Modes of Action of Pantoea agglomerans CPA-2, an Antagonist of Postharvest Pathogens on Fruits

Pantoea agglomerans CPA-2 is an effective antagonist against the postharvest pathogens Penicillium digitatum and Penicillium italicum on citrus fruits but its mode of action is unknown. Possible mechanisms studied in this work were antibiosis, induced resistance, competition and production of chitinolytic enzymes. P. agglomerans CPA-2 was unable to produce antibiotics or chitinolytic enzymes under the conditions tested. Induction of resistance by P. agglomerans CPA-2 was studied in oranges by measuring phenylalanine ammonia lyase and peroxidase enzyme activity in the orange peel at different time points after inoculation with the antagonist and/or the pathogen. No significant augmentation of enzyme activity after inoculation of oranges with P. agglomerans CPA-2 in the presence or absence of the pathogen was observed. P. agglomerans was effective only when it is in close contact with the pathogens. Competition for nutrients was studied using tissue culture plates with cylinder inserts, which allowed competition for nutrients to be studied without competition for space since physical contact between pathogen and antagonist was avoided. The presence of P. agglomerans in the tissue culture wells clearly decreased the germination of Penicillium conidia present in the cylinder when diluted orange peel extract or diluted potato dextrose broth was the nutrient source. Germination of Penicillium conidia, however, was almost completely inhibited when pathogen and antagonist were in physical contact. These results indicate that competition for nutrients is one of the modes of action of P. agglomerans CPA-2, but that physical contact between pathogen and antagonist is important for effective control.     

糖对稻曲病菌薄壁分生孢子萌发的影响

 以琼脂平板为萌发基质,测定了10种糖化合物(木糖、果糖、山梨糖、核糖、甘露糖、葡萄糖、麦芽糖、半乳糖、乳糖和蔗糖)对稻曲病菌薄壁分生孢子萌发的影响。结果发现,在浓度为1%时,除蔗糖外,其余9种糖对孢子萌发均有不同程度的抑制作用,其中木糖、果糖、山梨糖和核糖的抑制作用强烈;孢子经木糖或果糖处理6h后,并未丧失活力;多种糖混合仍表现为抑制孢子萌发。当各种糖与起刺激作用的马铃薯煮汁分别混合时,表现出明显相反的两种效应,一是马铃薯汁的刺激作用可掩盖甘露糖、葡萄糖、麦芽糖、半乳糖、乳糖和蔗糖的抑制作用;二是木糖、果糖、山梨糖和核糖的抑制作用可掩盖马铃薯汁的刺激作用。表明外源糖化合物及某些生命活性物质可影响薄壁分生孢子萌发。     

稻曲病菌分生孢子的生物学研究

 本文对稻曲病菌分生孢子的一些生物学特性进行了研究,结果表明,基质养分对分生孢子萌发影响较大,纯水不利于孢子萌发,PSA最适于孢子萌发,葡萄糖则强烈抑制孢子萌发,马铃薯煮汁既可抵消葡萄糖的抑制作用,又可刺激孢子萌发。分生孢子在琼脂面上比在液滴中萌发率高。分生孢子萌发的适宜温度为22~31℃,以28℃最好。分生孢子萌发对pH值敏感,以pH 6~7最适宜。用振荡培养法获取分生孢子,培养10 d后,孢子的萌发力开始下降。分生孢子的存活对水的依赖性强,在水中保存8 d萌发力不变,在100% RH中8 d萌发力略有降低,而在25% RH中5 h萌发力即迅速下降。根据这些特性,作者对分生孢子在田间的动态作了一些推测。     

拮抗菌P1、P5对葡萄灰霉病菌的抑制作用及其抗真菌谱的测定

从葡萄果穗附生微生物区系分离获得的拮抗细菌P1、P5,对葡萄灰霉病菌(Botrytis cinerea)具有很强的拮抗作用。通过对其抑菌效果、机制和抗真菌谱进行测定,结果表明,P1、P5和葡萄灰霉病菌对峙培养5 d后,均产生明显的抑菌带,抑菌带宽度为6.90～9.67 mm,培养10 d后,仍然保持稳定的抑菌效果;P1、P5的发酵原液对葡萄灰霉病菌孢子萌发具有显著的抑制作用,抑制率为97.51%～98.12%;P1、P5对病菌的作用机制表现为使病菌菌丝畸形、原生质浓缩、菌丝断裂、抑制病菌分生孢子的萌发、导致孢子芽管扭曲。对峙培养表明,P1、P5均具有较广的抗真菌谱,对10种供试病原真菌的菌丝生长抑制率都在50%以上。     

