The possible consequence of clove, cinnamon, mustard, Mentha piperita, eucalyptus, citronella, camphor, rose, lemon and lemon grass indispensable oils against Fungis identified from areca thenar foliages and palmyra thenar foliage sheath were investigated. The Fungi were identified by 18s rRNA sequencing method. An agar dilution method was employed to find the minimal repressive concentration ( MIC ) of indispensable oils. Zone suppression trials and the repressive consequence of the foliage and sheath dip treated and vapors treated with indispensable oils against those Fungis were examined. With an MIC of 0.
02 & A ; micro ; cubic decimeter milliliter-1Musturd indispensable oil had the strongest repressive consequence. The efficaciousness of mustard oil vapor on the fungous suppression was comparatively higher when compared to the liquid stage. It is besides relatively more possible than chemical antifungals.1. IntroductionIn southeast Asia and India, areca thenar (Areca catechu) leaf sheath dropped of course and Palmyra thenar (Borassus flabellifer) foliage have been traditionally used as an environmentally friendly nutrient helping and packaging stuff for centuries1.
Now a yearss with modern engineering, this natural and stiff stuff is compressed into different forms and used to function and pack nutrient. Over the past 20 old ages, the usage of bio-based packaging stuffs to protract the shelf-life and better the quality of fresh nutrient merchandises has been having increased attending2. Palmyra thenar foliages are employed in doing useful, aesthetic, artistic, originative, culturally attached, cosmetic, functional, traditional, sacredly and socially symbolic and important handcraft merchandises excessively.
The presence and growing of Fungi in the above stuffs may do nutrient spoilage and besides consequences in a decrease in quality and measure3. Aflatoxins produced byAspergillusspecies are known to be powerful hepatocarcinogens in animate beings and worlds. Therefore, the presence of toxigenic Fungis and mycotoxins in nutrients packing and functioning stuffs and handcraft merchandises present a possible jeopardy to human and carnal wellness.
Some chemical compounds are used as antifungals and they are normally made up of 90 % S and are really toxic. Copper sulphate ( bluestone ) is one of the commonly used antifungals among Cu signifiers. Bordeaux mixture a combination of Cu sulphate and calcium hydroxide is an oldest antifungal that successfully used for more than 150 old ages on ornamentals, fruits and veggies. Phosphorous acid another effectual antifungal that acts over the Fungis either by suppressing a peculiar procedure or by bring oning a defense mechanism response in the agent to demo inhibiting activity. Biphenyl is besides a antifungal that has the ability to suppress the monogenesis procedure of Fungi.As the chemical preservatives are considered responsible for many carcinogenic and teratogenic properties every bit good as residuary toxicity, consumers tend to be leery of chemical additives and therefore there is demand for natural and socially more acceptable preservatives4. Considerable involvement has developed on the usage of indispensable oils to efficaciously retard growing and mycotoxin production. A renewed involvement in natural saving appears to be stimulated by present nutrient safety concerns, turning jobs with microbic opposition, and a rise in production of minimally processed nutrient, together with green image policies of nutrient industries.
Numerous surveies have documented the fungicide5, 6effects of works indispensable oils. Natural merchandises may modulate the cellular effects of aflatoxins and grounds suggests that aromatic organic compounds of spices can command the production of aflatoxins7.The usage of the polymerase concatenation reaction ( PCR ) offers great advantage compared with conventional microbiological testing8, 9, 10. Fungal primers specific for the conserved sequence of 18s rRNA cistron common to all Fungis have been used to observe fungous specimens11.The aim of this work is to measure the fungicidal activity of 10 indispensable oils viz. split oil, cinnamon oil, mustard oil, Mentha piperita oil, eucalyptus oil, citronella oil, camphor oil, rose oil, lemon oil and lemon grass oil on Fungi normally found on the areca thenar foliage sheath and palmyra thenar foliage surface and compare them with the consequence of chemical antifungals.2. Materials and methods2.
1 Essential oil and foliage sheathFood grade indispensable oils ( split oil, cinnamon oil, mustard oil, Mentha piperita oil, eucalyptus oil, citronella oil, camphor oil, rose oil, lemon oil, and lemongrass oil ) derived from steam distillment were derived from Eastern Distributors, Coimbatore. The Areca thenar foliage sheath and toddy palm foliages used for doing handcraft merchandises were obtained from Natural Fibre Handicraft Centre, Punnayadi small town, Kanyakumari territory, Tamil Nadu, India.
