Biological Activity of the Isomeric Forms of Helminthosporium sacchari Toxin and of Homologs Produced in Culture

The effect of Helminthosporium sacchari (HS) toxin isomers and related, pathogen-produced compounds on dark CO₂ fixation in HS-susceptible sugar cane leaf slices was investigated. HS toxin consists of a mixture of three isomeric bis-5-O-(β-galactofuranosyl)-β-galactofuranosides (A, B, and C) differing in the position of one double bond in the sesquiterpene aglycone. Maximum inhibition of dark CO₂ fixation in susceptible sugar cane (CP52-68) occurred within 30 to 40 minutes, and amounts necessary to reach 50% inhibition values typically were approximately 1.7 micromolar for natural toxin mixture ( 2:3:5 mixture of isomers A:B:C) and 4, 6, and 0.7 micromolar for isomers A, B, and C, respectively. Other fractions from cultures of the pathogen consist of comparable mixtures of sesquiterpene isomers but have only 1, 2, or 3 galactofuranose units (HS₁, HS₂, HS₃) or two α-glucopyranose units as well as four β-galactofuranose units (HS₆). The lower toxin homologs were not toxic to clone CP52-68, but protected sugar cane from the effects of toxin. Minimum ratios of protectant: toxin giving 95% protection were approximately 50:1, 6:1, and 12:1 for HS₁, HS₂, and HS₃, respectively. HS₂ and HS₃ protected when added up to 12 minutes after toxin as well as when added with or before toxin. Some common plant galactopyranosides were not toxic and did not protect at 500:1 molar excess. The sample of HS₆ was toxic at 500 micromolar, and did not protect against HS toxin. With the availability of purified, homogeneous preparations of HS toxin, homologs, and chemically modified or synthetic analogs, the dark CO₂ fixation assay should prove to be a useful tool for understanding the mode of action of HS toxin.     

Interconversions of aglycone and host-selective toxin from Helminthosporium sacchari

Helminthosporium sacchari, a fungus that causes disease in sugarcane, produces oligosaccharide-sesquiterpene toxins (HS toxins A, B, and C) that are required for infection and disease development. Two free sesquiterpenes were isolated from mycelium but not from culture fluids of the fungus. One sesquiterpene was identified by HPLC and mass spectrometry as an aglycone of HS toxin C and could be obtained by enzymatic hydrolysis of this toxin. The other sesquiterpene appeared to be the 2-keto form of the first compound. The aglycone from toxin C hydrolysis was labelled with tritium by successive treatments with active manganese dioxide, sodium boro[³H]hydride, and lithium aluminium hydride. The labelled compound was fed to cultures of H. sacchari, radioactivity was incorporated into HS toxin C and into lower molecular weight homologues. The results suggest a metabolic route (aglycone → metabolite Y, → HS toxin → metabolite X) for the biosynthesis of HS toxin; metabolites X and Y are lower molecular weight homologues of the toxin.     

Eyespot Disease of Sugarcane : INDUCTION OF HOST-SPECIFIC TOXIN AND ITS INTERACTION WITH LEAF CELLS

Helminthosporium sacchari produces a toxin which is responsible for the symptoms of eyespot disease in Saccharum officinarum. A rapid and highly repeatable bioassay based on increase in conductivity of tissue leachates showed that the interaction of toxin with sugarcane obeys Michaelis-Menten hyperbolic saturation kinetics. There was no evidence for positive or negative cooperation interaction. Resistant and susceptible cultivars of sugar cane had distinctive conductivity characteristics. Co-cultures of H. sacchari and suspension cultures of sugarcane gave up to a 4,000-fold increase in toxin production.     

Conversion of Helminthosporium sacchari Toxin to Toxoids by beta-Galactofuranosidase from Helminthosporium

