Eucalyptus Staigeriana Research Articles

Antifungal potential of essential oils from different botanical sources against Penicillium digitatum: chemical composition and antifungal mechanisms of action by direct contact and volatile

Fungal deterioration is a recurring problem in citrus fruits, and the search for natural preservatives has been widely publicised. Therefore, the in vitro antifungal activity of clove, white thyme, tea tree, Eucalyptus citriodora, Eucalyptus staigeriana, green mandarin, sicilian lemon, sweet orange, and bergamot essential oils (EO’s) was evaluated against the mycelial growth of Penicillium digitatum isolated from contaminated citrus fruits, by direct contact and volatiles action. In addition, the chemical composition and volatile compounds of the EO’s were evaluated by gas chromatography. Clove, white thyme, and Eucalyptus citriodora EO’s inhibited 100% of the mycelial growth of P. digitatum by direct contact and volatile action at concentrations ≤ 2 µL/mL. On the other hand, EO’s from citrus sources could not inhibit 100% of mycelial growth by any mechanism of action. Thus, the clove, thyme, and Eucalyptus citriodora EO’s are promising for developing new ecologically correct products for controlling phytopathogens in citrus fruits.

Effect of carboxymethylcellulose-based edible coatings incorporating essential oil on Botrytis cinerea and strawberry attributes

The highly perishable strawberry is susceptible to postharvest fungal infections, such as gray mold caused by Botrytis cinerea. Current control methods involve synthetic fungicides, which can leave residues, harm the environment, and lead to resistance. There is a rising interest in safe and eco-friendly pesticide alternatives like essential oils (EOs). In the present study, the antifungal activity of the EOs from Eucalyptus staigeriana, Lippia sidoides, and Pimenta pseudocaryophyllus was evaluated against B. cinerea. An in vitro experiment assessed the direct contact between the fungus and various EOs, identifying the most potent antifungal EO. This EO underwent chemical composition analysis, along with investigations into its impact on pathogen morphology, in vivo activity with carboxymethylcellulose (CMC), and effects on postharvest and sensory quality of strawberries. In vitro, L. sidoides EO exhibited the most significant antifungal activity, characterized by its major compound thymol, which induced dehydration and rupture of pathogen hyphae. In the in vivo experiment, strawberries treated with CMC and L. sidoides EO showed reduced disease severity. During the 18-day storage period, the postharvest quality of strawberries demonstrated enhancement, characterized by increased antioxidant activity and decreased mass loss, while exhibiting minimal changes in sensory attributes.

INSECTICIDAL EFFECT OF SOME BOTANICAL OILS ON LARVAE OF CULEX PIPIENS (DIPTERA: CULICIDAE)

Resistance against commonly used insecticides is a serious obstacle against eliminating mosquito-borne diseases. There is a demand for novel safe insecticides to control mosquito populations. This study aimed to evaluate the larvicidal action of essential oils extracted from Citrus limon, Myristica fragrans, Citrus sinensis, Syzygium aromaticum, Carumcarvi, Prunusdulcis, Eucalyptus staigeriana, Rosmarinu sofficinalis, Jasminum officinale, and fixed oil extracted from Linum usitatissimum against Culex pipiens 3rd instar larvae. The LC50 value of lemon oil was the lowest and consequently it has been used for subsequent experiments. Lemon oil was characterised using gas chromatography/mass spectrometry (GC/MS), revealing 36 compounds. Treatment with sublethal lemon oil doses significantly reduced fecundity and fertility of the 3rd instar larvae. Transmission electron microscopy revealed histopathological changes in integument layers of lemon oil-treated larvae. The titre of octopamine was increased in 3rd instar larvae. Additionally, lemon oil demonstrated low cytotoxic effects in WI-38 human cells as evaluated. Given the relative non-lethality of lemon oil to non-targets, it is proposed as a promising and environmentally safe alternative to synthetic insecticides for controlling mosquito populations.