- Cultures
Three fungous strains were separated from areca thenar foliage sheath and Palmyra Palm foliage surfaces utilizing Agar Plate technique in Potato Dextrose Agar ( PDA ) medium and morphological designation was carried out. The stray Fungis were the major causes of impairment the leaf sheath, foliage and intermediate wet nutrient wrapped in them.
- Preparation of Inocula
Spores of the trial Fungi were obtained from mycelia grown on Potato Dextrose Agar ( PDA ) at 30 & A ; deg ; C for 14 yearss, and were collected by deluging the surface of the home bases with ~5ml unfertile saline solution ( NaCl,8.5 g/l H2O ) incorporating Tween 80 ( 0.1 % v/v ) .
After the spores were counted utilizing a haemocytometer, the suspension was standardized to concentrations of 107spores/ml by dilution made utilizing unfertile H2O before usage. The viability of all strains were checked utilizing quantitative settlement counts at 107CFU/ml.
- Fungal designation
Deoxyribonucleic acid was extracted from the fungous pellet and purified utilizing PureFast & A ; reg ; Fungal Genomic DNA purification kit. Fungal ITS forward and contrary primers were used in the PCR reaction Each 50 milliliter PCR reaction consisted of 2U of Taq DNA polymerase, 10X Taq reaction buffer, 2mM MgCl2, 1 & A ; mu ; cubic decimeter of 10mM dNTPs mix and PCR additives. PCR reactions were run utilizing the undermentioned parametric quantities: ( 1 ) 94 & A ; ordm ; C for 3 min, ( 2 ) 30cycles of 94 & A ; ordm ; C for 1 min, 58 & A ; ordm ; C for 1 min, and 72 & A ; ordm ; C for 1min, and ( 3 ) 72 & A ; ordm ; C for 5 min.
Clean PCR merchandises were sequenced utilizing ITS primers, assembled and edited. Nucleotide sequences were compared to those in the Gene Bank Database with the Basic Local Alignment Search Tool ( BLAST ) algorithm to place known closely related sequences. The 18s rDNA sequences were aligned and phylogenic tree was constructed by neighbour connection method.
- Inhibition of casts by Essential oils
Determination of minimum repressive concentration ( MIC ) of the indispensable oils on each trial fungus was performed by the agar dilution method. The indispensable oil was added aseptically to sterile PDA to do an agar solution with indispensable oil the concentration used was 50 & A ; micro ; l.
The ensuing agar solutions were instantly poured into petriplate dishes after whirl. The home bases were spot inoculated with 100 & A ; micro ; cubic decimeter ( 107spore/ml ) of each fungus. The vegetable oil ( olive oil ) was used as a control. The inoculated home bases were incubated at 25 & A ; deg ; degree Celsius for 3 yearss. At the terminal of the incubation period, the home bases were evaluated for the presence or absence of microbic growing. The MIC value was determined as the lowest concentration of the indispensable oil at which absence of growing was recorded12.
- Zone of Inhibition of leaf sheath Dip-Treated with Essential oils
The phonograph record diffusion method was employed to find a zone of suppression ( ZI ) of areca thenar foliage sheath and palmyra thenar foliage phonograph record treated with indispensable oils.
One hundred microlitres of a suspension incorporating 107( CFU ) /ml of single Fungi was spread on the PDA home base. Sets of 3 random triplicate specimens of the leaf sheath phonograph record of 6mm in diameter were dip-treated harmonizing to ASTM trial method D4445-91 ( American Society Testing Materials 1998 ) for 15s with each indispensable oil at its MIC obtained from suppression of fungi trials. Vegetable oil ( olive oil ) was used as a control. Different dilutions of indispensable oils were made with methyl alcohol. The phonograph record was placed in unfertile beaker that was covered tightly with a fictile sheet to forestall drying of the indispensable oil and stored in an sterile cabinet for 24h.
