Responses of Lentil to Co-Inoculation with Phosphate-Solubilizing Rhizobial Strains and Arbuscular Mycorrhizal Fungi

The purpose of this study was to evaluate the responses of lentil (Lens culinariscv. ‘Ziba’) to co-inoculation with arbuscular mycorrhizal (AM) fungi and some indigenous rhizobial strains varying in phosphorus (P)-solubilizing ability in a calcareous soil with high pH and low amounts of available P and nitrogen (N). A factorial experiment with completely randomized block design was conducted under controlled greenhouse conditions. The treatments consisted of (1) three inoculants of Rhizobium leguminosarum bv. viciae strains and a mixed rhizobial inoculant with an effective P-solubilizer strain of Mesorhizobium ciceri, (2) two AM fungal species, Glomus mosseae and Glomus intraradices, (3) two P sources, superphosphate and phosphate rock. Four replications were prepared for each treatment and a related control. After the growth period of three months, the dry matter of shoots plus seeds, their P and N contents, and percent of root colonized by AM fungus were measured. The results showed that the effects of AM fungi, rhizobial strains, and P fertilizers were highly significant (p < 0.01) for all the characteristics studied. The rhizobial strain with P-solubilizing ability showed a more beneficial effect on plant growth and nutrient uptake than the strain without this ability, although both strains had similar effectiveness for N₂-fixation in symbiosis with lentil. Synergistic relationships were observed between AM fungi and some rhizobial strains that related to the compatible pairing of these two microsymbionts. The P-uptake efficiency was increased when P fertilizers were applied along with AM fungi and/or P-solubilizer rhizobial strains.     

Effects of resident rhizobial communities and soil type on the effective nodulation of pulse legumes

Communities of resident rhizobia capable of effective nodulation of pulse crops were found to vary considerably over a range of soil environments. These populations from soils at 50 sites in Southern Australia were evaluated for nitrogen fixing effectiveness in association with Pisum sativum, Vicia faba, Lens culinaris, Vicia sativa, Cicer arietinum and Lupinus angustifolius. The values for nitrogen fixing effectiveness could be related to soil pH as determined by soil type and location. It was found that 33% of paddocks had sufficient resident populations of Rhizobium leguminosarum bv viciae for effective nodulation of faba bean, 54% for lentils, 55% for field pea and 66% for the effective nodulation of the vetch host plant. Mesorhizobium cicer populations were very low with only 7% of paddocks surveyed having sufficient resident populations for effective nodulation. Low resident rhizobial populations (<10 rhizobia g⁻¹ soil) of R. leguminosarum bv viciae and M. cicer were found in acid soil conditions. In contrast, Bradyrhizobium populations increased as soil pH decreased. Inoculation increased faba bean yields from 0.34 to 4.4 t ha⁻¹ and from 0.47 to 2.37 t ha⁻¹ for chickpeas on acid soils. On alkaline soils, where resident populations were large there was no consistent response to inoculation. Observations at experimental field sites confirmed the findings from the survey data, stressing the importance of rhizobial inoculation, especially on the acid soils in south-eastern Australia.     

Cultivar effects on nitrogen fixation in peas and lentils

Increasing nitrogen fixation in legume crops could increase cropping productivity and reduce nitrogen fertilizer use. Studies have found that crop genotype, rhizobial strain, and occasionally genotype-specific interactions affect N fixation, but this knowledge has not yet been used to evaluate or breed for greater N fixation in US crops. In this study five USDA varieties of lentils (Lens culinaris Medik.) and five varieties of peas (Pisum sativum L.) were tested with 13 to 15 commercially available strains of Rhizobium leguminoserum bv. viciae to identify the better N fixing rhizobial strains, crop varieties, and specific pairings. Peas and lentils inoculated with individual strains were grown in growth chambers for 6 week. Plants received (¹⁵NH₄)₂ SO₄ (5 at.%) starter fertilizer to measure N fixation by isotope dilution. Below- and above-ground biomass, numbers of nodules, and the proportion of plant N supplied by fixation (PNF) were determined. The percent of N fixed was significantly affected by crop variety and significantly correlated with number of nodules in both lentils and peas. This implies that one strategy for enhancing crop N fixation is developing varieties that have higher rhizobium infection rates. Total N fixation in lentils was significantly influenced by both crop variety and rhizobial strain. Eston variety lentil and Shawnee variety pea had the highest PNF of 80.8% and 91.3%, respectively. The different strains of R. leguminoserum affected PNF in lentils but not in peas. These findings suggest that N fixation improvement in lentils and peas may be addressed most effectively by breeding crops for greater N fixation hosting capacity.     

