fusarium isolates and compete for nutrients within the apoplasm (Bacon and Yates, 2006). Thus, the control of F. verticillioides may be achieved based on the use of this kind of antagonistic bacteria. In previous studies we selected B. amyloliquefaciens and M. oleovorans as potential antagonists of F. verticillioides within the maize agroecosystem (Pereira et al., 2011). Resistance of plants to fungal colonization is often expressed by the hypersensitive reaction (HR) of challenged plant cells, and is characterized by reactive oxygen species (ROS) production (Temple et al., 2005; Magbanua et al., 2007; Ashraf, 2009). The ROS excess causes damage to proteins, lipids, carbohydrates, DNA and ultimately results in cell death (Parvaiz et al., 2009). To mitigate this oxidative damage, plants have developed a complex defense antioxidative system including different enzymes such as catalases

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(CAT), peroxidases (POD) and superoxide dismutases (SOD), which are in charge of removing these activated species and modulate oxidative stress (Mittler, 2002; Kumar et al., 2009). Different studies assessed the impact of maize pathogens on antioxidative responses of the plants; e.g. Magbanua et al. (2007) found that CAT activity was significantly increased in lines resistant to Aspergillus flavus infection. However, there are no surveys available related to the antioxidative response of maize interacting with pathogens such us F. verticillioides, which can attain nonsymptomatic biotrophic states. The aim of the present work was to examine how inoculation with the biocontrol agents (BCAs) B. amyloliquefaciens and M. oleovorans may affect antioxidative responses of maize when challenged to F. verticillioides. This study also evaluated the impact of single inoculations of the fungus and the biocontrol agents as well as the impact of bacterial/fungal co-inoculations on the growth of maize seedlings and on the count of culturable bacteria and fungi naturally associated with the roots of the maize.

seeds submerged in a suspension 109 CFU ml−1 of M. oleovorans; T4 (Fv): maize seeds submerged in a suspension 107 spores ml−1 of F. verticillioides; T5 (FvBa): maize seeds submerged in a 1:1 suspension of 107 spores ml−1 of F. verticillioides and 109 CFU ml−1 of B. amyloliquefaciens and T6 (FvMo): maize seeds submerged in a 1:1 suspension of 107 spores ml−1 of F. verticillioides and 109 CFU ml−1 of M. oleovorans. All seeds were maintained in their respective suspensions for 20 min at 28 ◦ C and finally sown in plastic pots, 10.5 cm in diameter × 17.5 cm high, filled with sterile sandy loam soil obtained from an Argentinean maize field (pH 6.1 in water 1:1 (w/v), 1.4% organic matter, 86 ppm of nitrates). Soil was sterilized through dry heat treatment, 4 h at 180 ◦ C, twice. All pots were irrigated with sterile Hoagland solution (Hoagland and Arnon, 1950) at sowing time and at days 4 and 14 after sowing to half full soil water holding capacity. Pots were then incubated with 12 h photoperiod and at 28 ± 2 ◦ C until sampling.

2. Materials and methods

At days 10 and 20 after sowing eight seedlings were harvested from each of the assayed treatments. After determination of growth parameters root systems of collected plants were separated from shoots and immediately frozen in liquid nitrogen. Frozen tissues were then grounded in liquid nitrogen with mortar and pestle and stored at −80 ◦ C until determination of antioxidative enzymatic activities (CAT, SOD, APX and POD).