This allowed the draining of extra oil and clip for the indispensable oil to perforate into the leaf sheath before vaccination of the PDA. The inoculated home bases were so incubated at 25 & A ; deg ; degree Celsius for 3 yearss. The fungicidal activity of the treated foliage sheath was evaluated by mensurating the zone of suppression ( the breadth in millimeter, of the clear zone outside the phonograph record ) against the trial being12.2.
6 Mold Test on Areca thenar foliage sheath and palmyra thenar foliage utilizing Mustard oilSets of 3 triplicate specimens of all the 3 leaf sheath home base of 10mm in breadth and 70mm in length were dip-treated with mustard oil over the scope of 1-10 & A ; micro ; g/ml. Vegetable oil ( olive oil ) was used as a control. Essential oils and vegetable oil were diluted with methyl alcohol. Dip-treated specimens were held in a closed container overnight at room temperature before vaccination with spores of the trial fungus.The dip-treated specimens were inoculated with 1ml of each spore inoculants ( 107spores/ml ) and were incubated at 25 & A ; deg ; c with 100 % RH chamber for 45 yearss. The specimens were so separately rated for fungus growing screen. The specimens were so separately rated for fungus growing screen on a 0-5 graduated table ( 0, no growing ; 1, 20 % screen ; 2, 40 % screen ; 3, 60 % screen ; 4, 80 % screen ; 5, 100 % screen ) harmonizing to ASTM trial method D4445-91 ( American Society for Testing and Materials13) .
The per centum country of discoloration and fungus ( based on a control ) for each indispensable oil concentration was calculated as ( A/B ) x100, where A is the entire mark for each fungus at each concentration of indispensable oil, and B is the entire mark for each fungus in the controls12.2.7 Zone of suppression utilizing vapour stage of indispensable oilFor probe of the consequence of indispensable oil vapour 1ml of each fungal spore suspension of 107spores/ml was inoculated on a murphy dextroglucose agar and incubated at 25 & A ; deg ; C for 12 hour for acquiring the exponential growing stage of the Fungi. Then 20 & A ; micro ; cubic decimeter of each indispensable oil was put on the unfertile phonograph record ( 6mm ) in upper palpebra of the upside-down petriplates where it gets converted to indispensable oil vapor. The petriplate was sealed with parafilm and farther incubated for another 3 yearss at 25 & A ; deg ; C. Vegetable oil was used as the control. This allows the indispensable oil bluess to move against the Fungi.
Zone of suppression was observed.2.8 Inhibition of Fungis by AntifungalsDetermination of minimum repressive concentration ( MIC ) of the antifungals on each Fungi was performed by the agar dilution method.
The antifungals were added aseptically to sterile PDA. The ensuing agar solution was instantly poured into petriplate dishes after whirl. The home bases were inoculated with 100 & A ; micro ; cubic decimeter ( 107spores/ml ) of each spore inoculant. The home base that was non inoculated with the trial Fungi was used as a control. The inoculated home bases were incubated at 25 & A ; deg ; C for 3 yearss.
At the terminal of the incubation period, the home bases were evaluated for the presence or absence of fungus growing. The MIC value was determined as the lowest concentration of the antifungals at which absence of growing of the Fungis were observed.2.9 Zone of suppression of antifungals against FungiSpore suspension of each fungus at the concentration of 107spores/ml was inoculated on PDA home bases by dispersed home base technique. Discs dipped in selected antifungals ( CuSO4, OPA and Biphenyl ) were prepared and placed onto the inoculated PDA home bases, and incubated at 25 & A ; deg ; C for 3 yearss. After incubation zone of suppression was measured and tabulated.3. Consequences and Discussion3.
1 Fungal designationBased on the consequences of the 18s rRNA sequence analyses ( BLAST and evolutions ) and morphological comparings of the fungous isolates, the sequences of fungus 1 showed maximal per centum of similarity with the sequences of the speciesAspergillus Niger, while sequence of fungus 2 and 3 had higher per centum of similarity to the sequences ofAspergillus flavusandAspergillus oryzaeseverally ( Fig. 1 ) .3.2. Inhibition of Fungis by Essential oilsIn the agar dilution method ( Table 1 ) all 10 indispensable oils exhibited fungistatic consequence on the trial Fungi. Vegetable oil used at 10-500 milligram milliliter_1as a control showed no suppression on the trial Fungi.
