Rhizobium spp.: Isolation in quantitative culture; Molecular identification of species (PCR and sequencing)

Information 10-06-2019.

Rhizobia include bacteria of enormous agriculture value because their N2 fixation represents the major source of N input in agricultural soils. In addition to nitrogen fixation, many strains of rhizobia act as plant growth enhancers (PGPR: Plant-growth-promoting rhizobacteria) as they produce phytohormones, growth-regulating factors, siderophores, hydrolyzing enzymes, 1-aminociclopropane-1-carboxylic acid (ACC) deaminase, and solubilize the inorganic phosphate and potassium. This makes the rhizobia very valuable for both leguminous and non-leguminous plants. The application of rhizobia as biofertilizers ensures efficiency in crop productivity and reduces the need to use artificial fertilizers. On the other hand, these beneficial microorganisms also act as biocontrol agents for pests and diseases by inducing resistance

One well-studied example of a N2-fixing plant-microorganisms interaction is the legume-rhizobia symbiosis, in which the bacteria fix atmospheric nitrogen as endosymbionts inside root nodules in a nutrient-rich microenvironment. controlled oxygen microenvironment. This symbiosis is a host specific interaction, where rhizobia will only form nodules in a host species and with very few cross inoculations.

Recently, researchers have proposed the possibility of inoculating rhizobia in non-legume plants such as rice and isolated information has been published with the facilitation of seedling growth and grain production. However, the mechanism is not clear and is not due to the formation of nodules as in the Rhizobium-legume symbiosis. It has been postulated that the effect can be through the benefits of the production of phytohormones, the solubilization of phosphates, the improvement in the assimilation of soil nitrogen, or changing the morphology of the roots.

There are several genus of rhizobia: Rhizobium, Bradyrhizobium, Sinorhizobium, Mesorhizobium, Azorhizobium, Methylobacterium and Allorhizobium. Among them, Rhizobium species form an endosymbiotic association with the roots of legumes and Parasponia.

There are several different species of Rhizobium that are categorized based on the growth rate and the type of plant to which they are associated. Some species of Rhizobium are: R. leguminosarum, R. alamii, R. lentis, R. japonicum, R. metallidurans, R. smilacinae, R. phaseoli, R. trifolii.

The bacterium infects colonizes plant cells within root nodules, where they convert atmospheric nitrogen into ammonia, a biological form that can be directly used by the plant, such as glutamine or ureids using the enzyme nitrogenase. The plant, in turns provides the bacteria with organic compounds made by photosynthesis. This mutually beneficial relationship is true for all Rhizobia of which the genus Rhizobium is a typical example.

There are culture media, such as the YEMA (Yeast Extract Mannitol Agar), with Congo Red, which differentiates the Rhizobium colonies since they do not absorb the dye, which makes it possible to differentiate them by the color of the respective colonies. In addition, different inhibitors can subsequently be added to inhibit the growth of possible pollutants. Among them, the simultaneous addition of pentachloronitrobenzene (PCNB) and vancomycin in the PV-YEMA medium that allows a significant improvement in the efficiency of rhizobia enumeration effectively preventing the growth of fungi and gram-positive bacteria.

The identification of the rhizobia is usually carried out through biochemical and morphological tests of the isolated colonies in culture. However, in recent years, PCR identification methods have been applied. The use of molecular techniques by amplification and sequencing of the RNA16S gene has enabled the achievement of faster and more accurate identification of the isolates.


Biofertilizers are products based on beneficial microorganisms to the soil, mainly bacteria and/or fungi, which live in association with or in symbiosis with plants and help naturally to their nutrition and growth, constituting an alternative source to chemical fertilizers.

Nowadays, the indiscriminate use of chemical products in agriculture is a global problem. Although chemical fertilizers promoted a considerable increase in agricultural yields over several years, they are now the main cause of loss of soil fertility, contamination of groundwater and, in many cases, damage to human health. This situation is exacerbated by the growing demand for food and the progressive depletion of non-renewable resources on Earth. Therefore, the need to look for alternatives that allow agricultural sustainability becomes evident. The ability of legumes to use N2 from air is known, through the efficient symbiotic relationship established between these plants and nitrogen-fixing bacteria (diazotrophs). The process of colonization of these bacteria, although it occurs naturally, sometimes is not entirely efficient, which is due, among other causes, to the microbial load in soils is very low, as a result of erosion. In recent years the use of rhizobia as biofertilizers has increased, since the benefits for plants and soil are appreciable. The inoculation of plants with beneficial bacteria goes back centuries. At the end of the 19th century, the practice of mixing soil "naturally inoculated" with seeds became a recommended method of inoculating legumes in the USA. A decade later, the first plant inoculation patent was registered with Rhizobium spp. was registered.

In order to obtain a good yield in legume cultivation, the use of nitrogen fertilizers is necessary, which raise the production costs, affecting the economy of the producers and consumers. An alternative to this problem is the use of bacterial inocula containing viable and selected rhizobia for their symbiotic infectivity and effectiveness.

Tests carried out in IVAMI

  • Isolation in culture with quantification in differential media. 
  • Molecular identification of species (PCR and sequencing) 

Recommended samples

  • Roots of plants with nodules. 
  • Fertilizers (fertilizers): 25 mL / 25 g.

Conservation and sending of the sample:

  • Refrigerated (preferred) for less than 2 days.

Deadline for delivery of results:

  • 5 to 10 working days.

Cost of the test:

  • Quantification by isolation in crops: Consult ivami@ivami.com. 
  • Molecular identification of species (PCR and sequencing): Consult ivami@ivami.com.