Helminthosporium sacchari produces a host-selective toxin and structurally related nontoxic compounds, here referred to as `toxoids.' Toxin and the three toxoids were each isolated to a high level of purity and were hydrolyzed under acidic conditions. The released galactose was measured by a galactose oxidase/peroxidase assay. Toxin was found to contain four units of galactose per molecule, as previously reported. Toxoids I, II, and III contained one, two, and three units of galactose, respectively. In cultures of the fungus, toxin concentration peaked at 3 weeks, followed by a rapid decline; as toxin levels fell, the total amount of toxoids increased. An enzyme with β-galactofuranosidase activity was found in small amounts in the cultures of H. sacchari; the enzyme converted toxin to the toxoids in vitro. β-Galactofuranosidase was previously known from very few micro-organisms; therefore, several pathogenic Helminthosporia and other fungi were tested for production. β-Galactofuranosidase activity in culture filtrates and mycelia of H. victoriae, H. maydis, H. carbonum, and H. turcicum was much greater than in filtrates and mycelium of H. sacchari. More work is needed to determine the significance of enzyme production by these fungi. No β-galactofuranosidase was evident from Fusarium oxysporum and Cladosporium cucumerinum.     

Transfer of toxin susceptibility to plant protoplasts via the hemlmintosporoside binding protein of sugarcane.

The eyespot disease of sugarcane is caused by $\text{Helminthosporium sacchari}$. Helminthosporoside, a host-specific toxin produced by $\text{H. sacchari}$, is essential for the pathogenicity of this fungus. The presence of the helminthosporoside-binding protein in sugarcane likewise appears to be essential for susceptibility to the toxin. The results of this report show that leaf cell protoplasts of tobacco and toxin resistant sugarcane effectively adsorbed the toxin-binding protein derived from membranes of susceptible sugarcane. These protoplasts then became susceptible to the helminthosporoside. They also functioned to takeup raffinose, a trisaccharide structurally related to the toxin. Tobacco protoplasts were treated with [¹⁴C] - binding protein, ruptured, and fractionated on a sucrose density gradient column. A peak of radioactivity was associated with the enriched plasma membrane fraction. The results support the hypothesis that the binding protein is the primary recognition site governing susceptibility of sugarcane to helminthosporoside.     

Phenylpropenamide derivatives: anti-hepatitis B virus activity of the Z isomer, SAR and the search for novel analogs

Phenylpropenamides have been reported to be a class of non-nucleoside inhibitors of the hepatitis B virus (HBV). This class of compounds was explored with the objective of developing potent anti-HBV agents, with a novel mechanism of action, that could be combined with nucleos(t)ide analogs currently used to treat HBV infection. To accomplish this objective a series of substituted arylpropenamide derivatives were prepared and the E and Z geometrical isomers were separated. The structural identity of each of the E and Z isomers was determined by single crystal X-ray crystallography. Contrary to previous reports, the activity of this class of molecules resides in the Z isomer. Further structure-activity relationship studies around the active Z isomer identified compounds that displayed potent antiviral activity against HBV with EC₉₀ value of approximately 0.5 μM in vitro. Attempts to develop ring constrained analogs did not lead to active HBV inhibitors.     

Comparison of Activities of the Host-Specific Toxin of Helminthosporium maydis, Race T, and a Synthetic C(41) Analog

It previously had been proposed that the host-selective toxin of Helminthosporium maydis race T consists of a series of unusual linear (C₃₅ to C₄₅)polyketols, of equal toxicity on a weight or molar (10⁻⁸−10⁻⁹) basis. Previous laboratory synthesis of T-toxin analogs was limited to shorter (C₁₅ to C₂₆) versions which possessed the requisite specificity for susceptible corn (Zea mays) but were less toxic on a weight or molar (10⁻⁶−10⁻⁷) basis. In the present study, a C₄₁ analog with four β-ketol units spaced by CH₂ bridges as in native toxin has been synthesized. On a weight or molar basis, it is as effective as native toxin or its purified components in stimulating NADH oxidation of mitochondria from susceptible corn, thus providing firm evidence for the correctness of the proposed structures of T-toxin. Additional support derives from the observation that C₂₄ and C₂₆ analogs with -(CH₂)₄- and -(CH₂)₆- bridges between ketol groups are not as effective in stimulating NADH oxidation as are C₂₃ and C₂₅ analogs with the -(CH₂)₃- and -(CH₂)₅- bridges of native T-toxin.

It was calculated that a single molecule of the C₄₁ analog is at least 300 times more effective in stimulating mitochondrial oxidation than a molecule of the C₂₃ or C₂₅ analogs. This emphasizes the importance of chain length for toxicity, perhaps through perturbation of membrane functions of mitochondria and/or chloroplasts.

