Isolation and identification of phosphate solubilizing bacteria able to enhance the growth and aloin-A biosynthesis of Aloe barbadensis Miller

The effect of four phosphate solubilizing bacteria (PSB) was studied on growth and aloin-A content of Aloe barbadensis in soil containing tricalcium phosphate (TCP). PSB were identified based on 16S rRNA gene sequencing as Pseudomonas synxantha, Burkholderia gladioli, Enterobacter hormaechei and Serratia marcescens. These PSB solubilized 25-340 μg ml(-1) of TCP into the liquid phase. The treatment of plants with individual PSB or mixture of these increased soil available P, P uptake in plants and plant growth. The increase in aloin-A content due to higher plant biomass and unit biomass production was 673%, 294%, 276%, 119% and 108% in plants treated with a PSB consortium, P. synxantha, S. marcescens, B. gladioli, and E. hormaechei in TCP amended soil, respectively.     

Improvement of Arbuscular Mycorrhiza Development by Inoculation of Soil with Phosphate-Solubilizing Rhizobacteria To Improve Rock Phosphate Bioavailability ((sup32)P) and Nutrient Cycling

The interactive effect of phosphate-solubilizing bacteria and arbuscular mycorrhizal (AM) fungi on plant use of soil P sources of low bioavailability (endogenous or added as rock phosphate [RP] material) was evaluated by using soil microcosms which integrated (sup32)P isotopic dilution techniques. The microbial inocula consisted of the AM fungus Glomus intraradices and two phosphate-solubilizing rhizobacterial isolates: Enterobacter sp. and Bacillus subtilis. These rhizobacteria behaved as "mycorrhiza helper bacteria" promoting establishment of both the indigenous and the introduced AM endophytes despite a gradual decrease in bacterial population size, which dropped from 10(sup7) at planting to 10(sup3) CFU g(sup-1) of dry rhizosphere soil at harvest. Dual inoculation with G. intraradices and B. subtilis significantly increased biomass and N and P accumulation in plant tissues. Regardless of the rhizobacterium strain and of the addition of RP, AM plants displayed lower specific activity ((sup32)P/(sup31)P) than their comparable controls, suggesting that the plants used P sources not available in their absence. The inoculated rhizobacteria may have released phosphate ions ((sup31)P), either from the added RP or from the less-available indigenous P sources, which were effectively taken up by the external AM mycelium. Soluble Ca deficiency in the test soil may have benefited P solubilization. At least 75% of the P in dually inoculated plants derived from the added RP. It appears that these mycorrhizosphere interactions between bacterial and fungal plant associates contributed to the biogeochemical P cycling, thus promoting a sustainable nutrient supply to plants.     

Electron microscopy of some rock phosphate dissolving bacteria and fungi

BacteriaPseudomonas striata, Bacillus polymyxa, B. megaterium andB. pulvifaciens, and fungiAspergillus awamori, A. niger andPenicillium digitatum dissolve tricalcium phosphate and, much less, Mussorie and Udaipur rock phosphate. The solubilizing power of fungi was higher than that of bacteria, the highest being withA. awamori andA. niger, and withP. striata. Electron microscopy of the various cultures showed an electron-dense layer on the bacterial surface after negative staining. The size of phosphate particles decreased by the microbial action, with tricalcium phosphate from 140 — 250 to 30 — 90 nm after three weeks of incubation.     

