Endophytic selenobacteria and arbuscular mycorrhizal fungus for selenium biofortification and Gaeumannomyces graminis biocontrol

1021 RESEARCH ARTICLERESEARCH ARTICLE Endophytic selenobacteria and arbuscular mycorrhizal fungus for Selenium biofortification and Gaeumannomyces graminis biocontrol Paola Durán1*, Sharon Viscardi3 , Jacquelinne J. Acuña1 , Pablo Cornejo1 , Rosario Azcón2 María de la Luz Mora1 1Scientific and Technological Bioresource Nucleus, Universidad de La Frontera, Temuco-Chile. 2Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín (CSIC), Granada-Spain. 3Departamento de Procesos Diagnósticos y Evaluación, Facultad de Ciencias de la Salud, Universidad Católica de Temuco, Temuco, Chile *Corresponding author: [email protected] Abstract Selenium (Se) is an important antioxidant considered among the fertilization programs in developed countries. In Chile, chemical fertilization based in Se is inefficient due the physicochemical characteristics of Andisol that sustain the 60% of crop production. Andisol also are highly conductive to take-all disease caused by Gaeumannonyces graminis var tritici (Ggt). Here, we evaluated the effect of Bacillus sp.E5 and Acinetobacter sp.E6.2 and Claroideoglomus claroideum as potential inocula for Se biofortification and Ggt bicontrol in wheat. Plants inoculated with Acinetobacter sp.E6.2 showed major root growth and major Se content in shoots and grains. The antioxidant role of Se regarding DPPH activity was shown in Se-supplemented plants with small Se nanoparticles founded inside the roots. Mycorrhizal plants showed major SOD activity in shoots but no affected the Se uptake. Respect to pathogen biocontrol, plants inoculated with both bacteria showed an efficient control against Ggt independent to mycorrhization. Thus, our inocula could make important contributions to produce enriched Se flours for human nutrition and biocontrol against Ggt. Keywords: Selenium, biofortification, Green Fluorescent Protein, NanoSe, take-all disease 1. Introduction In recent years, several studies relating to the role of selenium (Se) in human health have been conducted because Se plays an essential antioxidant function in glutathione peroxides (GSH-Px), production of selenoproteins for antioxidant defence systems, inhibition of DNA damage and cancer prevention and progression (Xu et al., 2003). However, the level of Se intake worldwide is inadequate for the expression of important selenoproteins (Rayman, 2007). In developed Journal of Soil Science and Plant Nutrition, 2018, 18 (4), 1021-1035 1022 Microbial consortia to Gaeumannomyces graminis biocontrol Durán et al Journal of Soil Science and Plant Nutrition, 2018, 18 (4), 1021-1035 countries such as Finland and Australia, agronomic biofortification by the addition of inorganic Se to soil is commonly applied (Lyons et al., 2003). In Chile, this technology is inappropriate because in our Andisol, inorganic Se forms sodium selenite and is bound to soil constituents such as clays and oxy-hydroxides of aluminium (Al), iron (Fe) or manganese (Mn), remaining unavailable to plants (Mora et al., 2008), whereas sodium selenate can be leached during wet fall conditions (Govasmark and Salbu, 2011). Thus, while Se content in soils of the South of Chile is low, ranging between 21 and 180 µg kg-1 (Cartes et al., 2005) there are not viable strategies for Se fertilization. In addition, the mineralogical compositions of the Andisol provide limited conditions for root development; therefore, a decrease in plant performance is commonly observed (Mora et al., 2008). Studies previously conducted by our research group showed that microorganisms, such as selenobacteria, can metabolize and transform inorganic Se into nanospheres of elemental Se (NanoSe) (Acuña et al., 2009) and other organic Se forms. These Se forms can enhance the Se content in wheat plants through biofortification strategies based on efficient inoculation of rhizospheric bacteria with arbuscular mycorrhizal fungi (AMF) (Durán et al., 2013). In a recent study, we showed that the plant growth promoting (PGP) endophytic bacteria Bacillus sp.E5 and Acinetobacter sp.E6.2 have more important ecological advantages than rhizospheric bacteria in terms of Se tolerance and Gaeumannomyces graminis biocontrol (Durán et al., 