Number of Rhizobia and delayed inoculation influence nodulation of clovers

The relationship between numbers of rhizobia and nodulation response of legumes is of considerable practical importance. Experiments were done under controlled conditions to determine the influence of numbers of Rhizobium leguminosarum biovar. trifolii on nodulation of arrowleaf clover (Trifolium vesiculosum Savi.) and crimson clover (T. incarnatum L.). Numbers of rhizobia in excess of 1000 per seed did not substantially increase earliness of nodulation or total number of nodules formed on the taproot. Nodules, however, were formed nearer the top of the taproot as numbers of rhizobia increased to 100,000 per seed. Delayed inoculation experiments indicated that nodulation sites for these clovers only remained susceptible to infection for less than 1 day. Delaying inoculation for 4 days resulted in only a 1 to 2 day delay in nodulation for arrowleaf and crimson clovers respectively and no delay for subterranean clover (T. subterraneum L.). Apparently, larger seedlings nodulated faster.     

Evaluation of the symbiotic nitrogen-fixing potential of soils by direct microbiological means

A method for estimating the nitrogen-fixing capacity of a population of rhizobia resident in soil is presented. legume test plants, growing under microbiologically-controlled conditions in test tubes packed with a vermiculite substrate moistened with a nitrogen-free plant nutrient solution, are inoculated directly with a suspension of the soil under examination. Rhizobia in the soil nodulate the test plants, and the amount of foliage dry matter produced in the 28 days after inoculation is regarded as an index of their effectiveness. An inoculum of at least 30, and preferably 100, rhizobia is needed to ensure that nitrogen fixation is not masked by delayed nodulation. The new method is tentatively described as the ‘whole-soil inoculation’ technique. Appraisals were made withTrifolium subterraneum L. andRhizobium trifolii and withMedicago sativa L. andR. meliloti. Soil-borne pathogens did not interfere with plant growth. The whole-soil inoculation technique was less tedious and time-consuming than an alternative method which involved extracting representative isolates from the soil and testing their effectiveness individually, and appeared to give more realistic values for the nitrogen-fixing capacity of the soil as a whole. Used in association with a field experiment, the whole-soil inoculation technique confirmed microbiologically that there had been an agronomic response to surface application of inoculant to poorly-nodulatedT. subterraneum pasture. It is submitted that this technique for determining the effectiveness of rhizobia in soil, combined with a plant-infection method for counting rhizobia, can be a reliable guide to the need for inoculation in the field.     

Evaluation of two alternative metabolic pathways for creatinine degradation in microorganisms

The microbial decomposition of creatinine was found to proceed mainly via N-methylhydantoin or creatine as the first degradation product. Either N-methylhydantoin or urea or both were detected as metabolites derived from creatinine in various microorganisms, and creatinine deiminase and creatinine amidohydrolase activities were detected concomitantly. N-Methylhydantoin hydrolase and N-carbamoylsarcosine amidohydrolase were found to be formed inducibly in the presence of creatinine or N-methylhydantoin. Three microorganisms which decompose creatinine in different ways were screened from soil. Pseudomonas putida 77 rapidly metabolized creatinine solely via N-methylhydantoin. Degradation of creatinine proceeded with both creatine and N-methylhydantoin as the first degradation products at the same time in Pseudomonas sp. H21. Pseudomonas sp. 0114 was found to metabolize creatinine mainly via creatine and to also metabolize N-methylhydantoin. Changes in the metabolites of creatinine during a cultivation or enzyme reaction were found to be closely related to the enzyme activities of interest which are regulated by creatinine or N-methylhydantoin in different ways depending on the microbial strain.     