     

Serinol phosphate as an intermediate in serinol formation in sugarcane

A novel compound, serinol phosphate, was identified in sugarcane (Saccharum officinarum) clone 51NG97. It was produced by an enzyme-mediated transamination of dihydroxyacetone phosphate with either alanine, glutamate, aspartate, or glutamine serving equally well as an amino donor. Some detectable phosphatase activity was present in crude leaf enzyme preparation that hydrolyzed serinol phosphate. A proposal for a pathway of the biosynthesis of serinol in sugarcane was formulated.

Serinol can serve as an “activator” of toxin production in attenuated cultures of the sugarcane pathogen Helminthosporium sacchari and it is present in susceptible clone 51NG97. Resistant clone H50-7209 does not possess serinol and likewise no dihydroxyacetone phosphate transaminase activity could be demonstrated in enzyme preparations of this clone. The concept of toxin activation in attenuated fungus cultures is briefly discussed relative to disease resistance and susceptibility.

     

Stereochemistry of binding of thiophosphate analogs of ATP and ADP to carbamate kinase, glutamine synthetase, and carbamoyl-phosphate synthetase

Thiophosphate analogs of adenine nucleotides were used to establish the absolute stereochemistry of nucleotide substrates in the reactions of carbamate kinase (Streptococcus faecalis), unadenylylated glutamine synthetase (Escherichia coli), and carbamoyl-phosphate synthetase (E. coli). ³¹P NMR was used to determine that carbamate kinase uses the B isomer of Ado-5′-(2-thioPPP) in the presence of Mg²⁺. The stereospecificity of the reaction with carbamate kinase was not reversed by Cd²⁺ suggesting that the metal ion does not bind to the β-phosphoryl group or that both Mg²⁺ and Cd²⁺ bind to the sulfur atom. Carbamate kinase uses both A and B isomers of Ado-5′-(1-thioPP) with Mg²⁺ and Cd²⁺. We have previously reported that carbamoyl-phosphate synthetase uses the A isomer of Ado-5′-(2-thioPPP) at both ATP sites with Mg²⁺ (Raushel et al., 1978J. Biol. Chem.253, 6627). Current experiments show that the stereospecificity is reversed by Cd^2? and that both A and B isomers are used when Zn²⁺ is present. With Ado-5′-(1-thioPPP), the B isomer is used with Mg²⁺, the A isomer with Cd²⁺, and both isomers with Zn²⁺. Neither carbamate kinase nor carbamoyl-phosphate synthetase utilized Co(III)(NH₃)₄ATP as a substrate and thus we can only speculate that the Δ chelate ring configuration is the chelate structure utilized by carbamoyl-phosphate synthetase (based on the analogy between thiophosphate-ATP analogs and Co³⁺-ATP analogs utilized by hexokinase (E. K. Jaffe, and M. Cohn, 1978Biochemistry17, 652). If the sulfur of the β-phosphoryl of Ado-5′-(2-thioPPP) binds to the metal ion with carbamate kinase, then the Δ chelate ring is also used in this enzyme that catalyzes one of the steps in the overall reaction catalyzed by carbamoyl-phosphate synthetase. Glutamine synthetase reacts with the B isomer of both Ado-5′-(2-thioPPP) and Ado-5′-(1-thioPPP) in the presence of Mg²⁺. When Co²⁺ is used with this enzyme the A and B isomers of both thio-ATP compounds are substrates. Co(III)(NH₃)₄ATP is not a substrate for glutamine synthetase. Glutamine synthetase is therefore different from the two previously mentioned enzymes in that it used the opposite A ring configuration for the metal-ATP chelate.     

Degradation of Alpha-, Beta-, and Gamma-Hexachlorocyclohexane by a Soil Bacterium under Aerobic Conditions

A Pseudomonas sp., isolated from sugarcane rhizosphere soil, readily metabolized not only alpha and gamma isomers of hexachlorocyclohexane, but also the thermodynamically more stable beta isomer, under aerobic conditions. Bacterial degradation of the three isomers led to the accumulation of a transitory metabolite and eventual release of covalently linked chlorine as chloride in stoichiometric amounts.     

Sesquiterpene lactones of Calea zacatechichi and C. urticifolia

Calea zacatechichi yielded the sesquiterpene lactone zexbrevin and a new analog, several analogs of neurolenin B including calein A, two analogs of budlein A and the flavones acacetin and O-methylacacetin. Calea urticifolia contained additional analogs of neurolenin B as well as a series of epoxidation products. Structures were established by spectrometric techniques. The results are contrasted with previous findings.     