Aloe Vera coated Dextran Sulfate/Chitosan nanoparticles (Aloe Vera @ DS/CS) encapsulating Eucalyptus essential oil with antibacterial potent property

The goal of this work is to encapsulate Eucalyptus staigeriana essential oil in biopolymer matrices, to optimize the biological effects and the antibacterial properties of this oil. In this study, Eucalyptus extract was encapsulated in Aloe Vera coated Dextran Sulfate/Chitosan nanoparticles to form a hydrogel with potent properties. In this study, Eucalyptus extract was loaded on to Aloe Vera coated Dextran Sulphate/Chitosan nanoparticles to obtain a nano-hydrogel with potent properties. The characterization of nanoparticles was evaluated using transmission and scanning electron microscopes, dynamic light scattering, Fourier transform infrared spectroscopy, differential scanning calorimetry and antibacterial activity. The E. staigeriana release profile from the prepared nanoparticles was studied in vitro at a pH of 7.4. The results showed that this nano-carrier controls Eucalyptus release. Aloe Vera coated Dextran Sulfate/Chitosan nanoparticles encapsulated with E. staigeriana inhibited the bacteria by 47.27%. These investigations concluded that E. staigeriana loaded Aloe Vera coated Dextran Sulfate/Chitosan hydrogel could be used as a powerful dressing material to accelerate wound healing.

Essential Oils as In Vitro Ruminal Fermentation Manipulators to Mitigate Methane Emission by Beef Cattle Grazing Tropical Grasses.

There is increasing pressure to identify natural feed additives to mitigate methane emissions from livestock systems. Our objective was to investigate the effects of essential oils (EO) extracts star anise (Illicium verum), citronella (Cymbopogon winterianus), clove bud (Eugenia caryophyllus), staigeriana eucalyptus (Eucalyptus staigeriana), globulus eucalyptus (Eucalyptus globulus), ginger (Zingiber officinale), ho wood (Cinnamomum camphora), melaleuca (Melaleuca alternifolia), oregano (Origanum vulgare) and white thyme (Thymus vulgaris) on in vitro methane emissions from four rumen-cannulated Nellore cattle grazing a tropical grass pasture as inoculum donors. The semi-automated gas production technique was used to assess total gas production, dry matter degradability, partitioning factor, ammoniacal nitrogen, short-chain fatty acids and methane production. All essential oils were tested in four doses (0, 50, 250 and 500 mg/L) in a randomized block design, arranged with four blocks, 10 treatments, four doses and two replicates. Within our study, oregano and white Thyme EO reduced net methane production at 250 mg/L, without affecting substrate degradation. Essential oils from oregano and white thyme have the potential to modify ruminal fermentation and suppress rumen methanogenesis without negative effects on feed digestibility, indicating promise as alternatives to ionophores for methane reduction in beef cattle.

Control of anthracnose (Elsinoë ampelina) in grapevines with Eucalyptus staigeriana essential oil

Control of anthracnose (Elsinoë ampelina) in grapevines with Eucalyptus staigeriana essential oil

Antifungal activity of essential oil from Eucalyptus staigeriana against Alternaria alternata causing of leaf spot and black rot in table grapes.

Alternaria alternata causes leaf spot and black rot diseases in leaves and grapes of grapevines, respectively, and leads to huge economic losses in table grapes production. As natural antifungal agents, essential oils (EOs), which are generally recognized as safe substances, shows strong antifungal activity against fungal phytopathogens. The aim of this study was to determine the chemical composition of Eucalyptus staigeriana EO and its in vitro and in vivo effects against A. alternata. The major compounds of E. staigeriana EO were citral (34.32%, of which 21.83% geranial and 12.49% neral), limonene (20.60%) and 1,8-cineole (12.33%). E. staigeriana EO exhibited the highest inhibitory activity on mycelial growth and conidial germination at 1 µL mL-1. Moreover, the EO was able to reduce the incidence and severity of leaf spot disease in leaves and black rot disease in table grapes caused by A. alternata. These results represent a possible alternative to reduce the use of synthetic molecules for the control of diseases in postharvest of table grapes and in vineyard.

