Advances in plant growth-promoting bacterial inoculant technology: formulations and practical perspectives (1998–2013)

Background

Inoculation of plants to enhance yield of crops and performance of other plants is a century old, proven technology for rhizobia and a newer venue for plant growth-promoting bacteria and other plant symbionts. The two main aspects dominating the success of inoculation are the effectiveness of the bacterial isolate and the proper application technology.

Scope

An assessment of practical aspects of bacterial inoculants for contemporary agriculture and environmental restoration is critically evaluated from the point of view of their current technological status, current applications, and future use. This is done because there are windows of opportunity for new developments in applied research using renewable, non-contaminated natural resources and new venues for research. Special emphasis is given to formulations and polymeric carriers. This review concentrates on practical aspect of inoculation technology dating from 1998 to 2013. Earlier publications are mentioned only for clarification of a specific point.

Conclusions

This review discusses characteristics of a carrier for inoculants, formulations of inoculants including liquid, organic, inorganic, polymeric, and encapsulated formulations. Technical aspects include inoculation techniques (soil and seed application), mass culture production, bulk sterilization, seed coating, shelf-life, and effect of moisture. Future research venues needed are noted.     

Harnessing rhizobacteria to fulfil inter-linked nutrient dependency on soil and alleviate stresses in plants

Plant rhizo-microbiome comprises complex microbial communities that colonize at the interphase of plant roots and soil. Plant growth-promoting rhizobacteria (PGPR) in the rhizosphere provide important ecosystem services ranging from the release of essential nutrients for enhancing soil quality and improving plant health to imparting protection to plants against rising biotic and abiotic stresses. Hence, PGPR serve as restoring agents to rejuvenate soil health and mediate plant fitness in the facet of changing climate. Though it is evident that nutrient availability in soil is managed through inter-linked mechanisms, how PGPR expedite these processes remain less recognized. Promising results of PGPR inoculation on plant growth are continually reported in controlled environmental conditions, however, their field application often fails due to competition with native microbiota and low colonization efficiency in roots. The development of highly efficient and smart bacterial synthetic communities by integrating bacterial ecological and genetic features provides better opportunities for successful inoculant formulations. This review provides an overview of the interplay between nutrient availability and disease suppression governed by rhizobacteria in soil followed by the role of synthetic bacterial communities in developing efficient microbial inoculants. Moreover, an outlook on the beneficial activities of rhizobacteria in modifying soil characteristics to sustainably boost agroecosystem functioning is also provided.     

Alginate Beads as Synthetic Inoculant Carriers for Slow Release of Bacteria That Affect Plant Growth

Uniform synthetic beads were developed as carriers for the bacterial inoculation of plants. The beads are made of sodium alginate and skim milk and contain a large reservoir of bacterial culture which releases the bacteria at a slow and constant rate. The beads are biodegradable and produce no environmental pollution. The strength of the beads, the rate of bacterial release, and the time of their survival in the soil can be controlled by several hardening treatments. The final product, lyophilized beads, is simple to use and is applied to the seeds concomitantly with sowing. The released bacteria are available for root colonization immediately at seed germination. Dry beads containing bacteria can be stored at ambient temperature over a long period without loss of bacterial content; storage requires a limited space, and the quality control of a number of bacteria in the bead is simple. The level of plant inoculation with beads was similar to that with previously used peat inoculants, but the former method yielded more consistent results, as the frequency of inoculated plants was much higher. The former method provides a different approach for inoculation of plants with beneficial rhizosphere bacteria.     

Carrier-based Preparations of Plant Growth-promoting Bacterial Inoculants Suitable for use in Cooler Regions

Summary Carrier-based preparations of two plant growth-promoting rhizobacteria (PGPR) viz. Bacillus subtilis and Pseudomonas corrugata, developed in five formulations were evaluated for their growth promotion, rhizosphere colonization, and viability under storage. The effect of these formulations as fresh preparations, and after 6 months of storage at 4 °C and room temperature, was also determined. The bacterial inoculants in all the formulations were found to enhance the growth parameters of the test plant species; best results were obtained in case of alginate-based formulations. Maximum numbers of inoculated bacteria were recovered from the rhizosphere of alginate-based formulation-treated plants after 6 weeks of growth. Viability of bacterial inoculants was maximal in alginate beads, and alginate beads supplemented with skim milk formulations, after 180 days of storage at 4 °C.     