2.1. Plant material The experiments were carried out with maize seeds of DK684RR2 cultivar (Monsanto). Prior to treatment seeds were surface disinfected by immersion in ethanol 70% (3 min), followed by immersion in sodium hypochlorite 3% (5 min) and several rinses with sterile distilled water. Surface disinfection was considered to be achieved by the absence of CFUs in nutrient agar plates. 2.2. Microbial strains Bacterial isolates of B. amyloliquefaciens (GenBank accession number EU164542) and M. oleovorans (GenBank accession number EU164543) were used in the present study. Strains were originally isolated from maize plants grown at field (Pereira et al., 2007). Both isolates were deposited in the Banco Nacional de Microorganismos - Instituto de Investigaciones en Biociencias Agrícolas y Ambientales (INBA) and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) - accession numbers BNM0531 and BNM0532. Strains were stored at −20 ◦ C in glycerol (30%, v/v) and, when required for experimental use, were transferred to nutrient agar. Nutrient broths were also prepared, inoculated with each tested biocontrol agent and then incubated overnight with shaking (100 rpm) at 28 ◦ C until late log phase. After incubation total viable cells were counted by standard plate count method and suspensions of 109 CFU ml−1 were used to introduce bacteria as maize seeds coatings. The fungal strain used was F. verticillioides M7075, a fumonisin B1 producer previously isolated from maize in Argentina and deposited in the Fusarium Research Center Collection (Pennsylvania State University, University Park, PA, USA). Capellini–Peterson liquid medium was used to promote fungal sporulation (Capellini and Peterson, 1965). Broths were inoculated with 6 mm disks cut from F. verticillioides mycelia obtained from monosporic cultures grown in carnation leaf agar medium (Nelson et al., 1983). Inoculation was followed by an incubation period of 7 days at 25 ◦ C on an oscillatory shaker (150 rpm). Suspensions of 107 spores ml−1 were used to introduce F. verticillioides as maize seeds coating. 2.3. Treatments Six treatments were performed and maintained under greenhouse conditions. T1 control (C): maize seeds submerged in a sterile solution of NaCl 0.8% (w/v); T2 (Ba): maize seeds submerged in a suspension 109 CFU ml−1 of B. amyloliquefaciens; T3 (Mo): maize

2.4. Sampling procedures

2.5. Influence of treatments on maize agronomic parameters Parameters determined in maize seedlings were: germination percentage, stem length, root length, total seedling fresh weight, root system fresh weight and shoot/root ratios (S/R index). The concept of S/R ratios has been widely used to evaluate stress conditions in plants. The lower the ratio, the more stressed the plant (Holguin and Glick, 2001; Kozdrój et al., 2004). 2.6. Isolation and count of bacteria and fungi from maize roots Rhizospheric and endophytic bacterial and fungal isolates were recovered from maize roots as previously described by the authors (Pereira et al., 2009). Bacterial counts were performed in nutrient agar medium while fungal counts were performed in dichloran rose Bengal chloramphenicol agar (DRBC) and in Nash–Snyder agar. Counts were reported per treatment as log10 CFUs g−1 root. 2.7. Identification of fungal isolates The identification of fungal groups was performed as described by Samson et al. (2002). Further identification of isolates within Fusarium genus followed criteria established by Nelson et al. (1983) and Leslie and Summerell (2006). Fusarium isolates not assigned to Liseola section were named as Fusarium spp. 2.8. Protein extraction and enzyme assays CAT, SOD and APX were extracted from root samples (150 mg each), previously grounded and homogenized with liquid nitrogen, using 50 mM potassium phosphate buffer, pH 7.8 containing EDTA 0.5 mM and 25% (w/v) polyvinylpyrrolidone (PVP) to determine enzymatic activities. A 1:10 tissue/buffer ratio was used to perform extractions. Crude homogenates were centrifuged at 10,000 rpm for 30 min at 4 ◦ C. After centrifugation the pellets were discarded and the supernatants retained for protein quantification and enzymatic activities assays. Total soluble PODs were extracted from the root samples with 50 mM sodium acetate buffer pH 5.0, 1 M KCl, also using a ratio

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Table 1 Growth parameters of maize seedlings sampled 10 (a) and 20 (b) days after treatment. Treatment

