Characterization and pathogenicity of Colletotrichum species associated with anthracnose on chilli (Capsicum spp.) in Thailand

Fungal isolates from chilli ( Capsicum spp.) fruits in Thailand that showed typical anthracnose symptoms were identified as Colletotrichum acutatum , C . capsici and C . gloeosporioides . Phylogenetic analyses from DNA sequence data of ITS rDNA and β-tubulin ( tub 2) gene regions revealed three major clusters representing these three species. Among the morphological characters examined, colony growth rate and conidium shape in culture were directly correlated with the phylogenetic groupings. Comparison with isolates of C . gloeosporioides from mango and C . acutatum from strawberry showed that host was not important for phylogenetic grouping. Pathogenicity tests validated that all three species isolated from chilli were causal agents for chilli anthracnose when inoculated onto fruits of the susceptible Thai elite cultivar Capsicum annuum cv. Bangchang. Cross-infection potential was shown by C . acutatum isolates originating from strawberry, which produced anthracnose on Bangchang. Interestingly, only C . acutatum isolates from chilli were able to infect and produce anthracnose on PBC 932, a resistant genotype of Capsicum chinense . This result has important implications for Thai chilli breeding programmes in which PBC 932 is being hybridized with Bangchang to incorporate anthracnose resistance into chilli cultivars.     

Identification of colletotrichum species responsible for anthracnose and root necrosis of strawberry in Israel

ABSTRACT Strawberry anthracnose was observed for the first time in Israel in 1995. The disease reached epidemic proportions in Israeli nurseries and production fields in 1995 and 1996. Using morphological and cultural characteristics, the species responsible for anthracnose was identified as Colletotrichum acutatum. A reliable semi-selective medium, amended with iprodione and lactic acid, was used to isolate the fungus from infected tissues. In addition, C. acutatum was subsequently isolated from necrotic roots of stunted, chlorotic plants that exhibited no symptoms of anthracnose. High levels of the pathogen from naturally infested field soil and perlite growth substrate were quantified from the rhizosphere of diseased plants on the iprodione-amended medium. Both foliar- and rootinfecting isolates were equally pathogenic to strawberry, causing 95 to 100% plant mortality, when inoculated on roots and foliage. In complementation (heterokaryon) tests using nitrate nonutilizing mutants, 113 out of 115 isolates from different plant parts and locations belonged to a single vegetative compatibility group. Arbitrarily primed polymerase chain reaction of genomic DNA using four repetitive-motif primers produced nearly uniform amplified DNA banding patterns for 141 of the Israeli strawberry isolates from different sites, plots, plant tissues, and cultivars. When compared to reference isolates from the US, these band patterns suggested that a single introduction of C. acutatum was responsible for strawberry anthracnose on foliage and necrosis of roots in Israel.     

Development of protocols for detection of Colletotrichum acutatum and monitoring of strawberry anthracnose using real-time PCR

Real-time PCR (TaqMan®) assays were developed for the specific detection and discrimination of Colletotrichum spp., C. acutatum and C. gloeosporioides causing anthracnose in strawberry using the most divergent area of the internal transcribed spacers (ITS1 and ITS2) and 5·8S ribosomal RNA (rRNA) gene region. The specificity of the new assays was tested using DNA from six species of Colletotrichum and nine fungal species commonly found associated with strawberry material, and additionally by comparing the sequences with those from databases using a blast search. The sequences only showed identity with homologous sequences from the desired target organisms. The new assays were 10–100 times more sensitive than conventional PCR methods previously published for the diagnosis of strawberry anthracnose. When real-time PCR was compared with ELISA methods, PCR improved the sensitivity of the identification by obtaining positive results for samples of strawberry plant material that tested negative with ELISA. The development of C. acutatum was monitored using artificially infected strawberry crowns from two strawberry cultivars (Camarosa and Ventana) and a real-time PCR assay specific for this species between January and June 2006. The amount of C. acutatum detected using real-time PCR varied significantly by month ( P  < 0·001), but not by cultivar ( P  = 0·394). The new assays were shown to be useful tools for rapid detection and identification of these pathogens and to allow rapid and accurate assessment of the casual agents of anthracnose in strawberry.     

