Control de la    persistencia de una cepa genéticamente modificada de  Azorhizobium    caulinodans  en la rizosfera de  Triticum aestivum  L.

Jan Jacek Slaski; Daniel Joseph Archambault; John Vidmar; Carlos José Bécquer; Anthony Anyia

Untitled1 ARTÍCULO ORIGINAL

ISSN 2074-8647, RNPS: 2154 (Versión electrónica)
ISSN 1609-1841, RNPS: 0397 (Versión impresa)

Monitoring the persistence of a genetically modified strain of the Azorhizobium caulinodans in the rhizosphere of Triticum aestivum L.

Control de la persistencia de una cepa genéticamente modificada de Azorhizobium caulinodans en la rizosfera de Triticum aestivum L.

Jan Jacek Slaski¹*, Daniel Joseph Archambault¹⁺, John Vidmar¹, Carlos José Bécquer², Anthony Anyia¹. *Corresponding author.

¹Alberta Innovates_Technology Futures, Bioresource Technologies, Vegreville, Alberta, Canada, T9C 1T4. e-mail: slaski@arc.ab.ca
⁺Present address: Laurentian University, 935 Ramsey Lake Road, Sudbury, ON, Canada, P3E 2C6.
²Ministerio de la Agricultura, Instituto de Investigaciones de Pastos y Forrajes, Estación Experimental Sancti Spíritus, Cuba. Apdo 2255, Z.P. 1, Sancti Spíritus, Cuba.

ABSTRACT

A protocol was developed to monitor persistence and spread of a genetically modified strain of the growth promoting diazotroph Azorhizobium caulinodans used to inoculate field grown wheat. The protocol was used to identify an endogluconase (egl) overproducing strain of A. caulinodans isolated from inoculated soils. A detection limit of 2500 cfu g^-1 and 10⁸_10¹⁰cfu g^-1 of soil was determined using the BIOLOG^® fingerprinting method and PCR technique, respectively. Since none of the tested samples were positive for the bacteria or the Egl 1 gene construct, it was concluded that A. caulinodans ORS 571 pGV910-C1 did not persist or spread in any of the tested field locations. This result may be due to low soil temperatures and competition of indigenous microorganisms: environmental factors that were not favorable for the diazotroph to thrive in the test locations. The application of different inoculation methodologies, as well as the study of other rhizobial genera for the inoculation of wheat in further experiments, is strongly recommended.

Key words: detection, diazotroph, endogluconase, ORS 571, wheat

RESUMEN

Se llevó a cabo un protocolo para monitorear la persistencia y multiplicación de una cepa genéticamente modificada de la bacteria diazotrófica Azorhizobium caulinodans, la cual se usó para inocular trigo sembrado en condiciones de campo. El protocolo se utilizó para identificar una cepa productora de endogluconasa (egl), aislada del suelo inoculado. Se determinaron los límites de detección para los métodos de identificación BIOLOG® (2500 ufc/g^-1), y PCR (10⁸_10¹⁰ufc/g^-1 de suelo). Ya que ninguna de las muestras evaluadas resultó positiva para la bacteria o el gen Egl1, se concluye que A. caulinodans ORS 571 pGV910-C1 no persistió, ni se multiplicó en ninguno de los experimentos de campo evaluados. Este resultado puede ser debido a las bajas temperaturas del suelo y a la competencia de microorganismos nativos, lo cual constituye un factor ambiental que obstaculiza el desarrollo de la bacteria diazotrófica en los sitios experimentales. Se recomienda la aplicación de diferentes metodologías de inoculación, así como el estudio de otros géneros de rizobios para la inoculación de trigo en futuros experimentos.