一株高效解磷真菌新菌株的筛选鉴定及解磷特性

从辽宁省辽中县多年耕种的日光温室番茄根际土壤中筛选出一株解磷真菌,通过菌落形态特征和ITS rDNA序列对比,鉴定该菌株为草酸青霉菌的一株新菌株,将其命名为PSF1.该菌株能利用葡萄糖、蔗糖、乳糖、半乳糖、可溶性淀粉等多种碳源和硫酸铵、氯化铵、硝酸铵、硝酸钾、尿素等多种氮源进行生长代谢并表现出较强的解磷能力,在C/N 10∶1~60∶1、初始pH 7~8的条件下生长情况较好且解磷能力较高.该菌株有很强的产酸能力,在培养过程中培养液pH由7.00~7.50下降至2.06~4.87;在4种磷源培养液中的最高解磷量分别为磷酸三钙(869.62 mg·L^-1)>磷矿粉(233.56 mg·L^-1)>磷酸铝(44.77 mg·L^-1)>磷酸铁(28.42 mg·L^-1).Pearson相关分析表明,菌株在磷酸三钙、磷矿粉和磷酸铁培养液中的解磷量与pH的变化之间呈极显著负相关关系,在磷酸铝培养液中无显著相关关系.菌株PSF1解磷能力强,生长条件广,推测其在土壤中有较强的解磷能力.     

In vitro evaluation of some bacterial isolates as biofertilizers and biocontrol agents against the second stage juveniles of <Emphasis Type="Italic">Meloidogyne incognita</Emphasis>

Representative soil samples were collected from soil apart and the rhizosphere of different plant varieties grown in five Egyptian Governorates. The presence of plant parasitic nematodes (PPN) and free living nematodes (FLN) in the collected samples were estimated. In addition, one hundred and 65 bacterial cultures which well known as biofertilizers (some are able to fix nitrogen and the others solubilize either phosphate or potassium) were isolated. The isolated bacteria were screened depending on their rate of growth. Thirty-five cultures of fast growing nitrogen fixing bacteria (NFB), phosphate solubilizing bacteria (PSB) and potassium solubilizing bacteria (KSB) and their cultural filtrates were tested in vitro as biocontrol agents against the second stage juvenile (J_2s) of the Meloidogyne incognita. In general, higher mortality percentages of nematodes were recorded by bacterial cultures than their comparative cultural filtrates. The highest mortality percentages were recorded by cultures of NFB7, PSB2 and KSB2 (100% at dilution 1/10), while it was 99.3, 99 and 97.8% at dilution 1/100, respectively. NFB7 exhibited a high nitrogen fixation rate (4.2 μmole N₂/mL/h), while PSB2 and KSB2 effectively solubilized phosphate and potassium comparing with the control treatments (1.94 fold of available phosphate and 2.0 fold of available potassium, respectively). NFB7, PSB2 and KSB2 isolates showed the highest protease, gelatinase and chitinase activities which were thought responsible for their nematicidal effect. The three bacterial isolates were identified as Paenibacillus polymyxa, Bacillus megaterium and Bacillus circulans, respectively.     

Isolation and performance evaluation of halotolerant phosphate solubilizing bacteria from the rhizospheric soils of historic Dagong Brine Well in China

The use of halotolerant phosphate solubilizing bacteria as inoculants to convert insoluble phosphorus of salt-affected soils to an accessible form is a promising strategy to improve the phosphorus ingestion of plants in salt-affected agriculture. A total of four aerobic isolates with biggest clear halos on the 10% NaCl NBRIP medium plate containing tricalcium phosphate were isolated from the rhizospheric soils of native plants growing on the wall of Dagong Ancinet Brine Well, located in Sichuan of China. And these four isolates were classified to the same strain, named QW10-11, and closely related to Bacillus megatherium var. phosphaticum DSM 3228 and B. megaterium ATCC 14581 according to their phenotype and 16S rRNA. However, the Molecular evolutionary evidences of 16S-23S rRNA ISR further suggested that QW10-11, DSM 3228 and ATCC 14581 have respectively fall into the different sub-divisions in intra specific phylogeny. Strain QW10-11 has significantly better ability of tricalcium phosphate solubilization than that of lecithin solubilization. When it grows under pH 4.8–8.0, 24–33°C and 5–10% NaCl, it can exhibit the higher values of solubilized tricalcium phosphate between 59.3 and 71.4 μg ml⁻¹. Furthermore, its tricalcium phosphate solubilizing activity was associated with the release of organic acids. Taken together, our results indicted that QW10-11 from the rhizospheric soils of halobiot of Dagong Ancinet Brine Well is attractive as efficient phosphate solubilizing candidates in the salt-affected agriculture.     