Hordenine is responsible for plant defense response through jasmonate-dependent defense pathway

Organic agriculture does not rely on synthetic chemical fungicides. An alternative pest management strategy to chemical fungicides is the use of bioactive natural compounds. Hordenine [4-(2-dimethylaminoethyl)] is a phenethylamine alkaloid found in barley. Although hordenine has various pharmacological effects, including antibiotic activity against microorganisms, no studies have been carried out to investigate the inhibitory effects of hordenine on phytopathogenic fungal infection in host plants. Both grape downy mildew and strawberry anthracnose were suppressed by hordenine treatment. Hordenine had no effect on mycelial growth of phytopathogenic fungi, whereas plant defense response through the jasmonate-dependent defense pathway was enhanced in hordenine-treated plants. The concern over environmental pollution has led to the introduction of new pesticides, including bioactive natural compound based pesticide. Hordenine may be used in organic agriculture as an innovative elicitor of plant defense response to downy mildew and anthracnose.     

生物刺激素协同对草莓炭疽病防治及其生长发育的影响

草莓炭疽病是草莓定植时期最易造成死苗的真菌病害。本文研究了生物刺激素（木霉菌、海藻酸、聚谷氨酸）协同灌根对草莓炭疽病的防治以及促生作用。结果表明,与对照相比,木霉菌、海藻酸、聚谷氨酸单剂灌根和木霉菌+海藻酸、木霉菌+聚谷氨酸的协同灌根处理均能显著降低炭疽病造成的死苗率,其中木霉菌+海藻酸的防效最高,可达78.26%。相比对照,所有处理均能显著提高净光合速率、1,5-二磷酸核酮糖羧化酶活性和加氧酶活性等光合指标,以及土壤脲酶活性。协同灌根处理显著提高草莓株高、根茎粗、叶柄长、叶面积等生长指标达15.62%～47.00%。其中木霉菌+海藻酸处理的净光合速率、土壤脲酶活性最高。综合而言,木霉菌与海藻酸协同灌根在促进生长、促进光合、提高土壤供氮水平以及防控草莓炭疽病方面均表现出显著的增效作用,可作为生物防治的一种有效手段。     

Inheritance of Resistance to Colletotrichum acutatum in Fragaria x ananassa

ABSTRACT Anthracnose, caused by Colletotrichum acutatum, is a major disease of the octoploid cultivated strawberry, Fragaria x ananassa The inheritance of high and intermediate level plant resistances to C. acutatum, pathogenicity group 2, was investigated in an 8 x 8 factorial design. A single dominant gene (Rca2) controlled the high-level resistance, although minor genes may also contribute to resistance in cultivars such as Belrubi. The intermediate level of resistance was quantitative and controlled by minor genes. Analysis of 26 genotypes and cultivars from Fragaria spp. showed that the dominant gene was not rare in the germ plasm of F. x ananassa and that anthracnose resistance was also present in other species of Fragaria. These findings have important implications for anthracnose resistance breeding.     

Method to diagnose latent infection by <Emphasis Type="Italic">Glomerella cingulata</Emphasis> in strawberry plants using ethanol

Signs on strawberry leaves with latent infection by Glomerella cingulata became visible by a simple diagnostic method using ethanol immersion treatment (SDEI). Leaves treated with SDEI changed to dark brown or nearly black, and salmon-pink conidial masses were subsequently produced in the acervuli 5–10 days after incubation in moist petri dishes. The formation rate of conidial masses through SDEI was higher as the position of the leaves became lower. Conidial masses were produced more readily and abundantly when SDEI was performed at 28°C than at 22°, 25°, or 31°C. Latent infection was found to last 1–180 days. There was no difference in the time required for conidial production or in the rate of conidial formation regardless of isolate, cultivar, or leaf position. The varietal difference in resistance to strawberry anthracnose did not influence the rate of conidial mass formation after SDEI. SDEI is thus useful for detecting latent infections during the process of selecting disease-free plants.     

