Simultaneous P-solubilizing and biocontrol activity of microorganisms: potentials and future trends

Phosphate (P)-solubilizing microorganisms as a group form an important part of the microorganisms, which benefit plant growth and development. Growth promotion and increased uptake of phosphate are not the only mechanisms by which these microorganisms exert a positive effect on plants. Microbially mediated solubilization of insoluble phosphates through release of organic acids is often combined with production of other metabolites, which take part in biological control against soilborne phytopathogens. In vitro studies show the potential of P-solubilizing microorganisms for the simultaneous synthesis and release of pathogen-suppressing metabolites, mainly siderophores, phytohormones, and lytic enzymes. Further trends in this field are discussed, suggesting a number of biotechnological approaches through physiological and biochemical studies using various microorganisms.     

Optimizations of particle size and pulp density for solubilization of rock phosphate by a microbial consortium from activated sludge

Microbial solubilization of rock phosphate is getting more and more attention recently. However, the microorganisms used in previous studies were mostly single or known species, and seldom studies focused on the mixed microorganisms or microbial consortia from natural environments. In this study, a microbial consortium taken from activated sludge was used to solubilize two different mid-low-grade rock phosphates. The results showed that the microbial consortium could effectively solubilize the rock phosphates in National Botanical Research Institute’s phosphate growth medium and released soluble phosphorus in the broth. The biomass increased gradually, whereas the pH decreased sharply during the solubilizing process. The maximum phosphorus solubilization was recorded at particle size of 150?µm. Higher or lower than this optimal particle size, the phosphorus solubilization decreased. The phosphorus solubilization gradually decreased with a larger pulp density from 1 to 5%, and the optimal pulp density was 1%. The solubilization level of microbial consortium varied with different rock phosphates. The results revealed that the soluble phosphorus released from high-silicon ore was higher than which from high-magnesium ore. A strong positive correlation between biomass and phosphorus solubilization in the broth was observed from regression analysis results, and the phosphorus solubilization also had a significant negative correlation with pH in the broth.     

Stress-tolerant P-solubilizing microorganisms

Drought, high/low temperature, and salinity are abiotic stress factors accepted as the main reason for crop yield losses in a world with growing population and food price increases. Additional problems create nutrient limitations and particularly low P soil status. The problem of phosphate fertilizers, P plant nutrition, and existing phosphate bearing resources can also be related to the scarcity of rock phosphate. The modern agricultural systems are highly dependent on the existing fertilizer industry based exclusively of this natural, finite, non-renewable resource. Biotechnology offers a number of sustainable solutions that can mitigate these problems by using plant beneficial, including P-solubilizing, microorganisms. This short review paper summarizes the current and future trends in isolation, development, and application of P-solubilizing microorganisms in stress environmental conditions bearing also in mind the imbalanced cycling and unsustainable management of P. Special attention is devoted to the efforts on development of biotechnological strategies for formulation of P-solubilizing microorganisms in order to increase their protection against adverse abiotic factors.     

Plant growth promotion traits of phosphobacteria isolated from Puna,Argentina

The ability of soil microorganisms to solubilize phosphate is an important trait of plant growth-promoting bacteria leading to increased yields and smaller use of fertilizers. This study presents the isolation and characterization of phosphobacteria from Puna, northwestern Argentina and the ability to produce phosphate solubilization, alkaline phosphatase, siderophores, and indole acetic acid. The P-solubilizing activity was coincidental with a decrease in pH values of the tricalcium phosphate medium for all strains after 72 h of incubation. All the isolates showed the capacity to produce siderophores and indoles. Identification by 16S rDNA sequencing and phylogenetic analysis revealed that these strains belong to the genera Pantoea, Serratia, Enterobacter, and Pseudomonas. These isolates appear attractive for exploring their plant growth-promoting activity and potential field application.     