Palabras clave: diazotrófica, endogluconasa, identificación, ORS 571, trigo

INTRODUCTION

Azorhizobium caulinodans is a stem and root nodulating nitrogen-fixing bacterium, which was isolated from the stem and root nodules of the semiaquatic tropical legume Sesbania rostrata that grows in the Sahel region of West Africa (Dreyfus et al., 1988). Several studies have shown that this bacterium is able to colonize intercellular spaces of cortex, xylem and root meristems of several non-legumes through crack entry of emerging lateral roots (Sabry et al., 1997; Webster et al., 1997; Cocking, 2005). In the studies of Sabry et al. (1997), inoculation of aseptically grown wheat with A. caulinodans stimulated formation of large number of short(up to 3 mm) lateral roots accompanied by increased grain yield and nitrogen content. On the other hand, A. caulinodans were able to enter the roots of rice at emerging lateral roots (lateral root cracks) by crack entry and bacteria moved into intercellular space within the cortical cell layer of roots (Mia and Shamsuddin, 2010).

In greenhouse experiments, increased biomass, grain yield, leaf area, leaf area index and other agronomical variables of wheat cv. `CDC Teal' inoculated with the genetically modified strain of A. caulinodans, ORS571 pGV910-C1, was reported by Anyia et al. (2004), Bécquer et al. (2007) and Anya et al. (2009). This strain overproduces endoglucanase and shows a normal nodulation behavior on the legume host plant (Geelen et al., 1995).

The objective of the present work was monitoring of persistence and spread of a genetically modified strain of the growth promoting diazotroph Azorhizobium caulinodans used to inoculate field grown wheat with the intention of increasing grain yields.

MATERIALS AND METHODS

Strain used

One genetically modified rhizobial strain (ORS571 pGV910-C1), pertaining to Azorhizobium caulinodans was used. Strains purity was verified on yeast extract-mannitol agar (YMA) medium (Vincent, 1970) containing congo red (0.00125%). Stocks were prepared on tryptone-glucose-yeast-extract (TGYE) (Dreyfus et al., 1988) and kept at -70 ^oC (under 30% glycerol) for long-term storage and at 4 ^oC as source cultures.

Molecular procedures

The pGV910-C1plasmid was derived from the pGV910 plasmid vector by insertion of a 4.3 kb EcoR1 fragment of the Egl 1 gene (total = 19.9 kb) (Figure 1). The 4.3 kb fragment of the endoglucanase gene Egl1 is from A. caulinodans (Geelen et al., 1995). Expression of the endoglucanase gene is driven from the chloramphenicol resistance gene promoter. The gene can restore endoglucanase activity in Egl^- strains of A. caulinodans. The host plasmid pGV910 was originally constructed by Van den Eedeet al. (1992). It contains the ColE1
and pVS1 origins of replication and is stably maintained in A. caulinodans ORS571. Selection of the ORS571 pGV910-C1 strain is enhanced by antibiotic resistance genes including streptomycin/spectinomycin, ampicillin and chloramphenicol (Geelen et al., 1995).

Conditions of field experiments

A field study approved by the Canadian Food Inspection Agency was conducted at four sites located in different soil zones of Alberta. The sites included the following soils: Malmo series of an Eluviated Black Chernozem (at Vegreville), Lacorey soil series of an Orthic Gray Luvisol (St. Vincent), Black Loam Soil Zone, Niobe Silt Loam (80%), Penhold Meadow (20%) (at Innisfail) and Clay Loam within the Brown Soil Zone (at Taber, irrigated). On each of four trial sites, the experimental plots were secluded from other field operations with a 12 m fallowed buffer zone that isolated the treated crop and minimized treatment drift to non-target plants.

Experimental design and statistics for the field experiment

A factorial scheme in a randomized complete block design (RCBD) with four replicates per treatment was used. Three inoculation levels including inoculated (live inoculum), filtered (sterile filtered inoculum containing metabolites/compounds generated by A. caulinodans) and control (no inoculation) and four nitrogen levels (0%N, 25%N, 50%N and 100%N of recommended rates) were applied (Somasegaran and Hoben, 1994; Sabry et al., 1997). Duration of the experiment: 90 days.

Seed inoculation procedures

Pure culture of A. caulinodans (strain ORS 571 pGV910-C1) was applied to the seeds as a liquid inoculant in a tryptone, glucose yeast extract (TGYE) fermentation broth. The concentration of cells in the inoculant was 9.4 x 10⁸cfu ml^-1. The treatment (live inoculum and filtered inoculum) was applied at a rate of 75 ml/27 kg of wheat seed. In order to maximize the number of cells delivered to soil, a relatively high seeding rate of 265 kg ha^-1 was used. As a result, about 230 cells g^-1 soil was introduced to the top 30 cm of soil.