Mapping pathways of possible phage-mediated genetic interchange among soil bacilli

Bacillus pumilus (Strain W43) has been shown to sustain the growth of an unusually large number of different phages. From 22 isolates 16 distinguishable phages have been obtained. Phage BPPX which is similar to the defective particle PBSX of B. subtilis is induced by four of the phages. When tested against a variety of Bacillus spp 12 of the non-defective phages had host ranges crossing at least one species line: Phage K13 infected 14 of 25 strains, distributed among six species some of which are considered to be taxonomically distant. It is suggested that the relatively restricted host ranges noted for most Bacillus phages may result from the use. as test organisms, of bacteria isolated from soils ecologically distinct from the source(s) of phage. A genetic “circuit diagram” constructed from the host range table, maps possible genetic connexions between various soil bacilli made possible by the phages. These data are set in the context of recent theories which postulate that viruses are agents of accelerated cell evolution.     

Field response of legumes to inoculation with plant growth-promoting rhizobacteria

Carrier-based (soil/FYM, 1:1) plant growth-promoting rhizobacteria (PGPR) isolates (Bacillus subtilis, Klebsiella planticola and Proteus vulgaris) were tested individually and in combination with Bradyrhizobium japonicum and Rhizobium leguminosarum biovar viciae under field conditions on soybean and lentil crops, respectively, under field conditions. Inoculation of soybean (Glycine max) cv. Pusa 22 with B. subtilis produced maximum nodule number, mass and nitrogenase activity (acetylene reduction activity, ARA) followed by B. japonicum (SB 271). Maximum soybean yield was registered with the coinoculation of B. japonicum and B. subtilis over an uninoculated control. Maximum nodulation in the lentil (Lens culinaris) cv. L 4147 was obtained with a combination of R. leguminosarum (L-12-87) and P. vulgaris inoculation followed by a single inoculation with Rhizobium and B. subtilis. None of the PGPR isolates either singly or in coinoculation with R. leguminosarum could significantly influence the yield of the lentil crop.     

Interactions between bacteriophages and bacteria in soil

The population biology of certain phages for Bacillus species in soil is described. In soil at 37°C the growth profile of B. circulans phage ST1 shows a lag phase, a phase of exponential growth and a plateau phase, much as occurs in monoculture except that the time scale is transposed into hours rather than minutes. Individual strains of replicating bacteria have been identified by a phage typing technique. At 37°C the number of different phage-sensitive strains falls sharply during the first 6 h but has risen again by 10h. After 10 h at 37°C, 76% of the bacterial types scored represent new strains, as judged by their susceptibility to the eleven phages used for typing. The data presented indicate that a sensitive equilibrium exists between phage and host bacterial cells in soil and that this may be altered very quickly under selective pressures.     

Evaluation of a wild lentil collection for resistance to vascular wilt

Vascular wilt caused byFusarium oxysporum f. sp.lentis Vasud. & Srin. is the major disease of the cultivated lentil (Lens culinaris Medikus). Host plant resistance is the most practical method of disease management. Wild lentils represent an unexplored potential source for disease resistance and other characters. Screening 219 accessions of wildLens Miller and 2 accessions ofVicia montbretii Fisch. & Mey. (syn.Lens montbretii (Fisch et Mey) Davis et Plitm.) for resistance to a Syrian isolate of this fungus at the seedling stage was conducted under artificial inoculation in a plastic house. Resistance at the reproductive growth stage was confirmed in pots in a plastic house and in a wilt-sick plot. Three accessions each ofLens culinaris ssp.orientalis (Boiss.) Ponert andL. nigricans M.B. Godr. ssp.nigricans Godr. and 2 ofL. nigricans ssp.ervoides (Brign.) Lad. maintained their resistance at the reproductive growth stage in the plastic house. All accessions ofL. culinaris ssp.odemensis Lad. andV. montbretii were susceptible. However, in the sick-plot only three accessions (ILWL 79 & ILWL 113 ofL. culinaris ssp.orientalis and ILWL 138 ofL. nigricans ssp.ervoides) maintained a good level of resistance. Resistance at the seedling stage was often found in accessions collected from northern and western sites of the distribution of the genus at low elevations. The most resistant accessions in the field at the reproductive growth stage were from Syria and Turkey.     