ACC deaminase producing Pseudomonas putida strain PSE3 and Rhizobium leguminosarum strain RP2 in synergism improves growth, nodulation and yield of pea grown in alluvial soils

Plant growth promoting rhizobacteria affects the overall performance of plants by one or combination of mechanisms. However, little information is available on how ACC deaminase secreting bacteria enhance crop production. The present study aimed at identifying ACC deaminase producing and phosphate solubilizing bacterial strains and to assess their plant growth promoting activities. Additionally, the effect of two ACC deaminase positive bacterial strains Pseudomonas putida and Rhizobium leguminosarum on pea plants was determined to find a novel and compatible bacterial pairing for developing efficient inoculants for enhancing legume production and reducing dependence on chemical fertilizers. The isolated bacterial cultures were characterized biochemically and by 16S rRNA sequence analysis. The plant growth promoting activities was determined using standard microbiological methods. The impact of P. putida and R. leguminosarum, on pea plants was determined both in pots and in field environments. Of the total 40 bacterial strains, strain PSE3 isolated from Mentha arvenss rhizosphere and RP2 strain from pea nodules produced ACC deaminase, solubilized insoluble phosphate, synthesized indole acetic acid, ammonia, cyanogenic compounds, exopolysaccharides and had antifungal activity. The dual inoculation of P. putida strain PSE3 and R. leguminosarum strain RP2 had largest positive effect and markedly increased the growth, symbiotic characteristics, nutrient pool and quantity and quality of pea seeds. The measured parameters were further augmented when inoculated pea plants were grown in soils treated with urea or DAP. A significant variation in the measured parameters of pea plants was observed under both pot and field trials following microbial inoculation but the bacterial cultures did not differ significantly in growth promoting activities. The results suggest that ACC deaminase positive bacterial cultures endowed with multiple potential can be targeted to develop mixed inoculants for enhancing pea production and hence, to reduce dependence on synthetic fertilizers.     

Maintenance and assessment of cell viability in formulation of non‐sporulating bacterial inoculants

The application of beneficial, plant‐associated microorganisms is a sustainable approach to improving crop performance in agriculture. However, microbial inoculants are often susceptible to prolonged periods of storage and deleterious environmental factors, which negatively impact their viability and ultimately limit efficacy in the field. This particularly concerns non‐sporulating bacteria. To overcome this challenge, the availability of protective formulations is crucial. Numerous parameters influence the viability of microbial cells, with drying procedures generally being among the most critical ones. Thus, technological advances to attenuate the desiccation stress imposed on living cells are key to successful formulation development. In this review, we discuss the core aspects important to consider when aiming at high cell viability of non‐sporulating bacteria to be applied as microbial inoculants in agriculture. We elaborate the suitability of commonly applied drying methods (freeze‐drying, vacuum‐drying, spray‐drying, fluidized bed‐drying, air‐drying) and potential measures to prevent cell damage from desiccation (externally applied protectants, stress pre‐conditioning, triggering of exopolysaccharide secretion, ‘helper’ strains). Furthermore, we point out methods for assessing bacterial viability, such as colony counting, spectrophotometry, microcalorimetry, flow cytometry and viability qPCR. Choosing appropriate technologies for maintenance of cell viability and evaluation thereof will render formulation development more efficient. This in turn will aid in utilizing the vast potential of promising, plant beneficial bacteria as sustainable alternatives to standard agrochemicals.     

Survival and change in physiological state of Bradyrhizobium japonicum in soybean (Glycine max L. Merril) liquid inoculants after long-term storage

