Bradyrhizobium is a genus of gram-negative bacilli (rod shaped) originally described by Jordon in 1982 (Kuykendall, 2005). Bradyrhizobium bacteria tend to live in the soil and form symbiotic relationships with various legume plants such as soybeans, flowering plants, clover, peanuts, and beans. The symbiotic relationship is established as the bacteria fix atmospheric nitrogen into ammonia, a process not found in their plant hosts, in exchange of essential carbohydrates for bacteria survival. Therefore, Bradyrhizobium species are also classified as nitrogen-fixing bacteria and are known to reside in nodules (roots of legume plants). The Bradyrhizobium genus currently consists of nine rhizobia species and one non-rhizobia species (B. betae).
- B. canariense
- B. denitrificans – formally Blastobacter denitrificans
- B. elkanii
- B. iriomotense
- B. japonicum – formally Rhizobium japonicum
- B. jicamae
- B. lianingense
Provided by the current taxonomy of rhizobia webpage at this link below.
Bradyrhizobium bacteria are a gram-negative rod-shaped (bacilli) and are typically 0.5 to 0.9 μm wide and 1.2 to 3 μm long (rhizobia module 3). They require oxygen to survive even when living under the soil. Their motility is due to the presence of single polar flagella. Phenotypic variation of two B. japonicum strains USCA 110 and USDA 122, as proposed by Mullen and Wollen, depend on colony morphology, antibiotic resistance, growth in various media, and competition with other strains of Bradyrhizobium species (Mullen, 1989). According to their results, both strains grown in broth culture produced lower cell numbers and reduced extracellular polysaccharide production (Mullen, 1989).
Bradyrhizobium are commonly found in the soil or inside the root nodules of their host legumes (rhizobia module 3). The plants provide the bacteria with energy from photosynthesis in exchange for the nitrogen fixation completed by the bacteria. According to the literature, B. japonicum uses hypoxia conditions (low oxygen inside the cells) as the intracellular signal for expression of the symbiotic nitrogen fixation genes (Weidenhaupt, 1995). Besides soil oxygen levels, temperature and acidity of the soil are other abiotic factors that affect bacteria gene expression and may prevent the nodulation process when the bacteria is exposed to extreme temperatures and/or pH conditions. The different species of Bradyrhizobium may vary in tolerance to these abiotic factors. Additionally, in the laboratory, the bacteria are grown for 8 days on a yeast-mannitol agar (YMA) medium to produce an alkaline reaction, indicated by a pH indicator (BTB) to the petri dish (rhizobia module 3). Compared to the fast-growing rhizobium species, Bradyrhizobium species are slow-growing bacteria. This process of in-vitro growth takes only 2 days for rhizobium species, but 8 days for Bradyrhizobium species (rhizobia module 3).
Host-bacterium diversity is evidenced by the plant host specificity as the bacteria can either nodulate only one legume species or several legume species (Wagner, 2011). There is no evidence of any association between Bradyrhizobium and insect hosts (Brucker & Bordenstein, 2012). However, only seven species are known to from symbiotic relationships with legumes (Rivas, 2009).
- B. japonicum, B. elkanii, and B. liaoningense establish symbiosis with soybeans
- B. yuanmingese nodules Lespedeza (flowering plant in pea family)
- B. canariense nodulate endmic genistoid legumes
- Blastobacter denitrificans produces nodules in Aeschynomene indica (flowering plant)
- B. iriomotense is isolated from Entada koshuensis in Japan
The seven species listed above form well-understood symbiotic relationships with their legume hosts, however the Bradyrhizobium betae nodulation in sugar beets (Beta vulgaris) has an unknown symbiotic relationship (Rivas, 2009). It is possible that B. betae has a pathogenic function because it is labeled as a “tumor-forming bacteria” (Rivas, 2004).
Diseases and Parasites
Life History and Behavior
Members of this genus are mobile with single polar flagella. The symbiotic relationship between the legume plants and Bradyrhizobium are affected by two abiotic factors: high soil temperature and acidity. If soil temperatures are high and/or acidic soil conditions are present, then nitrogen fixation is prevented since the bacteria cannot attach themselves to the root hairs of their legume hosts (Radtke, 2003). To understand the effects of pH and temperature, we must first understand the multi-step nodulation process as shown below (Radtke, 2003).
1- Bradyrhizobium are attracted to flavonoids released by the host legume’s roots.
- Flavonoids (Latin for yellow “flavus”) are a class of plant secondary metabolites involved in a variety of functions: yellow flower coloration, attracting rhizobia bacteria, triggering secretion of Nod factors from rhizobia, and protecting plants from specific microbes, fungus, and insects.
