Effects of Kocide 101® on the bean (Phaseolus vulgaris L.)‐Rhizobium symbiosis

A glasshouse study was undertaken to investigate the effects of the copper fungicide Kocide 101 and its residues in soil on the growth, nodulation and nitrogen fixation of beans (Phaseolus vulgaris L.). The soil used was a sandy clay loam classified as Typic Rhodustalf. The bean variety SUA 90 was used as test crop. The bean rhizobia strains CIAT 899, PV, and a local isolate were used. Kocide 101 applied at the recommended rate (equivalent to 1.7 mg kg^‐1 soil) had no significant negative effects on the growth, nodulation or nitrogen fixation of bean plants. Higher levels of Kocide 101 significantly (P < 0.05) reduced plant growth, nodulation and nitrogen fixation. The bean plants inoculated with the “local isolate”; rhizobia had the highest dry matter weights, nodule numbers and nodule dry weights, and also had more N fixation. They were followed by those inoculated with the PV, strain and, lastly, those inoculated with CIAT 899. The growth and nodulation of bean plants were still curtailed by the Kocide 101 residues four months after the fungicide was first applied to the soil. Therefore, occurrence of high levels of Kocide 101 in soils can have long‐term effects on the performance of the bean‐rhizobia symbiosis.     

INTERACTIONS BETWEEN COMMON BEAN GENOTYPES AND RHIZOBIA STRAINS ISOLATED FROM MOROCCAN SOILS FOR GROWTH,PHOSPHATASE AND PHYTASE ACTIVITIES UNDER PHOSPHORUS DEFICIENCY CONDITIONS

The improvement of common bean production requires the selection of effective rhizobia strains and Phaseolus vulgaris genotypes adapted to available soil phosphorus limitations. The interactions between bean genotypes and rhizobia were studied in hydroponic culture using six genotypes and four strains, CIAT899 as reference and three strains isolated from nodule of farmer's fields in the Marrakech region. The phosphorus (P) sub-deficiency caused a significant reduction on shoot biomass in some bean genotype-rhizobia combinations. Nodule biomass is significantly more reduced under P limitation for several combinations tested. Bean plants inoculated with these local rhizobial strains showed higher nodulation and an increase of nodules phytase and phosphatase activities under phosphorus sub-deficiency especially for RhM11 strain. It was concluded that the studied bean-rhizobia symbiosis differ in their adaptation to phosphorus sub-deficiency and the nodule phosphatases and phytases activities may constitute a strategy of nodulated bean plants to adapt their nitrogen fixation to P deficiency.     

Genotypic Variation in Nodule Iron Content of Common Bean (Phaseolus Vulgaris L.) in Response to Phosphorus Deficiency

Common bean (Phaseolus vulgaris L.) can supply all of the iron that humans require for metabolism. Also, it fixes atmospheric nitrogen (N₂) in symbiosis with rhizobia. In order to analyze the relation between phosphorus (P) and iron (Fe) elements in nodules and their roles for the plant N₂-dependent growth, six common bean recombinant inbred lines (RIL) of the cross of BAT477 and DOR364 were inoculated with Rhizobium tropici CIAT 899 (originating from International Center of Tropical Agriculture, Colombia) and grown with sufficiency versus deficiency P supply in hydroaeroponic culture. Under P deficiency, the Fe content in nodules decreased in all studied genotypes and was significantly the highest for RIL 34. The nodule contents of Fe and P were significantly correlated under P deficiency. It is concluded that the regressions of nodule Fe content as a function of P content in nodules, roots and shoots, depend upon P supply and genotype.     

Effect of Sodium Chloride on Growth,Nutrient Accumulation,and Nitrogen Fixation of Common Bean Plants in Symbiosis with Isogenic Strains