Biotechnological Tools for Enhancing Microbial Solubilization of Insoluble Inorganic Phosphates

During recent years, many studies appeared on microbial solubilization of insoluble phosphates as an alternative of chemically based P-fertilizer production and bearing in mind the progressive increase in P-fertilizer prices based on high global P consumption and the scarcity of rock phosphate reserves. This biotechnological approach is mainly related to microbial production of organic acids such as citric, oxalic, gluconic, itaconic, and lactic acid, which react with the insoluble P-sources. The most applied and studied P-solubilizers are fungal microorganisms cultivated in conditions of submerged and solid-state fermentation systems. Therefore, the aim of this review is to summarize data on the effect of various abiotic factors on the fungal organic acid production. Nutrient medium components, fermentation process parameters, interaction between insoluble P-particles and microbial systems, and mode of fermentation are analyzed for their impact on both organic acid production and P-solubilization. Suggestions for further studies are also discussed.     

Immobilized cell technology applied in solubilization of insoluble inorganic (rock) phosphates and P plant acquisition

This paper reviews current knowledge of the production of organic acids by immobilized microorganisms with a simultaneous solubilization of rock phosphate in fermentation and soil conditions. The most widely applied methods are based on the passive immobilization in preformed porous carriers and entrapment of the microbial cells in natural gels. In general, immobilized systems show higher acid producing and rock phosphate solubilizing activity than freely suspended cells. The potential of gel-entrapped P-solubilizers and mycorrhizal fungi as microbial soil inoculants is also pointed out. Some advantages and constraints of using immobilized cells are discussed and a special emphasis on further research is given.     

4株溶磷细菌和真菌溶解磷矿粉的特性

溶磷微生物广泛地分布在土壤,根际等生态环境中,了解这些微生物溶解难溶性磷酸盐如磷矿粉的特性,对于开发利用这些微生物,提高磷素利用效率具有重要作用,研究发现;真菌比细菌溶解磷矿粉的能力要强得多,培养基中的铁,镁,锰,钠等成分可以提高真菌的溶磷量,但降低了细菌的溶磷量,培养基中磷矿粉用量越高,溶磷量越低;碳源物质浓度高于3%将显地降低溶磷量,微生物能够破坏磷矿粉的结构。使其中的磷在以后的培养过程中更加容易释放出来,可见利用微生物活化磷矿粉中的磷,具有良好前景。     

Biotechnological solubilization of rock phosphate on media containing agro-industrial wastes

Rock phosphate (RP) is an important natural material traditionally used for the production of phosphorus (P) fertilizers. Compared with chemical treatment, microbial solubilization of RP is an alternative environmentally mild approach. An overview of biotechnological techniques, mainly based on solubilization processes involving agro-industrial residues, is presented. Potential advantages of composting, solid-state fermentation, and liquid submerged fermentation employing free and immobilized microorganisms that produce organic acids and simultaneously solubilize RP are discussed. Subsequent introduction of the final fermented products into soil-plant systems promotes plant growth and P acquisition.     

Fluoride-Tolerant Mutants of Aspergillus niger Show Enhanced Phosphate Solubilization Capacity

P-solubilizing microorganisms are a promising alternative for a sustainable use of P against a backdrop of depletion of high-grade rock phosphates (RPs). Nevertheless, toxic elements present in RPs, such as fluorine, can negatively affect microbial solubilization. Thus, this study aimed at selecting Aspergillus niger mutants efficient at P solubilization in the presence of fluoride (F⁻). The mutants were obtained by exposition of conidia to UV light followed by screening in a medium supplemented with Ca₃(PO₄)₂ and F⁻. The mutant FS1-555 showed the highest solubilization in the presence of F⁻, releasing approximately 70% of the P contained in Ca₃(PO₄)₂, a value 1.7 times higher than that obtained for the wild type (WT). The mutant FS1-331 showed improved ability of solubilizing fluorapatites, increasing the solubilization of Araxá, Catalão, and Patos RPs by 1.7, 1.6, and 2.5 times that of the WT, respectively. These mutants also grew better in the presence of F⁻, indicating that mutagenesis allowed the acquisition of F⁻ tolerance. Higher production of oxalic acid by FS1-331 correlated with its improved capacity for RP solubilization. This mutant represents a significant improvement and possess a high potential for application in solubilization systems with fluoride-rich phosphate sources.     

