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.     

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.     

Trends in rhizobial inoculant production and use

Rhizobia inoculants have contributed to increase N₂ fixation and yield in legumes crops. However, most of the inoculants produced world-wide are of poor or suboptimal quality. We discuss here why some of them are poor products and how to improve their quality and efficacy. Reported data on the inoculation rate effect can be used to design good inoculants. Technologies are now available to produce inoculants with a shelf-life of more than 1 year. Available quality control methods can help to improve the quality of inoculants although they do not take into account the physiological satus of the rhizobia. Unfortunately quality control is not commonly used except in major inoculant companies and the quality of inoculants sold on the market is low. The need for an increase in quality standards is discussed especially for the number of rhizobia delivered per seed and for the presence of contaminants. Some new technologies which able to increase efficacy and reliability of inoculation are discussed.     

Limitations of the enzyme-linked immunosorbent assay for routine determination of legume inoculant quality

Peat inoculants containing strains of either Rhizobium or Bradyrhizobium spp. were used to determine correlations between cell numbers and A₄₀₅ values obtained with double antibody sandwich enzyme-linked immunosorbent assay (DAS ELISA) and indirect ELISA. ELISA values of inoculants containing strains of Rhizobium were weak and non-specific; with Bradyrhizobium spp. strains, readings were higher and cross-reactions negligible when heated inoculant suspensions were allowed to stand for 3 h before ELISA determinations were made. With soybean inoculant, correlation coefficients of r = 0.93 and 0.83 were obtained with DAS and indirect ELISA, respectively. A linear curve relating log cell numbers to A₄₀₅ values was used to determine the reliability of DAS ELISA values obtained over 2 years with tests on commercially produced soybean inoculants. In the range 5 times 10⁸-ca 3 times 10⁹ cells/g inoculant, DAS ELISA estimates closely followed plate counts but no significant correlation was found when inoculants contained >ca 3 times 10⁹ cells/g. With a minimum requirement of 1 times 10⁹ cells/g inoculant, discrepancies between DAS ELISA estimates and plate counts obtained with inoculants produced with gamma-irradiated peat would have resulted in the erroneous rejection or acceptance of 14.5% of all inoculants tested, based on DAS ELISA estimates. With inoculants produced with steam-sterilized peat, which was unfavourable for survival of strain WB1, 70.0% of the inoculants rejected because of low plate counts would have been acceptable on the basis of DAS ELISA estimates.     

Mineral Soils as Carriers for Rhizobium Inoculants

Mineral soil-based inoculants of Rhizobium meliloti and Rhizobium phaseoli survived better at 4°C than at higher temperatures, but ca. 15% of the cells were viable at 37°C after 27 days. Soil-based inoculants of R. meliloti, R. phaseoli, Rhizobium japonicum, and a cowpea Rhizobium sp. applied to seeds of their host legumes also survived better at low temperatures, but the percent survival of such inoculants was higher than peat-based inoculants at 35°C. Survival of R. phaseoli, R. japonicum, and cowpea rhizobia was not markedly improved when the cells were suspended in sugar solutions before drying them in soil. Nodulation was abundant on Phaseolus vulgaris derived from seeds that had been coated with a soil-based inoculant and stored for 165 days at 25°C. The increase in yield and nitrogen content of Phaseolus angularis grown in the greenhouse was the same with soil-and peat-based inoculants. We suggest that certain mineral soils can be useful and readily available carriers for legume inoculants containing desiccation-resistant Rhizobium strains.     

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.     

Screening and selection of lactic acid bacteria strains suitable for ensiling grass