Effect of the expression of Escherichia coli fhuA gene in Rhizobium sp. IC3123 and ST1 in planta: Its role in increased nodule occupancy and function in pigeon pea

The inability to utilize a fungal siderophore as source of iron nutrition by most of the rhizobial cultures isolated from pigeon pea, could be considered a negative fitness factor since hydroxamate siderophores are found in significant amounts in natural soils. Thus these cultures were engineered to use ferrichrome a prototype of hydroxamate type siderophore. Pigeon pea Rhizobium spp. IC3123 and ST1 harboring Escherichia coli fhuA gene, responsible for uptake of Fe³⁺-ferrichrome, were obtained by transformation with pGR1, a broad host range plasmid carrying the fhuA gene under the control of the lac promoter of E. coli. Expression of fhuA in transformed rhizobial strains IC3123::pGR1 and ST1::pGR1 was confirmed by the ability of the plasmid-bearing strains to utilize iron bound to ferrichrome. Inoculation of pigeon pea plants with fhuA expressing rhizobial strains in pot experiments showed a significant increase in plant growth as well as nodule density as compared to those inoculated with the parent as well as the empty vector-bearing strain. Inoculation of pigeon pea seedlings with IC3123::pGR1 and ST1::pGR1 led to marked increase in shoot fresh weight, nodule number per plant, chlorophyll content of leaves and effective nodule symbiosis when compared with plants inoculated with the parent strains IC3123 and ST1. The positive effect of IC3123::pGR1 and ST1::pGR1 treatment on plant growth was more significantly observed when ferrichrome producing Ustilago maydis, known to secrete ferrichrome, was co-inoculated along with the transformed rhizobia. The presence of fhuA gene in rhizobial strains also led to an increased survival and root colonization.     

Nitrous oxide emissions associated with nitrogen fixation by grain legumes

Nitrous oxide (N₂O) emissions and biological nitrogen (N₂) fixation by grain legumes are two major processes of N transformation in agroecosystems. However, the relationship between these two processes is not well understood. The objective of this study was to quantify N₂O emissions associated with N₂ fixation by grain legumes under controlled conditions. The denitrifying capability of two Rhizobium leguminosarum biovar viciae strains, 99A1 and RGP2, was tested in pure culture in the presence of nitrate and in symbiosis with lentil (Lens esculenta Moench) and pea (Pisum sativum L.), respectively, in sterile Leonard jars. Lentil and pea, either inoculated or N-fertilized, were grown in soil boxes under controlled conditions. Profile N₂O concentration and surface N₂O emissions were measured from soil–crop systems, and were compared with that of a cereal – spring wheat (Triticum aestivum L. ac. Barrie). Results indicated that: 1) neither R. leguminosarum strain, 99A1 or RGP2 was capable of denitrification in pure culture, nor in symbiosis with lentil and pea in sterile Leonard jars, suggesting that introducing these Rhizobium into soils through rhizobial inoculation onto lentil and pea will not increase denitrification or N₂O emissions; 2) soil-emitted N₂O from well-nodulated lentil and pea crops grown under controlled conditions was not significantly different than that from the check treatments, indicating that biological N₂ fixation by lentil and pea was not a direct source of N₂O emissions.     

Cryptic speciation in Lens culinaris

Summary Three crossability groups have been identified in the wild progenitor of lentil, Lens culinaris ssp. orientalis. The common one which is predominant in this taxon and in the cultigen, the unique one, which at present is known only from three populations in southern Turkey and northern Syria and the intermediate one, known from four populations in that general region. Crosses between members of the common and unique groups yield aborted seeds which can be rescued by embryo culture. Members of the intermediate group are cross-compatible with both other groups. Crossability potential seems to be controlled by a few major genes and minor genes of quantitative nature. One population of the unique group is characterized by novel karyotype and chromosome rearrangements and is reproductively isolated from any other population of Lens culinaris. The implication of the three crossability groups and the novel chromosome rearrangement for lentil taxonomy are briefly discussed.     