The effect of sodium chloride (NaCl)-salinity on growth responses and tissues organic solutes and mineral content was investigated in common bean plants inoculated with salt-tolerant Rhizobium tropici wild-type strain CIAT899 and four mutant derivatives having decreased salt-tolerance (DST). Under non-saline conditions two mutants formed partially effective (HB10, HB12) and another two almost ineffective (HB8, HB13) nodules. A great variation of NaCl tolerance in the different symbiosis tested was observed at harvest, 32 day after planting. Common bean plant responded to salinity by decreasing the content of dry plant biomass, nodule number and the nitrogen fixation, and increasing the root to shoot ratio. The salt dose of 25 mM produced an increase of total soluble sugar and free amino acids content. This result suggest that these metabolites might be related with a nodule osmotic adjustment response under saline conditions, however cannot be excluded that the increase of amino acids content could be a consequence of protein degradation. In the other hand, sodium, calcium and phosphorus contents in shoot increased under the saline treatments. Potassium (K) and calcium (Ca) contents, unlike phosphorus (P) content, in shoot were not related with the symbiotic efficiency of mutant, however the decrease of P content suggest that these symbioses have limited their P absorption process independently of the saline treatment. NaCl tolerance associated with a retention of sodium and maintenance of potassium selectivity seem to be a strategy used for the salt stressed common bean plants in symbiosis assayed here.     

PLANT GROWTH PROMOTING RHIZOBACTERIA INCREASE GROWTH,YIELD AND NITROGEN FIXATION IN PHASEOLUS VULGARIS

Nitrogen (N) fixation by legume-Rhizobium symbiosis is important to agricultural productivity and is therefore of great economic interest. Growing evidence indicates that soil beneficial bacteria can positively affect symbiotic performance of rhizobia. The effect of co-inoculation with plant growth-promoting rhizobacteria (PGPR) and Rhizobium, on nodulation, nitrogen fixation, and yield of common bean (Phaseolus vulgaris L.) cultivars was investigated in two consecutive years under field conditions. The PGPR strains Pseudomonas fluorescens P-93 and Azospirillum lipoferum S-21 as well as two highly effective Rhizobium strains were used in this study. Common bean seeds of three cultivars were inoculated with Rhizobium singly or in a combination with PGPR to evaluate their effect on nodulation and nitrogen fixation. A significant variation of plant growth in response to inoculation with Rhizobium strains was observed. Treatment with PGPR significantly increased nodule number and dry weight, shoot dry weight, amount of nitrogen fixed as well as seed yield and protein content. Co-inoculation with Rhizobium and PGPR demonstrated a significant increase in the proportion of nitrogen derived from atmosphere. These results indicate that PGPR strains have potential to enhance the symbiotic potential of rhizobia.     

Co-inoculation of selected nodule endophytic rhizobacterial strains with Rhizobium tropici promotes plant growth and controls damping off in common bean

Production of common bean(Phaseolus vulgaris)is limited by the occurrence of damping off(rhizoctoniosis),which is caused by the fungus Rhizoctonia solani.However,the co-inoculation of plant growth-promoting rhizobacteria(PGPR)involved in biological control along with diatomic nitrogen(N2)-fixing rhizobia can enhance N nutrition and increase production.In this context,finding microorganisms with synergistic effects that perform these two roles is of fundamental importance to ensure adequate yield levels.The aim of this study was to evaluate the effects of co-inoculation of nodule endophytic strains of the genera Bacillus,Paenibacillus,Burkholderia,and Pseudomonas with Rhizobium tropici CIAT 899,an N2-fixing rhizobial strain,on the biocontrol of damping off and growth promotion in common bean plants.Greenhouse experiments were conducted under axenic conditions using the common bean cultivar Pérola.The first experiment evaluated the potential of the 14 rhizobacterial strains,which were inoculated alone or in combination with CIAT 899,for the control of R.solani.The second experiment evaluated the ability of these 14 rhizobacterial strains to promote plant growth with three manners of N supply:co-inoculation with CIAT 899 at low mineral N supply(5.25 mg N mL^-1),low mineral N supply(5.25 mg N mL^-1),and high mineral N supply(52.5 mg N mL^-1).The use of rhizobacteria combined with rhizobia contributed in a synergistic manner to the promotion of growth and the control of damping off in the common bean.Co-inoculation of the strains UFLA 02-281/03-18(Pseudomonas sp.),UFLA 02-286(Bacillus sp.),and UFLA 04-227(Burkholderia fungorum)together with CIAT 899 effectively controlled damping off.For the common bean,mineral N supply can be replaced by the co-inoculation of CIAT 899 with plant growth-promoting strains UFLA 02-281/02-286/02-290/02-293.Nodule endophytes UFLA02-281/02-286 are promising for co-inoculation with CIAT 899 in the common bean,promoting synergy with rhizobial inoculation and protection against disease.     