Monitoring techniques and estimation of detection limits

Two independent techniques were used to monitor persistence of A. caulinodans in the fields used for experiments, including BIOLOG^®(Hayward, CA) fingerprinting employing morphology and substrate utilization patterns of A. caulinodans, and a PCR technique with specific primers designed to target highly conserved regions of the plasmid including the Egl1 gene. To establish the detection limit using BIOLOG^®, sterilized and unsterilized field soil was spiked with a serial dilution of A. caulinodans containing 0, 7.2 x 10³, 3.6 x 10⁴, 7.2 x 10⁴, 3.6 x 10⁵, 7.2 x 10⁵, and 3.6 x 10⁶cfu ml^-1. Five grams of the spiked soil was extracted with 50 ml of 0.05 M sodium phosphate buffer (Slaski et al., 2002). The soil extract (100 µl) was plated on TGYE plates containing ampicilin (50 µg ml^-1) and streptomycin (50 µg ml^-1) antibiotic markers to select for A. caulinodans ORS 571 pGV910-C1. Plates were incubated for 24 to 48 hours at 30°C±2. Some plates contained congo red (0.00125%). Circular and creamy single colonies typical of A. caulinodans on agar (Dreyfus et al., 1988) were counted. Isolated colonies, usually 2-10, were re-streaked on selective media and incubated for a further 48 hours to obtain pure cultures. The colonies from individual plates were then collected with sterile swabs and dissolved in BIOLOG^® inoculating fluid. One hundred microliter of the suspension was pipetted into 96 wells on the BIOLOG^®GN2 microplates. Plates were incubated for 24 to 48 h at 30°C±2 and read on an Emax BIOLOG^® plate reader. The identity of the colonies was confirmed by BIOLOG^® analysis of substrate utilization pattern. Since A. caulinodans was not present in the BIOLOG^® database, a visual comparison was made with the substrate utilization pattern produced by the pure culture of A. caulinodans ORS 571 pGV910-C1 obtained from Dr. E. Cocking (Nottingham, Univ of Nottingham, Biosciences Dept, UK). Isolates showing patterns similar to the original stock culture of A. caulinodans ORS 571 pGV910-C1 and wild strains of the bacterium were used to generate a database containing 36 entries, which served for identification of A. caulinodans in the monitoring process.

The lowest soil preparation contained 7 200 cells g^-1 of soil, which means that it was theoretically possible to extract 720 cells ml^-1 assuming 100% extraction efficiency (EF), (1g/10 ml extraction buffer). We successfully extracted 30 (sterile soil) and 20 (non-sterile soil) cfu ml^-1 with an EF of 4.2 and 2.8%, respectively. Using the relationship CFU = cells g^-1 soil / EF, it was possible to detect A. caulinodans ORS 571 pGV910-C1 using our BIOLOG^® assay in non-sterile soils at about 2500 cfu g^-1 of soil.