2014) due to endophytic microorganisms can produce antifungal metabolites. In recent studies, Bacillus sp.E5 and Acinetobacter sp.E6.2 were able to produce Se organic forms, such as seleno-methyl-selenocysteine (SeMeSeCys) and seleno-methionine (SeMet) (Durán et al., 2015), which can play a role in cancer prevention (Rayman, 2007). On the other hand, Gaeumannomyces graminis is a soil borne pathogen that affect wheat plant in Chile, causing losses around 50% of wheat production due to environmental and agronomic conditions as monoculture (Duran et al., 2017, 2018). However, further research involving agricultural plant species and using these endophytic bacteria and AMF are needed to elucidate their role in Se biofortification and Gaeumannomyces graminis biocontrol. Here, we hypothesized that endophytic bacteria could serve for Se augmentation inside the plant because bacteria can incorporate NanoSe into the cells and could have a synergic effect with mycorrhiza improving the antioxidant system and plant growth. Thus, the main objective of this study was to evaluate the efficiency of these microbial association in terms of Se increase, the antioxidant activity in plants and the capacity of biocontrol of Gaeumannomyces graminis. In addition, we studied the ability of NanoSe to enter plant cell and the endophytic capacity of bacteria inocula. Our results may be useful for the consideration of novel selenium biofertilizer with capacity of biocontrol of the main pathogen that affect the most consumed cereal in Chile and can to serve of basis to incorporate this important antioxidant inside the fertilization strategies. 2. Materials and Methods 2.1. Inocula preparation 2.1.1. Bacteria inococula preparation Two previously isolated endophytic selenobacteria strains identified as Acinetobacter sp.E6.2 and Bacillus sp.E5 (Durán et al., 2014) were used for inocula preparation. The isolates are deposited at the Chilean Culture Collection of Type Strains 1023 Microbial consortia to Gaeumannomyces graminis biocontrol Journal of Soil Science and Plant Nutrition, 2018,18 (4) ,1021-1035 (CCCT/UFRO), which is registered at the World Federation of Culture Collection under the number WDCM1111 and is hosted at the Scientific and Technological Bioresource Nucleus (BIOREN) from Universidad de La Frontera, Temuco, Chile with the codes: CCT 15.25 (Acinetobacter sp.E6.2) and CCT 15.23 (Bacillus sp.E5). Strains were grown in 200 mL of Luria Bertani media (LB) supplemented with 5mM sodium selenite (Na2SeO3, Merck, Inc.) according to Durán et al., (2013). After growth at 30 ºC for 24h with continuous shaking (150 rpm), the bacterial cells were collected by centrifugation at 10,000 x g for 10 min and rinsed twice with sterile saline solution (0.85% NaCl). The bacterial cell suspension was used as a selenobacteria inoculum for greenhouse experiments. 2.1.2. Mycorrhizal propagation Claroideoglomus claroideum (N.C. Schenck & G.S. Sm.) C. Walker & A. Schüßler, isolated from the rhizosphere of wheat plants growing in volcanic soils from Southern Chile was used for plant mycorrhization. For the Claroideoglomus claroideum (AMF) inoculum preparation, Sorghum bicolor plants cv Sordan (INIA) were inoculated with spores and mycelia of mycorrhiza and maintained for three months in a sand:vermiculite:peat (1:1:1; v:v:v) substrate for its propagation. Then, colonized roots and substrate were used as inocula (Durán et al., 2013). 2.1.3. Pathogen inocula preparation A highly pathogenic isolate of Gaeumannouyces graminis was used as inoculum inoculation. For this, oat kernel inoculum was prepared. Colonized oat were blended, sieved to a particle size of 0.5-1.0 mm, and stored at 5ºC until use (Duran et al., 2017). 