Rhizobium and phosphorus influence on lentil seed protein and lipid

Root nodulation by rhizobial bacteria and P fertilization may affect seed protein and lipid composition in plants by altering nitrogen (N) and phosphorus (P) nutrition or by eliciting metabolic responses by the host plant. This study was conducted to determine the effects of rhizobium and P fertilization on seed protein and lipid contents and yield of lentil (Lens culinaris Medik). Lentil was grown to maturity in a greenhouse with P levels of 0 (low) and 50 (high) mg kg^‐1 soil with or without inoculation with Rhizobium bacteria. At the low level of P, protein and lipid concentrations and protein contents were significantly higher in inoculated than in uninoculated plants. Seed dry weight and protein concentrations and contents were higher in inoculated than in uninoculated plants at the high level of P. Seed protein/lipid (Pro/L) concentration ratios varied between inoculated and uninoculated plants at both P levels, and was related to the intensity of root nodulation. Lipid and protein contents were highly correlated with P content in lentil seeds. Seed lipid and protein contents were lower at the high level of P in uninoculated than inoculated plants. The data indicate different patterns of seed P accumulation and different relationships between seed P content and protein and lipid contents in inoculated and uninoculated plants. This might indicate that the intensity of nodulation altered the response of seed protein and lipid metabolism to increasing P availability, which affected protein and lipid ratios.     

Effect of root temperature on nodule development of bean, lentil and pea

The rhizobia-legume symbiosis is the main source of fixed nitrogen for many agricultural systems. However, it is inhibited by low soil temperature. To date, research on nodulation has involved either qualitative or destructive analyses. The use of computer-based image analysis potentially allows nodules to be followed during the course of development. Seedlings of bean (Phaseolus vulgaris L.), lentil (Lens culinaris Medik.) and pea (Pisum sativum L.) were transplanted into plastic growth pouches suspended in water baths maintained at 10, 15, 20 or 25 °C. Two days after transplanting, all plants were inoculated with appropriate rhizobial strains. Seven days after inoculation, plant roots were scanned; this was repeated weekly for 7 weeks. Data on nodule length were collected through image analysis. Nodule length was correlated with nodule size and development. There were increases in the precision of estimates of environmental effects through observation of individual nodule development, as opposed to averages for populations of nodules. The effects of root temperature on nodulation and nodule development were observed both in the delayed onset of nodulation and in reduced subsequent nodule growth rate, resulting in effects on final nodule size.     

Characterization of a cowpea (Vigna unguiculata) rhizobiophage and its effect on cowpea nodulation and growth

A cowpea rhizobiophage (JRW 3 phage) from Jamaican soil was isolated and characterized. The phage has a polyhedral head and a non-contractile tail; maximum adsorption of the phage to the host occurred after 5 min. A one-step growth experiment revealed that the latent period, rise period and burst size of JRW3 phage were 12 h, 16 h, and 28 plaque-forming units/cell, respectively. The JRW 3 phage was highly sensitive to heat, but survived well between pH 5 and 8. The phage was stable in EDTA, though completely inactivated in sodium citrate. Host range analysis showed that 7 of the 40Rhizobium andBradyrhizobium strains tested were sensitive to phage infection. The phage significantly reduced nodule numbers and shoot dry weight of cowpea plants when inoculated with rhizobia in combination with the phage.     

Response of wild lentil to Ascochyta fabae f.sp. lentis from Syria

Summary Ascochyta blight induced by Ascochyta fabae f.sp. lentis is a major foliar disease affecting lentil. Screening 248 accessions of the ICARDA wild lentil germplasm collection for resistance to a Syrian isolate of this fungus was conducted under artificial inoculation in a plastic house. The reaction of resistant accessions was confirmed in a second trial. Twenty-four out of 86 accessions of Lens culinaris ssp. orientalis were resistant, as were 12 of 35 accessions of L. culinaris ssp. odemensis, 3 of 35 accessions of L. nigricans ssp. nigricans, 36 of 89 accessions of L. nigricans ssp. ervoides, and all 3 accessions of Vicia montbretii. Sixty-four per cent of resistant sources were from Syria and southeastern Turkey. Disease reaction was uncorrelated both to the altitude of collection and its annual average rainfall. A significant correlation (r = 0.281) between leaflet width and disease reaction was due more to the frequency of the resistant reaction within the narrow-leaved L. nigricans ssp. ervoides than as a function of small leaf area. Disease reaction was uncorrelated with a range of other morphological traits.     