Importance of iron use‐efficiency of nodules in common bean (Phaseolus vulgaris L.) for iron deficiency chlorosis resistance

Several studies suggest that the Fabaceae‐Rhizobium symbiosis is particularly sensitive to iron (Fe) deficiency with respect to NO₃^–‐dependent plants. The aim of this study, which is part of a screening program for common bean tolerance to Fe deficiency, was to study genotypical differences in Fe requirement and Fe use‐efficiency of common bean cultivars depending on symbiotic nitrogen fixation (SNF). Results show that ARA14 produces more whole plant dry matter and particularly more nodule biomass than Coco blanc. ARA14 is characterized by a high capacity of nitrogen fixation and a better Fe use‐efficiency for the growth and the function of the nodules.     

Native rhizobium strains are lacking in some agricultural soils in NE South Africa

ABSTRACT

Rhizobia is a group of gram-negative soil-borne bacteria with several beneficial strains for biological nitrogen fixation in legume crops. Rhizobium strains are found native in the soil but where they are absent, commercial strains are inoculated on crops. We assessed the availability of native rhizobia in chickpea fields at two sites, with contrasting soil types, in NE South Africa. Serial dilutions were used to identify bacteria from soil samples and chickpea nodules sampled before sowing and at flowering, respectively. Our results indicated the absence of rhizobia strains at both sites. Burkholderia cenocepacia, Klebsiella variicola, Bacillus subtilis and Ochrobactrum spp, which are not important in agriculture but are often reported in clinical environments, were identified. Therefore, inoculating chickpea with compatible rhizobia strains may be necessary in some soils in this region.     

Benefits of inoculation of the common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis>) crop with efficient and competitive<Emphasis Type="Italic"> Rhizobium tropici</Emphasis> strains

Cropping in low fertility soils, especially those poor in N, contributes greatly to the low common bean (Phaseolus vulgaris L.) yield, and therefore the benefits of biological nitrogen fixation must be intensively explored to increase yields at a low cost. Six field experiments were performed in oxisols of Paraná State, southern Brazil, with a high population of indigenous common bean rhizobia, estimated at a minimum of 10³ cells g^–1 soil. Despite the high population, inoculation allowed an increase in rhizobial population and in nodule occupancy, and further increases were obtained with reinoculation in the following seasons. Thus, considering the treatments inoculated with the most effective strains (H 12, H 20, PRF 81 and CIAT 899), nodule occupancy increased from an average of 28% in the first experiment to 56% after four inoculation procedures. The establishment of the selected strains increased nodulation, N₂ fixation rates (evaluated by total N and N-ureide) and on average for the six experiments the strains H 12 and H 20 showed increases of 437 and 465 kg ha^–1, respectively,in relation to the indigenous rhizobial population. A synergistic effect between low levels of N fertilizer and inoculation with superior strains was also observed, resulting in yield increases in two other experiments. The soil rhizobial population decreased 1 year after the last cropping, but remained high in the plots that had been inoculated. DGGE analysis of soil extracts showed that the massive inoculation apparently did not affect the composition of the bacterial community.     

抗春雷霉素大豆根瘤菌突变株的诱变及其生物固氮特性的分析

根瘤菌与豆科植物共生可以固定大量的氮。根瘤菌剂接种豆科作物是一项普遍推广应用、有效的农业技术。但由于大量土著根瘤菌的存在，产生竞争障碍，降低了接种菌剂的占瘤率。大多数的土著根瘤菌对春雷霉素敏感，因此接种抗春雷霉素的高效结瘤固氮根瘤菌，并用春雷霉素处理种子，可抑制土著根瘤菌，提高接种菌剂的占瘤率，从而达到高结瘤、高固氮和提高产量的目的。本文将探讨诱变对根瘤菌抗春雷霉素突变的作用，并对获得的抗性突变株的生物固氮特性进行分析。     

Genotypic diversity and symbiotic effectiveness of rhizobia isolated from root nodules of Phaseolus vulgaris L. grown in Tunisian soils