Length (mm) Roots

(a) C Ba Mo Fv FvBa FvMo (b) C Ba Mo Fv FvBa FvMo

Weight (g) Stem

74.50 150.75 109.93 90.50 90.25 52.66

± ± ± ± ± ±

13.89 ac 92.80 b 35.81 ab 13.81 abc 30.70 abc 29.68 c

109.95 115.46 112.38 98.11 104.60 104.19

± ± ± ± ± ±

18.35 a 22.56 a 25.58 a 23.39 a 12.83 a 21.48 a

Whole seedling

S/R index Roots

52.37 40.63 52.13 25.00 42.63 43.50

± ± ± ± ± ±

18.13 a 7.11 ab 7.45 a 12.01 b 18.01 ab 19.94 ab

1.32 1.64 1.67 1.11 1.04 1.00

± ± ± ± ± ±

0.34 a 0.88 a 0.34 a 0.43 a 0.45 a 0.54 a

0.75 1.64 1.06 0.87 0.65 0.59

± ± ± ± ± ±

0.21 a 0.61 a 0.34 a 0.35 a 0.30 a 0.33 a

0.78 0.62 0.66 0.29 0.67 0.79

± ± ± ± ± ±

0.28 a 0.20 ab 0.30 ab 0.19 b 0.29 ab 0.35 a

93.87 101.25 90.13 84.00 81.62 67.75

± ± ± ± ± ±

9.16 ab 8.97 b 10.08 ab 11.28 ac 12.79 ac 13.67 c

1.72 2.43 2.19 1.77 1.46 1.63

± ± ± ± ± ±

0.43 ac 0.59 b 0.57 ab 0.27 abc 0.37 c 0.52 ac

1.02 1.56 1.45 1.18 0.96 1.07

± ± ± ± ± ±

0.33 a 0.46 b 0.49 ab 0.19 ab 0.27 a 0.32 ab

0.72 0.58 0.57 0.51 0.54 0.54

± ± ± ± ± ±

0.12 a 0.17 a 0.13 a 0.08 a 0.07 a 0.14 a

Data are means and standard deviations of eight samples analyzed in duplicate. Different letters within a column indicate significant differences between treatments (p < 0.05, one-way ANOVA).

1:10 (tissue/buffer). The homogenates were vortexed for 10 s and then centrifuged at 10,000 rpm for 10 min at 4 ◦ C. The pellets were discarded and the supernatants retained for protein quantification and enzyme activity assay. Protein content of each sample was determined according to the Bradford method using bovine serum albumin as standard (Bradford, 1976). 2.8.1. CAT assay CAT activity was quantified based on the rate of disappearance of the substrate H2 O2 from a reaction medium containing the protein extract (Aebi, 1974). H2 O2 concentration in the reaction medium was quantified by the change in absorbance at 240 nm measured using a Spectronic Genesys 5 UV–visible spectrophotometer and the Milton Roy software for kinetics analysis (Ivyland, PA, USA). A cuvette containing 25 ␮l of the extract and 890 ␮l of 50 mM potassium phosphate buffer pH 7.0 was placed in the spectrophotometer and set to zero. A volume of 85 ␮l of 18 mM H2 O2 was then added to the extract solution and the absorbance was monitored for 1 min (at 10 s intervals). CAT activity (U mg−1 protein) was calculated using a molar absorption coefficient of 40 mM−1 cm−1 for H2 O2 . 2.8.2. SOD assay SOD activity was assayed by using the photochemical NBT method (Beauchamp and Fridovich, 1973). One milliliter of the reaction mixture contained 50 mM potassium phosphate buffer, pH 7.8, 0.1 mM EDTA, 20 ␮l of the extract, 75 ␮M NBT, 13 mM methionine and 4 ␮M riboflavin. One unit of SOD was defined as the quantity of enzyme required to inhibit the reduction of NBT by 50%. The enzyme activity (U mg−1 protein) was calculated after measuring the reaction mixture at 560 nm using a Beckman DU640 UV-visible Spectrophotometer (Beckman, Palo Alto, USA). 2.8.3. APX assay Total APX activity was measured spectrophotometrically by monitoring the decline in absorbance at 290 nm during oxidation of l-ascorbic acid (ε290 nm = 2.8 mM−1 cm−1 ), using the method described by Hossain and Asada (1984). One milliliter of the reaction mixture contained 50 mM potassium phosphate buffer, pH 7.0, 0.45 mM l-ascorbic acid, 0.3 mM H2 O2 and 30 ␮l of the extract. One unit of APX was defined as the quantity of enzyme required to consume 1 ␮M of substrate. The change in absorbance was monitored for 1 min using a Spectronic Genesys 5 UV-visible spectrophotometer and the Milton Roy software for kinetics analysis (Ivyland, PA, USA).