AIM: Lactic acid bacteria (LAB) strains shown to have broad-spectrum antimicrobial activity were screened for potential as grass silage inoculants. The strains capable of rapidly lowering the pH of the grass matrix and with low proteolytic activity were assessed in laboratory-scale silos in a grass matrix containing natural microbial flora. METHODS AND RESULTS: Screening of nine candidate strains was performed first in a grass extract medium. The four most promising strains were selected on the basis of growth rate in the medium, capacity to reduce pH and ability to limit the formation of ammonia-N. The efficiency of the selected strains was further assessed in a laboratory-scale ensiling experiment. Untreated (no additive) and formic acid served as controls. All tested inoculants improved silage quality compared with untreated. With one exception (Pediococcus parvulus E315) the fermentation losses in the inoculated silages were even lower than in the acid-treated control silage. Pure lactic acid fermentation was obtained in the timothy-meadow fescue silage with all inoculants. The results obtained in the ensiling experiments were consistent with those of the screening procedure, which appeared to predict correctly the potential of LAB as silage inoculants. The strains with a low ammonia production rate in the grass extract medium behaved similarly in the silage. Especially in this respect the strain Lactobacillus plantarum E76 was superior to the other candidates. CONCLUSIONS: The screening method using grass extract proved to be useful in strain selection. SIGNIFICANCE AND IMPACT OF THE STUDY: The rapid screening method developed for the LAB strains provides a useful tool for more systematic product development of commercial inoculant preparations. Time consuming and laborious ensiling experiments can be limited only to the most promising strains.     

Inoculants of plant growth-promoting bacteria for use in agriculture

An assessment of the current state of bacterial inoculants for contemporary agriculture in developed and developing countries is critically evaluated from the point of view of their actual status and future use. Special emphasis is given to two new concepts of inoculation, as yet unavailable commercially: (i) synthetic inoculants under development for plant-growth-promoting bacteria (PGPB) (Bashan and Holguin, 1998), and (ii) inoculation by groups of associated bacteria.This review contains: A brief historical overview of bacterial inoculants; the rationale for plant inoculation with emphasis on developing countries and semiarid agriculture, and the concept and application of mixed inoculant; discussion of microbial formulation including optimization of carrier-compound characteristics, types of existing carriers for inoculants, traditional formulations, future trends in formulations using unconventional materials, encapsulated synthetic formulations, macro and micro formulations of alginate, encapsulation of beneficial bacteria using other materials, regulation and contamination of commercial inoculants, and examples of modern commercial bacterial inoculants; and a consideration of time constraints and application methods for bacterial inoculants, commercial production, marketing, and the prospects of inoculants in modern agriculture.     

Polymer-entrapped rhizobium as an inoculant for legumes

Summary Field and cylinder experiments conducted in France and in Senegal showed that polyacrylamide, previously proposed as an entrapping gel for preparing Rhizobium inoculants, could be replaced by alginate (AER inoculant) or a mixture of xanthan and carob gum (XER inoculant). Semi-dried or dried AER and XER were used successfully provided that their storage time was less than 90 days. In soil inoculation trails, no marked differences were observed among semi-dried XER, dried AER, and dried XER. A number of seed inoculation experiments indicated that dried XER significantly outranked AER. Seeds preinoculated by up to 48 days with XER yielded plants which were comparable in nodulation and growth parameters to those derived from plant receiving peat inoculation at the time of planting.     

Antibacterial activity of lactic acid bacteria included in inoculants for silage and in silages treated with these inoculants

AIMS: To determine antibacterial activity in lactic acid bacteria (LAB) silage inoculants and in wheat and corn silages which were treated with these inoculants. METHODS AND RESULTS: Wheat and two corn silages were prepared in 0.25 l sealed glass jars. Inoculant treatments were prepared for each type of silage with each of 10 LAB silage inoculants at inoculation rate of 10(6) CFU g(-1). Untreated silages served as controls. Antibacterial activity was determined in the inoculants and in their respective silages with Micrococcus luteus and Pseudomonas aeruginosa. Antibacterial activity was detected in nine of the 10 inoculants whereas such activity in the silages varied. Control silages did not have antibacterial activity. CONCLUSIONS: Many LAB silage inoculants have antibacterial activity and in some cases this activity is imparted on inoculated silages. SIGNIFICANCE AND IMPACT OF THE STUDY: This study was conducted as part of a broader research objective, which is to find out how LAB silage inoculants enhance ruminant performance. The results of this study indicate that LAB silage inoculants produce antibacterial activity, and therefore, have a potential to inhibit detrimental micro-organisms in the silage or in the rumen.     