 One hundred and sixty isolates of rhizobia were sampled from the root nodules of common bean (Phaseolus vulgaris L.) cultivated in Tunisian soil samples originating from three geographically distinct fields. Plasmid profiling was used as a primary method to rapidly screen the isolates, and then 38 plasmid types were recorded among the 160 isolates. A sample representing the majority of plasmid types was chosen for further characterization by restriction fragment length polymorphism (RFLP) analysis of genomic DNA using chromosomal and symbiotic gene probes, and by their ability to nodulate a potential alternative host, Leucaena leucocephala. One third of the isolates showed a high similarity to Rhizobium gallicum isolated from common bean in France, another third showed the same characteristics as the R. etli-R. leguminosarum group, while the remaining isolates could not be related to any of the five species nodulating bean. When reexamined for nodulation, some of these isolates, showing similarities to R. tropici and Agrobacterium with respect to colony morphology and growth in different media, failed to nodulate their original host. The R. gallicum-like isolates, R. etli-like isolates, and R. leguminosarum-like isolates were recovered from regions where bean is frequently grown, while in fields which had not been cultivated with beans for at least the 10 previous years, solely unrecognized taxa of ineffective isolates were recovered. We detected variations in the symbiotic regions, but certain pSym RFLP patterns for nifH were conserved between Tunisian, French, and Austrian populations of bean rhizobia. Evaluation of symbiotic effectiveness showed that R. gallicum-like isolates and R. etli-like isolates were effective, whereas some R. leguminosarum-like isolates were ineffective. Furthermore, effective isolates were also found among the unrecognized taxa. Received: 10 March 1998     

Can introduced and indigenous rhizobial strains compete for nodule formation by promiscuous soybean in the moist savanna agroecological zone of Nigeria?

Promiscuous soybean lines have been bred on the basis that they would nodulate freely without artificial inoculation. However, our recent studies have demonstrated that the indigenous rhizobia are not able to meet their full nitrogen (N) requirement. Rhizobia inoculation might be necessary. We examined the competition for nodule formation among native Rhizobia spp. and two inoculated Bradyrhizobia strains (R25B indigenous strain and a mixture of R25B+IRj 2180A indigenous strain from soybean lines in the savanna of northern Nigeria), their effect on N fixation, and their contribution to the yield of four soybean cultivars, grown in the field in three different agroecological zones in the moist savanna of Nigeria. About 34% of nodules were formed by the mixture of introduced R25B+IRj 2180A, while R25B formed only about 24% of the nodules but did not influence biomass and grain yield production. The indigenous rhizobia strains that nodulated the soybean varieties fixed up to 70% of their accumulated total N, confirming the promiscuous nature of these soybean varieties. Even though these varieties fixed about 75 kg N ha ^-1; this was not enough to sustain their optimum grain yield, as earlier reported. However, the grain yield from inoculated soybean was not significantly higher than that from the uninoculated soybean, showing a degree of competitiveness among the introduced rhizobial strains and the native rhizobia population.     

Nodulation and growth of black locust (Robinia pseudoacacia) on a desurfaced soil inoculated with a local Rhizobium isolate

Nodulation and nitrogen fixation of black locust (Robinia pseudoacacia L.), a legume tree broadly used in Argentina for urban and agricultural afforestation, was studied in hydroponic culture. The development of seedlings inoculated with a local strain of Rhizobium, highly specific for R. pseudoacacia, was also compared with respect to non-inoculated but N-fertilised seedlings. This strain produced fast nodulation and high crop yield and leaf N content. Already nodulated plants with the local Rhizobium strain were assayed for growth in a greenhouse pot experiment with soil from a field where topsoil has been removed for industrial purposes, whilst pots with non-desurfaced soil from the same field were used as control. Non-inoculated plants were also grown in either control or desurfaced soil. Inoculated plants developed better than non-inoculated plants in desurfaced soil, and in control soil as well, suggesting that the symbiosis was able to overcome the nutrient limitation of the desurfaced soil. Non-inoculated plants were nodulated by native soil born Rhizobium, either in control or desurfaced soil, but they showed low final nitrogen leaf content and low nitrogen fixation activity, suggesting that native rhizobia were ineffective.     