Influence of Nutrient and Environmental Factors on Conidial Germination of Potebniamyces pyri

ABSTRACT Potebniamyces pyri is the causal agent of Phacidiopycnis rot, a postharvest disease of pears. Infection of fruit occurs in the orchard, and symptoms develop during storage. Conidial germination of P. pyri in response to nutrient, temperature, wetness duration, relative humidity (RH), and pH was determined in vitro. Conidia germinated by either budding or developing germ tubes in various concentrations of pear juice solutions. The mode of conidial germination was nutrient-dependent. Low nutrient levels favored budding, whereas high nutrient levels favored germ tube development. Conidia germinated at 0 to 30 degrees C but not at 35 degrees C, with optimum temperature between 20 and 25 degrees C. Wetness durations of 4 to 5 h and 6 to 8 h at optimum temperature were required for budding and developing germ tubes, respectively, and 20 to 24 h of wetness was required to reach germination peaks. Regardless of temperature, conidia germinated primarily by budding in 10% pear juice. Secondary conidia, produced by budding of conidia, initially increased their dimensions and later germinated at 0 to 25 degrees C in the same manner as mother conidia. No germination of secondary conidia occurred at 30 degrees C. Germ tubes from conidia elongated at 0 to 25 degrees C but not at 30 degrees C. No germination occurred at 相似文献   

Characterization of fludioxonil-resistant and pyrimethanil-resistant phenotypes of Penicillium expansum from apple

Penicillium expansum is the primary cause of blue mold, a major postharvest disease of apple. Fludioxonil and pyrimethanil are two newly registered postharvest fungicides for pome fruit in the United States. To evaluate the potential risk of resistance development in P. expansum to the new postharvest fungicides, one isolate of each of thiabendazole-resistant (TBZ-R) and -sensitive (TBZ-S) P. expansum was exposed to UV radiation to generate fungicide-resistant mutants. Four fludioxonil highly-resistant mutants (EC(50) > 1,000 microg/ml) and four pyrimethanil-resistant mutants (EC(50) > 10 microg/ml) were tested for sensitivities to thiabendazole, fludioxonil, and pyrimethanil, and fitness parameters including mycelial growth, sporulation on potato dextrose agar (PDA), sensitivity to osmotic stress, and pathogenicity and sporulation on apple fruit. The stability of resistance of the mutants was tested on PDA and apple fruit. Efficacy of the three fungicides to control blue mold incited by the mutants was evaluated on apple fruit. Six fungicide-resistant phenotypes were identified among the parental wild-type isolates and their mutants based upon their resistance levels. All four fludioxonil highly-resistant mutants were sensitive to pyrimethanil and retained the same phenotypes of resistance to TBZ as the parental isolates. All four pyrimethanil-resistant mutants had a low level of resistance to fludioxonil with a resistance factor >15. The two pyrimethanil-resistant mutants derived from a TBZ-S isolate became resistant to TBZ at 5 microg/ml. After 20 successive generations on PDA and four generations on apple fruit, the mutants retained the same phenotypes as the original generations. All mutants were pathogenic on apple fruit at both 0 and 20 degrees C, but fludioxonil highly-resistant mutants were less virulent and produced fewer conidia on apple fruit than pyrimethanil-resistant mutants and their parental wild-type isolates. Compared with the parental isolates, all four fludioxonil highly-resistant mutants had an increased sensitivity to osmotic stress on PDA amended with NaCl, while the pyrimethanil-resistant mutants did not. Pyrimethanil was effective against blue mold caused by fludioxonil-resistant mutants at both 0 and 20 degrees C. Pyrimethanil and fludioxonil reduced blue mold incited by pyrimethanil-resistant mutants during 12-week storage at 0 degrees C but were not effective at 20 degrees C. TBZ was not effective against pyrimethanil-resistant mutants derived from TBZ-S wild-type isolates at room temperature but provided some control at 0 degrees C. The results indicate that: (i) a fitness cost was associated with fludioxonil highly resistant mutants of P. expansum in both saprophytic and pathogenic phases of the pathogen but not pyrimethanil-resistant mutants; (ii) pyrimethanil possessed a higher risk than fludioxonil in the development of resistance in P. expansum; and (iii) triple resistance to the three apple-postharvest fungicides could emerge and become a practical problem if resistance to pyrimethanil develops in P. expansum populations.     