Management and cross-infectivity potential of Colletotrichum acutatum causing anthracnose on bell pepper in Florida

Early anthracnose caused by Colletotrichum acutatum has become an increasingly serious disease on green, unripe bell pepper fruit in Florida. This contrasts with earlier reports of anthracnose occurring on bell pepper primarily as a ripe-rot disease of mature, colored pepper fruit caused by Colletotrichum gloeosporioides. Management of anthracnose on green bell pepper fruit using fungicides and a commercial inducer of systemic acquired resistance, acibenzolar-S-methyl (ASM), was evaluated during three seasons. In two of the three trials, all the fungicides tested including azoxystrobin, fludioxonil + cyprodinil, mancozeb, famoxadone + cymoxanil, copper hydroxide, and ASM significantly increased the number of marketable fruit compared with control plants. These trials identified fungicides that could contribute to a successful pest management program on pepper for controlling anthracnose caused by C. acutatum. The cross-infectivity potential of C. acutatum was investigated on tomato and strawberry by in vitro and field inoculation. Anthracnose lesions formed readily on wound-inoculated detached fruits of all hosts in in vitro assays. Under field conditions, after inoculation, anthracnose lesions occurred on pepper fruit but no lesions of anthracnose were found on either ripe or unripe tomato or strawberry fruit in adjacent plots.     

哌珀霉素防治草莓炭疽病的研究

采用菌丝生长速率法,用橘绿木霉Trichoderma citrinoviride TR673在液体发酵过程中产生的一种次级代谢物——哌珀霉素（Peptaibols）,对多种植物病原菌进行了抑菌活性测试。结果显示,哌珀霉素对草莓炭疽病的病原菌胶孢炭疽菌Colletotrichum gloeosporioides有较高的抑菌活性,其EC₅₀和EC₉₀分别为1.480和22.193 mg/L。将含有哌珀霉素的发酵粗提物制成含12%哌珀霉素可湿性粉剂,在上海青浦和金山2个地区,进行防治草莓炭疽病的药效试验。当12%哌珀霉素的施用量≥450 g/hm²时,对草莓炭疽病的防治效果显著高于其他施用量处理和50%多菌灵可湿性粉剂的化学对照处理。在进行“安全性评价”测试时发现,用浓度为900、1800、3600 g/hm²的12%哌珀霉素可湿性粉剂对3个草莓品种进行施药,不仅对生长无影响,也无药害产生。     

Engineering flax with increased flavonoid content and thus Fusarium resistance

Flavonoids are a group of secondary plant metabolites important for plant growth and development, and thus the regulation of their biosynthesis is of special interest. We used a transgenic approach for flavonoid content manipulation. The multigene construct contained the cDNAs for chalcone synthase (CHS), and chalcone isomerase (CHI) and dihydroflavonol reductase (DFR) were prepared. Following flax plants transformation, the levels of the products of the enzyme overproduction were assessed in leaves and seeds. The simultaneous expression of genes resulted in a significant increase in the levels of flavanones, flavones, flavonols and anthocyanins, suggesting those three overproducing enzymes efficiently control the flavonoid route of the phenylpropanoid pathway.The increase in the flavonoid content in the transgenic flax plants might be the reason for observed, enhanced antioxidant capacity of those plants. The increased antioxidative properties of transgenic plants lead to improved resistance to Fusarium, the main pathogen of flax.The changes in phenylpropanoids accumulation in transgenic plants affect cell wall carbohydrate content. Immunochemical studies revealed significant increase in carbohydrates, constituents of pectin and hemicellulose. Since pectins contribute to flax stem retting, the compounds increase might affect fibre production. An increase in pectin and hemicellulose content leads to enhanced disease resistance of those plants.     

微生物制剂对草莓重要真菌病害的作用研究

为筛选防治草莓几种重要真菌病害的高效微生物制剂,本研究测定了以植物免疫蛋白、淡紫拟青霉、枯草芽胞杆菌和粉红黏帚霉等为活性成分的3种微生物制剂对草莓炭疽病、灰霉病和白粉病的防治作用及对草莓生长的影响。结果表明,3种微生物制剂对草莓炭疽病的防效均在65%以上,对灰霉病的防效在30%~55%。对于草莓白粉病害,沃丰康-克线散防治效果较好,达到64.1%,而普绿通粉剂和沃丰康-复合微生物菌剂防效不明显（<40%）。此外,3种微生物菌剂均表现出明显的促生、增产作用,其中沃丰康-复合微生物菌剂效果最为显著,植株干重增加57.3%,草莓干重增加83.1%;施用沃丰康-克线散粉剂植株和果实增幅分别为47.5%和76.1%,普绿通粉剂分别增加17.9%和66.2%。本研究表明,生防制剂可以替代化学农药用于草莓病害防治和有机生产。     