Application of free and Ca-alginate-entrapped Glomus deserticola and Yarowia lipolytica in a soil-plant system

This study was performed to investigate the applicability of microbial inoculants entrapped in alginate gel. Glomus deserticola (AM) was inoculated into soil microcosms, enriched with rock phosphate, as either free form or entrapped in calcium alginate alone or in combination with a P-solubilizing yeast culture (Yarowia lipolytica). Plant dry weight, soluble P acquisition, and mycorrhizal index were equal in treatments inoculated with free and alginate-entrapped AM. Dual inoculation with entrapped G. deserticola and free cells of Y. lipolytica significantly increased all analyzed variables. Highest rates of the latter were obtained when both fungal microorganisms were applied co-entrapped in the carrier. The yeast culture behaved as a 'mycorrhiza helper microorganism' enhancing mycorrhization of tomato roots. These results indicate that dual inoculation with an AM fungus and a P-solubilizing microorganism co-entrapped in alginate can be an efficient technique for plant establishment and growth in nutrient deficient soils.     

Organic acid production and plant growth promotion as a function of phosphate solubilization by Acinetobacter rhizosphaerae strain BIHB 723 isolated from the cold deserts of the trans-Himalayas

An efficient phosphate-solubilizing plant growth–promoting Acinetobacter rhizosphaerae strain BIHB 723 exhibited significantly higher solubilization of tricalcium phosphate (TCP) than Udaipur rock phosphate (URP), Mussoorie rock phosphate (MRP) and North Carolina rock phosphate (NCRP). Qualitative and quantitative differences were discerned in the gluconic, oxalic, 2-keto gluconic, lactic, malic and formic acids during the solubilization of various inorganic phosphates by the strain. Gluconic acid was the main organic acid produced during phosphate solubilization. Formic acid production was restricted to TCP solubilization and oxalic acid production to the solubilization of MRP, URP and NCRP. A significant increase in plant height, shoot fresh weight, shoot dry weight, root length, root dry weight, and root, shoot and soil phosphorus (P) contents was recorded with the inoculated treatments over the uninoculated NP₀K or NP_TCPK treatments. Plant growth promotion as a function of phosphate solubilization suggested that the use of bacterial strain would be a beneficial addition to the agriculture practices in TCP-rich soils in reducing the application of phosphatic fertilizers.     

Organic acid production in vitro and plant growth promotion in maize under controlled environment by phosphate-solubilizing fluorescent Pseudomonas

Background  

Phosphorus deficiency is a major constraint to crop production due to rapid binding of the applied phosphorus into fixed forms not available to the plants. Microbial solubilization of inorganic phosphates has been attributed mainly to the production of organic acids. Phosphate-solubilizing microorganisms enhance plant growth under conditions of poor phosphorus availability by solubilizing insoluble phosphates in the soil. This paper describes the production of organic acids during inorganic phosphate solubilization and influence on plant growth as a function of phosphate solubilization by fluorescent Pseudomonas.     

Phosphate solubilization potential and stress tolerance of Eupenicillium parvum from tea soil

Eupenicillium parvum was recorded for first time during isolation of phosphate-solubilizing microorganisms from the tea rhizosphere. The fungus developed a phosphate solubilization zone on modified Pikovskaya agar, supplemented with tricalcium phosphate. Quantitative estimation of phosphate solubilization in Pikovskaya broth showed high solubilization of tricalcium phosphate and aluminium phosphate. The fungus also solubilized North Carolina rock phosphate and Mussoorie rock phosphate, and exhibited high levels of tolerance against desiccation, acidity, salinity, aluminium, and iron. Solubilization of inorganic phosphates by the fungus was also observed under high stress levels of aluminium, iron, and desiccation, though the significant decline in phosphate solubilization was marked in the presence of aluminium than iron. The fungal isolate showed 100 % identity with E. parvum strain NRRL 2095 ITS 1, 5.8S rRNA gene and ITS 2, complete sequence; and 28S rRNA gene, partial sequence.     

Biosolubilization of poorly soluble rock phosphates by Aspergillus tubingensis and Aspergillus niger

Three isolates of Aspergillus tubingensis and two isolates of Aspergillus niger isolated from rhizospheric soils were tested on solubilization of different rock phosphates. All the isolates of Aspergillus were capable of solubilizing all the natural rock phosphates. A. tubingensis (AT1) showed maximum percent solubilization in all the rock phosphates tested in this study when compared to other isolates. This isolate also showed highest phosphorus (P) solubilization when grown in the presence of 2% of rock phosphate. A. tubingensis (AT1) seems to be more efficient in solubilization of rock phosphates compared to other isolates reported elsewhere. This is the first report of rock phosphate solubilization by A. tubingensis and might provide an efficient large scale biosolubilization of rock phosphates intended for P fertilizer.     