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。     

Bioremediation of Crude Oil–Contaminated Soil By Immobilized Bacteria on an Agroindustrial Waste—Sunflower Seed Husks

This study reports the immobilization and performance of a hydrocarbon-degrading Rhodococcus sp. strain (designated as QBTo) on sunflower seed husks (SH) for the bioremediation of soils polluted with crude oil. The SH performance as inoculants carrier was compared with peat, which is a vegetal material traditionally used in carrier-based inoculants production. The stability of the immobilized culture under storage conditions was assessed by viability at different times when stored at 25°C and 10°C. The catabolic activity of immobilized and free QTBo cells introduced into sandy loam soil, freshly contaminated with crude oil, was studied in microcosms. A higher number of viable QTBo cells were recovered from the inoculants formulated with SH (QTBo-SH) after prolonged storage at 10°C and 25°C. The microcosms amended with QTBo-SH inoculants showed a removal of about 66% of total petroleum hydrocarbons (TPH), whereas in those inoculated with QTBo-peat inoculants, the decrease was of about 47%. In the control microcosms (noninoculated) and liquid culture–amended soils, the TPH removal was about 28%. SH is a waste of edible oil industry, nontoxic, and biodegradable and has demonstrated to confer to the immobilized cultures greater potential to survive not only during storage but also in the soil environment, improving bioremediation process.     

Release of transgenic bacterial inoculants - rhizobia as a case study

The current debate on the release of genetically modified organisms to the environment must be informed by scientific data obtained from field studies. Many of the microorganisms that have potential applications outside the laboratory, especially in agriculture and horticulture, could be improved by genetic modification. Rhizobia, the bacteria that form N₂-fixing symbioses with leguminous plants, have a long history of safe use as seed inoculants, their biology is relatively well known, and they represent a relevant model system. There have been several field releases of genetically modified (GM) rhizobia in the USA and Europe, which provide information on various aspects of their ecology and efficacy. This review summarises the rationale for each release, details of the methods used for monitoring, and the results. Novel properties of rhizobia did not always have the predicted effects. Most studies revealed that rhizobial numbers dropped rapidly after application to soil or seeds but then numbers stabilised for months or years. The monitoring of survival and spread of rhizobia was greatly improved by the presence of novel marker genes. Tagging of rhizobia with marker genes provided more accurate information compared to the use of conventional strains, illustrating an important application of genetic modification, for tracking bacteria in the environment.     

The effect of cyanobacteria and azolla on the performance of rice under different levels of fertilizer nitrogen

Cyanobacteria and/or azolla were inoculated, with urea at 0, 72 or 144 kg N/ha, in plots in which azolla-free Indica rice var. IR 28 was grown. Productive tillers, yield and nitrogen contents of grain and straw positively responded to inoculation with cyanobacteria or azolla, even with fertilizer-N up to 144 kg N/ha. Inoculation improved colonization by cyanobacteria. Azolla were superior to the asymbiotic cyanobacteria in enhancing rice performance. Urea at a rate of 72 kg N/ha was found to support the best colonizations when applied with cyanobacteria or azolla or, to give maximum rice yields, both inoculants.     

Inoculant Maturity Influences Survival of Rhizobia on Seed

Survival of Rhizobium trifolii on seeds of arrowleaf clover (Trifolium versiculosum Savi) and subclover (Trifolium subterraneum L.) was affected by the maturity of peat-, vermiculite-, and charcoal-based inoculants. Ten times more rhizobia survived on seed 4 days after inoculation when inoculants were stored (cured) before being utilized as compared with uncured inoculants. Increasing the curing time of inoculants beyond 4 weeks had little effect on increasing survival of seed-applied rhizobia.     

Effect of Steam Sterilization and Gamma Irradiation of Peat on Quality of Rhizobium Inoculants

Data obtained by independent tests on each of 483 batches of Rhizobium inoculants for Glycine max, Medicago sativa, and Arachis hypogaea, manufactured commercially in South Africa, are reported and discussed. Whereas the average cell count per gram per batch was well in excess of 10⁹, inoculants for G. max and M. sativa manufactured with peat treated with gamma irradiation at a dose of 50 kGr contained significantly higher numbers of Rhizobium cells than inoculants from peat which received 25 kGr. Inoculants for M. sativa manufactured with steam-sterilized peat were similar in quality to those prepared with peat irradiated at a dose of 50 kGr. Contrary to the inoculants for G. max and M. sativa, the Rhizobium strain used in inoculants for A. hypogaea was apparently insensitive to the effect on peat of the higher gamma irradiation dosage.     