Symptomless infection by strawberry virus 1 (StrV-1) leads to losses in strawberry yields

High-throughput sequencing has opened up possibilities of detecting new plant pathogens, especially viruses, with relative ease. However, information about biological characteristics and relevance to plant production is usually lacking. Recently, a new strawberry cytorhabdovirus named strawberry virus 1 (StrV-1) was described and characterized at a molecular level. The virus can be transmitted by aphids and is commonly found in commercial plantations. Here, we studied the impact of a sole StrV-1 infection on production in two strawberry cultivars—Darselect and Karmen. All plants infected with StrV-1 remained symptomless during the experiment. In general, Darselect was more resilient to StrV-1 infection, and the average yield per plant was nonsignificantly lowered by 11.7% over the 2-year experiment. In contrast, StrV-1 infection in cultivar Karmen led to a significant yield loss of 31.8%, indicating that the genetic background plays a role. While the average fruit weight and diameter were relatively unaffected, lower yields in infected plants stemmed from a significantly reduced number of inflorescences, by 23.6% and 29.6% in Darselect and Karmen, respectively, which resulted in a reduction in the average number of flowers per plant (decreased by 16.5% and 27.3%, respectively), and hence the number of fruits per plant (by 14.5% and 27.6%, respectively). The StrV-1 load was higher in spring than in autumn, and generally higher in the second year after infection, which corresponded to higher losses in the second harvest, especially in cultivar Karmen.     

Use of Green Fluorescent Protein-Transgenic Strains to Study Pathogenic and Nonpathogenic Lifestyles in Colletotrichum acutatum

ABSTRACT Colletotrichum acutatum, which causes anthracnose disease on strawberry, can also persist on several other plant species without causing disease symptoms. The genetic and molecular bases that determine pathogenic and nonpathogenic lifestyles in C. acutatum are unclear. We developed a transformation system for C. acutatum by electroporation of germinating conidia, and transgenic isolates that express the green fluorescent protein (GFP) were produced. Details of the pathogenic and nonpathogenic lifestyles of C. acutatum were determined by using GFP-transgenic isolates. Major differences between colonization-mediating processes of strawberry and of other plants were observed. On the main host, strawberry, the germinating conidia formed branched, thick hyphae, and large numbers of appressoria were produced that were essential for plant penetration. In strawberry, the fungus developed rapidly, filling the mesophyll with dense mycelium that invaded the cells and caused necrosis of the tissue. In nonpathogenic interactions on pepper, eggplant, and tomato, the conidia germinated, producing thin, straight germ tubes. Appressoria were produced but failed to germinate and penetrate leaf tissue, resulting in epiphytic growth without invasion of the plant. Penetration of the plant occurred only several days after inoculation and was restricted to the intercellular spaces of the first cell layers of infected tissue without causing any visible damage. Much of the new fungal biomass continued to develop on the surface of inoculated organs in the nonpathogenic interaction. The differences in fungal development on strawberry compared with the other plant species suggest that signal molecules, which may be present only in strawberry, trigger appressorial germination and penetration of the primary host.     

Induction of a defense response in strawberry mediated by an avirulent strain of <Emphasis Type="Italic">Colletotrichum</Emphasis>

In the strawberry crop area of Tucumán (north-west Argentina) the three species of Colletotrichum causing anthracnose disease (C. acutatum, C. fragariae and C. gloeosporioides) were detected. Among all isolates characterized, one of them identified as C. acutatum (M11) and another as C. fragariae (F7) were selected due to their conspicuous interaction with the strawberry cultivar Pájaro. Whereas isolate M11 produced a strong compatible interaction in cv. Pájaro with clear disease symptoms (DSR = 5.0), the isolate F7 brought about a typical incompatible interaction (DSR = 1.0). When plants of cv. Pájaro were inoculated with F7 prior to the inoculation with M11, the former avirulent strain prevented the growth of the latter virulent pathogen. Experimental evidence indicated that the time elapsed between the first inoculation with the avirulent pathogen and the second inoculation with the virulent one was crucial to inhibit the growth of the latter. The growth of F7 on the plant without provoking damage and the fact that there was no in vitro antagonistic effect between the pathogens, suggests that the avirulent strain triggers a plant defensive response against M11. The defense response was further confirmed by the detection of an early oxidative burst occurring within 4 h after the first inoculation and by the observation of anatomical changes associated with defense mechanisms that lasted 50 days after the inoculation with F7. Results obtained support the hypothesis that the plant resistance against the virulent strain M11 is elicited by one or more diffusible(s) compound(s) produced by the avirulent strain F7.     