Responses of Pinus halepensis growth, soil microbial catabolic functions and phosphate-solubilizing bacteria after rock phosphate amendment and ectomycorrhizal inoculation

We examined the effects of an ectomycorrhizal (EM) fungus, Pisolithus sp., on of the growth of Pinus halepensis (Allepo pine) seedlings, soil microbial functions and rock phosphate solubilization in a un-disinfected soil amended or not with a Moroccan rock phosphate. Allepo pine seedlings were inoculated with an EM fungus (Pisolithus sp. strain PH4) isolated from a P. halepensis plantation and selected for its high ability to mobilize P from an inorganic form of phosphate. After 4 month’s culture in a disinfected substrate, plants were transferred in 10 l-containers filled with a natural forest soil and amended or not with rock phosphate powder. After 12 month’s culturing, the growth, needle nutrient concentrations of P. halepensis plants were measured. Soil microbial catabolic diversity was assessed by measuring CO₂ production of substrate induced respiration responses. Fluorescent pseudomonads were isolated from each soil treatment and tested in axenic conditions for their ability to solubilize a source of inorganic phosphate. The results clearly showed that (i) P. halepensis growth was greatly promoted by the presence of the ectomycorrhizal fungus Pisolithus strain PH4 in a disinfected soil/vermiculite mixture and in a non disinfected soil, (ii) ectomycorrhizal inoculation induced significant changes in the functions of soil microbial communities and selected microorganisms potentially beneficial to the plant growth (i.e. phosphate-solubilizing fluorescent pseudomonad) and (iii) rock phosphate solubilisation was mainly dependent on EM inoculation and mycorrhizosphere microorganisms. These results were in accordance with previous studies where it was demonstrated that EM symbiosis has a beneficial effect on plant growth through a direct effect on the host plant but also an indirect effect via a selective pressure on soil microbiota that favours microorganisms potentially beneficial to plant growth.     

一株新的溶磷草生欧文氏菌的分离、鉴定及其溶磷效果的初步研究

从湖南、湖北、云南等地磷矿开采场的土壤样品中筛选到一株溶磷能力较强的菌株P21,结合生理生化指标和16S rDNA序列分析鉴定其属于草生欧文氏菌菠萝变种(Erwinia herbicola var.ananas).该菌能溶解磷酸三钙、羟基磷灰石、磷酸铁、磷酸锌,其中对磷酸三钙和羟基磷灰石的每升液体培养基溶磷量(P2O5)分别高达1206.20mg、529.67mg.溶磷菌草生欧文氏菌菠萝变种P21对产地不同的8种磷矿石溶解能力不同,对云南晋宁和昆阳、四川雅安、江苏锦屏等地磷矿石有较强的溶解能力,每升液体培养基溶磷量分别为96.64mg、78.46mg、67.07mg、65.24mg,对其它产地的磷矿石溶解能力较差.实验表明,培养液的pH下降与溶磷菌P21的溶磷量无直接关系.     

Plant Growth-Promoting Effect of the Chitosanolytic Phosphate-Solubilizing Bacterium Burkholderia gladioli MEL01 After Fermentation with Chitosan and Fertilization with Rock Phosphate

Phosphate-solubilizing bacteria (PSB) are important plant growth-promoting rhizobacteria that can increase soil fertility through the solubilization of insoluble inorganic phosphate and organophosphorus. In this study, a PSB, Burkholderia gladioli MEL01, was isolated and identified from rice–wheat rotation rhizosphere soil. MEL01 had an excellent phosphate-solubilizing capacity (reaching 107.69 mg/L) toward insoluble inorganic phosphate rock phosphate. HPLC analysis revealed that the mechanism of phosphate solubilization of MEL01 was probably due to secreted oxalic acid and gluconic acid transformation of phosphate from insoluble to soluble. MEL01 also exhibited 4030 U/L specific chitosanase activity when cultured with chitosan fermentation medium. Interestingly, the chitosan hydrolysis product chitooligosaccharide could significantly enhance the MEL01 phosphate-solubilizing capacity. Pot experiments showed that MEL01 chitosan medium fermentation liquor (MCMFL) could promote improvement of soil available phosphorus and pakchoi growth when supplemented with phosphate rock phosphate as the phosphate fertilizer. In addition, pot experiments demonstrated that MCMFL could also promote the growth of wheat, which could decrease the amount of compound fertilizer used. Microbial diversity analysis showed that the genera Pseudomonas, Burkholderia, Mycoplana, and Cellvibrio were enriched, which might participate in synergetic phosphate solubilization. Therefore, after fermentation with chitosan and fertilization with rock phosphates, MEL01 has potential as a phosphate biofertilizer in ecological agricultural production.