Nodulation and growth response ofAllocasuarina andCasuarina species to phosphorus fertilization

To examine how soil phosphorus status affects nitrogen fixation by the Casuarinaceae —Frankia symbiosis,Casuarina equisetifolia and two species ofAllocasuarina (A. torulosa andA. littoralis) inoculated or fertilized with KNO₃ were grown in pots in an acid soil at 4 soil phosphate levels. InoculatedC. equisetifolia nodulated well by 12 weeks after planting and the numbers and weight of nodules increased markedly with phosphorus addition. Growth ofC. equisetifolia dependent on symbiotically fixed nitrogen was more sensitive to low levels of phosphorus (30 mg kg^–1 soil) than was growth of seedings supplied with combined nitrogen; at higher levels of phosphorus, the growth response curves were similar for both nitrogen fertilized and inoculated plants. The interaction between phosphorus and nitrogen treatments (inoculated and nitrogen fertilized) demonstrated that there was a greater requirement of phosphorus for symbiotic nitrogen fixation than for plant growth when soil phosphorus was low.WithAllocasuarina species, large plant to plant variation in nodulation occurred both within pots and between replicates. This result suggests genetic variation in nodulation withinAllocasuarina species. Nodulation ofAllocasuarina species did not start until 16 weeks after planting and no growth response due toFrankia inoculation was obtained at the time of harvest. Addition of nitrogen starter is suggested to boost plant growth before the establishment of the symbiosis. Growth ofAllocasuarina species fertilized with nitrogen responded to increasing levels of phosphorus up to 90 mg P/kg soil after which it declined by 69% forA. littoralis. The decrease in shoot weight ofA. littoralis, A. torulosa, C. equisetifolia andC. cunninghamiana at high phosphorus was confirmed in a sand culture experiment, and may be atributable to phosphorus toxicity.     

Complete genome sequence of Rhizobium leguminosarum bv. trifolii strain WSM1325, an effective microsymbiont of annual Mediterranean clovers

Rhizobium leguminosarum bv trifolii is a soil-inhabiting bacterium that has the capacity to be an effective nitrogen fixing microsymbiont of a diverse range of annual Trifolium (clover) species. Strain WSM1325 is an aerobic, motile, non-spore forming, Gram-negative rod isolated from root nodules collected in 1993 from the Greek Island of Serifos. WSM1325 is produced commercially in Australia as an inoculant for a broad range of annual clovers of Mediterranean origin due to its superior attributes of saprophytic competence, nitrogen fixation and acid-tolerance. Here we describe the basic features of this organism, together with the complete genome sequence, and annotation. This is the first completed genome sequence for a microsymbiont of annual clovers. We reveal that its genome size is 7,418,122 bp encoding 7,232 protein-coding genes and 61 RNA-only encoding genes. This multipartite genome contains 6 distinct replicons; a chromosome of size 4,767,043 bp and 5 plasmids of size 828,924 bp, 660,973 bp, 516,088 bp, 350,312 bp and 294,782 bp.     

Symbiotic nitrogen fixation of Rhizobium (Galega) in acid soils,and its survival in soil under acid and cold stress

Summary Goat's rue (Galega orientalis) is a potential perennial forage legume for northern temperate acid soils. Greenhouse experiments were performed to compare symbiotically nitrogen fixing goat's rue with plants receiving mineral nitrogen in five different acid soils. Soil acidity had the same effect on yields of symbiotically grown plants as on plants receiving mineral nitrogen, suggesting that the acid sensitivity of the symbiosis was not limiting plant growth, even under very acidic conditions. The survival of an antibiotic resistant Rhizobium (Galega) strain in acid soil and freezing conditions was also studied. The survival of the bacteria was not affected at 15°C, when the pH of the soil (measured in 0.01M CaCl₂) was 5.2 or 4.9. In pH 3.4, and after freezing to –5°C, the population density decreased from 3×10⁸ to 1×10⁵/g in a few weeks. It is concluded that goat's rue, its symbiotic nitrogen fixation and R. (Galega) are tolerant of moderately acid agricultural soils, but that harsh winters may reduce bacterial numbers in the soil.     

Effects of intercropping white cabbage with clovers on pest infestation and yield

During two consecutive years the effects of intercropping fresh market white cabbage with two species of clover on pest populations and yield were studied. White cabbage cv. Minicole was intercropped withTrifolium repens (white clover) andTrifolium subterraneum (subterranean clover) as compared to the monocrop. During the season observations were made on pest population developments, especially ofMamestra brassicae L. (cabbage moth),Brevicoryne brassicae L. (cabbage aphid),Delia brassicae L. (cabbage root fly), and evaluation of caterpillar feeding injury. At harvest the yield in quantity and quality was determined to be able to assess the gross financial result. Intercropping effects in terms of suppression of oviposition and larval populations of various pests were found. Although no pesticides were used and competition reduced the weight, the quality of the intercropped cabbages lead to a better financial result compared to the monocropped cabbage crop. The results are discussed in the perspective of the practical implications in the context of IPM.     