For the PCR analysis, 5 g of soil was initially extracted as described by Yeates et al. (1998). To improve DNA yield and extraction efficiency, the freshly prepared extracts were processed with MoBioUltraClean^TM Soil DNA Isolation kits (Mo Bio Lab. Inc., Carlsbad, CA), with the addition of two modifications, at the extraction step the use of glass beads for increase cell rupture and AlNH₄(SO₄)₂and aurintricarboxylic acid for improved DNA recovery (Gulden et al., 2005). The PCR detection limit of A. caulinodans ORS 571 pGV910-C1 in soil was established from soil spiked with different dilutions of the bacterium culture ranging from 10 to 1.0 x 10⁶cfu g^-1 of soil. The spiked soil was extracted using the procedure described above. Once DNA amplifiability using the universal 16S primer was determined, specific oligonucleotides designed to target the endoglucanase-chloramphenicol construct were used to detect the Egl 1 gene in the soil DNA extract. The primers used were Chlor F1 (5'-ACCTATAACCAGACCGTTCAGC-3' forward primer, which hybridized to the chloramphenicol sequence-Genbank accession # LO8855) and Egl R24 (5'-AGTGTGAGAGAGAGGGTCTCG-3' reverse primer, which hybridized to the endoglucanase sequence-Genbank accession # Z48958). This primer set had high specificity and sensitivity resulting in the generation of a single 900 bp amplification product at annealing temperatures from 50-60°C. Amplification conditions were established on an Eppendorf Mastercycler EP thermocycler using the following PCR program:1: 95 ^oC for 9 min. 2: 95 ^oC for 1 min. 3: 60 ^oC for 1 min. 4: 72 ^oC for 1 min. 5: Cycle back to step 2; 39 cycles. 6: 72 ^oC for 4 min. 7: 4 ^oC hold. These PCR parameters resulted in the production of 900 bp DNA fragment at the lowest dilution tested (10¹⁰cfu g^-1 soil). Because of the specificity and sensitivity of the primers, detection of A. caulinodans at lower dilution is possible, with the only limitation being the DNA extraction efficiency. Using the Chlor F1 and EglR24 primers, direct PCR of serial dilution of pure culture of A. caulinodans ORS 571 pGV910-C1 yielded the expected 900 bp product at a minimum cell concentration of 10⁸cfu ml^-1.

Protocol procedures

Once detection limits of the BIOLOG^® and PCR methods were established, the protocols were used to analyze soil samples from plots of each trial site seeded with wheat cv. CDC Teal inoculated with A. caulinodans ORS 571 pGV910-C1. Time zero soil samples were taken from inoculated and uninoculated plots as a reference for comparison. The same plots were sampled every 30 days throughout the growing season until the ground froze and into the following seasons until the organism could no longer be detected. Additionally, soil was sampled at 1 m intervals in a perimeter approximately 5 m into the buffer around the experimental site. These samples were used primarily to monitor for possible dispersion from the treated plots. In addition to the extensive soil sampling program, three wheat plants from each inoculated plot were randomly collected. DNA was extracted from root, leaf and grains and tested for the presence of A. caulinodans or the Egl 1 gene using the Chlor F1 and EglR24 primers combination. For plant tissue DNA extraction, the QiagenDNeasy^® Plant Mini kit (Mississauga, ON) was used.

RESULTS AND DISCUSSION

Several antibiotic resistant bacteria were found in soil extracts. From these, some produced colonies exhibiting similar morphological characteristics to A. caulinodans ORS 571 pGV910-C1: circular and creamy single colonies; in a media that included congo red in the formulation, no absorption of this dye was observed (Dreyfus et al., 1988) (Fig. 2, Fig. 3).

None of these antibiotic resistant bacteria were confirmed to be A. caulinodans using the BIOLOG^® substrate utilization pattern protocol or the PCR analysis. A search of the BIOLOG^®GN database suggests that most of the isolates were Pseudomonas, Burkholderia and Xanthomonas (99-100% statistical probability) (Table 1).

The origin of the multi-antibiotic resistance in those isolates is unknown. However, the fact that these bacteria were present in time zero samples suggests that resistance was not acquired from A. caulinodans ORS 571 pGV910-C1. The pGV910 plasmid is not self-transmissible and can only be mobilized with the helper plasmid pRK2013 in tri-parental mating (Van den Eede et al., 1992). The fact that the indigenous antibiotic resistant bacteria were highly prevalent in the soil samples suggests that they might have out competed the introduced A. caulinodans ORS 571 pGV910-C1, which appears not to have thrived under the field conditions. Perhaps linked to this fact, Bécquer et al. (2012) obtained experimental results that suggested a marked influence of the indigenous rhizosphere microorganisms as compared with inoculated rhizobial strains in different variables assessed in Triticum aestivum and T. secale under field conditions.

The monitoring protocol described in this communication was used to test over 1200 soil and 400 plant samples collected from the 4 field locations used for experiments with A. caulinodans ORS 571 pGV910-C1. Since none of the tested samples were positive for the bacteria or the Egl 1 gene construct, it is obvious that A. caulinodans ORS 571 pGV910-C1 did not persist or spread in any of the tested field locations.