2- Bradyrhizobium attach to root hairs in a 2-step process (chemotaxis)
- Step 1: Bacteria use binding proteins to attach to root hairs of plant. This step is prevented by acidic soil or high temperature soil. The favorable conditions of pH range from 6-8 and soil temperatures at 25° to 30°C (rhizobia module 3).
- Step 2: Bacteria express bacterial nodulation (nod) genes that can be host-specific or general for a variety of plant hosts.
3- These nod factors cause root hairs to curl allowing bacteria to enter into the root cortex. Once it is inside the plant host, the bacteria loss their cell walls and specializes into bacteroids as they function in nitrogen fixation.
- The membrane that surrounds the bacteria is plant-derived and allows nutrients to flow (malate, succinate, iron, sulfur, molybdenum) that are needed in the nitrogen-fixation process.
- In return, the bacteroids provide amino acids to plant needed for asparagine synthesis, therefore there is a symbiotic relationship between the bacterium and plant host.
Another environmental factor influencing Bradyrhizobium species is the amount of rainfall: rhizobia prefer moist soils in areas that receive adequate rainfall (rhizobia module 3). Another factor is the presence of vegetation positively correlates with more rhizobia (note: vegetation includes legume or non-legumes). The immediate area between plant roots and the rhizobia niche is named the rhizosphere (rhizobia module 3).
Evolution and Systematics
Bradyrhizobium species cannot be distinguished by genomic analysis of comparing 16rRNA gene sequences, because these sequences are highly conserved and have high sequence similarity between the various species (Rivas, 2009). Bradyrhizobium species were initially distinguished using SDS-PAGE of total cellular proteins and/or amplified fragment length polymorphism (AFLP) analysis (Willems, 2001). The AFLP method revealed a few species, but was very difficult and time-consuming. The alternative method to distinguish the species is using either DNA-DNA hybridization techniques and/or combined analysis of several housekeeping genes (Rivas, 2009):
- atpD gene encodes the beta-subunit of the membrane ATP synthase
- dnaK gene encodes a conserved Hsp70 class chaperone
- gyrB gene encodes a topoisomerase II (prevents supercoiling of bacterial DNA during replication)
- rpoB gene encodes a product involved in transcription
General distinguishing features of Bradyrhizobium genus:
- Gram-negative bacilli (rod-shaped) bacteria
- DNA GC content: approx. 64% (NCBI)
- Abiotic factors: Soil temperature and acidity (Radtke, 2003)
- Mobile bacteria due to presence of a single flagella
- Presence of Nod gene expression when plant host releases flavonoids compounds (Radtke, 2003)
Physiology and Cell Biology
Bradyrhizobium bacteria can survive the low oxygen content in the soil. The BORS278 stain can utilize sunlight as an energy source (via photosynthesis), but the other strains use carbohydrates and nutrients provided by their plant hosts (Genoscope). Bradyrhizobium bacteria developed antibiotic resistance to a combination of streptomycin, neomycin, rifampicin, kanamycin and rifampicin (Mueller et al., 1988).
Molecular Biology and Genetics
Only four whole genome sequencing have been completed by the National Center for Biotechnology Information (NCBI). The other species have incomplete full genome sequencing analysis.
- Bradyrhizobium japonicum
- Genome size = 9.1 Mb
- GC content = 64.1%
- Bradyrhizobium elkanii
- Genome Size = 8.68 Mb
- GC content = 63.6%
- Bradyrhizobium sp. ORS278
- Genome size: 7.46 Mb
- GC content: 65.5%
- Nucleotides: 918 bp linear DNA
- Bradyrhizobium sp. BTAi1
- Genome size: 8.53 Mb
- GC Content: 65.5%
- Bradyrhizobium canariense
- Bradyrhizobium denitrificans
- Bradyrhizobium iriomotense
- Bradyrhizobium jicamae
- Bradyrhizobium lianingense
- Bradyrhizobium batae
- Bradyrhizobium yuanmingense
- Bradyrhizobium liaoningense
Relevance to Humans and Ecosystems
Photosynthetic species of Bradyrhizobium genus, such as the ORS278 strain, are found to associate with wild rice (Oryza brevillguiata) and increase plant growth and seed production, suggesting how specific strains may increase the rice yield (Genoscope). Other Bradyrhizobium species is expected to enhance soybean plant growth and nodulation without the use of fertilizers (Mishra et al., 2009). The symbiotic relationship provides a great agricultural benefit, such that today we isolate the bacteria from their plant nodules and culture them, such that these rhizobia inoculants can be sold commercially (rhizobia module 3). Also, scientists have found specific Bradyrhizobium strains with more efficient in the nitrogen fixation mechanisms, which means farmers can select those general host-bacterial strains to improve their plant growth (rhizobia module 3).