The role of active Bradyrhizobium japonicum in iron stress response of soybeans

The objective of this study was to identify the sites of H-ion exudation and Fe(III) reduction along both inoculated and non-inoculated roots of A7 and T203 soybeans. A split-root system was used in which half the roots of each plant were inoculated and actively fixing nitrogen and the other half were not. Expectedly, the Fe-stress response was strong on both sides of the split-root system in the +N-Fe treatment of variety A7 (inactive nodules) but not of variety T203. The Fe-stress response of A7 was enhanced by the presence of active nodules. Variety T203 is Fe inefficient and normally fails to produce any Fe-stress response, but in the absence of nitrogen and iron (–N–Fe), inoculated roots responded to Fe stress with exudation of both H-ions and reductants. Intact split-root systems were embedded in agar to determine the location of H-ion exudation and Fe(III) reduction. On the inoculated side of the –N–Fe and –N+Fe treatments (active nodules) of both soybean varieties, H-ion production was associated mainly with the active nodules. However, quantities of H-ion release were much greater under Fe stress (–N–Fe) than with adequate Fe (–N+Fe). Reduction of Fe(III) to Fe(II) was found only on the nodulated side with T203, but on both sides with A7. In variety T203 the Fe reduction was associated with younger roots located just below the nodule clusters on the inoculated side of the –N treatments. Active nodules appear to play a key role in the Fe-deficiency stress response of T203 soybean.     

酸性旱地红壤无机氮同化菌株筛选及其全基因组分析

微生物执行的无机氮同化作用可固定施入土壤后未被作物直接吸收的化学氮肥,有效减少化学氮肥损失、降低环境氮素污染风险。土壤无机氮同化作用不是由大量冗余微生物共同执行的,而是由一小部分功能微生物优先执行。【目的】对酸性旱地红壤中的优势无机氮同化细菌进行富集、菌株分离鉴定及全基因组测序,并明确菌株在土壤中的氮同化能力,为酸性土壤化学氮肥应用及其转化过程研究提供菌株资源和理论依据。【方法】在酸性旱地红壤中添加KNO3或(NH4)2SO4作为无机氮源,以葡萄糖作为碳源,在好氧条件下进行富集预培养,采用稀释分离法筛选出优势无机氮同化细菌菌株;将菌株回接至土壤中从而验证其无机氮同化能力,并通过全基因组测序分析菌株的氮素代谢途径及相关功能基因。【结果】酸性旱地红壤经富集预培养一周后,优势无机氮同化微生物的16SrRNA基因相对丰度从0.20%–0.94%增长至20.2%–30.2%;分离筛选后得到的3株优势无机氮同化细菌菌株,鉴定为伯克霍尔德氏菌(Burkholderia sp.) M6-3、索状芽孢杆菌(Bacillus funiculus) M2-4和节杆菌(Arthrobacter sp.) M7...     

Competition between a native isolate of Rhizobium leguminosarum bv trifolii and two commercial inoculant strains for nodulation of clover

Field experiments were conducted to determine the influence of six commercial herbicides and of hand-hoeing on symbiotic parameters and grain yield of lentil (Lens culinaris Med.). The herbicides were sprayed at the pre-emergence stage. Number of nodules, nodule dry weight and nitrogenase activity were found to decrease with the use of Oxyfluorfen, Linuron, Metribuzin and Oxadiazon, whereas the effects of Terbutryn and Methabenzthiazuron were similar to that of hand-hoeing. Any harmful effect of herbicides on symbiotic parameters was less when seeds were inoculated with Rhizobium leguminosarum.     

