Plant growth promoting bacteria

The interaction of crop plants with soil microbes is an important area of study for boosting crop nutrition. The symbiotic interaction between leguminous plants and Rhizobia is well known, where the bacteria colonising root nodules supplies biologically fixed N in return for a carbon source, and allows for very low inputs of N fertiliser. It has been of interest to find equivalent plant growth promoting rhizobacteria (PGPR) that can interact with major non-leguminous cereal crops to boost plant nutrition and permit reductions in inputs of chemical fertiliser.

Bacterial nitrogen fixation from non-symbiotic, (endophytic or root-associative) bacteria, particularly Azospirillum brasilense and Gluconacetobacter diazotrophicus, was determined in the 1990s to be a factor enabling the low input growth of tropical crops such as sugar cane, and significant colonisation of rice plants by Azospirillum species was found in the 1970s. More recent studies by Embrapa Agrobiology in Brazil have reported absorption and anchoring of Azosprillum strains to the roots of wheat seedlings, principally located in the cracks between epidermal cells .

Nitrogen fixing rhizobacteria are common in soils around the world and many research groups have isolated strains from the wheat rhizosphere that are suspected to have a plant growth promotion effect. Azospirillum itself has been found in a meta-analysis of previous studies to increase wheat grain yields by up to 30%, but only in conditions where N fertilisation was at a low rate of typically 50-60 kg N/ha . A lack of reproducibility has been a hallmark of field studies with Azospirillum and other PGPR, though this may be due to lack of control of other soil factors in the trials.

Mechanisms of plant growth promotion

Although the rhizobacteria studied all contain the nitrogenase enzyme responsible for biological nitrogen fixation, it is becoming increasingly clear that nitrogen fixation is not the only mechanism responsible for any observed plant growth promotion. 15N studies have indicated that only around 7% of N supplied to wheat by Azospirillum is due to biological nitrogen fixation, the remainder being increased uptake of soil N, though this percentage may increase where less N fertiliser is applied.

Insensitivity of the nifA transcriptional activator, responsible for nitrogen fixation, to excess soluble N has long been known for Azospirillum, though this is not necessarily the case for all PGPR. Although only a small proportion of fixed nitrogen may be directly available for plant growth, further benefits from non-symbiotic N2-fixation may arise on a larger time scale due to long-term liberation of nitrogen sequestered in microbial biomass by microfaunal grazers, a process with outstanding importance in the rhizosphere .

Other important mechanisms may be mobilisation of N, P and other mineral nutrients in the soil, suppression of pathogens, and excretion of phytohormones that promote the growth of larger and more efficient root systems. Even if nitrogen fixation is only responsible for part of a growth promotion effect, there can still be significant value in increasing the efficiency of uptake of applied fertiliser nutrients, especially for nutrients with low mobility in the soil as P. It is feasible that PGPR provided in inocula to wheat root systems can modulate these processes, and along with a possible contribution of nitrogen fixation and protective effects against plant pathogens, provide an additional boost to plant yield and quality in a low-input cropping system.

Engineering of PGPR strains

RHIBAC partner K.U.Leuven is experienced in the development and characterisation of mutant strains of Azospirillum for understanding better its interaction with plants. Azospirillum spp. are known to excrete auxins, cytokinins and gibberellins, with auxin IAA being quantitatively the most important. IAA engineered Azospirillum brasilense strains have been developed . By altering expression of the ipdC gene that plays a key role in biosynthesis of IAA, it has been possible to modulate the growth of wheat root hairs in vitro.

Overexpression of ipdC has been linked with significant enhancement of grain yield and N-use efficiency compared with wild type strains, while abolishing the effect with an Azospirillum mutant deficient in ipdC. The best effects were obtained with a plant inducible promoter, and interestingly, the effect of high applied N fertiliser appears to be less inhibitory. These results indicate a strong contribution of IAA in plant growth promotion due to Azospirillum brasilense, although this IAA overproduction may be quite unique among PGPR.

A further study by K.U.Leuven using a glnA mutant Azospirillum brasilense which excretes NH4+, gives a positive effect on plant growth in pot trials with lower levels of N fertilisation, compared with the wild type Sp7 strain. This suggests a significant contribution from biological nitrogen fixation in agreement with results of previous 15N dilution studies.

Interactions of PGPR with plants

There are clear indications for differences in the capacity of plant genotypes to support PGPR in the rhizosphere as demonstrated for rice, maize and wheat . Commercial crop varieties have been shown to substantially differ in their nutrient efficiency, and there is experimental evidence that in some cases associative rhizobacteria are involved in expression of this trait. Besides the release root exudates that may by themselves enhance N and P mobilization in the rhizosphere, symbiosis with PGPR or mycorrhizal fungi may contribute to this effect. A successful attraction of beneficial microorganisms, however, depends on the release of signaling compounds, by which plants communicate their identity to the microsymbionts.

Although the application of such signaling compounds can increase microbial inoculation rates and yield, surprisingly little is known about the released chemoattractants that are relevant for PGPR and to what extent plant cultivars differ therein. Relevant components of root exudates may include signal compounds for chemo-attraction of PGPR via chemotaxis, acting as carbon source or altering the redox conditions in the rhizosphere, precursors for bacterial synthesis of phytohormones or interactions with the production of bacterial exopolysaccharides.

Encapsulation of PGPR and coated seeds

As a non spore-former, Azospirillum has proved fragile for handling, and it may be that strains originating in the tropics do not establish well in temperate soils. Spore-forming PGPR that are easier to handle and preserve, for example from spore-forming bacteria of the Bacillus group, will be more practical for application in European and other temperate climates. Azospirillum, Azotobacter and other non spore-formers excrete poly-?-hydroxybutyrate to self-encapsulate and form cysts under stress, and this natural process may be manipulated to improve shelf life.

Survival in the rhizosphere can also be influenced by practical measures, including methods of inoculation that can be dependent on local climate conditions, encapsulation of inocula, and coating of inocula directly onto the seed. In the MicroNfix project and also independently at partner UACh, alginate capsules for PGPR have been successfully developed that enhance shelf life and in initial tests appear to assist the plant growth promotion effect, presumably through assisting establishment in the rhizosphere.

This technology will be optimised further in RHIBAC and experiments performed to understand release behaviour of the capsules or coated seeds under different conditions and how this approach contributes to colonisation of the rhizosphere by PGPR.

Interactions with other beneficial organisms

PGPR do not act in isolation and several studies notably the EU project IMPACT have investigated effects of microbial inoculants on soil microbial diversity. IMPACT found no significant changes from inoculation, with more disruption caused by typical agricultural practices like conventional tilling. RHIBAC will investigate the possibility of synergies of PGPR with other microbial agents. An example has been shown in a recent study which reported that co-application with nematodes (C. elegans, A. thornei, Cruznema sp.) can increase rhizosphere colonisation by PGPR. RHIBAC will seek to substantiate these results and test other combinations.