2.8.4. POD assay POD activity was determined spectrophotometrically using odianisidine and H2 O2 as described by Agostini et al. (1997). One milliliter of the reaction mixture contained 0.63 mM o-dianisidine, 5 mM H2 O2 , 1 M acetate buffer pH 5.5, and 2.5 ␮l of crude extract. POD activity was measured spectrophotometrically, following the increase in absorbance at 470 nm produced by oxidation of odianisidine at 37 ◦ C (ε470 nm = 11.3 mM−1 cm−1 ). One enzyme unit (U) was defined as the amount of enzyme that generated 1 ␮mol of product in 1 min in the conditions described. Patterns of POD isoforms of the homogenates were analyzed by both cationic (Reisfeld et al., 1962) and anionic (Davis, 1964) electrophoresis. Gels were stained with benzidine and H2 O2 to reveal POD activity as described by Sosa Alderete et al. (2009). 2.9. Data analysis Data were analyzed applying one-way ANOVA using SYSTATSigmastat for Windows version 3.1 software (SPSS, Chicago, IL, USA). The Tukey test was used for posteriori comparisons between treatments. A p < 0.05 significance level was used throughout. 3. Results and discussion The antagonistic activities of B. amyloliquefaciens and M. oleovorans as well as their role in the antioxidative response of maize to inoculation with F. verticillioides were tested in roots of seedlings grown under greenhouse conditions. 3.1. Influence of treatments on maize agronomic parameters Inoculations did not affect maize germination; percentages of 100% were obtained in all treatments. All seedlings grown from seeds inoculated with the fungus and with the BCAs (single inoculations or co-inoculations BCA-F. verticillioides) grew well and did not show symptoms of disease after 20 days of cultivation in pots (data not shown). Roots weight did not differ between control seedlings (C) and seedlings treated with either BCAs (Ba and Mo) or with F. verticillioides alone (Fv) or combined with BCAs (FvBa and FvMo) after 10 days of cultivation in pots (Table 1a). On the other hand, a significant increase in the root length of T2 seedlings (Ba) was observed with respect to control values during this first sampling period. Length of the stems was significantly lower after inoculation with F. verticillioides (Fv). In addition, 10 days old seedlings of T4 (Fv) were stressed with respect to control (C), based on S/R indexes

a a a b ab ab Data are means and standard deviations of eight samples analyzed in duplicate. Different letters within a column indicate significant differences between treatments (p < 0.05, one-way ANOVA).

1.56 × 105 ± 0.21 × 101 2.58 × 104 ± 0.24 × 101 3.45 × 104 ± 0.46 × 101 1.91 × 106 ± 0.54 × 101 3.42 × 105 ± 0.63 × 101 2.30 × 105 ± 0.42 × 101 a a a b b b 5.15 × 106 ± 0.14 × 101 5.25 × 107 ± 0.13 × 101 1.64 × 106 ± 0.47 × 101 1.87 × 107 ± 0.13 × 101 2.18 × 107 ± 0.12 × 101 3.02 × 107 ± 0.11 × 101 8.17 × 107 ± 0.80 × 101 3.29 × 109 ± 0.11 × 101 5.92 × 108 ± 0.18 × 101 2.07 × 108 ± 0.18 × 101 1.24 × 108 ± 0.30 × 101 9.18 × 108 ± 0.12 × 101 9.66 × 109 ± 0.14 × 101 b 9.18 × 1010 ± 0.36 × 101 a 2.19 × 1011 ± 0.15 × 101 a 1.55 × 1011 ± 0.11 × 101 a 8.07 × 1010 ± 0.14 × 101 ab 2.52 × 1011 ± 0.20 × 101 a

bc a abc bc c ab

3.29 × 108 ± 0.94 × 101 1.58 × 109 ± 0.70 × 101 5.02 × 108 ± 1.50 × 101 3.60 × 107 ± 0.69 × 101 8.05 × 107 ± 1.22 × 101 2.58 × 109 ± 0.39 × 101 5.57 × 109 ± 0.26 × 101 ab 1.64 × 109 ± 0.30 × 101 a 6.11 × 1010 ± 0.33 × 101 b 3.77 × 1010 ± 1.32 × 101 ab 3.19 × 109 ± 0.80 × 101 ab 2.20 × 109 ± 0.37 × 101 a

(a) C Ba Mo Fv FvBa FvMo (b) C Ba Mo Fv FvBa FvMo

RIT RP

ab a ab b ab ab

bc abc c ab a a

4.37 × 106 ± 0.17 × 101 6.93 × 106 ± 0.16 × 101 1.25 × 106 ± 0.17 × 101 2.56 × 107 ± 0.14 × 101 2.60 × 107 ± 0.15 × 101 2.75 × 107 ± 0.13 × 101