Low root zone temperature effects on bean (Phaseolus vulgaris L.) plants inoculated with Rhizobium leguminosarum bv. phaseoli pre-incubated with methyl jasmonate and/or genistein

Abstract

Methyl jasmonate (MeJA) has recently been shown to act as a plant-to-bacteria signal. We tested the hypothesis that pre-induction of Rhizobium leguminosarum bv. phaseoli cells with genistein and/or MeJA would at least partially overcome the negative effects of low root zone temperature (RZT) on bean nodulation, nitrogen fixation and plant growth. Otebo bean plants were grown at constant air temperature (25^oC) and two RZT regimes (25 and 17^oC) and inoculated with R. leguminosarum bv. phaseoli pre-induced with MeJA and/or genistein. Our results indicate that low RZT inhibited nodulation, nitrogen fixation and plant growth. The plants growing at low RZT began fixing nitrogen seven days later compared to those at higher RZT. The low RZT plants had fewer nodules, lower nodule weight, less N fixation, slower plant growth, fewer leaves, smaller leaf area, and less dry matter accumulation comared to plants at a higher RZT. Rhzobium leguminosarum bv. phaseoli cells induced with genistein and/or MeJA enhanced bean nodulation, nitrogen fixation and growth at both optimum and suboptimum RZTs. The results of this study indicate that MeJA improves bean nitrogen fixation and growth at both optimum and suboptimum RZTs, and can be used alone or in combination with genistein to partially overcome the low RZT induced inhibitory effects on nodulation and nitrogen fixation.     

Alleviation of drought stress in the common bean (Phaseolus vulgaris L.) by co-inoculation with Paenibacillus polymyxa and Rhizobium tropici

A greenhouse experiment was performed to evaluate the influence of Rhizobium when co-inoculated with each of two Paenibacillus polymyxa strains, singly and in mixture on growth, nitrogen content, phytohormone levels and nodulation of the common bean (Phaseolus vulgaris L.) under three levels of drought stress. Stress was applied continuously by the control of matric potential (ψ_m) through a porous cup. Bean plants cv. Tenderlake were grown in pots with Fluvic Neosol eutrophic soil under three different ψ_m (S₁ −7.0; S₂ −70.0 and S₃ < −85 kPa). The seeds were inoculated with Rhizobium tropici (CIAT 899) and each of P. polymyxa (DSM 36) and P. polymyxa Loutit (L) singly and in mixture (CIAT 899 + DSM36 + Loutit). Co-inoculation of bean with Rhizobium and both Paenibacillus strains resulted increased plant growth, nitrogen content and nodulation compared to inoculation with Rhizobium alone. This was particularly evident at the most negative ψ_m (S₃ < −85 kPa) we used. Drought stress triggered a change in phytohormonal balance, including an increase in leaf abscisic acid (ABA) content, a small decline in indole acetic acid (IAA) and gibberellic acid (GA₃) and a sharp fall in zeatin content in bean leaves. The content of endogenous Cks decreased under water stress, possibly amplifying the response of shoots to increasing ABA content. We hypothesize that co-inoculation of bean with R. tropici (CIAT 899) and P. polymyxa strains (DSM 36) and Loutit (L) mitigates some of the negative effects of drought stress on bean.     

新开垦土壤上构建玉米/蚕豆-根瘤菌高效固氮模式

为了在新开垦土壤上构建高效种植模式,本文采用温室盆栽和大田试验相结合的方法,选用4种根瘤菌接种方式(保水剂拌种、清水拌种、三叶期灌根和种子丸衣化)接种4种不同蚕豆根瘤菌(NM353、CCBAU、G254和QH258),分析接菌后新开垦土壤上玉米/蚕豆间作体系的生产潜力、地上部氮素吸收和结瘤特性以及生物固氮等方面的优势,拟为该体系筛选出高效的根瘤菌及其接种技术。结果表明:接种NM353后,玉米/蚕豆间作体系中蚕豆籽粒产量比单作平均增加152.84%,而玉米保持相对稳产;以保水剂拌种的方式接种NM353的间作蚕豆地上部氮素积累量最高,蚕豆结瘤数、瘤重、固氮比例和固氮量均高于本试验中其他3种方式接种的根瘤菌。在盛花期和盛花鼓粒期,接种NM353蚕豆的固氮比例比接种CCBAU的分别高19.1%和11.1%,在各个生育时期两者固氮量之间差异均达显著水平;接种NM353与接种其他菌种间固氮量和固氮比例差异更显著。因此,在新开垦土壤上,用保水剂拌种的方式对间作蚕豆接种NM353根瘤菌,构建玉米/蚕豆-根瘤菌高效固氮体系,为新开垦土壤合理开发利用的可持续发展模式。     