Differential response in growth and mycorrhizal colonisation of soybean to inoculation with two isolates ofGlomus clarum in soils of different P availability

Soybean (Glycine max cv. Mikiwashima) seedlings were inoculated with two tropical isolates of the vesicular-arbuscular mycorrhizal fungusGlomus clarum (Gc); isolateGcA was isolated from soils of low phosphate (P) and isolateGcB from soils of high P availability. In soil with low P,GcA was more efficient in increasing growth, nodulation and nitrogenase activity ofG. max thanGcB. Upon contact with the root surface, pre-infection hyphae ofGcA penetrated the root directly and rapidly colonised the cortical cells, while those ofGcB grew extensively on theroot surface with little host penetration. Mycorrhizal colonisation was higher in roots inoculated withGcA. Dual inoculation with the two isolates did not increase the effect of the single inoculation withGcA. In soils of high P status, both isolates formed pre-infection hyphae with few entry ooints and percentage mycorrhizal root colonisation was consequently low. The variation in efficacy of the isolates emphasizes the significance of evaluating host specificity when selecting efficient VA mycorrhizae strains for field studies.     

Calcium magnesium imbalance in clovers: A cause of negative yield response to liming

Three pot experiments, in which causes of negative yield responses to liming were investigated, are reported. The soil used, Waimumu silt loam (Fragiochrept), differed from others that have been reported to show negative yield response to liming, in that it is only moderately weathered and leached, only moderately acid and has previously shown positive yield responses to liming. Deficiencies of Zn and Mg were identified, but limed (pH 6.8) soils still showed a 40% yield depression even where all nutrients were supplied daily. Phosphorus availability was little affected by liming, and despite Mg and Zn addition, yields were depressed at high lime (pH 6.4) and high P while plants showed leaf symptoms of Mg deficiency. Neither plant nor soil analyses indicated low Mg levels but Ca: Mg ratios in soil were 22:1. When a pH range 5.2–6.1 was produced by liming with CaCO₃ and MgCO₃ at ratios between 100:0, and 50:50 on an equivalent basis, negative yield response was eliminated at Ca:Mg of 50:50. There was no evidence that Mg was fixed or rendered unavailable at the higher pH levels. A Ca induced Mg deficiency arising when exchangeable Ca:Mg>20 is suggested as the cause. The role of variable surface charge in converting soils that respond positively to lime to a negative response condition is discussed.     

Two new entodiniomorphid Triplumaria ciliates from the intestine of the wild African white rhinoceros

Two new species of Triplumaria in the order Entodiniomorphida, T. alluvia n. sp. and T. grypoclunis n. sp., are described from the large intestine of the wild African white rhinoceros. T. alluvia has three bud-shaped caudalia, one broad skeletal plate with a wavy left dorsal edge, and an axe-shaped tail flap. T. grypoclunis has three short arched caudalia, two broad skeletal plates, and a pointed and ventrally curved tail flap. These two new species have a C-shaped adoral polybrachykinety, a slender perivestibular polybrachykinety, and paralabial kineties in their retractable adoral ciliary zone. In T. alluvia, the perivestibular polybrachykinety is joined to both ends of the adoral polybrachykinety and paralabial kineties along the ventral side of the adoral polybrachykinety, showing the same arrangement as in Cycloposthium species. In T. grypoclunis, the perivestibular polybrachykinety is joined only to the right end of the adoral polybrachykinety and paralabial kineties along the left ventral side of the adoral polybrachykinety, showing an arrangement analogous to the Tripalmaria species.     

Contribution to the knowledge of the genus Phyllopodopsyllus T. Scott (Copepoda,Harpacticoida) from Africa with description of two new species

Two new species of marine, sand-dwelling harpacticoids are described: Phyllopodopsyllus pallaresae n. sp. from Namibia, and P. hartmannorum n. sp. from Tanzania. The hitherto unknown male of P. xenus (Kunz) is described from Namibia. Further remarks are made on P. furciger Sars from Tanzania.Zwei neue sandbewohnende Harpacticoiden-Arten werden beschrieben: Phyllopodosyllus pallaresae n. sp. von Namibia und Phyllopodopsyllus hartmannorum n. sp. von Tansania. Außerdem wird das bisher unbekannte Männchen von Phyllopodopsyllus xenus (Kunz) von Namibia beschrieben. Morphologische Merkmale von Phyllopodopsyllus furciger Sars werden anhand eines Fundes von Tansania ergänzt.     