3.73 × 105 ± 0.13 × 101 a 4.58 × 104 ± 0.37 × 101 a 6.46 × 104 ± 0.88 × 101 a 4.55 × 105 ± 0.28 × 101 a 3.18 × 105 ± 0.48 × 101 a 2.57 × 105 ± 0.50 × 101 a

bc abc a ab

4.56 × 103 ± 0.93 × 101 0.000 ± 0.000 c 5.53 × 102 ± 0.58 × 101 4.93 × 103 ± 2.16 × 101 2.51 × 105 ± 0.44 × 101 3.21 × 105 ± 0.25 × 101 3.93 × 103 ± 0.66 × 101 2.63 × 104 ± 0.26 × 101 4.57 × 103 ± 0.98 × 101 8.95 × 103 ± 0.42 × 101 1.57 × 105 ± 0.43 × 101 7.89 × 104 ± 0.21 × 101 c c ac abc ab b 3.93 × 105 ± 0.65 × 101 3.21 × 105 ± 0.38 × 101 1.57 × 106 ± 0.26 × 101 2.54 × 106 ± 0.21 × 101 1.04 × 107 ± 0.21 × 101 1.57 × 107 ± 0.19 × 101 6.98 × 105 ± 0.19 × 101 9.61 × 105 ± 0.28 × 101 1.86 × 106 ± 0.20 × 101 1.91 × 106 ± 0.21 × 101 5.47 × 106 ± 0.29 × 101 1.33 × 107 ± 0.20 × 101

c c ac ac ab b

DRBC

RIT

Nash–Snyder RP

Fungal count (log10 CFU g−1 root) Bacterial count (log10 CFU g−1 root) Treatment

Table 2 Total count of bacterial and fungal isolates recovered from the rhizoplane and root inner tissues of maize seedlings sampled 10 (a) and 20 (b) days after treatment.

DRBC

c abc c ac b ab

Nash–Snyder

abc

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concept. This response of maize seedlings was not observed after co-inoculation of the fungus and BCAs (FvBa and FvMo) neither 10 (Table 1a) nor 20 days after treatment (Table 1b). In agreement with obtained results, Kozdrój et al. (2004) found that S/R ratios of maize seedlings obtained after inoculation with Pseudomonas chlororaphis IDV1 and Pseudomonas putida RA2 were age-dependent. Available information regarding the impact of maize inoculation with F. verticillioides demonstrates that plant growth responses do not present a regular pattern, mainly due to factors such as the plant cultivar used (Yates et al., 2005; Presello et al., 2008; Nayaka et al., 2009), the characteristics of fungal isolates (toxigenic potential, inoculum size, type of inoculation, etc.) (Williams et al., 2007) and climatic conditions (soil and air temperature, mean rainfall values, etc.) (Schjøth et al., 2009). Twenty days-old seedlings of T2 (Ba) presented a significant increase in total weight when compared with control weights (Table 1b). This result may be indicative of a plant growth promoting feature of B. amyloliquefaciens. Increases in maize growth and yield after treatment with different Bacillus isolates has been extensively reported (Idriss et al., 2002; Egamberdiyeva, 2007; Adesemoye and Kloepper, 2009). However, we did not observe growth promoting characteristics of this isolate in previous greenhouse and field studies (Pereira et al., 2009, 2010). Probably, the use of different kinds of soil, sterile vs non sterile, in the different contrasted surveys influenced the performance of introduced BCAs. 3.2. Bacterial and fungal count of isolates recovered from the roots of maize seedlings Root tissues form a morphologically, physically and chemically complex microcosm that provides different habitats for diverse communities of microorganisms. This microcosm is not stable, and changes over space and time are expected due to different biotic and abiotic factors affecting the boundaries between soil, rhizosphere, and living roots (Foster et al., 1983). Total count of bacteria inhabiting the rhizoplane and root inner tissues of maize seedlings grown under the different assayed treatments are presented in Table 2. Mean values of total count of bacteria isolated from the rhizoplane (RP) of 10 days old seedlings ranged from 1.6 × 109 to 6.1 × 1010 , while count from the root inner tissues (RIT) ranged from 3.6 × 107 to 1.5 × 109 (Table 2a). No significant treatment effects were observed in these parameters between seedlings of the first sampling. On the other hand, rhizospheric and endophytic bacterial counts of 20 days old seedlings were significantly higher in T2 (Ba) when compared with control values (Table 2b). This result may suggest that introduced BCA survived in the rhizosphere of maize and remained associated to the roots of seedlings after germination; or, possibly, its addition stimulated growth of other seed-borne bacteria. Furthermore, 20 days old seedlings of T2 were significantly weightier than controls and may have produced larger amount of root exudates, thus stimulating growth of bacterial populations. Count of bacteria recovered from the rhizoplane of 20 days old seedlings ranged from 9.7 × 109 to 2.5 × 1011 , while count of endophytic bacteria maintained the same range observed in the seedlings of the first sampling (107 to 109 ). 3.3. Count of rhizospheric and endophytic fungi. Identification of Fusarium isolates Diverse fungal groups growing in the surface or in the inner tissues of maize seeds may affect germination, growth and yield of resultant plants. Among these groups, Fusarium is the prevailing genus reported as the main causal agent of several maize rots. Thus, estimation of fungal population sizes and composition is of a great