Agro-industrial waste materials and wastewater sludge for rhizobial inoculant production: a review

Inoculating legumes with commercial rhizobial inoculants is a common agriculture practice. Generally, inoculants are sold in liquid or in solid forms (mixed with carrier). The production of inoculants involves a step in which a high number of cells are produced, followed by the product formulation. This process is largely governed by the cost related to the medium used for rhizobial growth and by the availability of a carrier source (peat) for production of solid inoculant. Some industrial and agricultural by-products (e.g. cheese whey, malt sprouts) contain growth factors such as nitrogen and carbon, which can support growth of rhizobia. Other agro-industrial wastes (e.g. plant compost, filtermud, fly-ash) can be used as a carrier for rhizobial inoculant. More recently, wastewater sludge, a worldwide recyclable waste, has shown good potential for inoculant production as a growth medium and as a carrier (dehydrated sludge). Sludge usually contains nutrient elements at concentrations sufficient to sustain rhizobial growth and heavy metals are usually below the recommended level. In some cases, growth conditions can be optimized by a sludge pre-treatment or by the addition of nutrients. Inoculants produced in wastewater sludge are efficient for nodulation and nitrogen fixation with legumes as compared to standard inoculants. This new approach described in this review offers a safe environmental alternative for both waste treatment/disposal and inoculant production.     

Occurrence of two cell subpopulations with different cell-cycle durations in the central and peripheral zones of the vegetative shoot apex of Sinapis alba L.

The cell-cycle duration and the growth fraction were estimated in the vegetative shoot apical meristem of Sinapis alba L. The length of the cell cycle was about 86 h, i.e. 2.5 times shorter than the cell-doubling time (M. Bodson, 1975, Ann. Bot. 39, 547–554) and the growth fraction was between 32 to 41%. These data demonstrated that the cell population of this meristem was heterogeneous, including one subpopulation of rapidly cycling cells and one subpopulation of non-cycling cells, i.e. cells with a very long cell cycle compared with that of the rapidly cycling cells. Non-cycling cells had no particular localization within the meristem. Both the central and peripheral zones of the meristem were mosaics of rapidly cycling and non-cycling cells.Abbreviations G₁ pre-DNA-synthesis phase - G₂ post-DNA-synthesis phase - GF growth fraction - M mitosis phase - PLM pulse-labelled-mitoses method - S DNA-synthesis phase - T cell-cycle duration - TdR thymidine     

Filter Mud as a Carrier for Rhizobium Inoculants

Laboratory tests indicated that filter mud, a waste product from sugar cane mills, proved to be a suitable carrier for Rhizobium inoculants, provided it was not dried by heating. Autoclaving air dried material was not detrimental and ensured high survival when held at room temperature. The adverse effect of oven drying, which was increased by subsequent autoclaving or gamma irradiation, lessened with time.     

Characterization of two novel Rhizobium leguminosarum bacteriophages from a field release site of genetically-modified rhizobia

Two Rhizobium leguminosarum biovar viceae bacteriophages with contrasting properties were isolated from a field site in which the survival of genetically modified R. leguminosarum inoculants had been monitored for several years. Inoculant strain RSM2004 was used as the indicator for phage isolation and propagation. One phage, RL1RES, was temperate and could not replicate in any of the 42 indigenous R. leguminosarum field isolates tested although nested PCR indicated that phage sequences were present in six of the isolates. The second phage, RL2RES, was virulent, capable of generalised transduction, contained DNA with modified cytosine residues, and was capable of infecting all field isolates tested although the GM inoculant strain CT0370 was resistant. Sequence with homology to RL2RES was detected by nested PCR in six of the 42 field-isolates. These were not the same isolates that showed homology to RL1RES. The implication of these findings for the survival of rhizobial inoculants, and the ecology of phages and their host bacteria, are discussed.