Commercial liquid inoculants for soybean, stored at 20 °C for 1–8 years in 400 ml bottles or in 5000 ml containers, were assessed for their efficacy and changes in the physiological activity of Bradyrhizobium japonicum. A decrease in viable counts and in bacterial survival on seeds was observed in inoculants stored for several years. The number of nodules produced per plant in a growth chamber decreased and was correlated to the number of bacteria surviving on the seeds. Changes in physiological properties were assessed using biochemical, physiological and microscopic methods. The cell total sugars content decreased with increased storage of the inoculants. High calculated ratios of suspended solid dry matter/carbon/nitrogen/proteins weight per c.f.u. strongly suggested the presence of dead or viable but non-culturable (VBNC) cells in the inoculants. This was confirmed in a study of bacterial respiratory activity, using p-iodonitrotetrazolium reduction. The time of colony appearance on plates increased in the old inoculants stored for a long time, especially on yeast-free culture medium. The heterogeneity in colony size also increased with storage length. Inoculants stored for more than 2 years could be differentiated from the others by using nalidixic acid against cellular division. Nucleic acid staining of cells showed that the percentage of membrane-compromised bacteria in all the inoculants increased with increased storage length, whatever the type of packaging used for the inoculants. These results demonstrated that the physiological activity of B. japonicum cells in commercial liquid inoculants changes after storage. To complete c.f.u. determination, three methods were proposed to assess the fitness of stored bradyrhizobia, but they remain to be checked for reliability on a variety of commercial inoculants.     

Plant growth-promoting bacteria as inoculants in agricultural soils

Plant-microbe interactions in the rhizosphere are the determinants of plant health, productivity and soil fertility. Plant growth-promoting bacteria (PGPB) are bacteria that can enhance plant growth and protect plants from disease and abiotic stresses through a wide variety of mechanisms; those that establish close associations with plants, such as the endophytes, could be more successful in plant growth promotion. Several important bacterial characteristics, such as biological nitrogen fixation, phosphate solubilization, ACC deaminase activity, and production of siderophores and phytohormones, can be assessed as plant growth promotion (PGP) traits. Bacterial inoculants can contribute to increase agronomic efficiency by reducing production costs and environmental pollution, once the use of chemical fertilizers can be reduced or eliminated if the inoculants are efficient. For bacterial inoculants to obtain success in improving plant growth and productivity, several processes involved can influence the efficiency of inoculation, as for example the exudation by plant roots, the bacterial colonization in the roots, and soil health. This review presents an overview of the importance of soil-plant-microbe interactions to the development of efficient inoculants, once PGPB are extensively studied microorganisms, representing a very diverse group of easily accessible beneficial bacteria.     

有机肥和解磷固氮菌配施对缺碳黄棕壤养分特性的协同效应

研究不同有机肥配比下解磷固氮细菌对土壤养分和相关酶活性的动态影响,可为筛选合适的菌肥类型、有机肥配比和施肥周期提供参考。试验设置0%、4%、8%、12%(质量比)4种有机肥配比,选择巨大芽孢杆菌、荧光假单胞菌2种解磷菌和褐球固氮菌、巴西固氮螺菌2种固氮菌,以亚热带贫瘠黄棕壤为研究对象,在控制条件下(28 ℃、避光)持续土壤培养60 d,以探讨不同有机肥水平和不同取样时间(培养第3、8、16、30、45、60天)4种细菌单独或混合接种对土壤有效态养分和相关酶活性的影响。结果表明: 随着有机肥含量的增加,菌肥处理土壤的有效氮和有效磷含量逐渐提高,表现为12%>8%>4%>0%。随着培养时间的延长,各处理土壤养分基本呈现先增加后降低的趋势。相对于单施有机肥,配施有机肥和菌肥能够显著提高和延长菌肥对土壤养分含量和酶活性的增益。菌肥对土壤养分特性的影响在不同有机肥配比下呈现不同的规律: 有机肥含量较低(0%～4%)时,虽然菌肥显著提高了土壤有效养分含量,但在初期各菌肥间差异并不显著;随着有机肥含量的提高和培养时间的延长,解磷菌(尤其是巨大芽孢杆菌)显著提高了有效磷含量,固氮菌(尤其是巴西固氮螺菌)显著提高了有效氮含量。4种细菌混合菌肥主要呈现解磷功能,比单施解磷菌具有更明显的优势,而固氮功能未得到显著提高。相关分析表明,土壤养分含量和酶活性大小呈显著正相关关系,而酶活性受土壤培养时间和碳氮比的影响。因此,针对该土壤类型,菌肥能够在短期内显著提高土壤养分含量,但菌肥特定功能的发挥依赖于有机碳的输入和土壤碳氮比的大小,配施合适比例的有机肥(8%～12%)能够提高并延长菌肥的改良效果;每45～60 d需进行菌肥复施,以保证其存活率和持续效果。     