Óleo essencial de Eucalyptus staigeriana no controle de podridões fúngicas no período pós-colheita e na análise sensorial de uvas

Abstract The objective of this work was evaluate the effect of Eucalyptus staigeriana essential oil on Colletotrichum gloeosporioides and Greeneria uvicola mycelial growth and conidia germination on grapes, as well as its potential for the control of postharvest rot diseases and its effect on the organoleptic properties of grapes. The essential oil (EO) showed in vitro antifungal activity against both pathogens, with fungicidal effect on mycelial growth and on conidia germination at the concentrations of 1.0 and 0.5 μL mL−1, respectively. The EO volatile compounds had a fungistatic effect on the mycelial growth of C. gloeosporioides and a fungicidal effect on G uvicola. At postharvest, the EO reduced the incidence of ripe rot up to 75% and 86% in the preventive and curative treatments, respectively, and the incidence of bitter rot up to 54% in the curative treatment. Since the EO does not affect significantly grape sensory properties, it does not affect the consumption intention of grapes treated with the EO. The EO of E. staigeriana is efficient in the in vitro control of both pathogens; moreover, it is also efficient in the control of the incidence of postharvest fungal rot diseases, mainly in the curative treatment.

Eucalyptus staigeriana essential oil can control downy mildew in grapevine

Downy mildew (Plasmopara viticola) is the primary disease in viticulture worldwide, and your control requires synthetic fungicides applications to avoid quality and yield loss in the grapevines. However, alternatives to reduce synthetic fungicides are needed to ensure the consumer’s health and the environment. Essential oils (EOs) are amongst the most promising natural plant protection alternatives because of their antifungal properties on several crop diseases. The present study objective was to determine the effect of Eucalyptus staigeriana EO in vitro and in vivo against P. viticola. The EO exhibited the highest activity in vitro, inhibiting 90% of the incidence and severity of disease caused by P. viticola in leaves of grapevines in the greenhouse. In the field (in vivo), treatment with EO could not control the disease; however, treatment with EO in consortium with conventional treatment reduced approximately 50% of the incidence and more than 90% of the severity of downy mildew disease in leaves, decreasing the application of synthetic fungicides by 50%.

Nanoparticles as Alternatives for the Control of Haemonchus contortus: A Systematic Approach to Unveil New Anti-haemonchiasis Agents

Haemonchus contortus is an infectious gastrointestinal nematode parasite of small ruminants. This study addresses the in vitro/in vivo anti-haemonchiasis potential, toxicological effects, and mechanism of action of nanoparticles. Online databases were used to search and retrieve the published literature (2000 to 2021). A total of 18 articles were selected and reviewed, out of which, 13 (72.2%) studies reported in vitro, 9 (50.0%) in vivo, and 4 (22.2%) both in vitro/in vivo efficacy of different nanoparticles. Mostly, organic nanoparticles (77.7%) were used including polymeric (85.7%) and lipid nanoparticles (14.3%). The highest efficacy, in vitro, of 100% resulted from using encapsulated bromelain against eggs, larvae, and adult worm mortality at 4, 2, and 1 mg/ml, respectively. While in vivo, encapsulated Eucalyptus staigeriana oil reduced worm burden by 83.75% and encapsulated Cymbopogon citratus nano-emulsion by 83.1%. Encapsulated bromelain, encapsulated Eucalyptus staigeriana oil, and encapsulated Cymbopogon citratus nano-emulsion were safe and non-toxic in vivo. Encapsulated bromelain damaged the cuticle, caused paralysis, and death. Nanoparticles could be a potential source for developing novel anthelmintic drugs to overcome the emerging issue of anthelmintic resistance in H. contortus. Studies on molecular effects, toxicological consequences, and different pharmacological targets of nanoparticles are required in future research.

English

Small ruminants farming is a traditional activity mostly practiced by local populations in developing countries since several centuries. Nowadays, due to many biotic and climatic factors, it faces various problems which damage smallholders’ income especially those related to gastrointestinal parasites. In opposite to the chemical drugs use in controlling those parasites, medicinal plants have been investigated with fewer side effects on both the meat quality and the environment. This current study aimed at reviewing Haemonchus contortus prevalence in small ruminants across the world and present medicinal plants that have been investigated in the last decades. H. contortus is identified as the most significant nematode parasite in small ruminants due to its high prevalence reported by many studies. Its presence in small ruminants results in a loss of feed absorption and disturbance of nutrient metabolism, which lead to poor performance and significant economic loss in the herds, especially in rural areas of developing countries. For the past decades, its control was mainly based on the use of chemical anthelmintics; whose use has been limited due to several factors like the irrational and misuse. Recently, the use of medicinal plants has been identified as alternatives methods of its control with conclusive results. Parts of plants or the whole plants of several plant species were reported to be relevant to control H. contortus infection in small ruminants such as: Bridelia ferruginea, Mitragyna inermis, Combretum glutinosum, Hagenia abyssinica, Chenopodium ambrosioides, Leucaena leucocephala, Phytolacca icosandra, Eucalyptus staigeriana, Carica papaya, Newbouldia laevis and Zanthoxylum zanthoxyloïdes. Key words: Economic losses, gastrointestinal nematodes, chemical anthelmintics, medicinal plants, poor performance.