棘孢木霉GYSW-6m1对草莓炭疽病的生防机制及其防病促生作用研究

探究棘孢木霉GYSW-6m1对草莓炭疽病的生防机制、盆栽防治效果及其促生作用,为筛选用于草莓炭疽病绿色防控的高效生防菌提供资源及应用技术。采用平皿培养法、显微镜观察法和盆栽灌根法测定了棘孢木霉GYSW-6m1对草莓炭疽病的生防机制、盆栽防效及其对草莓幼苗的促生效果。结果显示,棘孢木霉GYSW-6m1对草莓炭疽菌LC0220具有较强的竞争、抗生和重寄生作用,菌株GYSW-6m1能迅速占领营养空间,对峙培养3、5和7 d对病菌LC0220抑制率分别为68.06%、71.03%和79.03%,病菌LC0220在含有菌株GYSW-6m1发酵代谢产物平板上培养3、5和7 d抑菌率分别为79.18%、75.81%和76.13%,菌株GYSW-6m1挥发代谢物对病菌LC0220菌丝生长几乎没有抑制作用,菌株GYSW-6m1菌丝可通过接触、缠绕和穿透病菌LC0220的菌丝并引起菌丝消解断裂。菌株GYSW-6m1对草莓炭疽病具有较好的盆栽防治效果,处理LC0220+GYSW-6m1防效最显著,防效高达87.23%,处理GYSW-6m1（3 d）+LC0220和LC0220（3 d）+GYSW-6m1防效分别为52.72%和44.76%。棘孢木霉GYSW-6m1对草莓幼苗具有明显的促生效果,株高、根长、植株总鲜重和植株总干重均显著增加,促生率分别为7.96%、10.42%、22.54%和34.21%。棘孢木霉GYSW-6m1可以作为一种重要的生防资源用于草莓炭疽病的防治,具有一定的实用开发潜力。     

Role of the Insect Vector, Pseudotheraptus devastans, in Cassava Anthracnose Disease Development

The aim of this study was to investigate the role of Pseudotheraptus devastans in cassava anthracnose disease transmission and development. P. devastans, Dist (Het. Coriedae) insects were collected from cassava (Manihot esculenta Crantz) field plots at the International Institute of Tropical Agriculture, Ibadan, Nigeria and reared in large cages. The insects were separated at different developmental growth stages of eggs, first to fifth instar nymph, and adults. The different stages of P. devastans showed the presence of Colletotrichum gloeosporioides both externally and internally. Isolates of C. gloeosporioides derived from the insects produced cassava anthracnose disease symptoms (necrotic lesions, wilt and defoliation) 8 weeks after inoculation on two cassava clones. Re-infectivity of cassava plants by the insect-derived fungus established that P. devastans was a potential vector in anthracnose transmission. Except for the first and second instar nymphs, all nymph stages and adult insects produced significant anthracnose symptoms on cassava plants. Defoliation and lesion diameters were greatest using fifth instar nymphs and adult insects. The association between P. devastans feeding and C. gloeosporioides f.sp. manihotis, showed that feeding by P. devastans followed by fungal inoculation and vice versa resulted in more severe anthracnose symptoms than insect feeding or fungal inoculation alone. It was also observed that the influence of P. devastans damage/infection on the development of anthracnose depended on cassava cultivar resistance to both the fungus and the insect feeding.     

Identification of Random Amplified Polymorphic DNA Markers Linked to the Co-4 Resistance Gene to Colletotrichum lindemuthianum in Common Bean

