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Diversity and functions of free-living and associated rhizobacteria in wheat rhizosphere and their influence on soil quality and productivity

Michel Aragno, Professor
Pierre Rossi, Coordinator, April 2000 to December 2002
David Roesti, Ph.D. student and coordinator since January 2003
Nathalie Fromin, Expert
Noam Shani, Msc student
Gwenaël Imfeld, Msc student
Noémie Duvanel, Techniciam

Keywords : wheat, rhizosphere, root, micro-organisms, diversity, soil management, PGPR, mycorrhiza, Indo-Swiss project

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Background

The Indo-Gangetic Plains (IGP) occupies nearly one-sixth of the total geographical area of the sub-continent and includes nearly 42% of the total population of 1.3 billion of South Asia. Rice-wheat rotations are practised on nearly 13.5 million ha (Ladha, 2000) with another 12 million ha in China. Demand for rice and wheat will grow at 2.5% per year over the next 20 years (Hobbs and Gupta, 2001). The degradation of arable land is a world-wide major threat to the sustainability of the agricultural production. During the last 40 years, nearly one third of the world’s arable land has been lost by erosion and continues to be lost at a rate of more than 10 million ha per year (Piementel, 1995). In India, 2400 tons of silts are estimated to be carried away through the rivers every year, and, furthermore 4,5 million ha are affected by salinization and 6 million ha by waterlogging due to agricultural mis/-management (Pingali et al. 1996). Thus, future growth in food production will have to come from yield increases integrating a more holistic and long-term approach to food production in sustainable agro-ecosystems.

Photo D. Roesti : Wheat field in Bhavanipur UP	Photo site : www.cymmit.org

The IGP's subhumid climate with two distinct seasons -wet monsoon summer and a dry cool winter- allows rice and wheat to be grown in a double cropping pattern in one year rice in the summer and wheat in the winter. Soils range in textures from loamy sands to silty clay loams. The IGP is endowed with extensive canal irrigation systems using water storage reservoirs in the Himalayan mid-hills. Such areas will require a integrated management including the use of biotechnology, improving not only the crop (creation of high-yielding varieties), but also the interaction of roots with its soil microbial partners (bacterial community and vesicular-arbuscular mycorrhizal fungi). The results of this research will hopefully help improving cropping methods. Soil management and the introduction of beneficial microorganisms will also be included. This approach is therefore aimed to help sustainable development, by ensuring long-term soil maintenance and even improvement. The regional productivity rate’s decrease can be mainly explained by the emergence of weeds, pathogens and pest populations adapted to the specific conditions of the system. A pest control based on culture specificity and aiming to protect regional natural resources leads to an improvement of wheat health during growth.

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Partners

This project is part of an Indo-Swiss collaboration in biotechnology which main goals are to develop partnerships between public and private institutions according to the bilateral agreements between the two countries. LAMUN is in mainly in collaboration with the Microbiology department in Pantnagar University, Uttranchal state. Other collaborations include the Center for Mycorrhizal Research, TERI New Dehli and the Institute of Botany in the University of Basel. The addresses are indicated below :

Department of Microbiology
Pantnagar University of Agriculture and Technology
Pantnagar, India
Prof. Bhavdish Johri

Center for Mycorrhizal Research
Tata Energy Research Institute (TERI)
New Delhi, India
Dr Alok Adholeya

Institute of Botany
University of Basel
Prof. Andres Wiemken

Indo-Swiss Collaboration in Biotechnology (ISCB)
Katharina Jenny

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Framework of the study

The rhizosphere is generally defined as the soil region under the influence of the root. It includes the rhizoplane (surface of the root) and the endorhizosphere (intercellular space between the root tissues inhabited by non-symbiotic bacteria). The rhizodeposition, one of the main factors influencing the rhizosphere is the organic or inorganic production of the root within the soil. It corresponds to 15-40% of the total photosynthetic production of the plant and secretes an important carbon and energetic source towards the micro-organisms of the rhizosphere. It comprises sloughed of cells, secreted mucilage (facilitates water exchanges), soluble exudates (e.g. sugars, amino acids, attractants or antibiotics). The root also influences the rhizosphere by creating a negative oxygen gradient (root respiration), by absorbing water (increasing the soil air conductivity) and by absorbing mineral salts.