Investigation into the ability of Gluconacetobacter sacchari to live as an endophyte in sugarcane

The relatively low numbers and sporadic pattern of incidence of the acetic acid bacterium Gluconacetobacter sacchari with the pink sugarcane mealybug (PSMB) Saccharicoccus sacchariCockerell (Homoptera: Pseudococcidae) over time and from different sugarcane-growing regions do not indicate that Glac. sacchari is a significant commensal of the PSMB, as has been previously proposed. This study was conducted to investigate the hypothesis that Glac. sacchari is, like its closest relative Glac. diazotrophicus, an endophyte of sugarcane (Saccharum officinarum L.). In this study, bothGlac. sacchari and Glac. diazotrophicus were isolated from internal sugarcane tissue, although the detection of both species was sporadic in all sugarcane-growing regions of Queensland tested. To confirm the ability of Glac. sacchari to live endophytically, an experiment was conducted in which the roots of micropropagated sugarcane plantlets were inoculated with Glac. sacchari, and the plantlets were subsequently examined for the presence of the bacterium in the stem cells. Pure cultures of Glac. sacchari were grown from homogenized surface sterilized sugarcane stems inoculated withGlac. sacchari.Electron microscopy was used to provide further conclusive evidence that Glac. sacchari lives as an endophyte in sugarcane. Scanning electron microscopy of (SEM) sugarcane plantlet stems revealed rod-shaped cells of Glac. sacchari within a transverse section of the plantlet stem cells. The numbers of bacterial cells inside the plant cell indicated a successful infection and colonization of the plant tissue. Using transmission electron microscopy, (TEM) bacterial cells were more difficult to find, due to their spatial separation. In our study, bacteria were mostly found singularly, or in groups of up to four cells inside intercellular spaces, although bacterial cells were occasionally found inside other cells.     

Identification and genetic diversity of Fusarium sacchari associated with pokkah boeng disease of sugarcane in Northeast Brazil

Isolates from the Fusarium fujikuroi species complex, mainly F. sacchari, have been reported to be the causal agents of pokkah boeng in sugarcane in Brazil. However, inadequate information was available on the occurrence and genetic diversity of F. sacchari in Northeast Brazil, which is a limiting factor on management. Thus, isolates of F. subglutinans sensu lato from sugarcane plants with symptoms of pokkah boeng were evaluated using the sexual cross-fertility to determine species. All the isolates produced black perithecia when they were crossed with the test isolates of F. sacchari. Three weeks after the crossing, the formation of fertile ascospores cirri was observed. Thirty-four isolates were self-sterile hermaphrodites, while 21 were fertile only as males. Five isolates were homothallic. The effective size [N_e(f)] of the population as a function of the frequency of hermaphrodites and female sterile strains was 95.5%. The F. sacchari isolates were separated into four genetic groups independent of geographic location. The mean of polymorphism among all populations was 79%, and the average unbiased genetic diversity (uh) was considered moderate (0.31). This study in addition to confirming that F. sacchari as the main species associated with pokkah boeng in sugarcane in Northeast Brazil, reveals the relationship of mating type and genetic diversity of F. sacchari. The unrestricted gene flow between regions is probably the best explanation for the low geographic correlation. This knowledge will help in the adoption of management measures with fungicides or resistant cultivars.     

Characteristics of a Galactose-adapted Sugarcane Cell Line Grown in Suspension Culture

Although d-galactose is normally toxic to sugarcane (Saccharum sp.) cells, a cell line that grows on 100 mm galactose has been propagated. Nonadapted cells in a medium containing galactose instead of sucrose accumulate UDP-galactose; these cells also have much lower UDP-galactose 4-epimerase (EC 5.1.3.2) activity than do adapted cells. This enzyme may determine whether or not galactose will cause toxicity symptoms to develop. The growth rate of galactose-adapted cells is similar to most cell lines on several other carbohydrates. The galactose-adapted cells are also similar to sucrose stock cells in cell wall composition and sugar phosphate concentrations, but, like the nonadapted cells, accumulate free galactose.     