Nodulation competitiveness of Rhizobium leguminosarum bv. phaseoli and Rhizobium tropici strains measured by glucuronidase (gus) gene fusion

Summary The nodulation competitiveness of 17 Rhizobium leguminosarum bv. phaseoli and 3 R. tropici strains was analysed in growth pouches, at pH 5.2 and 6.4. All 20 strains were coinoculated with a gus ⁺ strain of R. leguminosarum bv. phaseoli strain KIM5s. The gus⁺ phenotype, carrying the glucuronidase gene, was used to type nodules directly in the growth pouches. Nodule occupancy ranged from 4% for the least competitive to 96% for the most competitive R. leguminosarum bv. phaseoli strain. The R. tropici strains showed low rates of nodule occupancy at pH 6.4 but their competitiveness improved significantly under acid conditions. CIAT 895 was the only R. leguminosarum bv. phaseoli strain that was less competitive (P<0.05) at the lower pH. The competitiveness of all the other R. leguminosarum bv. phaseoli strains was unaffected by pH. Various physiological and genetic properties of the strains were analysed in search of correlations with nodulation competitiveness. Hybridisation patterns with three different DNA probes (nif KDH, common nod genes, and hup genes) and the metabolism of 53 different C sources were compared. No general correlations were found between hybridisation or growth pattern and competitiveness. The less competitive R. tropici strains had a unique DNA hybridisation pattern and were not able to use shikimate, ferulate, coumarate, or asparagine as C sources. Most of the less competitive R. leguminosarum bv. phaseoli strains could not metabolize either ferulate or coumarate. This might indicate a relationship between nodulation competitiveness and the ability to degrade aromatic compounds.     

Genetic diversity of rhizobia associated with indigenous legumes in different regions of Flanders (Belgium)

We investigated the diversity of rhizobia isolated from different indigenous legumes in Flanders (Belgium). A total of 3810 bacterial strains were analysed originating from 43 plant species. Based on rep-PCR clustering, 16S rRNA gene and recA gene sequence analysis, these isolates belonged to Bradyrhizobium, Ensifer (Sinorhizobium), Mesorhizobium and Rhizobium. Of the genera encountered, Rhizobium was the most abundant (62%) and especially the species Rhizobiumleguminosarum, followed by Ensifer (19%), Bradyrhizobium (14%) and finally Mesorhizobium (5%). For two rep-clusters only low similarity values with other genera were found for both the 16S rRNA and recA genes, suggesting that these may represent a new genus with close relationship to Rhodopseudomonas and Bradyrhizobium. Primers for the symbiotic genes nodC and nifH were optimized and a phylogenetic sequence analysis revealed the presence of different symbiovars including genistearum, glycinearum, loti, meliloti, officinalis, trifolii and viciae. Moreover, three new nodC types were assigned to strains originating from Ononis, Robinia and Wisteria, respectively. Discriminant and MANOVA analysis confirmed the correlation of symbiosis genes with certain bacterial genera and less with the host plant. Multiple symbiovars can be present within the same host plant, suggesting the promiscuity of these plants. Moreover, the ecoregion did not contribute to the separation of the bacterial endosymbionts. Our results reveal a large diversity of rhizobia associated with indigenous legumes in Flanders. Most of the legumes harboured more than one rhizobial endosymbiont in their root nodules indicating the importance of including sufficient isolates per plant in diversity studies.     

Effects of local environmental variables and geographical location on the genetic diversity and composition of Rhizobium leguminosarum nodulating Vicia cracca populations

Different legume populations are known to accommodate different genotypes of Rhizobium leguminosarum. However, in contrast to interspecific diversity and composition, very little is known regarding which environmental factors drive the genetic diversity and genetic composition of a single Rhizobium species. Based on chromosomal and plasmid genes, we quantified the genetic diversity and compositional differences of R. leguminosarum biovar viciae genotypes associated with twenty-four different Vicia cracca populations across a wide environmental and geographical range. Long-term soil nitrogen availability had a positive effect on chromosomal and plasmid diversity, whereas salinity had a negative effect on chromosomal diversity. Soil pH and geographic distance were the main factors driving compositional differences among populations. In contrast to differences in chromosomal composition, differences in the symbiotic plasmid composition were primarily related to geographic distance or unmeasured related environmental factors (e.g. host plant genetic differentiation). We propose different hypotheses to explain how long-term soil nitrogen availability affects rhizobial genetic diversity. Furthermore, our findings demonstrate that ecological processes that are known to operate at the interspecific level do not necessarily result in the same patterns at the intraspecific level.     