Phosphate-solubilization mechanism and in vitro plant growth promotion activity mediated by Pantoea eucalypti isolated from Lotus tenuis rhizosphere in the Salado River Basin (Argentina)

Aims: To isolate and characterize phosphate‐solubilizing strains from a constrained environment such as the Salado River Basin and to assess their phosphate‐solubilizing mechanisms, to further selection of the most promising strains to inoculate and improve the implantation and persistence of Lotus tenuis in the most important area devoted to meat‐cow production in Argentina. Methods and Results: Fifty isolates were obtained and through BOX‐PCR analysis, 17 non‐redundant strains were identified. Subsequently, they were found to be related to Pantoea, Erwinia, Pseudomonas, Rhizobium and Enterobacter genera, via 16S rRNA gene sequence analysis. This was in agreement with the clusters obtained by antibiotic resistance analysis. All isolates were tested for their phosphate‐solubilizing activity and selected strains were inoculated onto L. tenuis plants. The most efficient isolate, was identified as Pantoea eucalypti, a novel species in terms of plant growth‐promoting rhizobacteria. Conclusions: The isolates obtained in this study showed a significant in vitro plant‐growth promoting activity onto Lotus tenuis and the best of them solubilizes phosphate mainly via induction of the metabolism through secretion and oxidation of gluconic acid. Singnificance and Impact of the Study: The use of these bacteria as bioinoculants, alone or in combination with nitrogen‐fixing micro‐organisms, could be a sustainable practice to facilitate the nutrient supply to Lotus tenuis plants and preventing negative side‐effects such as eutrophication.     

Revegetation of a lakeside barren area by the application of plant growth-promoting rhizobacteria

The growth stimulation of wild plants by several bacterial species showing plant growth-promoting capabilities was examined in a barren lakeside area at Lake Paro, Korea. Microbial numbers and activities in the field soil were monitored for 73 days after inoculation of the bacteria. The acridine orange direct counts for the total soil bacterial populations ranged between 2.0-2.3x10(9) cells/g soil and 1.4-1.8x10(9) cells/g soil in the inoculated and uninoculated soils, respectively. The numbers of Pseudomonas spp., which is known as a typical plant growth-promoting rhizobacteria, and the total microbial activity were higher in the inoculated soil compared to those in the uninoculated soil. The average shoot and root lengths of the wild plants grown in the inoculated soil were 17.3 cm and 12.4 cm, respectively, and longer than those of 11.4 cm and 8.5 cm in the uninoculated soil. The total dry weight of the harvested wild plants was also higher in the inoculated soil (42.0 g) compared to the uninoculated soil (35.1 g). The plant growth-promoting capabilities of the inoculated bacteria may be used for the rapid revegetation of barren or disturbed land, and as biofertilizer in agriculture.     

Enriching vermicompost by nitrogen fixing and phosphate solubilizing bacteria

The effect of inoculation of vermicompost with nitrogen-fixing Azotobacter chroococcum strains, Azospirillum lipoferum and the phosphate solubilizing Pseudomonas striata on N and P contents of the vermicompost was assessed. Inoculation of N2 fixing bacteria into vermicompost increased contents of N and P. Enriching vermicompost with rock phosphate improved significantly the available P when inoculated with P. striata. During the incubation period, the inoculated bacterial strains proliferated rapidly, fixed N and solubilized added and native phosphate.     