It is possible that the organism was outcompeted by indigenous microbes and moreover, since it was isolated from tropical soils, it can be assumed that environmental conditions were not favorable for it to thrive in the test locations. In a non-sterile soil, native microorganisms may interfere with the root colonization of introduced bacteria (Okon, 1982). According to Van Berkum and Bohlool (1980), since it is impossible to simulate the plethora of interactions that operate in nature, superior strains and improved mutants manufactured in laboratories may have little ecological relevance and even less agronomic value.

found by Wartiainen et al. (2008). These authors suggested that failure in the identification of the microorganism might be due to template discrimination in the PCR reactions, or low abundance of cyanobacteria compared to heterotrophic nitrogen-fixing bacteria. There also is a possibility that the method of inoculation used in this experiment, might have not ensured a satisfactory amount of rhizobial cells per seed to succeed competition with the indigenous microbial population in the rhizosphere of wheat.

CONCLUSIONS

It is concluded that A. caulinodans ORS 571 pGV910-C1 did not persist or spread in any of the tested field locations probably due whether to the negative influence of several stress environmental conditions in the test locations, or the disadvantageous competition with the indigenous microbial population, or even the combination of both factors.

It is strongly recommended to apply different methodologies of seeds inoculation in further studies, as well as to study other rhizobial genera for the inoculation of wheat.

Low soil temperature was suggested as an important environmental factor drastically affecting survival of A. caulinodans in temperate climatic conditions of UK soils (Bullard, 1999). The author reported that moderate temperatures 18/8 ^oC reduced introduced populations of A. caulinodans by an order of magnitude in 35 days. Although other environmental factors could have a negative influence on the persistence of ORS 571 pGV910-C1, authors consider that the above mentioned negative factors constituted the most relevant in this experiment since according to previous studies, they could directly or indirectly hamper the normal development of bacteria. For instance, there are arguments on how low temperatures may influence uptake of organic and inorganic substrates in different bacterial communities (Nedwell, 1999). According to Wiebe et al. (1993), even mesophilic marine bacteria, isolated from the southeastern subtropical shelf waters of the USA, were inhibited by lack of substrate at temperatures near their minimum for growth.

REFERENCES

Anyia, AO, Archambault DJ, Slaski JJ (2004) Growth promoting effects of the diazotroph Azorhizobium

Therefore, it appears that low spring temperatures in Alberta with frozen soils at depths below 40 cm observed at times in April may have resulted in the cell rupture and death of A. caulinodans.

Similar results, on the identification of cyanobacteria in a rice paddy rhizosphere, were caulinodans on Canadian wheat cultivars. Proc. 4th Intl. Crop Sci. Congress, Brisbane, Australia

Anya, AO, Archambault DJ, Bécquer CJ, Slaski JJ (2009) Plant growth-promoting diazotrophs and productivity of wheat on the Canadian prairies. In: MS Khan et al. (Eds) Microbial strategies for crop improvement. Chapter 4, pp. 287-300. Springer-Verlag Berlin Heidelberg

Bécquer,CJ, Salas Beatriz, Archambault D, Slaski J, Anyia A(2007) Inoculation of wheat (Triticum aestivum L.) with rhizobia adapted to livestock ecosystems of Alberta, Canada. Pastos y Forrajes 30: 133-141

Bécquer, CJ, Nápoles JA, Alvarez Orquidia, Ramos Yamilka, Quintana Maribel, Galdo Yaldreisy (2012) Response of different varieties of cereals to inoculation with Bradyrhizobium sp. Mexican J. Agri. Sci. 3: 187-200

Bullard, M (1999) Further studies with Azorhizobium caulinodans as a novel diazotrophic symbiont in temperate cereals. Final Report # CE0153, UK Ministry Agric Fish Food. [Online] Available: randd.defra.gov.uk/Document.aspx? Document=CE0153_1787_FRP.doc [Consulted 2009 October 4]

Cocking, E (2005) OBPC Symposium: Maize 2004 & beyond-Intracellular colonization of cereals and other crop plants by nitrogen-fixing bacteria for reduced inputs of synthetic nitrogen fertilizers. In Vitro Cell. Dev. Biol. Plant 41: 369-373