2.2. Greenhouse assay 1 A completely randomized experimental design was adopted. Wheat seeds cv Fritz were surface-disinfected by dipping in 0.8% (v:v) NaClO solution for 15 min. Then, seeds were placed in a pot containing 1 kg of soil from Maquehue belonging to Freire series (Table 1). For AMF incolated plants, pots including AMF inoculum:soil (1:1) were used. After 7 days, the bacteria inoculum was directly injected into the rhizosphere of wheat with two millilitres of 109 CFU mL-1 of each strain grown in selenite, for the consortia preparation the same number of cell of both bacteria were considered. The treatments were as follows: (1) control, (2) Acinetobacter sp.E6.2, (3) Acinetobacter sp.E6.2 grown in selenite, (4) Bacillus sp.E5, (5) Bacillus sp.E5 grown in selenite, (6) consortium (Acinetobacter sp. + Bacillus sp.), and (7) consortium grown in selenite. These treatments were applied to both AMF inoculated (with Claroideoglomus claroideum) and non-AMF inoculated pots at 14 and 30 days, thus we considered a total of 14 treatments. Plants were irrigated every 10 days with Taylor and Foy nutrient solution (Taylor and Foy, 1985) and maintained for 4 months under greenhouse conditions at day/night cycle of 16/8 h, 21/15 °C and 50% relative humidity. Mycorrhizal colonization in root plants was measured by using 10% KOH and staining with 0.05% Trypan blue in lactic acid (v/v) and was quantified according to the grid-line intersect method (Giovannetti and Mosse, 1980). In addition, colonized roots were observed by using an Olympus CX31 optical microscope. 2.3. Antioxidant activity determination Enzymatic extracts were obtained according to Lin et al., (2009). Total protein concentration was determined by the Coomassie blue G-250 dye-binding assay, using bovine serum albumin as a standard (Bradford, 1976). 1024 Microbial consortia to Gaeumannomyces graminis biocontrol Durán et al Journal of Soil Science and Plant Nutrition, 2018, 18 (4), 1021-1035 2.3.1. Superoxide dismutase activity (SOD) The activity of SOD was assayed spectrophotometrically by measuring its ability to inhibit the photochemical reduction of nitroblue-tetrazolium (NBT) (Medha et al. 2013). A set of cuvettes was covered with a black cloth as a control. The other set was placed approximately 30 cm below a bank of two 15-W fluorescent lamps. The reaction was initiated by turning the light on for 10 min. Following light exposure, the tubes were covered (Abassi et al., 1998). One enzyme unit was defined as the amount of free extract that inhibited the reduction of nitrobluetetrazolium by 50% at pH 7.0. The total specific enzyme activity was expressed as U mg protein-1. The scavenging ratio of samples was expressed as Trolox equivalents per gram of plant samples (Wong et al., 2006). Thus, 0.1g of vegetal material (wheat shoot) was homogenized with methanol. Then, 0.25mL of samples were mixed with 0.75 mL of 0.25mM DPPH ethanol. The reaction mixture (1mL) was added to the automatic injector SynergyTM HT multimode microplate reader. The DPPH solution absorbance was measured at 517nm. 2.4. Total Se determination For Se determination, two phenological steps were considered. The first step was at 30 days after seedling, when plants were in the middle tillering, and the second step was at 4 months (dry grain). The Se content was measured by Atomic Absorption Spectrophotometry (AAS) with an HG 3000 Hydride generator (GBC Scientific Equipment Ltd.) using NaBH4 solution as the reducing agent (Kumpulainen, 1983). Two Se-enriched flour samples supplied by the Department of Applied Chemistry and Microbiology of Helsinki University (Finland) were used for reference. 2.5. NanoSe penetration in plants analysed by transmission electron microscopy (TEM) As the main Se form found in the inocula was NanoSe (Durán et al., 2015), the ability of NanoSe Table 1. Chemical parameters of the Maquehue soil (Freire series) used in the assay. Variable Value N (mg kg-1) 29 P (mg kg-1) 27 K (mg kg-1) 344 pH (water 1:5, w:v) 5.75 Organic Matter (%) 17 K (cmol+ kg-1) 0.88 Na (cmol+ kg-1) 0.15 Ca (cmol+ kg-1) 7.61 Mg (cmol+ kg-1) 1.77 Al (cmol+ kg-1) 0.05 Al sat (%) 0.48 CICE (cmol+ kg-1) 10.46 Bas. sat (cmol+ kg-1) 10.41 1025 Microbial consortia to Gaeumannomyces graminis biocontrol Journal of Soil Science and Plant Nutrition, 2018,18 (4) ,1021-1035 to penetrate cell walls was visualized by highmagnification TEM (model JEOL/JEM 1200 EX II) using root collected from plants with and without exposure to Se (Dhanjal and Cameotra, 2010). Ultra-thin sections were cut using an ultra-microtome, and the sections were stained with alcoholic uranyl acetate (saturated solution in ethanol) for 2 min and subsequently stained in lead citrate for 2 min before examining the grids by TEM. Plants without Se supplementation were used as controls and processed similarly. 