The rhizosperic microflora is able to :

• solubilise or metabolise minerals
• fix atmospheric N2
• secrete exopolysacharides
• secrete antibiotics or compounds promoting plant growth
• compete or induce plant pathogen resistance

Some bacteria originating from the rhizospheric microflora : Plant Growth Promoting Rhizobacteria as Azospirillum, Pseudomonas and Bacillus species display a plant beneficial effect on :

• nutrition (nitrogen fixation, phosphate solubilization, etc…)
• Resistance to telluric pathogens (antibiotic production as phenazine, DAPG, etc…)
• Root growth

Among the secondary metabolites produced by the rhizobacteria, the 2.4-DAPG (2,4-diacetylphloroglucinol) is one of the most effective and plays an important role in the suppression of telluric-borne diseases (antifungal, antibacterial and antihelminthics properties). The production of this phenolic compound has been found in several bacterial strains of the genus Pseudomonas. The phlD gene is involved in the biosynthesis of DAPG and is currently used as a genetic marker in the detection of potential DAPG-producing rhizobacteria.

Chemical structure of 2.4-DAPG

Arbuscular mycorrhizal fungi (AMF) are root symbiotic fungi improving plant stress resistance to abiotic factors such as phosphorus deficiency or deshydratation.

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Project objectives

This project aims to understand plant-rhizobacterial interactions at the wheat rhizospheric scale. A special attention is given to the PGPR strain characterisation and the interactions between rhizobacteria and arbuscular mycorrhizal fungi. Other aspects like the impact of agricultural practices on bacterial populations as well as the microbial diversity of potential DAPG producers will be included.

Another aspect of the project is to isolate and characterise potential bacterial inoculants capable of:

• improving wheat growth
• stimulate plant defences (ISR)
• colonise and maintain in the root
• interacting favourably with the spontaneous microflora of the rhizosphere (including AMF)

Advanced molecular tools of microbial ecology (DGGE, hybridisation, cloning-sequencing, gfp tagging, etc…), as well as classical methods are used in order to ensure a global approach of the wheat rhizosphere system.

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Actual experiments

1. Bacterial community study of three wheat fields in the Bhavanipur region (U.P. Inde)

The wheat variety UP 2338 is cultivated during the dry season in rotation with rice culture during the monsoon. Three fields differing from their fertilisation levels or yields have been chosen.

Table 1: Fertilisation level and yield of the three studied wheat fields

Figure 1 : Nitrogen and phosphorus rate before wheat cultivation in the fields in 2001

During the wheat season 2001 and 2002, sampling has been performed according to the different wheat growth stages.

Figure 2 : Sampling according to the wheat growth stages

Physico-chemical analysis (C, N, P, enzymatic activities,….) were undertaken on bulk soil (B) before and after wheat cultivation. Total cultivable bacterial counts on Angle’s medium as well as PGPR bacterial counts (siderophore producers, phosphate solubilizers, nitrogen fixators…) on selective media were performed on the rhizospheric fraction (Rh) and the rhizoplan/endorhizosphère (RE). In parallel, molecular analysis of the bacterial community’s structure on three fractions (B, Rh, RE) by DGGE fingerprinting on the 16SrDNA, were also accomplished. The resulting patterns were in-depth analysed using ecological numerical statistical methods (PCA, CA, CCA, clustering, Fischer’s tests, etc…).

In summary, DGGE analysis on the 16SrDNA region as well as the bacterial counts show that the growth stage of the plant was the most important factor influencing the bacterial community structure and that the level of fertilisation level had a minor influence on it.

2. Influence of arbuscular mycorrhizal fungi on the wheat rhizosphere’s bacterial community in a microcosm system

Experiments in microcosms have been undertaken in order to study the impact of AMF on the bacterial community at three different fractions: bulk soil (B), rhizospheric (Rh) and rhizoplan/endorhizosphere (RE). The soil matrix is a 1:1 mix of sans and soil originating from a organic practised wheat field. The mix has been sterilised by tyndallisation.