Low recovery frequency of <Emphasis Type="Italic">Gluconacetobacter diazotrophicus</Emphasis> from plants and associated mealybugs in Cuban sugarcane fields

This study was aimed to isolate and identify the N₂-fixing bacterium Gluconacetobacter diazotrophicus from 11 sugarcane varieties, grown under field conditions in four Cuban provinces, and from their associated mealybugs Saccharicoccus sacchari. Identification was based on morphological and biochemical tests and PCR-amplification of 16S rRNA genes using species-specific primers. From all sugarcane varieties and numerous mealybug colonies sampled, G. diazotrophicus isolates were recovered from inside sugarcane stems of only three varieties, and one from S. sacchari colony. These four isolates showed acetylene reduction activity in nitrogen-free media and contained nifH genes which were PCR-amplified using specific primers. ERIC-PCR fingerprinting was used to compare the Cuban G. diazotrophicus isolates with type and reference strains of N₂-fixing Gluconacetobacteria. The very low frequency of G. diazotrophicus isolates recovered is probably related with the high doses of nitrogen fertilizers applied to the sugarcane in the Cuban fields for almost 30 years. Some genetic differences, using ERIC-PCR, were detected among G. diazotrophicus strains, which could be related with its source.     

Degradation of Phthalic Acids by Denitrifying, Mixed Cultures of Bacteria

Mixed cultures of bacteria, enriched from aquatic sediments, grew anaerobically on all three isomers of phthalic acid. Each culture grew anaerobically on only one isomer and also grew aerobically on the same isomer. Pure cultures were isolated from the phthalic acid (o-phthalic acid) and isophthalic acid (m-phthalic acid) enrichments that grew aerobically on phthalic and isophthalic acids. Cell suspension experiments indicated that protocatechuate is an intermediate of aerobic catabolism. Pure cultures which grew aerobically on terephthalic acid (p-phthalic acid) could not be isolated from the enrichments, and neither could pure cultures that grew anaerobically on any of the isomers. Cell suspension experiments suggested that separate pathways exist for the aerobic and anaerobic oxidation of phthalic acids. Each enrichment culture used only one phthalic acid isomer under anaerobic conditions, but all isomers were simultaneously adapted for the anaerobic catabolism of benzoate. Cells grown anaerobically on a phthalic acid immediately attacked the isomer under anaerobic conditions, whereas there was a lag before aerobic breakdown occurred, and, for phthalic and terephthalic acids, chloramphenicol stopped aerobic adaptation but had no effect on anaerobic catabolism. This work suggests that phthalic acids are biodegradable in anaerobic environments.     

In vitro generation of somaclonal variant plants of sugarcane for tolerance to Fusarium sacchari

Key message

A combination of in vitro culture and mutagenesis using ethyl methanesulfonate (EMS) followed by culture filtrate-mediated selection produced variant sugarcane plants tolerant and resistant to Fusarium sacchari.

Abstract

Eldana saccharina is a destructive pest of the sugarcane crop in South Africa. Fusarium sacchari PNG40 (a fungal strain harmful to E. saccharina) has the potential to be an endophytic biological control agent of the stalk borer. However, the fungus causes Fusarium stalk rot in sugarcane. In the current study, sugarcane plants tolerant and resistant to F. sacchari PNG40 were produced by exposing embryogenic calli to the chemical mutagen ethyl methanesulfonate (EMS), followed by in vitro selection during somatic embryogenesis and plantlet regeneration on media containing F. sacchari culture filtrates (CF). The incorporation of 100 ppm CF in the culture media at the embryo maturation stage, at germination, or at both, resulted in callus necrosis and consequent reduced plantlet yield. Subsequent trimming of the roots of regenerated plants and their exposure to 1,500 ppm CF served as a further selection treatment. Plants produced from EMS-treated calli displayed improved root re-growth in the presence of CF pressure compared with those from non-treated calli. The tolerance of CF-selected plants was confirmed in greenhouse tests by inoculation with F. sacchari PNG40, re-isolation of Fusarium spp. from undamaged tissue of asymptomatic plants and establishment of the identity of fungal isolates as PNG40 using molecular analysis. The restriction of PNG40 presence to the inoculation lesion in some plants suggested their resistance to the fungus. Genotypes exhibiting symptomless endophytic colonization by PNG40 were identified and will be utilised for testing biological control strategies against E. saccharina.     