Prestorage heat treatment reduces pathogenicity of Penicillium expansum in apple fruit

Penicillium expansum is one of the main postharvest pathogens of apples in Israel. Heating apple fruit inoculated with P. expansum for 96 h at 38°C completely inhibited decay development. Fruit held for 24 h at 42°C or 12 h at 46°C had significantly reduced decay after an additional 14 days incubation at 20°C, compared with unheated inoculated control fruit. Mycelial growth and percentage spore germination in vitro were inversely proportional to length of time of exposure to various temperatures. The ET₅₀ for spore germination was 42, 34 and 20 h at 38, 42 and 46°C, respectively, while the ET₅₀ for mycelial growth was 48, 44 and 36 h at those temperatures. When Penicillium spores were incubated on crude extract prepared from the peel of apple fruits held 4 days at 38°C, germ tube elongation was significantly reduced, while the walls of the tubes were thicker, compared with germ tubes from spores incubated on crude extract prepared from peel of non-heated fruit. The evidence presented here supports the hypothesis that the effect of heating on the decay of apples caused by P. expansum is not only the result of direct inhibition of fungal germination and growth by high temperature, but is also partly due to the formation of an inhibitory substance in the heated peel.     

Effect of Germination Initiation on Competitive Capacity of Trichoderma atroviride P1 Conidia

ABSTRACT Trichoderma biocontrol isolates are most effective as highly concentrated inocula. Their antagonism to other fungi may be a result of pregermination respiration. In a nutrient-rich medium, almost all Trichoderma atroviride P1 (P1) conidia initiated germination processes and increased respiration, even in dense suspensions. When 1 x 10(7) P1 conidia/ml were coinoculated with 1 x 10(5) Botrytis cinerea conidia/ml, dissolved oxygen fell to <1% within 2 h and the pathogen failed to germinate. More dilute P1 suspensions consumed oxygen slowly enough to allow coinoculated B. cinerea to germinate. On nutrient-poor media, fewer P1 conidia initiated germination. Oxygen consumption by the inoculum and inhibition of B. cinerea were enhanced when P1 conidia were nutrient activated before inoculation. Pregermination respiration also affected competitive capacity of the antagonist on solid substrates, where respiratory CO(2) stimulated germination rate and initial colony growth. These parameters were directly correlated with inoculum concentration (R(2) >/= 0.97, P < 0.01). After initiating germination, Trichoderma conidia became more sensitive to desiccation and were killed by drying after only 2 h of incubation on a nutrient-rich substrate at 23 degrees C. These results indicate that nutrient-induced changes preceding germination in Trichoderma conidia can either enhance or decrease their biological control potential, depending on environmental conditions in the microhabitat.     

Antagonism of Nutrient-Activated Conidia of Trichoderma harzianum (atroviride) P1 Against Botrytis cinerea

ABSTRACT The effect of preliminary nutrient activation on the ability of conidia of the antagonist Trichoderma harzianum (atroviride) P1 to suppress Botrytis cinerea was investigated in laboratory, greenhouse, and field trials. Preliminary nutrient activation at 21 degrees C accelerated subsequent germination of the antagonist at temperatures from 9 to 21 degrees C; at >/=18 degrees C, the germination time of preactivated T. harzianum P1 conidia did not differ significantly from that of B. cinerea. When coinoculated with B. cinerea, concentrated inocula of preactivated but ungerminated T. harzianum P1 conidia reduced in vitro germination of the pathogen by >/=87% at 12 to 25 degrees C; initially quiescent conidia achieved this level of suppression only at 25 degrees C. Application of quiescent T. harzianum P1 conidia to detached strawberry flowers in moist chambers reduced infection by B. cinerea by >/=85% at 24 degrees C, but only by 35% at 12 degrees C. Preactivated conidia reduced infection by >/=60% at 12 degrees C. Both quiescent and preactivated conidia significantly reduced latent infection in greenhouse-grown strawberries at a mean temperature of 19 degrees C, whereas only preactivated conidia were effective in the field at a mean temperature of 14 degrees C on the day of treatment application. An antagonistic mechanism based on initiation of germination in sufficiently concentrated inocula suggests that at suboptimal temperatures the efficacy of Trichoderma antagonists might be improved by conidia activation prior to application.     