Efficacy of Essential Oils in In Vitro Control of Acinetobacter baumannii

Although Acinetobacter baumannii is common in nature and considered human skin and respiratory tract commensal, it has also been associated to serious infectious diseases, such as pneumonia, urinary tract infection, endocarditis, wound infection, meningitis and septicemia. The present study aimed to evaluate the intrinsic antimicrobial activity of essential oils on the Acinetobacter baumannii ATCC 17978 strain. We used essential oil of rosemary, artemisia, cinnamon, camphor, citronella, Indian clove, Eucalyptus globulus, Eucalyptus staigeriana, Eucalyptus citriodora, tea tree (also known as melaleuca), mint, oregano and sage, in concentrations that ranged from 0 to 100%. The antibacterial activity was determined through the broth microdilution method and through bactericidal kinetics of essential oils. All the oils performed antibacterial activity against Acinetobacter baumannii. citronella, Eucalyptus staigeriana and mint oils presented lower minimum inhibitory concentration and minimum bactericidal concentration. Oregano, tea tree and Indian clove oils presented higher bacterial death rate, and they canceled the microbial load of A. baumannii in up to 60 minutes. Keywords: Medicinal plants, Essential compounds for treatment, Antimicrobials, Mechanisms of action, Acinetobacter baumannii, Infectious diseases. Read more →

Essential oil as sustainable alternative for diseases management of grapes in postharvest and in vineyard and its influence on wine

This study aimed to determine the chemical composition of Eucalyptus staigeriana essential oil (EO) and its effect in vivo against Botrytis cinerea and Colletotrichum acutatum in postharvest of grapes and in a vineyard. Moreover, grapes collected from de field experiments were used to evaluate the impact of the alternative control on the alcoholic fermentation and wine composition. The major compounds of E. staigeriana EO were citral (30.91%), 1,8-cineole (24.59%), and limonene (19.47%). In the postharvest experiment, EO was efficient, reducing the incidence and severity of disease caused by B. cinerea and the incidence of disease caused by C. acutatum, both in preventive and curative treatment. Moreover, this EO reduced the incidence and severity of gray rot caused by B. cinerea and the severity of ripe rot caused by C. acutatum in the field. The alternative control did not significantly influence the alcoholic fermentation and volatile composition of wines, except for the residual presence of 1,8-cineole that can contribute to the aroma complexity of ‘Isabella’ wine. These results are promising and indicate that E. staigeriana EO might be further investigated as a natural alternative to control fungal rots on grapes.

Essential oils from Eucalyptus staigeriana F. Muell. ex Bailey and Eucalyptus urograndis W. Hill ex Maiden associated to carboxymethylcellulose coating for the control of Botrytis cinerea Pers. Fr. and Rhizopus stolonifer (Ehrenb.:Fr.) Vuill. in strawberries

The fungi Rhizopus stolonifer (Ehrenb.:Fr.) Vuill. (R. stolonifer (Ehrenb.:Fr.) Vuill.) and Botrytis cinerea Pers. Fr. (B. cinerea Pers. Fr.) are causal agents of important post-harvest diseases in strawberries. For their control, the essential oil (EO) extracted from the leaves of Eucalyptus staigeriana Muell. ex Bailey (E. staigeriana F. Muell. ex Bailey) and Eucalyptus urograndis W. Hill ex Maiden (E. urograndis W. Hill ex Maiden) can be a good alternative to the synthetic fungicides. Thus, the antifungal activity of the EOs from E. staigeriana F. Muell. ex Bailey and from E. urograndis W. Hill ex Maiden was evaluated in vitro on the fungi B. cinerea Pers. Fr. and R. stolonifer (Ehrenb.:Fr.) Vuill.. The effects on pathogen morphology and the in vivo antifungal activity of the EO that presented the highest activity in vitro, associated with carboxymethylcellulose (CMC), were also verified. The chemical composition of the essential oils evaluated was determined. The EO from E. staigeriana F. Muell. ex Bailey was the most efficient in the in vitro assays on the two fungi. This EO, whose major compound is limonene, caused dehydration and rupture of the fungal hyphae, suggesting its action is on the cell wall and membrane. The incorporation of this EO to CMC in the curative way reduced the severity of soft rot, caused by R. stolonifer (Ehrenb.:Fr.) Vuill.; nevertheless, it did not present an effect on the inhibition of B. cinerea Pers. Fr.. These results suggest that E. staigeriana F. Muell. ex Bailey EO, associated to CMC coating, can be an alternative to the use of synthetic fungicides for the control of R. stolonifer (Ehrenb.:Fr.) Vuill., mainly.