Rock phosphate solubilization and colonization of maize rhizosphere by wild and genetically modified strains of <Emphasis Type="Italic">Penicillium rugulosum</Emphasis>

Maize root colonization and phosphate solubilizing activity of the fungus Penicillium rugulosum were assessed in a greenhouse trial using soil-plant microcosms. The bacterial gene hph conferring resistance to hygromicin B was introduced by electroporation in the wild-type strain IR-94MF1 of P. rugulosum and one transformant, w-T3, was selected. Maize plants were grown for 5 weeks in a P-poor soil and fertilized with a Florida apatite mineral, with Navay, an apatite rock deposit from Venezuela, or with simple superphosphate. Inoculation treatments included strain IR-94MF1, transformant w-T3 and two IR-94MF1 UV-induced mutants with enhanced (Mps++) or reduced (Mps-) in vitro mineral phosphate solubilizing activity. In the absence of P fertilization, inoculation with any P. rugulosum isolate significantly reduced the size of the total and P-solubilizing bacterial community present in maize rhizosphere. The bacterial community significantly increased in maize inoculated with IR-94MF1 and w-T3 when P was added as apatites Navay or Florida. All P. rugulosum strains were able to stimulate the growth of maize plants as indicated by 3.6 to 28.6% increases in dry matter yields. In the presence of rock phosphate, P uptake by maize plants inoculated with the two mutants Mps++ and Mps- was not always in agreement with their P-solubilizing phenotypes. Strain IR-94MF1 and transformant w-T3 increased P assimilation by the plants fertilized with Navay rock phosphate by 26 and 38%, respectively. In this treatment, w-T3 showed its highest significant maize rhizosphere colonization. With the simple superphosphate treatment, w-T3 increased P uptake in plants by 8% over the uninoculated control and also decreased significantly the community size of total bacteria, total fungi, and P-solubilizing fungi in the rhizosphere.     

Critical Evaluation of Phosphate Solubilizing Pseudomonads Isolated from a Partially Recultivated Potash Tailings Pile

Ten phosphate solubilizing pseudomonads isolated from a partially recultivated potash tailings pile in Germany were characterized and tested for their potential to assist in the ongoing recultivation process. Despite fertilization, the plants which are grown for recultivation show phosphate deficiency symptoms, and therefore the isolates are intended to be used as biofertilizer inoculants. On agar plates incubated at five different temperatures, some of the strains showed a temperature-dependent ability to solubilize tricalcium phosphate, while others performed the same at any given temperature. In liquid medium, the isolates solubilized between 271 and 730 μg ml(-1) of phosphate from tricalcium phosphate. Both the weakest (designated S10) and the strongest solubilizing strain (S06) were further tested for their viability during solubilization. In an assay over the course of 1 week, both strains released their maximum amount of phosphate after 2-4 days. At that later point of time, however, viable cells of isolate S06 were no longer detectable, whereas the weaker strain S10 could be cultured after 1 week in broth. Taking all in vitro observations into account, the usability of the isolates as biofertilizers is critically discussed regarding both the in situ conditions on the tailings pile and the lowered viability due to the excess production of organic acids.     

Comparison of protein profiles and enzymes in non-mycorrhizal and mycorrhizal roots of Pennisetum pedicellatum

Pennisetum pedicellatum plants were inoculated with Glomus mosseae, G. aggregatum and Gigaspora margarita. There were both quantitative and qualitative changes in the protein pattern of inoculated plants. Gi. margarita induced increase in protein in the plants. Acid phosphatase, alkaline phosphatase, superoxide dismutase and chitinase activities were high at the beginning of infection, but declined as the infection advanced. Gi. margarita was an efficient fungus in enhancing enzyme activity and proteins in roots compared with G. mosseae and G. aggregatum. Protein profile revealed the presence of 12 peaks in mycorrhizal plants compared with 8 in nonmycorrhizal plants.     

Can immobilization of Bacillus megaterium cells in alginate beads protect them against bacteriophages?