The survival and saprophytic competitive ability of the Botrytis spp. antagonist Ulocladium atrum in lily canopies

In 1995, conidia of Ulocladium atrum were applied to a canopy of green lily (Lillium spp.) leaves in order to investigate its survival, colonisation of artificially induced necrotic leaf tissues and competitive ability against Botrytis spp. and naturally occurring saprophytes. U. atrum conidia density cm^-2 at the top and middle canopy levels was not significantly different following application of the antagonist with a propane powered backpack sprayer. In repeat experiments, conidia density on leaves at the lower canopy level was 18% to 20% of that deposited onto leaves at the top of the lily canopy. There was a significant (P < 0.001) linear decline of U. atrum conidia over time and after 21 days conidia density had declined by up to 73%. Germination of U. atrum on green leaves in the field reached a maximum of 81%, seven days after antagonist application. Conidial viability, measured as germination potential, declined slightly (100% to 88%) after seven days exposure to field conditions but there were no further changes in the germination potential even after 21 days of field exposure. The germination potential was not affected by canopy level. The ability of surviving U. atrum conidia to colonise necrotic tissues, artificially induced with paraquat, was measured. U. atrum colonisation was consistently highest on necrotic leaves at the top level of the canopy and consistently lower on leaves from the bottom canopy level. Necrotic leaf colonisation by U. atrum decreased over time from 51% (necrosis induced immediately after antagonist application) to 21% when necrosis was induced 21 days after antagonist application. A significant (P < 0.001) linear relationship (R² = 0.713) between colonisation of necrotic tissues and conidia density prior to induction of necrosis was detected. When necrosis was induced immediately after antagonist application, U. atrum outcompeted commonly occurring saprophytic Alternaria spp. and Cladosporium spp. The ability of U. atrum to significantly reduce colonisation by Alternaria spp. was maintained for up to 21 days. Botrytis spp. did not occur in these field experiments. It was concluded that U. atrum had the ability to survive and persist in the phyllosphere for up to 21 days in the field and provided further evidence that U. atrum has the necessary survival characteristics to be a successful biological control agent of Botrytis spp.     

RETRACTED ARTICLE: Effect of calcium chloride on the biocontrol efficacy of two antagonistic yeasts against <Emphasis Type="Italic">Penicillium expansum</Emphasis> on apple fruit

Two antagonistic yeasts, Candida membranaefaciens and Pichia guilliermondii, were evaluated for the control of the blue mold of apple caused by Penicillium expansum. Dual culture, cell-free metabolite and volatile tests were used for in vitro assay. Yeast strains of two genera inhibited growth of P. expansum; inhibition varied from 30.27% to 44.19% in dual culture, from 79.40% to 90.57% in the volatile metabolite test, and from 72.99% to 88.77% in the cell-free metabolite test. Calcium chloride (2% w/v) significantly inhibited the growth of the pathogen P. expansum, but did not affect the colony-forming units (CFU) of the yeasts C. membranaefaciens and P. guilliermondii in potato dextrose broth. The concentration of yeast suspension influenced spore germination and germ tube growth of P. expansum in vitro, as well as disease incidence and lesion development in fruits. There were significant negative relationships between the suspension concentrations of the yeasts and the growth as well as infectivity of the pathogen. The addition of calcium resulted in lower spore germination rates and slower growth of germ tubes in vitro, as well as in lower disease incidences and smaller lesion diameters compared with treatments with yeast antagonists alone. When yeast cell suspensions reached a concentration of 10⁷ CFU ml^-1, growth of the pathogen was completely limited in vitro, and no infection was found in apple fruits treated with or without calcium. This article has been retracted because part of the data shown has already been published before, by different authors. An erratum to this article can be found at     