Eco-friendly synthesis of an alkyl chitosan derivative

Chitosan (CH) was N-alkylated via Schiff base formation and further reduced via sodium borohydride. The reaction was carried out at room temperature, in a homogeneous aqueous medium, using as a source of alkyl group an essential oil (Eucalyptus staigeriana) containing an unsaturated aldehyde (3,7-dimethylocta-2,6-dienal). Derivatives were characterized by Infrared Spectroscopy, proton and carbon Nuclear Magnetic Resonance, XRD, particle size distribution and zeta potential. Chitosan hydrophobization evidence was given by FTIR as new bands at 2929 cm−1 due to methyl groups, along with the presence of strong band at 1580 cm−1 owing to N substitution. Moreover, carbon and proton NMR corroborated the insertion of methyl groups in chitosan backbone. The degree of substitution was found to be in the range 0.69–1.44. X-ray diffractograms revealed that the insertion of alkyl substituents in chitosan backbone led to a less crystalline material. Data from antibacterial activity revealed that chitosan and derivatives were effective against Gram-positive bacteria, whereby derivatives exhibited greater inhibitory effect than CH. Derivatives are likely candidates for use as carriers for active principles of interest of food, pharmacy and medicine.

Alternative control of grape rots by essential oils of two Eucalyptus species.

Essential oils (EOs) are volatile natural compounds produced by plant secondary metabolism, and some of them exhibit antimicrobial activity. The objective of the present study was to determine the chemical composition the EOs of Eucalyptus staigeriana and Eucalyptus globulus, and their effect in vitro and in vivo against Botrytis cinerea and Colletotrichum acutatum, the most important fungal rot diseases of grapes. Moreover, grapes collected from field experiments were used to evaluate the impact of the alternative control on the alcoholic fermentation and wine composition. The major compound of E. staigeriana EO were citral 30.91% (19.74% geranial, 11.17% neral), 1.8-cineole (24.59%) and limonene (19.47%), while 1.8-cineole represented 68.26% of E. globulus EO. The two EOs showed in vitro antifungal activity against both pathogens. Eucalyptus staigeriana EO exhibited the highest activity inhibiting mycelial growth (MG) and conidial germination at 0.5 μL mL-1 . Moreover, this EO was able to reduce the incidence and severity of grey rot caused by B. cinerea and the severity of ripe rot caused by C. acutatum The alternative control did not significantly influence alcoholic fermentation, the physicochemical characteristics, and the volatile composition of wines. These results are promising and indicate that E. staigeriana EO might be further investigated as a natural alternative for the control of fungal rots on wine grapes. © 2019 Society of Chemical Industry.

Molecular Encapsulation of Eucalyptus staigeriana Essential Oil by Forming Inclusion Complexes with Hydroxypropyl-β-Cyclodextrin

This study investigated the inclusion complex of Eucalyptus staigeriana essential oil (ESEO) formed with hydroxypropyl-β-cyclodextrin (HPCD) as a model system to determine which essential oil (EO) components were encapsulated in the complex. The ESEO/HPCD inclusion complex was prepared in aqueous solution and consequently analyzed by gas chromatography-mass spectrography (GC-MS), ultraviolet–visible spectroscopy (UV-VIS), thermogravimetric–differential thermal analysis (TG-DTA), 1H ROESY NMR, and molecular modeling. The results clearly showed that inclusion complexes had been successfully formed between ESEO and HPCD. It was demonstrated that the EO components did not form an inclusion complex with HPCD in equal proportions, but certain EO components were preferentially encapsulated. Components such as alcohols and aldehydes were preferentially encapsulated by HPCD, leading to higher concentrations of these compounds in the complex. This implies that encapsulation of EOs by forming inclusion complexes with HPCD may alter their composition in the complexes with potential changes in their properties such as antimicrobial activity.