Dreyfus, B, Garcia JL, Gillis M (1988) Characterization of Azorhizobium caulinodans gen. nov., sp. nov., a stem nodulating nitrogen-fixing bacterium isolated from Sesbania rostrata. Int. J. System. Bacteriol.38: 89-98

Okon, Y (1982) Recent progress in research on biological nitrogen fixation with non leguminous crops. Phosphorous and Agriculture 82: 3-8

Geelen, D, Montagu MV, Holsters M (1995) Cloning of an Azorhizobium caulinodans endoglucanase gene and analysis of its role in symbiosis. Appl. Environ. Microbiol. 61: 3304-3310

Gulden, RH, Lerat S, Hart MM, Powell JR, Trevors JT, Pauls KP, Klironomos JN, Swanton CJ (2005) Quantitation of transgenic plant DNA in leachate water: real-time polymerase chain reaction analysis. J. Agric. Food Chem. 53: 5858-5865

Mia, MAB, Shamsuddin ZH (2010) Rhizobium as a crop enhancer and biofertilizer for increased cereal production. African Journal of Biotechnology 37: 6001-6009

Nedwell, DB (1999) Effect of low temperature on microbial growth: lowered affinity for substrates limits growth at low temperature. FEMS Microbiology Ecology 30: 101-111

Sabry, SRS, Saleh SA, Batchelor CA, Jones J, Jotham J, Webster G, Kothari SL, Davey MR, Cocking EC (1997) Endophytic establishment of Azorhizobium caulinodans in wheat. Proc. R. Soc. Lond. B. 264: 341-346

Slaski, JJ, Archambault DJ, Li X, Macyk T (2002) BIOLOG® system as a tool for the evaluation of functional diversity of microbial communities in soils, pp. 204-209. 39thAlberta Soil Science Workshop, Nisku

Somasegaran P, Hoben HJ (1994) Handbook for rhizobia. Springer-Verlag. New York, USA. 450 p.

Van den Eede, G, Deblaere R, Goethals K, Van Montagu M, Holsters M (1992) Broad host range and promoter selection vectors for bacteria that interact with plants. Mol. Plant Microbe Interact. 5: 228-234

Van Berkum, P, Bohlool BB (1980) Evaluation of nitrogen fixation by bacteria in association with roots of tropical grasses. Microbiol. Rev. 44: 491-517

Vincent, JM (1970) A manual for the practical study of root nodules bacteria. Blackwell Scientific Publications, Oxford and Edinburgh.164 p.

Wartiainen, I, Eriksson T, Zheng W, Rasmussen U (2008) Variation in the active diazotrophic community in rice paddy-nifHPCR-DGGE analysis of rhizosphere and bulk soil. Appl. Soil Ecol. 39: 65-75

Webster, G, Gough C, Vasse J, Batchelor CA, O'Callaghan KJ, Kothari SL, Davey MR, Denarie J, Cocking EC (1997) Interactions of rhizobia with rice and wheat. Plant Soil 194: 115-122

Wiebe, WJ, Sheldon Jr. WM, Pomeroy LR (1993). Evidence for enhanced substrate requirement by marine mesophilic bacterial isolates at minimal growth temperatures. Microb. Ecol. 25: 151-159

Yeates, C, Gillings MR, Davison AD, Altavilla N, Veal DA (1998) Methods for microbial DNA extraction from soil for PCR amplification. Biol. Proced. Online 1: 40-47

Recibido: 23-10-2012
Aceptado: 19-12-2012

Copyright (c) 2016 Biotecnología Vegetal

Biotecnología Vegetal eISSN 2074-8647, RNPS: 2154. ISSN 1609-1841, RNPS: 0397 Editada por: Instituto de Biotecnología de las Plantas. Universidad Central Marta Abreu de Las Villas. Carretera a Camajuaní km 5.5, Santa Clara, Villa Clara, Cuba CP 54 830 Tel: 53 42200124, e-mail: info@ibp.co.cu

Biotecnología Vegetal está bajo una Licencia Creative Commons Atribución-NoComercial 4.0 Internacional.