Simple method of genomic DNA extraction from Rhizobia in dried nodules of Phaseolus vulgaris for strain differentiation by PCR-based DNA fingerprinting

Repetitive DNA peR fingerprinting of bacterial genomic DNA is a useful tool for typing and differentiation of rhizobial strains. The method was reported to be suitable for strain differentiation of Rhizobia present in individual root nodules of some leguminous plants without the need for isolation and cultivation of the strains, in which rhizobial genomic DNA was extracted directly from each fresh or frozen nodule. We developed a new protocol of rhizobial genomic DNA extraction/purification from dried nodules of Phaseolus vulgaris for generating repetitive DNA peR fingerprints of Rhizobia present in the nodules. The simplified protocol consists of only three major steps, heat extraction of genomic DNA from rhizobial cells prepared from dried nodules, ethanol precipitation of the DNA and Sephadex G-50 column purification of the DNA, and generated fingerprints with good quality for differentiation of Rhizobia strains. The protocol will be useful to examine the nodule occupancy of inoculated rhizobial strains in field experiments.     

Competition and persistence of Rhizobium tropici and Rhizobium etli in tropical soil during successive bean (Phaseolus vulgaris L.) cultures

Strains of Rhizobium tropici IIB, CIAT899 and F98.5, both showing good N₂ fixation, and a R. etli strain W16.3SB were introduced into a field which had no history of bean culture. Plant dilution estimates showed that in the presence of its host (Phaseolus vulgaris cv. Carioca) during the cropping seasons and the subsequent fallow summer periods, the bean rhizobial populations increased from less than 30 to 10³ g^–1 dry soil after 1 year and to 10⁴ g^–1 dry soil after 2 years. In the 1st year crop, the inoculated strains occupied most of the nodules, which resulted in a higher nodulation and C₂H₂ reduction activity. Without reinoculation for the second and third crops, however, little R. tropici IIB was recovered from the nodules and the bean population consisted mainly of R. etli, R. leguminosarum bv. phaseoli, and R. tropici IIA. Reinoculation with our superior R. tropici IIB strains before the second crop resulted in R. tropici IIB occupying the main part of the nodules and a positive effect on nodulation and C₂H₂ reduction activity, but reintroduction of the inoculant strain in the third season did not have any effect.     

Sources of resistance to ascochyta blight in wild species of lentil (<Emphasis Type="Italic">Lens culinaris</Emphasis> Medik.)

Cultivated lentil (Lens culinaris Medik. subsp. culinaris) has a relatively narrow genetic base and many commercial cultivars are susceptible to ascochyta blight caused by Ascochyta lentis Vassilievsky. A total of 375 accessions of six wild species of lentil received from ICARDA and 18 cultivated genotypes were screened for resistance to A. lentis under both field and greenhouse conditions in Saskatoon, Canada. A mixture of three monoconidial isolates of A. lentis was used as an inoculum and the level of infection rated using the Horsfall-Barratt scale (0–11). Accessions with resistance to A. lentis were observed in all wild species except for L. culinaris subsp. tomentosa (Ladiz.) Ferguson et al. showing no resistant accessions. Several consistently resistant accessions were found among entries of L. ervoides (Brign.) Grande and L. nigricans, (M. Bieb.) Godr., both of which belong to the secondary gene pool and a few in L. culinaris subsp. orientalis (Boiss.) Ponert and L. culinaris subsp. odemensis (Ladiz.) Ferguson et al. belonging to the primary gene pool. Some accessions of L. ervoides exhibited lower disease ratings and AUDPC values than the resistant control cv. ‘Indianhead.’ Thirteen accessions, previously reported as resistant to Syrian isolates of A. lentis were also resistant to the Canadian isolates; some also had resistance to anthracnose. The highest frequency of resistance was found in accessions of L. ervoides which originated from Syria and Turkey. These wild accessions represent a useful and untapped source for improving disease resistance in lentil.