稗草生防菌新月弯孢菌株J15（2）的生物学特性

研究了分离自稗草上的一个致病菌新月弯孢Curvularia lunata菌株J15（2）的基本生物学特性。结果表明,该菌菌丝生长、产孢的最适温度为28～32℃,pH为6～8。菌丝生长对光照无要求,黑暗利于增加产孢量,培养至15d可达产孢高峰。碳、氮、磷和硫等元素是该菌菌丝体生长、产孢的必需元素,钾、镁和铁对菌丝体的生长、产孢有极大的促进作用。分生孢子萌发的适宜温度范围为20～35℃,最适温度28℃;适宜培养基初始pH值在4～10之间。在58℃下,分生孢子10min失活。     

Mechanisms involved in the biological control ofBotrytis cinerea incited diseases

Mechanisms involved in the biological suppression of infection and inoculum potential ofBotrytis cinerea are numerous and variable and the involvement of two or more mechanisms has been demonstrated in several systems. Reported combinations include antibiosis with enzyme degradation ofB. cinerea cell walls; competition for nutrients followed by interference with pathogenicity enzymes of the pathogen or with induced resistance; and alteration of plant surface wettability combined with antibiosis. Since germinatingB. cinerea conidia are dependent on the presence of nutrients, competition for nutrients is regarded as important in systems where biocontrol is involved. Conidial viability and germination capacity are also potentially affected by the presence of antibiotics produced by biocontrol agents and present in the phyllosphere. Slower in action are mechanisms involving induced resistance in the host plant and production of hydrolytic enzymes that degradeB. cinerea cell walls. The latter has been demonstrated much more convincinglyin vitro than in the phyllosphere. Biocontrol in established lesions and reduction of sporulation on necrotic plant tissues is a means to minimize the pathogen inoculum.Abbreviations BCA bio-control agent - Bc Botrytis cinerea - PG polygalacturonase - PL Pectin lyase - PME Pectin methyl esterase - PR pathogenesis related - VPD vapour pressure deficit     

玉米灰斑病菌生物学特性研究

 玉米灰斑病菌(Cercospora zeae-maydis Tehon and Doniels)适合分离的培养基有花生叶斑病尾孢菌培养基、PDA、V8汁、V8汁+蔗糖20 g和Richard培养基。适宜菌丝生长的pH 4~12,最适宜的pH 6~8,适宜温度为20~25℃。病菌对葡萄糖、麦芽糖、乳糖和对酵母膏、硝酸钾、牛肉膏的利用好于其它碳源或氮源。分生孢子萌发的温度为20~30℃,pH范围广,RH ≥ 81%以上分生孢子萌发,随湿度增加萌发率增高。营养对孢子萌发无影响。     

Effects of Ca2+ and Mg2+ on Botrytis cinerea and Penicillium expansum in vitro and on the biocontrol activity of Candida oleophila

Calcium salts have been reported to play an important role in the inhibition of postharvest decay of apples and in enhancing the efficacy of postharvest biocontrol agents. Therefore, the present study was conducted in order to examine and compare the effects of calcium and magnesium salts on the germination and metabolism of the postharvest pathogens Botrytis cinerea and Penicillium expansum , and to determine the effects of these salts on the biocontrol activity of two isolates (182 and 247) of the yeast Candida oleophila. Increasing concentrations of CaCl₂ (25–175 mM) resulted in decreased spore germination and germ-tube growth of both pathogens. The greatest effect was observed in the case of B. cinerea. The inhibitory effect could be overcome by the addition of glucose to the germination medium. MgCl₂ (25–175 mM) had no effect on germination or germ-tube growth of either pathogen, indicating that the calcium cation rather than the chloride anion was responsible for the inhibition. The pectinolytic activity of crude enzyme obtained from the culture medium of both pathogens was also inhibited by 25–175 mM CaCl₂, with the greatest effect on the crude enzyme from P. expansum. Biocontrol activity of isolate 182 was enhanced by the addition of 90 or 180 MM CaCl₂, whereas there was no effect on the biocontrol activity of isolate 247. This was apparently due to the inability of isolate 247 to proliferate in apple wounds. It is postulated that enhanced biocontrol activity of isolate 182 of the yeast C. oleophila in the presence of Ca²⁺ ions is directly due to the inhibitory effects of calcium ions on pathogen spore germination and metabolism, and indirectly due to the ability of isolate 182 to maintain normal metabolism in the presence of"toxic" levels of calcium.     