The ability of free and alginate-immobilized cells of Bacillus megaterium to dissolve tricalcium phosphate as well as their susceptibility to phages were compared in pure liquid cultures and in pot experiment with maize. In both liquid culture and cultivated soil, alginate-immobilized cells of B. megaterium exhibited much higher efficiency in increasing the availability of phosphorus than the free cells. Bacteriophages of B. megaterium were found to be common in soil. Specific bacteriophages, in the presence of free cells of B. megaterium, completely inhibited the phosphate-dissolving activity of the bacteria in pure liquid culture and markedly decreased their number in rhizosphere of maize plants. The phosphorus content of maize plants inoculated with free cells of B. megaterium decreased in the presence of their specific bacteriophages, whereas, when alginate-immobilized cells were used as inoculum, no effect of the presence of bacteriophages on phosphate-dissolving activity of bacterial cells was detected.     

Disruption of the Paenibacillus polymyxa levansucrase gene impairs its ability to aggregate soil in the wheat rhizosphere

Inoculation of wheat roots with Paenibacillus (formerly Bacillus ) polymyxa CF43 increases the mass of root-adhering soil. We tested the role of levan, a fructosyl polymer produced by strain CF43, in the aggregation of soil adhering to wheat roots. The P. polymyxa gene homologous to the Bacillus subtilis sacB gene encoding levansucrase was cloned and sequenced. The corresponding gene product synthesises high molecular weight levan. A P. polymyxa mutant strain, SB03, whose sacB gene is disrupted, was constructed using heterogramic conjugation. Effects of wheat inoculation with the wild type and the mutant strain were compared using two different cultivated silt loam soils in four independent pot experiments. Roots of wheat plantlets inoculated with CF43 or SB03 were colonized after 7–14 days at the same level, and root and shoot masses were not significantly different from those of the non-inoculated control plants. The ratio of root-adhering soil dry mass to root tissue dry mass was significantly higher for plants inoculated with strain CF43 than for those inoculated with mutant strain SB03: + 30% in Orgeval soil and + 100% in Dieulouard soil. Thus the levan produced by P. polymyxa is implicated in the aggregation of root-adhering soil on wheat.     

Microcosm study for revegetation of barren land with wild plants by some plant growth-promoting rhizobacteria

Growth promotion of wild plants by some plant growth-promoting rhizobacteria (PGPR) was examined in the microcosms composed of soils collected separately from a grass-covered site and a nongrass-covered site in a lakeside barren area at Lake Paro, Korea. After sowing the seeds of eight kinds of wild plants and inoculation of several strains of PGPR, the total bacterial number and microbial activity were measured during 5 months of study period, and the plant biomasses grown were compared at the end of the study. Acridine orange direct counts in the inoculated microcosms, 1.3-9.8 x 10(9) cells x g soil(-1) in the soil from the grass-covered area and 0.9-7.2 x 10(9) cells x g soil(-1) in the soil from the nongrass-covered site, were almost twice higher than those in the uninoculated microcosms. The number of Pseudomonas sp., well-known bacteria as PGPR, and the soil dehydrogenase activity were also higher in the inoculated soils than the uninoculated soils. The first germination of sowed seeds in the inoculated microcosm was 5 days earlier than the uninoculated microcosm. Average lengths of all plants grown during the study period were 26% and 29% longer in the inoculated microcosms starting with the grass-covered soil and the nongrass-covered soil, respectively, compared with those in the uninoculated microcosms. Dry weights of whole plants grown were 67-82% higher in the inoculated microcosms than the uninoculated microcosms. Microbial population and activity and growth promoting effect by PGPR were all higher in the soils collected from the grass-covered area than in the nongrass-covered area. The growth enhancement of wild plants seemed to occur by the activities of inoculated microorganisms, and this capability of PGPR may be utilized for rapid revegetation of some barren lands.     

Effect of phosphate-solubilizing fungi Aspergillus awamori S29 on mungbean (Vigna radiata cv. RMG 492) growth

A phosphate solubilizing fungus, Aspergillus awamori S29 was isolated from rhizoshpere of mungbean. The phosphate solubilizing activity of A. awamori S29 in liquid was 1,110?mg/L for tricalcium phosphate (TCP). The organism was able to solubilize various inorganic forms of phosphate at a wide range of temperatures. Among various insoluble phosphate sources tested, di-calcium phosphate was solubilized the most, followed by TCP. A. awamori S29 had significant effect (p?<?0.05) on mungbean growth, total P and plant biomass under pot conditions, although no obvious difference in available P in soil and number of leaves was found compared to the control.     