With an MIC of 0.02 & A ; micro ; cubic decimeter milliliter-1mustard oil was the most powerful inhibitor. Eucalyptus oil and lemon grass oil had the least MIC of 50 & A ; micro ; cubic decimeter milliliter-1and 30 & A ; micro ; cubic decimeter milliliter-1severally. Cinnamon oil, split oil and lemon oil had MIC of 2 & A ; micro ; cubic decimeter milliliter-1, 4 & A ; micro ; cubic decimeter milliliter-1and 8 & A ; micro ; cubic decimeter milliliter-1severally.
Mustard oil, eucalyptus oil, lemon grass oil, cinnamon oil, split oil and lemon oil had MICs similar in all the three trial Fungi. Citronella oil camphor oil, rose oil and Mentha piperita oil had different MIC for each trial Fungi. Citronella oil, camphor oil and Mentha piperita oil had MIC of 6 & A ; micro ; cubic decimeter milliliter-1forAspergillus flavusand rose oil had MIC of 8 & A ; micro ; cubic decimeter milliliter-1. Rise oil and Mentha piperita oil had similar MIC forAspergillus oryzae( 8 & A ; micro ; cubic decimeter milliliter-1) , citronella oil and camphor oil had 2 & A ; micro ; cubic decimeter milliliter-1and 12 & A ; micro ; cubic decimeter milliliter-1MIC severally. MIC againstAspergillus Nigerwas 12 & A ; micro ; cubic decimeter milliliter-1, 10 & A ; micro ; cubic decimeter milliliter-1, 4 & A ; micro ; cubic decimeter milliliter-1and 8 & A ; micro ; cubic decimeter milliliter-1for citronella oil, camphor oil, rose oil and Mentha piperita oil severally.
Strong fungicidal activity of these oils has been reported by many writers.14, 15, 163.3 Inhibition of Fungis by antifungalsCopper sulfate inhibited all three Fungis with an MIC of 600 & A ; micro ; g ml-1in PDA. Orthophosphoric acid was besides a strong inhibitor with an MIC of 2 & A ; micro ; cubic decimeter milliliter-1,12 & A ; micro ; cubic decimeter milliliter-1and 14 & A ; micro ; cubic decimeter milliliter-1againstAspergillus Niger, Aspergillus oryzaeandAspergillus flavusseverally.
But biphenyl was a weak antifungal, when compared with other antifungals with an MIC of 260 milligrams milliliter-1against all three Fungis ( Table 2 ) .3.4. Zone of Inhibition of leaf sheath dipped in Essential oilsZone of suppression ( ZI ) of the indispensable oils at their MICs obtained via. the disc diffusion method confirmed that all the indispensable oils at their MICs inhibited the three Fungi. Even though all indispensable oils used exhibited ZI, comparatively big suppression was found in mustard oil ( Table 3 ) . It is confirmed that mustard oil was the strongest inhibitor compared to all other indispensable oils.
Cinnamon oil had the following highest zone of suppression in all the three Fungis. Clove oil besides acted as a powerful inhibitor.Aspergillus oryzaewas more sensitive to mustard oil, lemon oil, split oil, lemon grass oil and cinnamon oil.Aspergillus NigerandAspergillus flavuswas extremely sensitive to mustard oil and cinnamon oil.Aspergillus flavuswas sensitive to lemon oil excessively. Peppermint oil showed medium suppression inAspergillus oryzaeand was a weak inhibitor inAspergillus NigerandAspergillus flavus.
Rise oil acted as a medium inhibitor in all the three Fungis.Aspergillus oryzaewas extremely sensitive to lemongrass oil whileAspergillus flavusandAspergillus Nigerwere reasonably sensitive.All the three Fungis were immune to citronella oil.Aspergillus NigerandAspergillus flavuswere immune to eucalyptus oil. In the instance of camphor oil inhibitory consequence was observed onAspergillus oryzaeandAspergillus flavuswhen toddy palm foliage was employed while the same oil inhibited the growing of all the three Fungis while areca thenar foliage sheath was used.