     

Novel P-Solubilizers from Calcium Bound Phosphate Rich Pine Forest of Lower Himalaya

Phosphorus, an essential element for life, is continuously depleting from soils and thus demands sustainable management particularly in agriculture and forestry. Inorganic P constitutes the major proportion as tricalcium phosphate in soils of lower Himalayan region of Pakistan. We sampled these soils and screened for P-solubilizing microbes. A range of culturable microbial community (bacteria and fungi) was isolated and molecularly characterized which make the P available from mineral phosphates. There was an increase in abundance of phosphate solubilizing bacteria (PSB) at a 6-inch depth of the pine rhizosphere compared to the surface soil samples. Moreover, the isolates from lower Himalaya have higher abundance and better efficiency to solubilize the inorganic P than the ones from non-Himalaya. Most likely the P-solubilization done by our P-solubilizing microbes is via acidification as we observed the decrease in pH of the medium of microbial growth. Furthermore, the majority of isolated PSB belong to gammaproteobacterial class of Gram negative bacteria. Most interestingly, 13% of our isolated PSB were psychrotolerant (physiologically active at cold environment, i.e., 4°C) and able to solubilize inorganic P as efficiently as at ambient temperature. This study is unique in reporting the P-solubilizing microbes, particularly the psychrotolerant bacterial strains, of Lower Himalaya. Therefore the isolated bacterial and fungal strains have potential and may serve as biofertilizers in the region to increase the P availability in soils.     

Solubilization of low-grade Indian rock phosphates and inorganic phosphates byBacillus licheniformis

Bacillus licheniformis solubilized a range of inorganic phosphates and five different Indian rock phosphates (low grade) to varying extent in broth culture and in soil. Statistical evaluation indicated that broth cultures alone were inadequate indicators of the potential for microbial solubilization of rock phosphates.     

Pyrroloquinoline quinone biosynthesis gene pqqC, a novel molecular marker for studying the phylogeny and diversity of phosphate-solubilizing pseudomonads

Many root-colonizing pseudomonads are able to promote plant growth by increasing phosphate availability in soil through solubilization of poorly soluble rock phosphates. The major mechanism of phosphate solubilization by pseudomonads is the secretion of gluconic acid, which requires the enzyme glucose dehydrogenase and its cofactor pyrroloquinoline quinone (PQQ). The main aim of this study was to evaluate whether a PQQ biosynthetic gene is suitable to study the phylogeny of phosphate-solubilizing pseudomonads. To this end, two new primers, which specifically amplify the pqqC gene of the Pseudomonas genus, were designed. pqqC fragments were amplified and sequenced from a Pseudomonas strain collection and from a natural wheat rhizosphere population using cultivation-dependent and cultivation-independent approaches. Phylogenetic trees based on pqqC sequences were compared to trees obtained with the two concatenated housekeeping genes rpoD and gyrB. For both pqqC and rpoD-gyrB, similar main phylogenetic clusters were found. However, in the pqqC but not in the rpoD-gyrB tree, the group of fluorescent pseudomonads producing the antifungal compounds 2,4-diacetylphloroglucinol and pyoluteorin was located outside the Pseudomonas fluorescens group. pqqC sequences from isolated pseudomonads were differently distributed among the identified phylogenetic groups than pqqC sequences derived from the cultivation-independent approach. Comparing pqqC phylogeny and phosphate solubilization activity, we identified one phylogenetic group with high solubilization activity. In summary, we demonstrate that the gene pqqC is a novel molecular marker that can be used complementary to housekeeping genes for studying the diversity and evolution of plant-beneficial pseudomonads.