Design of the experiment :

Mycorrhizosphere AMF and roots Hyphosphere Inoculated and stérile	Rhizospheric Only roots Control No wheat
Fig.5 : Compartments microcosme experiment

4 compartments were defined : Mycorrhizosphere containing wheat roots and mycorrhiza (Mycorrhizal inoculum = non sterilised soil), rhizospheric fraction containing only wheat roots, hyphosphere (containing a 30 mm double nylon membrane mesh containing only the hyphae and the control without any wheat plant. A bacterial suspension prepared from the filtrated soil ( in order to eliminate hyphae or AMF spores) was added to all the compartments except the sterile hyphosphere. 3 wheat growth stages were studied: emergence, flowering and maturity.

Standard physico-chemical analysis, mycorrhizal infection rates, bacterial counts on PGPR media as well as DGGE community profiles were performed on these samples. All was then analysed using numerical ecology statistical methods

3. Root colonization assays of PGPR strains isolated from
   indian wheat fields

This experiment allows to evaluate the rhizospheric competence of a PGPR bacterial strain. For this purpose, different PGPR strains were tagged with the fluorescent protein GFP. The green fluorescent protein (GFP) is a 27kD protein isolated from a bioluminescent jellyfish and absorbs blue light at 295nm and emits green light at 510nm. The tagged bacteria producing this protein will be visible on a epifluorescence microscope. It enables us therefore to observe the localisation of the transformed strains as well as their number in the roots.

4. Impact of a new agricultural practice on phlD gene pool

1. Resources conserving technologies

Studies realized the last ten years leaded to the assessment of a positive repercussion of new agricultural technologies at the forming scale. Among them the practices called “no-tillage” (absence or reduction of tillage), combining with no puddling (no land preparation before rice cropping generating a compaction of the soil layers) are particularly relevant and actually in extension process. In the broad context of a transient process towards “sustainable” agriculture, this agricultural practice presents many advantages in wheat cropping:

• Improvement of regional water management: because of a residual persistent humidity dues to the last cropping, practices including no-tillage promote a reduction of the irrigation’s level. Moreover, a pre-irrigation before wheat cropping is not necessary. The do saved water value is estimated to about 1 million litters per hectares (rwc, 2000).
• Erosion control and improvement of a sustainable soil structure: particularly in sub-tropical areas where upper layer of the soil is subjected to erosion process, an excessive tillage increase dramatically soil
• Weed control efficiency : because seed’s exposition and germination is reduced, wheat emergence is prior to weed apparition, improving consequently a manual control and efficient control of weed.

One agricultural practice called « raised beds » allows an optimisation in the targeted objectives. Basic principle in simple: furrows existing between artificially raised band of land flooded during the Monsoon period (Summer) and allow the irrigation in winter. Consequently, the upper parts of the raised beds remain in oxic condition throughout the year.

Bed planter system (for wheat cropping)
Photo : site web de RWC (Rice-Wheat Consortium : www.rwc.org)

The best option concerning the application of the practice in the rice-wheat cropping system seems to be permanent and progressively reconstructed raised beds throughout several rotations combing the seedling of the new crop in the straws of the previous one

2. Screening, function and identification of PGPR of the wheat rhizosphere in a rice-wheat cropping system

Rhizobacteria of the genus Pseudomonas spp. Represent a major component of the bacterial population of the wheat rhizosphere. The group of the Pseudomonas fluorescents is generally studied for their involvement in biocontrol of telluric diseases
The present study, realized from two cropping rotations (four seasons), leaded to the development of molecular tools (membrane hybridisation, double step PCR) allowing the detection of phlD gene possessing rhizobacteria. The assessment of the diversity, the abundance and the characterisation of bacteria integrating the fields experimental conditions is actually carried out.

Screening process of putatively 2,4-DAPG producing rhizobacteria :

 

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last update : 06.08.2004