Bacteria isolated from soils of the western Amazon and from rehabilitated bauxite-mining areas have potential as plant growth promoters

Several processes that promote plant growth were investigated in endophytic and symbiotic bacteria isolated from cowpea and siratro nodules and also in bacterial strains recommended for the inoculation of cowpea beans. The processes verified in 31 strains were: antagonism against phytopathogenic fungi, free-living biological nitrogen fixation, solubilization of insoluble phosphates and indole acetic acid (IAA) production. The resistance to antibiotics was also assessed. Sequencing of the partial 16S rRNA gene was performed and the strains were identified as belonging to different genera. Eight strains, including some identified as Burkholderia fungorum, fixed nitrogen in the free-living state. Eighteen strains exhibited potential to solubilize calcium phosphate, and 13 strains could solubilize aluminum phosphate. High levels of IAA production were recorded with l-tryptophan addition for the strain UFLA04-321 (42.3 μg mL^?1). Strains highly efficient in symbiosis with cowpea bean, including strains already approved as inoculants showed the ability to perform other processes that promote plant growth. Besides, these strains exhibited resistance to several antibiotics. The ability of the nitrogen-fixing bacteria to perform other processes and their adaptation to environmental conditions add value to these strains, which could lead to improved inoculants for plant growth and environmental quality.     

Tolerance of Bradyrhizobium japonicum E109 to Osmotic Stress and the Stability of Liquid Inoculants Depend on Growth Phase

Salinity and drought induce osmotic stress in plants and nodulating bacteria. The introduction of soybean in areas with higher soil salt contents or periods of drought pose a challenge for the rhizobial inoculants used to improve nodulation and enhance nitrogen fixation. Bradyrhizobium japonicum is a slow-growing rhizobium used for soybean inoculation that was previously regarded as salt-sensitive. We tested the survival ability of cultures of B. japonicum E109 at the exponential and stationary phases of growth in liquid culture medium against different concentrations of NaCl. We found that stationary-phase cells could tolerate higher levels of salt than exponential-phase cells. This result suggested that the physiological manipulation of the cultures could improve the salt tolerance of this strain. Nonetheless, we also found that exponential-phase cells adapted significantly better to two key situations that a commercial product must face, survival in liquid formulations and survival in soil microcosms resembling conditions of drought. These results suggest that the use of actively growing cells could be an improvement in the production of inoculants. However, it is not cost-effective, because bacteria should be harvested at a time when cell density is lower than that of early stationary-phase cultures, which are normally used in the industry. To overcome this drawback we proved that a fed-batch system can produce exponential-phase cultures with higher cell densities and able to produce liquid inoculants with acceptable survival rates.     

Inoculation with selected strains of Azospirillum brasilense and A. lipoferum improves yields of maize and wheat in Brazil

Interest in the use of inoculants containing bacteria that promote plant growth is likely to increase in the coming years, due to higher costs of fertilizers, concerns over pollution and emphasis on sustainable agriculture. Although Brazil has a long tradition in research on nitrogen fixation in Azospirillum-grass associations, it has not led to recommendations of strains for use in commercial inoculants. In this study, we report the selection and evaluation of Azospirillum strains for the maize (Zea mays L.) and wheat (Triticum aestivum L.) crops, following protocols established by the Brazilian legislature, i.e. field experiments have to be performed in at least two different localities representing the crop growing regions, and for at least two seasons. In a first set of nine trials performed at Londrina and Ponta Grossa, southern Brazil, nine Azospirillum strains were evaluated after application to seeds as peat-based inoculants. A. brasilense strains Ab-V4, Ab-V5, Ab-V6 and Ab-V7 increased grain yields of maize by 662–823 kg ha^?1, or 24–30%, in relation to non-inoculated controls. Two A. lipoferum strains were tested in two of these experiments and promising results were also obtained. With wheat, A. brasilense strains Ab-V1, Ab-V5, Ab-V6 and Ab-V8 were the most effective, increasing yields by 312–423 kg ha^?1, or 13–18%. In a second trial set with eight field experiments at Londrina an Ponta Grossa, liquid and peat-based inoculants carrying a combination of A. brasilense strains Ab-V5 and Ab-V6 increased maize and wheat yields by 27% and 31%, respectively. Effects of inoculation were attributed to general increases in uptake of several macro and micronutrients and not specifically to biological nitrogen fixation. All experiments received only a low N-fertilizer starter at sowing (24 kg and 20 kg of N ha^?1 for the maize and wheat, respectively) and although yields can be globally considered low, they were compatible with Brazilian mean yields. This study resulted in the identification of the first Azospirillum strains authorized for the production of commercial inoculants in Brazil.     