枯草芽孢杆菌BS80-6对苹果采后炭疽病的控病效果及作用机制

为提高枯草芽孢杆菌的生防效果,通过离子束诱变筛选出BS80-6菌株.利用生物测定方法研究BS80-6菌株及不同处理方法在不同贮藏温度下对采后苹果炭疽病的控制效果和作用机制.结果表明,BS80-6能显著提高对苹果炭疽菌的抑制作用.枯草芽孢杆菌BS80-6对苹果果实炭疽病的防治效果以活菌液效果最好,菌丝生长抑制率为80.14%,孢子萌发抑制率为98.65%,控病效果为60.34%;灭菌液和过滤液对苹果炭疽菌也有一定的抑制效果.贮藏温度明显影响BS80-6对苹果炭疽病的防治效果;接种方式对控病效果影响显著,先接拮抗菌再接病菌的防效好于先接病菌再接拮抗菌.     

Differential responses of germinating Venturia inaequalis conidia to kresoxim-methyl

A new class of agricultural fungicides derived from strobilurins act as respiration inhibitors by binding to mitochondrial cytochrome b. The effects of the strobilurin, kresoxim-methyl, on conidia germination, mycelial growth and the protection of apple leaves from scab development were investigated for two isolates of Venturia inaequalis randomly selected from a culture collection. Inhibition of mycelial growth required relatively high doses of kresoxim-methyl (ED₅₀=1 μg ml^-1) for both isolates. In comparison, germination of conidia was highly sensitive for one of the isolates (ED₅₀=0·005 μg ml^-1), while the level of inhibition achieved for the second isolate was 60-fold less (ED₅₀=0·3 μg ml^-1). As deduced from identical sequences of cytochrome b cDNAs prepared from both isolates, the different responses of germinating conidia to kresoxim-methyl were not caused by differences in the sequence of cytochrome b as the target site for strobilurin action. Strong synergistic effects of salicylhydroxamic acid on kresoxim-methyl inhibitory potency suggested that the reduced kresoxim-methyl sensitivity observed for germinating conidia was caused by interference of the alternative respiratory pathway with inhibitor action. However, this mechanism of target site circumvention in germinating conidia had no adverse effects on the protection of apple leaves from scab infection by kresoxim-methyl. © 1998 Society of Chemical Industry     

<Emphasis Type="Italic">Paecilomyces lilacinus</Emphasis>, a potential biocontrol agent on apple rust mite <Emphasis Type="Italic">Aculus,schlechtendali</Emphasis> and interactions with some fungicides <Emphasis Type="Italic">in vitro</Emphasis>

The apple rust mite Aculus schlechtendali (Nal.) (Acari: Eriophyidae), is a main pest in apple-growing areas in Ankara, Turkey, and chemical control applications have some limitations. Entomopathogenic fungi have a potential for biological control of mites. In this study, an entomopathogenic fungus, Paecilomyces lilacinus (Thom) Samson (Deuteromycota: Hyphomycetes), was first isolated from the mite cadavers on Japanese crab apple leaves and pathogenicity of the fungus was observed in different inoculum densities and relative humidities. The pathogen caused up to 98.22% mortality of the mite population. The effects of some fungicides on the entomopathogenic fungus were determined in in vitro studies. Carbendazim, penconazole and tebuconazole were the most effective fungicides on mycelial growth of P. lilacinus, with EC₅₀ values under 3 μg ml⁻¹. In spore germination tests, captan, mancozeb, propineb were the most effective fungicides, followed by tebuconazole, penconazole, nuarimol and chlorothalonil. Sulphur could not inhibit the conidia germination totally at 5,000 μg ml⁻¹. Copper oxychloride and fosetyl-al prevented conidia formation at concentrations above 1,000 μg ml⁻¹.     

微重力对扩展青霉孢子萌发、菌丝生长和致病力的影响

 After being onto 22nd recoverable satellite of China for 18-day spaceflight under microgravity condition,me growth and pathogenicity of PeniciUium expansum were investigated.Spore germination rate of the spaceflight pathogen was insignificantly lower than that of the ground control.After germination,germ tube elongation of spaceflight pathogen was slower,as well as mycelia growth.However,there was no significant difference according to independent-samples T-test.The consistent results were obtained in vivo.The space. flight pathogen exhibited a little weaker pathogenicity in peach fruit.These findings suggested that the microgravity reduced the growth and pathogenicity of P.expansum.but the effect Was not marked.