Response of Soybean to dual Inoculation with Rhizobium japonicum and Phosphate dissolving Bacteria

A field experiment in silty clay soil was carried out to evaluate the effect of dual inoculation with Rhizobium japonicum and phosphate dissolving bacteria (PDB) on nitrogen and phosphorus uptakes as well as seed yield of soybean. Seed inoculation with PDB before planting did not induce marked enrichment of PDB counts in the rhizosphere. Inoculation with R. japonicum alone increased the dry weight, N-uptake and seed yield significantly. Further significant increases in P-uptake and seed yield were recorded by inoculation with rhizobia and fertilization with superphosphate. However, the inoculation with PDB in combination with R. japonicum in the presence of rock phosphate or superphosphate did not show significant increases in dry weight, N and P uptakes or seed yield as compared with the treatments inoculated with rhizobia alone.     

Characterization of local rhizobia in Thailand and distribution of malic enzymes

TWenty-six isolates were obtained from nodules of various legume plants (Glycine max, Vigna sinensis, Arachis hypogaea, Desmanthus virgatus, Acacia mangium, Centrosema pascuorum, Pterocarpus indicus, Xylia xylocarpa, and Sesbania rostrata) in Thailand. After confirming their nodulation and nitrogen-fixing abilities, they were identified by 16S rRNA gene analysis as Bradyrhizobium japonicum, Bradyrhizobium elkanii, Rhizobium leguminosarum, Rhizobium gallicum, and Rhizobium galegae. Using these local isolates, the distribution of the activities of both NAD⁺-dependent (DME: EC 1.1.1.39) and NADP⁺-dependent (TME: EC 1.1.1.40) malic enzymes was surveyed. The malic enzyme activities were present in all the isolated rhizobia and in other 17 local Bradyrhizobium strains in Thailand. In almost all the rhizobia, the DME activity predominated whereas the TME activity predominated only in the Rhizobium gallicum strains that were major symbionts of Sesbania rostrata. Southern hybridization analysis was performed to survey the distribution of the malic enzyme genes among the local rhizobia, which are similar to those of B. japonicum. DNA probes (ME1 for DME and ME2 for TME) were prepared by polymerase chain reaction (PCR) using degenerated primers from conserved regions of the protein sequences of bacterial malic enzymes. Southern blot analysis with ME1 as a probe showed a single band in about half of the isolates, especially in B. japonicum and R. leguminosarum strains, suggesting the wide distribution of such DME genes among local rhizobia. In contrast, Southern blot analysis with ME2 as a probe detected a single band only in five B. japonicum strains, suggesting that the TME genes, which are similar to those of B. japonicum, would be unique in a group of B. japonicum.     

Inoculation with Native Bradyrhizobia Strains Improved Growth of Cowpea Plants Cultivated on a Saline Soil

Selection of osmotolerant rhizobia gained importance because of increasing soil salinity in many regions. A field experiment in the Cauto Valley was conducted to determine the effect of seed inoculation with two native strains of Bradyrhizobium sp. on biological nitrogen (N)-fixation of cowpea (Vigna unguiculata) on a salt affected soil. Inoculation with both strains resulted in higher numbers of nodules, specific N-fixation, and yields of cowpeas than the control without inoculation. The strain VIBA-1 (Bradyrhizobium liaoningense) was partly superior to VIBA-2 (Bradyrhizobium yuanmingense). This study indicates that the strains inoculated are competitive against other natural strains and able to nodulate cowpea.     

The effect of acidity on the distribution and symbiotic efficiency of rhizobia in Lithuanian soils

The distribution and symbiotic efficiency of nodule bacteria Rhizobium leguminosarum_bv. trifolii F., Sinorhizobium meliloti D., Rhizobium galegae L., and Rhizobium leguminosarum bv. viciae F. in Lithuanian soils as dependent on the soil acidity were studied in the long-term field, pot, and laboratory experiments. The critical and optimal pH values controlling the distribution of rhizobia and the symbiotic nitrogen fixation were determined for every bacterial species. The relationship was found between the soil pH and the nitrogen-fixing capacity of rhizobia. A positive effect of liming of acid soils in combination with inoculation of legumes on the efficiency of symbiotic nitrogen fixation was demonstrated.