2.6. Fluorescent labelling of endophytic selenobacteria Bacillus sp.E5 and Acinetobacter sp.E6.2 were tagged by parental conjugation (Lambersten et al., 2004) with pBK-mini-Tn7-gfp (pUC19-derived delivery vector for mini-Tn7-gfp, gentamycin resistant, chloramphenicol acetyltransferase, β-lactamase, Mob+, Koch et al., 2001) and the helper plasmid pUX-BF13 (donor of Tn7 transposase tns ABCDE R6K, ampicillin resistant, β-lactamase, Mob+, Bao et al., 1991). Bacteria were grown on Luria-Bertani (LB) medium containing gentamycin as antibiotic (10 µg mL-1). 2.7. Confocal laser scanning microscopy The ability of bacteria to colonize the roots of wheat plants was checked by confocal laser scanning microscopy (CLSM). For this purpose, wheat plants were growth in soil from Maquehue (Table 1) and 5 mL of 109 CFU mL-1 grown in LB were inoculated in each pot at 7 days. The inoculum was injected into the rhizosphere of wheat. The treatments were: (1) control (non inoculated), (2) Acinetobacter sp.E6.2, (3) Bacillus sp.E5, and (4) consortium (Acinetobacter sp. + Bacillus sp.). Then, roots were carefully washed and the presence of tagged bacteria was confirmed by the CLSM Olympus Fluoview 1000. 2.8. Biocontrol greenhouse assay 2 In order to evaluate the capacity of biocontrol against Gaeumannonyces graiminis of microbial inoculant using in this study another assay using substrate sand:vermiculite:peat (1:1:1) to avoid the effect of soil was made. The treatments were 1) control, (2) Acinetobacter sp.E6.2, (3) Bacillus sp.E5 and consortium (Acinetobacter sp. + Bacillus sp.). These treatments were applied to both AMF inoculated (with Claroideoglomus claroideum) and non-AMF inoculated pots and with Ggt and without Ggt, thus we considered a total of 14 treatments. All treatments in triplicates. Briefly, experiment was conducted in pots filled with 80g of sterile soil (121°C 15 min, for 3 days), wheat seedlings var. Fritz were superficially disinfected and pre-germinated as previously described. Wheat plants, CV Fritz were watered each three days and nutrient Taylor and Foyd solution was applied each 15 days. Plants were grown in greenhouse conditions and collected after 45 days. Then, the percentage of root blackening against a white background, on a 0-100% scale, was recorded. Shoots carefully separated to roots and dried at 70 °C for 72 H to obtain the shoot dry weight 2.9. Statistical analysis Data sets were analysed by one-way analysis of variance (ANOVA), and mean comparisons were performed using Tukey’s multiple range test with the SPSS software (SPSS, Inc.). All experiments were performed in triplicate, and the values were given as the means ± SE. Differences were considered to be significant when the P value was less than or equal to 0.05. 1026 Microbial consortia to Gaeumannomyces graminis biocontrol Durán et al Journal of Soil Science and Plant Nutrition, 2018, 18 (4), 1021-1035 3.2. Antioxidant activity Higher SOD activity was found in the shoots of plants inoculated with both bacteria, mainly in mycorrhizal plants supplemented with Se respect to the rest of treatments. No significant differences between noninoculated and inoculated plants (Figure 2a) were observed for SOD of roots. Plants inoculated with Se-supplemented bacteria, either Acinetobacter or Bacillus, showed higher DPPH activity in both shoots 3. Results 3.1. Plant growth The roots of wheat plants inoculated with Acinetobacter sp.E6.2, particularly when Se was supplemented, showed higher growth than plants of other treatments (Figure 1). No significant differences were found in plants inoculated with Bacillus sp.E5 compared with the control plants in all treatments. Wheat plants were effectively colonized by the mycorrhizal inoculum (from 40 to 54%) but this colonization reduced radical growth significantly in control plants and plants inoculated with Acinetobacter sp.E6.2. However, no significant differences in shoot yield were found between mycorrhizal and non-mycorrhizal plants. Figure 1. Dry weight (g) of shoots and roots of wheat plants inoculated with endophytic bacteria (Bacillus sp.E5 or Acinetobacter sp.E6.2 and dual consortia) and the arbuscular mycorrhizal fungus (AM) Claroideoglomus claroideum, supplemented or not with Se. Tukey’s post-hoc test was used to compare treatment means. Values followed by the same letter do not differ at P≤0.05 (n= 3). Bars denote means ± S.E. c (control), cAM (control+mycorrhiza), E5 (Bacillus sp.E5), E5AM (Bacillus sp.E5+mycorrhiza), E5Se (Bacillus sp.E5+selenium), E6.2 (Acinetobacter sp.E6.2), E6.2AM (Acinetobacter sp.E6.2+mycorrhiza), E6.2Se (Acinetobacter sp.E6.2+selenium), Cons (consortia), ConsAM (consortia+mycorrhiza), ConsSe (consortia+selenium). 