一株高效解磷肠膜明串珠菌的分离鉴定及解磷能力研究

【背景】植物根际土壤含有多种溶磷微生物,但是具有溶磷能力的肠膜明串珠菌未见报道。【目的】从脐橙根际土壤分离高效解磷菌,研究其解磷应用。【方法】通过初筛和复筛从23株菌中筛选解磷能力较强的菌株,同时采用钼蓝比色法测定磷含量。通过测定发酵液中小分子有机酸含量、磷酸酯酶酶活及pH值的变化,探究菌株的解磷机理。【结果】经过筛选得到9株具有一定解磷能力的菌株。通过菌种16S rRNA基因序列分析和生理生化实验确定其中一株菌为肠膜明串珠菌,命名为肠膜明串珠菌G7。培养基初始pH6.0、碳源为葡萄糖、氮源为硫酸铵时G7的解磷能力较佳。G7发酵过程中产生大量有机酸,而其酸性磷酸酯酶活性高于碱性磷酸酯酶。【结论】碳源、氮源以及初始pH值都能影响G7的解磷能力,其解磷能力主要缘于在发酵过程中产生了大量小分子有机酸,关于G7的解磷机理还需要更深入的研究。     

Alterations in plant growth and in root hormone levels of lodgepole pines inoculated with rhizobacteria.

The presence of other soil microorganisms might influence the ability of rhizobacterial inoculants to promote plant growth either by reducing contact between the inoculant and the plant root or by interfering with the mechanism(s) involved in rhizobacterially mediated growth promotion. We conducted the following experiments to determine whether reductions in the extent of growth promotion of lodgepole pine mediated by Paenibacillus polymyxa occur in the presence of a forest soil isolate (Pseudomonas fluorescens M20) and whether changes in plant growth promotion mediated by P. polymyxa (i) are related to changes in P. polymyxa density in the rhizosphere or (ii) result from alterations in root hormone levels. The extent of plant growth, P. polymyxa rhizosphere density, and root hormone concentrations were determined for lodgepole pine treated with (i) a single growth-promoting rhizobacterial strain (P. polymyxa L6 or Pw-2) or (ii) a combination of bacteria: strain L6 + strain M20 or strain Pw-2 + strain M20. There was no difference in the growth of pines inoculated with strain L6 and those inoculated with strain L6 + strain M20. However, seedlings inoculated with strain Pw-2 had more lateral roots and greater root mass at 12 weeks after inoculation than plants inoculated with strain Pw-2 + strain M20. The extent of growth promotion mediated by P. polymyxa L6 and Pw-2 in each treatment was not correlated to the average population density of each strain in the rhizosphere. Bacterial species-specific effects were observed in root hormone levels: indole-3-acetic acid concentration was elevated in roots inoculated with P. polymyxa L6 or Pw-2, while dihydrozeatin riboside concentration was elevated in roots inoculated with P. fluorescens M20.     

Factors determining rock phosphate solubilization by microorganisms isolated from soil

Forty two soil isolates (31 bacteria and 11 fungi) were studied for their ability to solubilize rock phosphate and calcium phosphate in culture medium. Eight bacteria and 8 fungi possessed solubilizing ability. Pseudomonas cepacia and Penicillium purpurogenum showed the highest activity. There was a correlation between final pH value and titratable acidity (r=–0.29 to –0.87) and between titratable acidity and soluble phosphate (r=0.22 to 0.99). Correlation values were functions of insoluble phosphate and of the group of microorganisms considered. A high correlation was observed between final pH and soluble phosphate only for the rock phosphates inoculated with the highest concentration of solubilizing bacteria (r=–0.73 to –0.98).