Response of Lotus rhizobia to acidity and aluminium in liquid culture and in soil

Measurements of multiplication in liquid culture indicated that fast-growing Lotus rhizobia (Rhizobium loti) were tolerant of acidity and aluminium (at least 50 μM A1 at pH 4.5). Slow-growing Lotus rhizobia (Bradyrhizobium sp. (Lotus)) were less tolerant of acidity but equally tolerant of A1. Both genera were able to nodulateLotus pedunculatus in an acid soil (pH 4.1 in 0.01M CaCl₂) and the slow-growing strains were more effective than the fast-growing strains in this soil over 30 days.     

Leaf photosynthesis,fluorescence response to salinity and the relevance to chloroplast salt compartmentation and anti-oxidative stress in two poplars

In a 16-day study, the effect of increasing soil NaCl on leaf photosynthesis, chlorophyll a fluorescence, chloroplast ion compartmentation, variations of SOD (superoxide dismutase) and POD (peroxidase) isoenzymes and the relevance to salt resistance were investigated in seedlings of Populus euphratica Oliv. (P. euphratica) (salt-resistant) and rooted cuttings of P. “popularis 35–44” (P. popularis) (salt-sensitive). Initial salinity caused a rapid decline of net photosynthetic rate (Pn) and unit transpiration rate (TRN) in P. euphratica, resulting from the NaCl-induced stomatal closure. In a longer-term of salinity, CO₂ assimilation in P. popularis was severely reduced whereas stressed P. euphratica maintained a relatively higher and constant level of Pn. Pn–Ci curves showed that salt stress (12 days) reduced CO₂ saturation point (CSP), CO₂ saturated Pn (CSP _n ), and carboxylation efficiency (CE), but increased CO₂ compensation point (CCP) in the two genotypes. Similarly, salinity lowered light saturation point (LSP), light saturated Pn (LSP _n ), and apparent quantum yield (AQY) in both genotypes but the inhibitory effect of NaCl on light reaction was more pronounced in P. popularis, as compared to P. euphratica. Chlorophyll a fluorescence data indicated that a longer-term of salt stress (12 days) exhibited a marked influence on fluorescence parameters of P. popularis in both dark- and light-adapted states: (a) NaCl inhibited the maximal efficiency of PSII photochemistry (Fv/Fm) due to the salt-induced increase of Fo (the minimal fluorescence) and the marked decline of Fm (the maximal fluorescence); (b) salinity decreased coefficient of photochemical quenching (qP) but markedly elevated coefficient of nonphotochemical quenching (qN) in the light-adapted state. In contrast, there were no corresponding changes of chlorophyll a fluorescence in salinised P. euphratica. X-ray microanalysis results showed that salinity caused salt accumulation in the chloroplasts of P. popularis in which Na⁺ and Cl⁻ increased up to 42 and 221 mmol dm⁻³, respectively. Great buildup of Na⁺ and Cl⁻ in chloroplasts of P. popularis may exhibit direct and indirect restrictions on dark and light reactions. The activity of SOD isoenzymes (CuZn-SOD I and CuZn-SOD II) and POD isoenzymes in P. popularis decreased with increasing exposure period, and leaf malondialdehyde (MDA) content and membrane permeability (MP) increased correspondingly. In contrast to P. popularis, stressed P. euphratica maintained activity of SOD and POD isoenzymes and there was no significant increase of MDA and MP during the period of salt stress. In conclusion, P. euphratica plants exhibited a higher capacity to maintain the activity of anti-oxidant enzymes and restrict salt accumulation in the chloroplasts, the photosynthesis processes were less restricted consequently.     