1027 Microbial consortia to Gaeumannomyces graminis biocontrol Journal of Soil Science and Plant Nutrition, 2018,18 (4) ,1021-1035 and roots than the control plants (Figure 3b). The lowest DPPH activities were observed in roots and shoots of plants inoculated with only Acinetobacter sp.E6.2, without Se (Figure 2b), this activity was improved by AM colonization for this treatment. Figure 2. (A) Superoxide dismutase and (B) α,α-diphenyl-β-picrylhydrazyl-radical activity in shoots and roots of wheat plants inoculated with endophytic bacteria (Bacillus sp.E5 or Acinetobacter sp.E6.2 and dual consortia) and the arbuscular mycorrhizal fungus (AM) Claroideoglomus claroideum, supplemented or not with Se. Tukey’s post-hoc test was used to compare treatment means. Values followed by the same letter do not differ at P≤0.05 (n= 3). Bars denote means ± S.E. c (control), E5 (Bacillus sp.E5), E5Se (Bacillus sp.E5+selenium), E6.2 (Acinetobacter sp.E6.2), E6.2Se (Acinetobacter sp.E6.2+selenium), Cons (consortia), ConsSe (consortia+selenium). 1028 Microbial consortia to Gaeumannomyces graminis biocontrol Durán et al Journal of Soil Science and Plant Nutrition, 2018, 18 (4), 1021-1035 3.3. Total selenium content In general, mycorrhizal plants showed lower Se content in the shoots than non-mycorrhizal plants, whereas no significant differences were found in grain, except in plants inoculated with Acinetobacter sp., where major Se content was observed in non-mycorrhizal plants (Figure 3c). Higher Se content was observed in shoots of plants inoculated with Acinetobacter sp.E6.2 than in those inoculated with Bacillus sp.E5 in both stages, at 30 days (tillering) and 4 months (mature grain). Shoots and grains showed similar tendencies with regard to Se concentration (Figure 3a, 3b, 3c). Thus, at both tillering and mature grain, a positive and significant correlation between Se concentration in shoots of plants was found (R=0.420; P<0.05 and R=0.744; P<0.01), and we also observed a positive correlation regarding Se content between grain and waste spikes (Figure 3d, R=0.877; P<0.01). 3.4 NanoSe penetration into plant cells analysed by transmission electron microscopy (TEM) Differences in NanoSe size (~20-200nm) were observed in the lyophilized powder of bacteria inocula (Figure 4a, b). We found NanoSe inside and scattered around the cells, both as free deposits and as aggregates attached to the bacterial cell mass in Se supplemented plants (Figure 5c, d), but no in plants without Se treatments (Figure 4e). Moreover, the electron diffraction patterns of single NanoSe particles confirmed the occurrence of amorphous Se in lyophilized selenobacteria (Figure 4a). Figure 3. Se content in (A) wheat shoots at stage 1 (tillering), (B) wheat shoots at stage 2 (mature grain), (C) dry grain and (D) waste spikes of plants inoculated with endophytic bacteria (Bacillus sp.E5 or Acinetobacter sp.E6.2 and dual consortia) and AM, supplemented or not with Se. Tukey’s post-hoc test was used to compare treatment means. Values followed by the same letter do not differ at P≤0.05. Bars denote means ± S.E. 1029 Microbial consortia to Gaeumannomyces graminis biocontrol Journal of Soil Science and Plant Nutrition, 2018,18 (4) ,1021-1035 Figure 4. Transmission electron microscopy images of (A) bacteria supplemented with Se biosynthesized as elemental nanospheres, (B) root cells of wheat seedlings without Se supplementation and (C) vacuoles from root cell of Se-supplemented wheat. Micrographs were taken after 10 days of bacterial inoculation. Figure 5. Confocal laser scanning microscopy images of endophytic colonization: (A) control bacteria without GFP tag, (B) GFP-tagged Bacillus sp.E5, (C) GFP-tagged Acinetobacter sp.E6.2, (D) roots of wheat plants without bacterial colonization, (E) roots of wheat plants colonized by GFP-tagged Bacillus sp.E5 and (F) roots of wheat plants colonized by GFP-tagged Acinetobacter sp.E6.2.