ac ac bc b bc

Data are means and standard deviations of eight samples analyzed in duplicate. Different letters within a column indicate significant differences between treatments (p < 0.05, one-way ANOVA).

0.00 ± 0.00 a 5.73 × 104 ± 2.47 × 101 1.00 × 105 ± 8.22 × 101 2.30 × 105 ± 2.56 × 101 2.09 × 106 ± 1.52 × 101 8.53 × 105 ± 1.03 × 102 bc 0.00 ± 0.00 a 0.00 ± 0.00 a bc 0.00 ± 0.00 a c 1.80 × 107 ± 2.89 × 102 b 0.00 ± 0.00 a ab 2.21 × 107 ± 7.41 × 102 b 4.72 × 105 ± 1.64 × 101 0.00 ± 0.00 a 3.73 × 105 ± 8.22 × 101 1.00 × 106 ± 5.05 × 102 0.00 ± 0.00 a 6.11 × 104 ± 7.36 × 101 ab ab b c ac ac 8.07 × 106 ± 1.14 × 102 6.25 × 106 ± 2.75 × 101 2.87 × 106 ± 1.67 × 101 3.67 × 107 ± 1.04 × 102 2.77 × 107 ± 1.29 × 102 2.64 × 107 ± 1.09 × 102 ab ab bc c bc 4.19 × 106 ± 1.56 × 102 7.80 × 106 ± 1.61 × 101 1.11 × 106 ± 5.19 × 101 2.98 × 107 ± 4.68 × 101 1.87 × 107 ± 3.90 × 101 0.00 ± 0.00 c

bc ab bc a a

0.00 ± 0.00 a 5.82 × 104 ± 2.21 × 101 1.00 × 105 ± 9.62 × 101 2.32 × 105 ± 6.44 × 101 1.93 × 106 ± 2.60 × 102 8.61 × 105 ± 2.10 × 101

a ab

7.55 × 102 ± 0.86 × 101 0.00 ± 0.00 c 1.05 × 102 ± 0.33 × 101 0.00 ± 0.00 c 9.04 × 105 ± 1.28 × 102 4.45 × 105 ± 3.19 × 101

1.88 × 106 ± 1.76 × 102 ab 7.78 × 104 ± 1.41 × 101 a 5.21 × 106 ± 7.18 × 101 b 0.00 ± 0.00 b 0.00 ± 0.00 b 0.00 ± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 b 0.00 ± 0.00 b 0.00 ± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 b