Bradyrhizobium is a genus of Gram-negative soil bacteria, many of which fix nitrogen. Nitrogen fixation is an important part of the nitrogen cycle. Plants cannot use atmospheric nitrogen (N2) they must use nitrogen compounds such as nitrates.
Bradyrhizobium species are Gram-negative bacilli (rod shaped) with a single subpolar or polar flagellum. They are a common soil dwelling microorganism that can form symbiotic relationships with leguminous plant species where they fix nitrogen in exchange for carbohydrates from the plant. Like other rhizobia, they have the ability to fix atmospheric nitrogen into forms readily available for other organisms to use. They are slow growing in contrast to Rhizobium species, which are considered fast growing rhizobia. In a liquid media broth, it takes Bradyrhizobium species 3–5 days to create a moderate turbidity and 6–8 hours to double in population size. They tend to grow best with pentoses as a carbon source. Some strains (for example, USDA 6 and CPP) are capable of oxidizing carbon monoxide aerobically.
Nodules are a growth on the roots of leguminous plants where the bacteria reside. The plant roots secrete amino acids and sugars into the rhizosphere. The rhizobia move toward the roots and attach to the root hairs. The plant then releases flavanoids, which induce the expression of nod genes within the bacteria. The expression of these genes results in the production of enzymes called Nod factors that initiate root hair curling. During this process, the rhizobia are curled up with the root hair. The rhizobia penetrate the root hair cells with an infection thread that grows through the root hair into the main root. This causes the infected cells to divide and form a nodule. The rhizobia can now begin nitrogen fixation.
There are over 55 genes known to be associated with nodulation. NodD is essential for the expression of the other nod genes. There are two different nodD genes: nodD1 and nodD2. Research has found that only nodD1 is needed for successful nodulation.
Bradyrhizobium and other rhizobia take atmospheric nitrogen and fix it into ammonia (NH3) or ammonium (NH4+). Plants cannot use atmospheric nitrogen; they must use a combined or fixed form of the element. After photosynthesis, nitrogen fixation (or uptake) is the second most important process for the growth and development of plants. The levels of ureide nitrogen in a plant correlate with the amount of fixed nitrogen the plant takes up.
Nif and fix are important genes involved in nitrogen fixation among Bradyrizobium species. Nif genes are very similar to genes found in Klebsiella pneumoniae, a free living diazotroph. The genes found in Bradyrhizobia have similar function and structure to the genes found in K. pneumoniae. Fix genes are important for symbiotic nitrogen fixation and were first discovered in rhizobia species. The nif and fix genes are found in at least two different clusters on the chromosome. Cluster I contains most of the nitrogen fixation genes. Cluster II contains three fix genes located near nod genes.
This genus of bacteria can form either specific or general symbioses. This means that one species of Bradyrhizobium may only be able to nodulate one legume species, whereas other Bradyrhizobium species may be able to nodulate several legume species. Ribosomal RNA is highly conserved in this group of microbes, making Bradyrhizobium extremely difficult to use as an indicator of species diversity. DNA-DNA hybridizations have been used instead and show more diversity. However, there are few phenotypic differences and therefore not many species have been named.
Grain legumes are cultivated on about 1.5 million km2 of land per year. The amount of nitrogen fixed annually is about 44–66 million tons worldwide, providing almost half of all nitrogen used in agriculture.
Bradyrhizobium has also been identified as a contaminant of DNA extraction kit reagents and ultra-pure water systems, which may lead to its erroneous appearance in microbiota or metagenomic datasets. The presence of nitrogen fixing bacteria as contaminants may be due to the use of nitrogen gas in ultra-pure water production to inhibit microbial growth in storage tanks.
Bradyrhizobia fix more nitrogen than the plant can use. The excess nitrogen is left in the soil and available for other plants or later crops. Intercropping with a legume has the potential to decrease the need for applied fertilizer. There are commercial inoculants of Bradyrhizobium available. These can be applied as a peat or liquid directly to the seed before planting.
- Bradyrhizobium betae was isolated from tumor like root deformations on sugar beets; they have an unknown symbiotic status.
- Bradyrhizobium elkanii, Bradyrhizobium diazoefficiens, and Bradyrhizobium liaoningense establish symbiosis with soybeans.
- Bradyrhizobium japonicum nodulates soybeans, cowpeas, mung beans, and siratro.
- Bradyrhizobium yuanmingense nodulates Lespedeza.
- Bradyrhizobium canariense nodulates Genistoid legumes endemic to the Canary Islands. It has also been found in Lupin and serradella nodules in western Australia and southern Africa.
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