ABSTRACT New cultivars of the common bean (Phaseolus vulgaris) with durable resistance to anthracnose can be developed by pyramiding major resistance genes using marker-assisted selection. To this end, it is necessary to identify sources of resistance and molecular markers tightly linked to the resistance genes. The objectives of this work were to study the inheritance of resistance to anthracnose in the cultivar TO (carrying the Co-4 gene), to identify random amplified polymorphic DNA (RAPD) markers linked to Co-4, and to introgress this gene in the cultivar Rudá. Populations F(1), F(2), F(2:3), BC(1)s, and BC(1)r from the cross Rudá x TO were inoculated with race 65 of Colletotrichum lindemuthianum, causal agent of bean anthracnose. The phenotypic ratios (resistant/susceptible) were 3:1 in the F(2) population, 1:1 in the BC(1)s, and 1:0 in the BC(1)r, confirming that resistance to anthracnose in the cultivar TO was monogenic and dominant. Six RAPD markers linked to the Co-4 gene were identified, four in the coupling phase: OPY20(830C) (0.0 centimorgan [cM]), OPC08(900C) (9.7 cM), OPI16(850C) (14.3 cM), and OPJ01(1,380C) (18.1 cM); and two in the repulsion phase: OPB03(1,800T) (3.7 cM) and OPA18(830T) (17.4 cM). OPY20(830C) and OPB03(1,800T), used in association as a codominant pair, allowed the identification of the three genotypic classes with a high degree of confidence. Marker OPY20(830C), which is tightly linked to Co-4, is being used to assist in breeding for resistance to anthracnose.     

pHBA对菜心炭疽病的诱导抗性及植株生理特性的影响

 以菜心品种为材料,设置不同浓度对羟基苯甲酸(pHBA)处理,并通过人工接种炭疽病菌,研究pHBA对菜心炭疽病的诱导抗性作用机理。结果表明,在菜心生长发育过程中根系可以分泌出pHBA。pHBA对菜心的生长、产量形成和炭疽病的发生有明显的影响。适宜浓度的pHBA处理可促进菜心生长,提高植株对炭疽病的抗性,显著地降低病情指数(DI),提高菜薹产量,但当pHBA积累到一定浓度时,就对菜心植株产生自毒作用,提高DI和降低菜薹产量。炭疽病菌的感染破坏了植株细胞膜,增强细胞膜透性水平,诱导产生病程相关蛋白(几丁质酶、β-1,3-葡聚糖酶)和细胞壁物质(木质素、富含羟脯氨酸糖蛋白(HRGP))。适宜浓度的pHBA处理可抑制炭疽病菌对细胞膜透性的伤害,加强诱导提高几丁质酶活性、β-1,3-葡聚糖酶活性、木质素含量和HRGP含量,引起细胞壁木质化加强,增加了菜心植株对炭疽病的诱导抗病能力,从而阻止病原菌进一步侵入和扩散,抑制炭疽病菌对菜心产量造成的损失。     

Distribution of Pseudomonas solanacearum in the stem tissues of tomato plants with different levels of resistance to bacterial wilt

Ten tomato cultivars, from the USA, Taiwan and French Antilles, were compared for resistance to bacterial wilt as measured by disease and pathogen population invasiveness and density in the stem at the collar and midstem regions. On the basis of disease incidence, cultivars ranked from highly susceptible to totally resistant. By contrast, no significant difference was observed in bacterial population in wilting plants, regardless of the cultivar. All symptomless plants were latently infected at the collar level. Percentage of symptomless plants with bacteria at the midstem level was significantly correlated with the degree of resistance; the more resistant, the lower the stem colonization. Restriction of Pseudomonas solanacearum invasiveness in the vascular tissues of the stem is associated with resistance properties in tomato. This observation may be useful in developing improved criteria for selecting stable resistance to the disease.     

Effects of nitrogen, phosphorus, potassium and calcium nutrition on strawberry anthracnose

The effects of a range of concentrations of four nutrients – nitrogen, phosphorus, potassium and calcium – in fertilizer solutions on the severity of anthracnose on strawberry cv. Nyoho cultivated under a noncirculation hydroponics system were determined after inoculation with Colletotrichum gloeosporioides . Crop growth and tissue nitrogen, phosphorus, potassium and calcium contents of the entire above-ground parts of the plant were also investigated. Elevated nitrogen and potassium concentrations in the fertilizer solution increased disease severity in contrast to phosphorus and calcium. Treatment with either NH₄ or NO₃ nitrogen was not significantly different. The dry weight of the strawberry plants increased significantly with elevated concentrations of nitrogen ( R ² = 0·9078) and phosphorus ( R ² = 0·8842), but was not influenced by the elevated amounts of potassium ( R ² = 0·8587) and calcium ( R ² = 0·6526) concentrations.