RP

bc 7.67 × 105 ± 9.06 × 101 3.33 × 105 ± 1.33 × 102 2.88 × 105 ± 1.23 × 102 a 6.01 × 106 ± 5.09 × 101 ab 4.46 × 106 ± 2.76 × 102 ab 8.53 × 106 ± 8.05 × 101 bc 6.35 × 103 ± 2.52 × 101 0.00 ± 0.00 c bc 0.00 ± 0.00 c ab 5.93 × 105 ± 1.12 × 101 ab 9.44 × 104 ± 1.05 × 101 a 5.78 × 104 ± 1.29 × 101 abc 3.21 × 105 ± 8.91 × 101 0.00 ± 0.00 c ab 3.80 × 105 ± 7.60 × 101 bc 3.69 × 106 ± 1.60 × 102 a 3.95 × 106 ± 6.78 × 102 1.39 × 107 ± 5.50 × 102 8.04 × 104 ± 0.19 × 101 0.00 ± 0.00 c 1.62 × 105 ± 2.75 × 101 1.91 × 104 ± 0.56 × 101 2.47 × 106 ± 3.20 × 101 0.00 ± 0.00 c a a ab ab b b 0.00 ± 0.00 a 0.00 ± 0.00 a 4.74 × 105 ± 9.12 × 101 1.11 × 105 ± 2.64 × 101 7.83 × 104 ± 1.90 × 101 ab 0.00 ± 0.00 a 6.81 × 105 ± 2.11 × 101 b 3.40 × 102 ± 0.46 × 101 ab 2.40 × 106 ± 1.48 × 102 0.00 ± 0.00 a 0.00 ± 0.00 a 2.77 × 106 ± 9.89 × 101 1.66 × 107 ± 1.64 × 102 2.71 × 106 ± 1.15 × 102 b 0.00 ± 0.00 a 0.00 ± 0.00 a 4.44 × 105 ± 3.43 × 101 b 2.61 × 107 ± 1.50 × 102

(a) 1 2 3 4 5 6 (b) C Ba Mo Fv FvBa FvMo

RP

RIT Fusarium verticillioides

RP

RIT Fusarium proliferatum

RIT RP

Fusarium subglutinans

Treatment Fusarium species count (CFU g−1 root)

Table 3 Identification and count of Fusarium species isolated from the rhizoplane and root inner tissues of maize seedlings sampled 10 (a) and 20 (b) days after treatment.

Fusarium spp.

bc c c ab ab a

RIT

bc

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abc

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importance in order to detect unfavorable biotic factors that may affect plant growth and development. Total fungal counts obtained in general count medium (DRBC) are shown in Table 2. The rhizoplane of 10 days old seedlings hold a culturable fungal population that ranged from 7.0 × 105 to 1.3 × 107 while at the root inner tissues the range was between 3.9 × 103 to 1.6 × 105 (Table 2a). There were no significant differences in total rhizospheric or endophytic fungal count between seedlings of BCAs treatments (Ba–Mo) and control (C). On the other hand, no count of Fusarium species was obtained from the root inner tissues of T2 seedlings (Ba) when using Nash–Snyder selective medium for isolation. Identified Fusarium within Liseola section isolated from the roots of 10 days old maize seedlings belonged to F. subglutinans, F. proliferatum and F. verticillioides species (Table 3). Identified species are in good agreement with those reported by Fandohan et al. (2003) when reviewing main Fusarium groups that infect maize. Fungal species such as F. verticillioides and F. proliferatum, and related species within the Liseola section, are notorious mycotoxin producers on cereal grains and foodstuffs (Rheeder et al., 2002). Therefore, studies on maize colonization by Fusarium species are necessary in order to develop control measures for this group of fungi that may alter the quantity and phytosanitary quality of food supplies. Count of endophytic F. verticillioides propagules was significantly higher than control (C) in seedlings grown from F. verticillioides treated seeds (Fv) during the first sampling period, while this difference was not evidenced in 20 days old maize seedlings (Table 3b). On the other hand, rhizospheric count of this fungal species was significantly higher in T4 seedlings with respect to control as seedlings grew (20 days old vs 10 days old seedlings). Total fungal count at the rhizoplane of 20 days old seedlings ranged from 1.6 × 106 to 5.2 × 107 while at the root inner tissues the range was from 4.6 × 104 to 4.5 × 105 ; when using DRBC medium for isolation. This result indicates that there were no major differences in the root fungal population size between 10 and 20 days old maize seedlings. The cultivation approach has been widely applied to assess fungal populations, but a problem of this methodology is the fastidious nature of several fungal species. Thus, fungi such as rust or smut fungi or arbuscular mycorrhizae are difficult or impossible to grow in axenic media under laboratory conditions (Marcial Gomes et al., 2003). On the other hand, although morphophysiological characterizations seem to be replaced by molecular techniques, they have been widely used to achieve the identification of fungal and bacterial species and have not lost their usefulness. In addition, there is some evidence on the high correspondence between both techniques (Morales-Rodríguez et al., 2007).