高效光合细菌菌剂对土壤微生物的影响

为了筛选在盆栽试验条件下适宜的高效光合细菌菌剂浓度以促进番茄生长,研究了高效光合细菌菌剂的3种浓度对番茄种植土壤中微生物特性的影响。试验结果表明：高效光合细菌菌剂的施用可以显著提高土壤中细菌和放线菌的数量,降低土壤中真菌数量;不同程度提高土壤脲酶、酸性磷酸酶和过氧化氢酶的活性。其中以稀释100倍的光合细菌菌剂+化肥处理效果最明显,稀释100倍的菌剂+化肥处理最有利于土壤中微生物的生长繁殖,对土壤环境的改善效果最显著。     

促分解菌剂对还田玉米秸秆的分解效果及土壤微生物的影响

为了探明促分解菌剂的应用对还田玉米秸秆的促分解效果及对土壤微生物群落结构的影响,选用3组促分解菌剂,编号依次为ND、NK和NS,于2009年10月至2010年4月期间,在河北省农林科学院辛集实验站冬小麦-玉米轮作田对玉米秸秆还田地进行了接种试验。在接种后的15、25、145和160 d分别测定秸秆残重率和秸秆残渣中C/N,结果表明与未施菌剂对照(CK)相比,3组菌剂均在一定程度上加快了玉米秸秆的分解,其中以菌剂ND促分解效果最好,NK次之,NS较差,三者的最高促分解效果分别比CK提高了14.3%、7.7%和1.6%,主要促分解效果都出现在早期(前25 d),且菌剂促进秸秆残渣中C/N降低的效果也在早期明显。采用变性梯度凝胶电泳技术(DGGE)检测菌剂对玉米秸秆降解过程中土壤细菌和真菌群落结构的影响,结果表明,与不接种CK相比,接种菌剂主要在早期对土壤细菌和真菌的群落结构产生较大的影响,而后期对土壤微生物群的影响不明显。秸秆还田后接种促分解菌剂,能在接种早期有效加快秸秆分解,而随接种后时间的推进,其促进效果逐渐减弱,与之对应,土壤微生物群落结构早期差异明显,其后差异逐渐减小。     

Real time PCR detection targeting nifA gene of plant growth promoting bacteria Azospirillum brasilense strain FP2 in maize roots

The plant growth promoting bacteria (PGPB) Azospirillum brasilense has been recommended for use in commercial inoculants in Brazil. Effective methods are necessary to monitor PGPB strains in the rhizosphere. Our purpose was to develop a real time PCR method for detection of A. brasilense strain FP2 in maize seedlings targeting nifA. Primer pairs were designed and their specificity was verified using DNA from 12 different bacterial species. Standard curves were prepared for DNA quantification using serial dilution of A. brasilense DNA extracts. PCR efficiencies and correlation coefficient presented values within the acceptable range for qPCR, mean PCR efficiency was 95 % and correlation coefficient was 0.98, indicating that nifA gene was suitable for the quantitative analysis of the target bacterial genome. Inoculated maize seedlings were grown in vitro or in pots, bacterial DNA copy number per gram of fresh root was quantified 1, 4, 7 and 10 days after inoculation. The developed primers targeting nifA will be useful for monitoring Azospirillum brasilense strain FP2 in crops.     