Phosphate-solubilizing microorganisms isolated from rhizospheric and bulk soils of colonizer plants at an abandoned rock phosphate mine

The abandoned “Monte-Fresco” rock phosphate mine in Táchira, Venezuela, was sampled to study the biodiversity of phosphate-solubilizing microorganisms (PSM). Rhizosphere and bulk soils were sampled from colonizer plant species growing at a mined site where pH and soluble P were higher than the values found at a near by unmined and shrubby soil. Counting and isolating of PSM choosing strains showing high solubilization halos in a solid minimal medium with hydroxyapatite as phosphate source were evaluated using ammonia or nitrate as nitrogen sources and dextrose, sucrose, and mannitol as carbohydrate sources. A larger number of PSM were found in the rhizospheric than in the bulk soil. Six fungal strains belonging to the genus Penicillium and with high hydroxyapatite dissolution capacities were isolated from bulk soil of colonizer plants. Five of these strains had similar phenotypes to Penicillium rugulosum IR-94MF1 but they solubilized hydroxyapatite at different degrees with both nitrogen sources. From 15 strains of Gram-negative bacteria isolated from the rhizosphere of colonizer plants, 5 were identified as diazotrophic free-living encapsulated Azotobacter species able to use ammonium and/or nitrate to dissolve hydroxyapatite with glucose, sucrose and/or mannitol. Different nitrogen and carbohydrate sources are parameters to be considered to further characterize the diversity of PSM.     

Variation in wild populations of annual beet (Beta,Chenopodiaceae)

Among the morphological variation in wild annual populations of sect.Beta only tepal characters show a geographic pattern, and hence can be used to distinguish different taxa. Two morphological types correspond with taxa already described:B. macrocarpa (incl.B. bourgaei) has a Macaronesian and W. to E. Mediterranean andB. adanensis an E. Mediterranean distribution area. A third type in the Aegean region is not well known yet and possibly has to be included inB. macrocarpa. Both diploid and tetraploid (x = 9) cytotypes are found withinB. macrocarpa, the latter exclusively on the Canary Islands.     

Pelotomaculum terephthalicum sp. nov. and Pelotomaculum isophthalicum sp. nov.: two anaerobic bacteria that degrade phthalate isomers in syntrophic association with hydrogenotrophic methanogens

An anaerobic phthalate isomer-degrading strain (JT^T) that we previously isolated was characterized. In addition, a strictly anaerobic, mesophilic, syntrophic phthalate isomer-degrading bacterium, designated strain JI^T, was isolated and characterized in this study. Both were non-motile rods that formed spores. In both strains, the optimal growth was observed at temperatures around 37°C and neutral pH. In syntrophic co-culture with the hydrogenotrophic methanogen Methanospirillum hungatei, both strains could utilize two or three phthalate isomers for growth, and produce acetate and methane as end products. Strain JT^T was able to grow on isophthalate, terephthalate, and a number of low-molecular weight aromatic compounds, such as benzoate, hydroquinone, 2-hydroxybenzoate, 3-hydroxybenzoate, 2,5-dihydroxybenzoate, 3-phenylpropionate in co-culture with M. hungatei. It could also grow on crotonate, hydroquinone and 2,5-dihydroxybenzoate in pure culture. Strain JI^T utilized all of the three phthalate isomers as well as benzoate and 3-hydroxybenzoate for growth in co-culture with M. hungatei. No substrates were, however, found to support the axenic growth of strain JI^T. Neither strain JT^T nor strain JI^T could utilize sulfate, sulfite, thiosulfate, nitrate, fumarate, Fe (III) or 4-hydroxybenzoate as electron acceptor. Phylogenetically, strains JT^T and JI^T were relatively close to the members of the genera Pelotomaculum and Cryptanaerobacter in ‘Desulfotomaculum lineage I’. Physiological and chemotaxonomic characteristics indicated that the two isolates should be classified into the genus Pelotomaculum, creating two novel species for them. Here, we propose Pelotomaculum terephthalicum sp. nov. and Pelotomaculum isophthalicum sp. nov. for strain JT^T and strain JI^T, respectively. The type strains are strains JT^T (= DSM 16121^T = JCM 11824^T = NBRC 100523^T) and JI^T (= JCM 12282^T = BAA-1053^T) for P. terephthalicum and P. isophthalicum, respectively.Nucleotide sequence accession number: The GenBank/EMBL/DDBJ accession numbers of the 16S rRNA gene sequences of strains JT^T and JI^T are AB091323 and AB232785, respectively