Aspergillus Nigerwas immune to camphor oil, citronella oil and eucalyptus oil. Citronella oil had no repressive consequence exceptAspergillus flavus( areca thenar leaf sheath ) in which it showed a really mild activity. While the consequence of eucalyptus oil was observedAspergillus oryzaeandAspergillus flavuswere sensitive when areca thenar foliage sheath was employed while the same oil inhibited the growing of all the three Fungis while palmyra foliage was used.The antimicrobic agents in indispensable oils may either base on balls into the agar medium by diffusion or be released through vaporization in the headspace within the home base. This complex mechanism warrants farther probe.Table 3: Zone of suppression of leaf sheath dipped in indispensable oils.
3.5 Zone of suppression of antifungals against FungiAspergillus Niger Aspergillus oryzaeandAspergillus flavuswere opposition to biphenyl. CuSO4 and orthophosphoric acid were possible antifungals.Aspergillus Nigerwas sensitive to CuSO4 and orthophosphoric acid with a zone of measuring 15.33 & A ; plusmn ; 0.57 millimeter and 10 & A ; plusmn ; 1 millimeter severally. In the instance ofAspergillus oryzae,the zone of suppression was 15.33 & A ; plusmn ; 0.
57 millimeter and 34.66 & A ; plusmn ; 8.33 millimeter in CuSO4 and OPA severally. Whereas,Aspergillus flavuswas reasonably sensitive with a zone of 23.66 & A ; plusmn ; 1.52 millimeter for CuSO4 and 15.33 & A ; plusmn ; 0.
57 millimeter for orthophosphoric acid ( Table 4 ) .3.6.
Fungi growing on the foliage sheathEssential-oil-coating on the surface is required for effectual bar of Fungi on the surface of the areca thenar foliage sheath and Palmyra thenar foliage, and so dip-treatment was employed for fungous testing17. Growth of Fungi on leaf sheaths dip-treated with mustard oil and vegetable oil was examined after incubation for 45 yearss. The mean evaluation for mustard oil showed no growing, while the vegetable oil ( control ) showed growing in the three Fungis within few yearss. Thus the indispensable oil showed fungi opposition for up to 45 yearss.It should be noted that although oil intervention might hold some effects on exclusion of wet from the trial specimens, fungous spores were able to shoot and turn on the control specimens dip-treated with vegetable oil. As a effect, mechanisms other than moisture exclusion that are caused by constituents in indispensable oils should play a cardinal function in suppression of fungous spore sprouting.3.7.
Zone of Inhibition utilizing vapour stage of Essential oilThe consequences tabulated in Table 5 reveals the growing of Fungis on the PDA plates showed a zone of suppression ( ZI ) at 25 & A ; deg ; degree Celsius for 3 yearss. More effectual consequences were observed againstAspergillus oryzaein mustard oil, split oil, Mentha piperita oil and lemon oil. Rise oil had no consequence onAspergillus oryzae.Peppermint oil and lemon oil had really low consequence onAspergillus Niger, while the mustard oil had the highest consequence. Mustard oil and Mentha piperita oil were really effectual againstAspergillus flavusthan other oils. Vapour stage of mustard oil was efficient against all the three fungous agents. Vegetable oil ( control ) showed no suppression. The usage of indispensable oils in vapour stage was reported before by Amit and Anushree18.
4. DecisionClove oil, cinnamon oil, mustard oil, Mentha piperita oil, eucalyptus oil, citronella oil, camphor oil, rose oil, lemon oil and lemon grass oil were inhibitory to three Fungis antecedently isolated from areca thenar foliage sheaths and palmyra foliage. They were the fungous agents chiefly responsible for the impairment of the foliage merchandises and spoilage of nutrient packed in them. These indispensable oils at their MICs were capable of suppressing spore sprouting and growing of these Fungis on leaf sheath and foliage phonograph record for at least 45 yearss in storage at 25 EsC and 100 % RH. Areca palm foliage sheath and palmyra thenar foliage treated with indispensable oils can hence be considered to hold good potency as an fungicidal bio-based stuff for usage in nutrient packaging and other handcraft merchandises. Among the 10 indispensable oils employed for showing, mustard oil was more possible than others.
However, consumer sensory trials will be needed to find concentrations of indispensable oils suited for specific merchandises.RecognitionThe writers appreciatively acknowledge the support rentered by Sri Paramakalyani College, Alwarkurichi and Department of Science and Technology, New Delhi.