3.4. Treatment effect on antioxidant enzyme activities The production of ROS by plants takes place, at low rates, under growth favorable conditions while most environmental stresses induce an enhanced production of these compounds (Ashraf, 2009). Thus, the role of ROS-scavenging enzymes such as SOD, CAT, POD and APX is crucial to achieve a steady-state concentration of ROS, preventing oxidative damage at cellular and subcellular levels (Hernández et al., 2010). SOD activity was significantly increased after treatment of maize seeds with F. verticillioides, either alone (Fv) or co-inoculated with the BCAs (FvBa, FvMo), when compared with T2 (Ba) and control (C) values (Fig. 1a). On the other hand, POD activity values were significantly lower after inoculation with F. verticillioides alone (Fv) and did not differ from control ones (C) when the fungus was coinoculated with the BCAs (FvBa, FvMo). However, none of these variations were found in 20 days old seedlings (Fig. 1b).

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Fig. 1. Superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX) and catalase (CAT) activities in roots of maize seedlings sampled 10 (a) and 20 (b) days after treatment. Data are means and standard errors of eight samples analyzed in duplicate. Different letters indicate significant differences between treatments (p < 0.05, one-way ANOVA).

Latha et al. (2009) reported that some bacterial strains such as P. fluorescens and B. subtilis increased POD activity in tomato tissues (Lycopersicon esculentum Mill.) and Xu et al. (2008) found that antagonistic yeasts significantly stimulated POD activity of stored

peach fruits. They concluded that the increased levels of antioxidant enzymes elicited by the BCAs may play a key role in reducing damage cause by pathogens. However, in F. verticillioides-maize interactions the antagonistic role of different BCAs could rarely

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P. Pereira et al. / Applied Soil Ecology 51 (2011) 52–59

Fig. 2. Cationic (a) and anionic (b) gel electrophoresis of POD isoforms detected in roots of maize seedlings grown from inoculated and non-inoculated seeds. First lines within each treatment (1, 3, 5, 7, 9 and 11) belong to root extracts of 10 days old maize seedlings while the remaining lines (2, 4, 6, 8, 10 and 12) belong to root extracts of 20 days old maize seedlings.

be evidenced by damage reduction due to the endophytic asymptomatic state that the fungus can attain. Inoculation with M. oleovorans (Mo) resulted in a variable antioxidative response that included e.g. an increase in SOD activity and a decrease in APX activity in seedlings of the first sampling. These variations were not observed in roots of 20 days old seedlings (Fig. 1b), thus indicating a lower production of ROS and a tendency to stabilization of the antioxidative response as the seedlings grew. On the other hand, different surveys show that POD isoforms of maize may change under stressful factors (Giannakoula et al., 2010; Vuletic´ et al., 2010). However, we did not find any differences in the number of isoforms detected between infected and non-infected plants (Fig. 2), thus indicating no alteration of normal isoenzyme expression in response to assayed treatments. Most of the changes observed in response to maize inoculation with either BCAs alone or in combination with F. verticillioides occurred in 10 days old seedlings; suggesting an early response of the plants. Possibly, if we had performed determinations in seedlings of less than 10 days old we would have observed significant changes in growth and antioxidative response of seedlings. However, the first sampling took place when the roots possessed the enough minimum weight to perform specified determinations. The reduction of F. verticillioides infection in roots of maize seedlings after treatment with B. amyloliquefaciens, added to the absence of changes in the antioxidant response of such seedlings, indicate that this bacterial agent may be effective in preventing horizontal transmission of the pathogen without affecting normal growth of the plant. Further studies on the antioxidative response of maize to inoculation with F. verticillioides under variable environmental conditions will be necessary for determining whether infection of maize by this fungus may become a stressful factor affecting growth and development of the crop. Acknowledgements This work was carried out through grants from Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT, FONCyT-PICT 1372/08). SECyT-UNRC 2009–2010. Res. 807/09.

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