A new species of Burkholderia isolated from sugarcane roots promotes plant growth

Sugarcane is a globally important food, biofuel and biomaterials crop. High nitrogen (N) fertilizer rates aimed at increasing yield often result in environmental damage because of excess and inefficient application. Inoculation with diazotrophic bacteria is an attractive option for reducing N fertilizer needs. However, the efficacy of bacterial inoculants is variable, and their effective formulation remains a knowledge frontier. Here, we take a new approach to investigating diazotrophic bacteria associated with roots using culture‐independent microbial community profiling of a commercial sugarcane variety (Q208^A) in a field setting. We first identified bacteria that were markedly enriched in the rhizosphere to guide isolation and then tested putative diazotrophs for the ability to colonize axenic sugarcane plantlets (Q208^A) and promote growth in suboptimal N supply. One isolate readily colonized roots, fixed N₂ and stimulated growth of plantlets, and was classified as a new species, Burkholderia australis sp. nov. Draft genome sequencing of the isolate confirmed the presence of nitrogen fixation. We propose that culture‐independent identification and isolation of bacteria that are enriched in rhizosphere and roots, followed by systematic testing and confirming their growth‐promoting capacity, is a necessary step towards designing effective microbial inoculants.     

Formulations useful in applying beneficial microorganisms to seeds

Microbial inoculants are very likely to have an increasing role in agriculture as the trend away from chemical treatments for plants continues. For microbial inoculation to be effective, it must be compatible with agricultural needs. This usually means that the microorganisms must be formulated with agents that facilitate packaging, extend shelf-life and render the preparation easy to handle. This article addresses these general points and considers formulations that may be useful in applying beneficial microorganisms to seeds.     

How to monitor released rhizobia

Existing methods for detection and identification of rhizobia are reviewed. Some perspectives for development of new and more effective techniques for monitoring of rhizobia in soil and in inoculants are presented. The advantages of the recently developed approach — PCR-genome fingerprinting, by use of arbitrary and repetitive primers, for precise bacterial identification are described. The possible application of this technique for developing taxon-specific rhizobial probes for direct detection of these bacteria in environmental samples is discussed.     

Comparison of application methods to prolong the survival of potential biocontrol bacteria on stored sugar-beet seed

AIMS: To develop bacterial inoculation treatments on sugar-beet seed that will maintain a commercially acceptable degree of viability for a minimum of 4 months storage at ambient temperature. METHODS AND RESULTS: Single rifampicin-resistant (Rif(+)) strains of both Gram-positive and negative bacterial isolates (mostly pseudomonads) were applied in turn to sugar-beet seed in a comparative study by seed soaking, encapsulation in alginate, pelleting using an inoculated peat carrier or seed priming. The treated seed was assessed for bacterial survival over a time course by plating out homogenized samples onto a selective medium. Priming inoculation offered a significant improvement over all the other application strategies tested. After pelleting with fungicides and drying at 40 degrees C, Pseudomonas marginalis/putida P1W1 maintained populations of >6.6 log(10) CFU g(-1) seed during 4 months storage at 15 degrees C. Subsequent experiments verified a stabilized population under these storage conditions with commercial pellets at <7% moisture content. CONCLUSION: An inoculation method was established which allowed the survival on seed of a Gram-negative bacterium at ambient temperature with little loss in viability. SIGNIFICANCE AND IMPACT OF THE STUDY: This has promising implications for the delivery of beneficial bacteria, especially Gram-negative strains, on sugar beet.     

Soil and rhizosphere as habitats for Pseudomonas inoculants: new knowledge on distribution,activity and physiological state derived from micro-scale and single-cell studies

Pseudomonas spp. comprise an important group of bacteria used for biological control of microfungi in the plant rhizosphere. Successful performance of microbial inoculants requires both establishment, proliferation and activity under in situ conditions. To identify the factors controlling fate and performance of the inoculants, small-scale analyses are needed due to the heterogeneity characterizing the complex soil and rhizosphere environments. Direct staining techniques and advanced microscopy have provided the first detailed single-cell images of root colonization by these bacteria using fluorescent antibodies, fluorescent in situ hybridization and marker gene technology. These tracking methods have, in conjunction with activity assays, provided high-resolution data on the metabolic activity and growth of the inoculants. Finally, Pseudomonas reporter bacteria constructed to sense phosphorus, nitrogen, iron, and oxygen limitations have provided new insight into the significance of growth-limiting factors in the soil and along the root. The present work reviews the current knowledge on Pseudomonas inoculants in soil and rhizosphere based on these modern techniques. Finally, some perspectives for future studies are discussed.