Toxicity and repellency of essential oils in the management of Sitophilus zeamais

ABSTRACT This study had the following objectives: to identify and quantify the constituent compounds of essential oils from Cymbopogon winterianus Jowitt, Eucalyptus globulus Labill., Eucalyptus staigeriana F. Muell. ex F.M. Bailey, Foeniculum vulgare Mill., Ocimum basilicum L., Ocimum gratissimum L., and Piper hispidinervum C. DC., investigate their toxicity and repellency to S. zeamais and evaluate the toxicity of P. hispidinervum to immature S. zeamais. Individual tests for each essential oil were conducted with a completely randomized design with five concentrations for each oil and four replicates. Gas chromatography-mass spectrometry revealed the presence of citronellal in C. winterianus, 1,8-cineole in E. globulus, limonene in E. staigeriana, limonene in F. vulgare, linalool in O. basilicum, (E)-anethole in O. gratissimum, and safrole in P. hispidinervum. The median lethal concentration required to kill 50% of the insect (LC50) in contact and ingestion toxicity tests ranged from 5.12 to 78.89 μL 40g-1 corn in P. hispidinervum and C. winterianus, respectively. In adult fumigation tests, the LC50 ranged from 2.1 to 19.4 µL L-1 air, and in immature fumigation tests, the egg stage was susceptible to essential oil, whereas larval and pupal phases were tolerant. All of the oils repelled S. zeamais.

Antimicrobial and antibiofilm activity of the essential oil from dried leaves of Eucalyptus staigeriana

ABSTRACT: In recent years, compounds with biological properties produced by plants have received attention as an alternative to control microorganisms. Essential oils extracted from green leaves of Eucalyptus sp. have been demonstrated to have antimicrobial activities, but so far there are no reports of antimicrobial activity of essential oils extracted from dried leaves of Eucalyptus staigeriana. So, the objectives of this study were to determine the chemical composition of the essential oils obtained from dried leaves of E. staigeriana (EOdlES) and to evaluate in vitro antimicrobial and antibiofilm activities of EOdlES against gram-positive and gram-negative, resistance and multiresistant Enterococcus faecalis isolated from food and clinical samples. The characterization of EOdlES was performed by gas chromatography-mass spectrometry (GC/MS). For this study, 26 bacterial strains were used, which included 11 reference strains and 15 antibiotic resistant and multiresistant E. faecalis strains. Antimicrobial activities of EOdlES against gram-positive and gram-negative were determined using the disc diffusion method. The minimum inhibitory concentration (MIC) value was evaluated by a microbroth dilution technique. The antibiofilm effects were assessed by microtiter plate method. As a result, 21 compounds were identified, being oxygenated monoterpenes (69.58%) the major chemical family. EOdlES showed only antimicrobial activity against gram-positive strains. E. faecalis resistant and multiresistant strains show the lowest MIC (3.12 to 6.25%), when compared with reference E. faecalis strain. EOdlES has the ability to inhibit the biofilm formation, but little or none ability to inhibit the preformed biofilm. This study demonstrates that EOdlES is a promising alternative to control important foodborne and clinic gram-positive resistant bacteria.

Control of Rhizopus stolonifer in strawberries by the combination of essential oil with carboxymethylcellulose

Strawberry has a limiting postharvest shelf life, especially because of soft rot. The antifungal activity of the essential oils (EOs) of Eucalyptus staigeriana, Lippia sidoides and Pimenta pseudocaryophyllus was tested in vitro against plant pathogen Rhizopus stolonifer. The chemical composition of the EO with the highest activity and its effects on pathogen morphology were verified. The in vivo antifungal activity of this EO associated with carboxymethylcellulose (CMC) coating, in preventive and curative applications, was also evaluated. L. sidoides EO presented the highest in vitro antifungal activity. The analysis of the chemical composition of this EO showed a prevalence of the compound thymol and the scanning and transmission electron microscopy showed that L. sidoides EO was able to cause damage to the cell wall and the intracellular components of the pathogen. Strawberries treated with L. sidoides EO associated with CMC presented a reduction in disease severity, especially when treated in a curative way.