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Use of rhizobial Nod factors as biofertilizers for legumes

Dominika KidajJerzy Wielbo

M.Curie-Sk odowska University, Lublin, Poland

July 11-17, 2010Mikkeli, Finland

Agricultural importance of symbiotic nitrogen fixation

The availability of reduced nitrogenous compounds is a major limiting factor in agricultural productivity.The microbiological process that converts atmospheric dinitrogen (N2) into plant-accessible species is known as biological nitrogen fixation (BNF).Total global N2 fixation from BNF has been estimated to 100-290 million tones N/year, compared with 83 million tones N fixed industrially in fertilizer production. Among the N2-fixing bacteria, the most important are the symbiotic systems of leguminous plants and rhizobial species belonging to -proteobacteria of the genera Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium, and Sinorhizobium.

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Agricultural importance of symbiotic nitrogen fixation

Rhizobia constitute „common soil inhabitants” in all climatic zones, from arctic to the tropics, and are commonly found in different types of soils.The rhizobial symbioses with 18,000 legume species contribute nearly half of the annual quantity of BNF in soil ecosystems.Rotations of legumes with other non-nitrogen-fixing plants enrich the soil with fixed N and increase the productivity and sustainability of agricultural systems.The nitrogen derived from legume sources are less susceptible to losses than chemical fertilizer N.

The events involved in Rhizobium –legume symbiosis

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Rhizobial genomes and nodulation genes

The genes involved in nodulation and N2-fixation are clustered on symbiotic plasmid (pSym), or incorporated into the chromosome as symbiotic islands.The architecture of rhizobial genomes may directly underlie the great genetic and physiological variation of rhizobial strains.

Fig. 1. Genetic organization of nod genes in (a) R. leguminosarum bv. viciae

(b) R. leguminosarum bv. trifolii (c)Sinorhizobium meliloti

Nodulation genes and Nod factorsNodulation genes have been classified into three categories:

1. the common nodulation genes (nodABC);2. the host-specificity nodulation genes (nodFE, nodH, nodG,

nodPQ, and several others);3. the family of regulatory nodD genes.

The moieties controlled by the host-specificity nod gene products make the Nod factors specific for target plant hosts.

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Biological activity of Nod factors (LCOs)

Application of purified Nod factors to legume roots at nanomolar concentrations can elicit:

1. root hair deformation,2. cortical cell divisions,3. in some cases, the formation of nodule-like outgrowth.

Natural and synthetic LCOs improve germination and early growth of legume and nonlegume plants, such as carrot (Daucus carota) tobacco (Nicotiana tabacum) and Norway spruce (Picea abies) in the absence of auxin and cytokinin.*The chitin pentamer did not elicit such responses, which demonstrated that the structure of Nod factor plays a role in specificity of its biological activity.

* 1. De Jong et al.,1993, Plant Cell 5:615–620

2. Schmidt et al., 1993, Plant J 4:651–658

3. Dyachok et al., 2000, Plant Cell Rep 19:290–297

Rhizobial biofertilizers for legumes

The most essential characteristics in rhizobial strains, that can be used as biofertilizers:

1. the ability to form nodules and to fix nitrogen in the host plant in a range of environmental conditions;

2. the ability to compete for nodulation with indigenous rhizobial population.There is a need to investigate the strain competitiveness for nodulation as part of the process of converting a „potentially usefull strain” into a „commercial inoculant strain”.

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Nod factors as biofertilizers –experimental part

Using Rhizobium leguminosarum bv. trifolii and viciae nodule isolates, it was found that competitive strains more readily responded to a wide range of synthetic and natural flavonoids present in seed exudates in comparison to uncompetitive strains.*Next, two competitive and highly responsive to flavonoid induction strains: R. leguminosarum bv. trifolii KO17 and R. leguminosarum bv. viciae GR09 were used as producers specific Nod factors and inoculants for clover and pea.

The experimental part of this study was conducted at:1. The Department of Genetics and Microbiology, University M. Curie-Sklodowska, Lublin, Poland;2. The Institute of Soil Science and Plant Cultivation, Pulawy, Poland.

* 1. Wielbo et al., 2007, Can J Microbiol 53:957–9672. Maj et al., 2010, Microbiol Res 165:50–60

The effect of R. leguminosarum bv. trifolii KO17 Nod factor on the germination of clover seeds

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Control 10-8M 10-9M 10-10M

10-11M 10-12M 10-13M

Clover germination in the presence of LCOs – 24 h

Clover germination in the presence of LCOs – 48 h

Control 10-8M 10-9M 10-10M

10-11M 10-12M 10-13M

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The effect of R. leguminosarum bv. viciae GR09 LCOs on the germination

of pea seeds

The effect of R. leguminosarum bv. trifoliiKO17 on the growth of clover grown in

sterile sand under greenhouse conditions

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The effect of R. leguminosarum bv. trifoliiKO17 on the growth of clover grown in

sterile sand under greenhouse conditions

Clover (T. pratense) growth assays in the presence of LCOs

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The effect of R. leguminosarum bv. viciaeGR09 LCOs on the germination of pea seeds and the growth of pea plants grown in sterile

sand

The effect of R. leguminosarum bv. viciaeGR09 LCOs on the germination of pea seeds and the growth of pea plants grown in sterile

sand

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The effect of LCOs, produced by R. leguminosarumbv. trifolii KO17 on the growth and nodulation of

clover inoculated with KO17, grown in soil (outdoor experiment)

Outdoor experiment – clover plants

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The effect of LCOs, produced by R. leguminosarumbv. viciae GR09, on the growth and nodulation of

pea inoculated with GR09, grown in soil (outdoor experiment)

Outdoor experiment – pea plants

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Outdoor experiment – pea plants

Conclusions

Significant increase in the germination of seeds and growth of plant seedlings was observed after LCOs application.The most important effect of LCOs seed treatment was significant increase of plants fresh masses and percentage of nodule numbers.The LCOs treatment did not affect the competitiveness of KO17 and GR09 inoculant strains.Purified LCOs that are capable of initiating complete nodule structures at submicromolar concentrations could be use as effective biofertilizers for legumes.Our „green fertilizers” make possible the indigenous rhizobia to invade more effectively the legume roots.

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