Structural and activity changes in three bioactive anuran peptides when Asp is replaced by isoAsp

The Asp and isoAsp isomers of three bioactive peptides, Crinia angiotensin 11 [APGDRIYHPF(OH)], uperin 1.1 [pEADPNAFYGLM(NH₂)] and citropin 1.1 [GLFDVIKKVASVIGGL(NH₂)] were tested for changes in (i) susceptibility towards proteolytic cleavage, (ii) activity (smooth muscle activity for Crinia angiotensin 11 and uperin 1.1 isomers, and antimicrobial activity for the two isomers of citropin 1.1), and (iii) 3D structures in water, trifluoroethanol-d₃/water (1:1) and DPC micelles as determined by 2D nuclear magnetic resonance spectroscopy. Proteolytic cleavage with trypsin was identical for each pair of Asp/isoAsp isomers. Cleavage with chymotrypsin was the same for the Crinia angiotensin and uperin 1.1 isomeric pairs, but different for the two Asp/isoAsp citropin 1.1 isomers. Chymotrypsin cleaved at Phe3 (adjacent to Asp4) for citropin 1.1, but not at Phe3 (adjacent to isoAsp4) for isoAsp citropin 1.1. The smooth muscle activity of the isoAsp isomer of Crinia angiotensin 11 was less than that of the Asp isomer. The smooth muscle activity of isoAsp3-uperin 1.1 is greater than that of the Asp isomer at low concentration (<10⁻⁹ M) but no different from the Asp isomer at concentrations > 10⁻⁹ M. Citropin 1.1 is a wide-spectrum antibiotic against Gram positive organisms, while the isoAsp isomer is inactive against the test pathogens Staphylococcus aureus and Bacillus subtilis. The observed changes in activity are accompanied by changes in the 3D structures of isomers as determined by 2D nuclear magnetic resonance spectroscopy.     

Cyclic fatty esters: Stereochemistry of monounsaturated products from the hydrogenation and reduction of 9-(6-propyl-3-cyclohenxenyl)-8-nonenoic acid or its ester

Diunsaturated, C-18 cyclic fatty acid methyl esters (CFAME) were previously synthesized as model derivatives for characterization and biological evaluation of cyclic fatty acids (CFA) formed in heat-abused vegetable oils. The propyl substituted, diunsaturated CFMAE (I) was selectively reduced to prepare two monounsaturated, positional isomers with the double bond located either in the ester substituent (alkene isomer II) or in the ring (cyclohexene isomer III). The stereochemistry of these monounsaturated products was investigated by capillary GLC and NMR. Capillary GLC showed that each positional isomer was a mixture of two ‘ring’ isomers (i.e. a mixture of two isomers with side chains either cis or trans). The ring double bond in diene I was readily hydrogenated with various metal catalysts, and no cyclohexene isomer III was detected in the product. Platinum oxide poisoned with Ph₃P was the most selective catalyst examined to convert diene I to monoene II. Diimide reduction was the only method foud to reduce selectively the double bond in the ester side chain of diene I. This diimide reduction was facilitated when the Z-double bond in the side chain was isomerized to E-double bond with p-toluenesulfinic acid. Cyclohexene isomer III and alkene isomer II were separated by argentation HPLC. These two isomeric monoenes were characterized by GC-MS, capillary GLC, micro-ozonolysis, IR and NMR. Catalytic hydrogenation with Ph₃P-poisoned PtO₂ and diimide reduction of the diunsaturated cyclic ester may provide useful methods to synthesize and label monounsaturated cyclic fatty esters.     

A novel aryl-hydrazide from the marine lichen Lichina pygmaea: Isolation,synthesis of derivatives,and cytotoxicity assays

A new aryl-hydrazide l-glutamic acid derivative, pygmeine (3), was isolated from a methanolic extract of Lichina pygmaea, a marine lichen. Synthetic derivatives obtained via a two-step coupling of l-glutamic acid with phenylhydrazine moieties were useful to elucidate the structure of 3 and to carry out biological assays. Thus, the cytotoxicity of the ortho-, meta-, and para-hydroxyl isomers along with their respective benzyl intermediates, and a natural methoxylated analog, were evaluated on murine and human melanoma cells (B16, A375). The para-hydroxyl isomer 6 was found to be the most active (IC₅₀ = 1.6 μM) on B16 cells.